Antagonism, Thyroid Receptor

60-56-0

PMRYVIKBURPHAH-UHFFFAOYSA-N

Methimazole

2H-Imidazole-2-thione, 1,3-dihydro-1-methyl- 1,3-Dihydro-1-methyl-2H-imidazole-2-thione 1-Methyl-1,3-dihydroimidazole-2-thione 1-Methyl-1H-imidazole-2-thiol 1-Methyl-2-mercapto-1H-imidazole 1-Methyl-2-mercaptoimidazole 1-Methyl-4-imidazoline-2-thione 1-Methylimidazole-2(3H)-thione 1-Methylimidazole-2-thiol 1-Methylimidazole-2-thione 2-Mercapto-1-methyl-1H-imidazole 2-Mercapto-1-methylimidazole 2-Mercapto-N-methylimidazole 4-Imidazoline-2-thione, 1-methyl- Basolan Danantizol Favistan Frentirox Imidazole-2-thiol, 1-methyl- Mercaptazole Mercazole Mercazolyl Metazolo Methimazol Methylmercaptoimidazole Metothyrin Metothyrine Metotirin N-Methyl-2-mercaptoimidazole N-Methylimidazolethiol NSC 38608 Strumazol Tapazole Thacapzol Thiamazol thiamazole Thycapzol Thymidazol Thymidazole tiamazol

DTXSID4020820

51-52-5

KNAHARQHSZJURB-UHFFFAOYSA-N

6-Propyl-2-thiouracil

6-Propyl-2 thiouracil (PTU) 4(1H)-Pyrimidinone, 2,3-dihydro-6-propyl-2-thioxo- 2,3-Dihydro-6-propyl-2-thioxo-4(1H)-pyrimidinone 2-Mercapto-4-hydroxy-6-n-propylpyrimidine 2-Mercapto-4-hydroxy-6-propylpyrimidine 2-Mercapto-6-propylpyrimidin-4-ol 2-Thio-4-oxo-6-propyl-1,3-pyrimidine 2-Thio-6-propyl-1,3-pyrimidin-4-one 6-n-Propyl-2-thiouracil 6-n-Propylthiouracil 6-Propyl-2-thio-2,4(1H,3H)pyrimidinedione 6-Propylthiouracil NSC 6498 NSC 70461 Procasil Propacil propiltiouracilo Propycil Propyl-Thiorist Propylthiorit propylthiouracil Propylthiouracile Propyl-Thyracil Prothiucil Prothiurone Prothycil Prothyran Protiural Thiuragyl Thyreostat II URACIL, 4-PROPYL-2-THIO- Uracil, 6-propyl-2-thio-

DTXSID5021209

UBERON:0002421

UBERON hippocampal formation

GO:0045202

GO synapse

GO:0010468

GO regulation of gene expression

GO:0007420

GO brain development

GO:0007268

GO chemical synaptic transmission

GO:0007611

GO learning or memory

GO:0050890

GO cognition

WIKI abnormal

WIKI morphological change

WIKI decreased

Methimazole

2016-11-29T18:42:19

Propylthiouracil

2016-11-29T18:42:22

Iodine deficiency

2017-03-26T11:37:44

WCS_9606

common toxicological species human

10090

NCBI mouse

10116

NCBI rats

10116

NCBI rat

WCS_9606

common toxicological species humans

Antagonism, Thyroid Receptor

TR Antagnoism

Molecular

Thyroid hormone receptors (TR) are a nuclear receptors that are activitated by binding of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). The majority of TH bound to TR being T3 due to its 10-fold higher affinity.    Bound receptors, homodimerized or heterodimerized with retinoic acid, bind to thyroid response elements and regulate gene expression by either increasing or decreasing tragent gene transcription activity. Important to note is ligand free TR can form complexes with corepressors to inhibit gene expression.  There are <span style="background-color:#ffffff; color:#212121; font-family:BlinkMacSystemFont,-apple-system,"Segoe UI",Roboto,Oxygen,Ubuntu,Cantarell,"Fira Sans","Droid Sans","Helvetica Neue",sans-serif; font-size:16px">two major thyroid hormone receptor subtypes, thyroid receptor alpha(TRα) and thyroid receptor beta (TRβ). There are two subtypes for each, TRb1, TRb2, TRa1, and TRa2.  Notably, the carboxy-terminal structure of TRalpha2 prevents hormone binding and transscription (Sinha and Yen, 2018).   There are a large number of genes regualated by TH. These include genes involved in <span style="background-color:#ffffff; color:#212121; font-family:BlinkMacSystemFont,-apple-system,"Segoe UI",Roboto,Oxygen,Ubuntu,Cantarell,"Fira Sans","Droid Sans","Helvetica Neue",sans-serif; font-size:16px">Both TRa and TRb are known to be expressed during neurodevelopment (ref).  <span style="background-color:#ffffff; color:#212121; font-family:BlinkMacSystemFont,-apple-system,"Segoe UI",Roboto,Oxygen,Ubuntu,Cantarell,"Fira Sans","Droid Sans","Helvetica Neue",sans-serif; font-size:16px">The predominate TR form during brain develop is TRa1expression of the Sinha R, Yen PM. Cellular Action of Thyroid Hormone. [Updated 2018 Jun 20]. In: Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK285568/

High Mixed

High

During development and at adulthood

Not Specified Not Specified AOPWiki 2019-06-08T17:38:33

2021-01-11T10:14:39

Hippocampal gene expression, Altered

Tissue

Thyroid hormones control genes in the developing brain by classical ligand (T3) activation of thyroid receptors which leads to DNA binding and subsequent transcription and translation (for a review of TH rols in brain development see, Bernal 2015). Gene expression profiles have been published for the developing human and rodent hippocampus (Zhang et al., 2002; Mody et al., 2001). In both humans and rodents, the hippocampus undergoes typical stages of neurodevelopment found in most brain regions, including: cell proliferation, migration, differentiation, synapse formation, and the maturation of synaptic function. In the rodent, peak windows during pre- and post-natal periods have been identified during which major cellular and physiological events occur (see Figure 1). Each window expresses distinct patterns of gene transcription and clusters of genes increase their expression corresponding to the progression of events of hippocampal ontogeny (see Mody et al., 2001).  Tables of gene clusters associated with these phases can be found in Supplementary Tables of Mody et al. (2001). <img alt="" 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cHxGu86juOsra0JIRoaGjg+foHGAJMnT87Nzc3Pzy8qKqqoqLC0tCSEiIqKwtIVuHDhAl6qxgRcNq3GDBgwgOO43bt3w0dTU1Mw9eTn56urq0OPf/Xq1fv374U1BmsuLi5++fJl4WYh1GjbeI3BG5VeavD5fGgcHo+HC8RNmzZhhsjISBgdunTpUlJSUudZ0xrTAI3XGGlpaZwZ3L17FwOjJ02aBIm0xvTv3//9+/ccx5WXl2PIv4KCQlpaGsdxaWlpaOFZsWIFfV6oDRs2bIDE0aNHQ4qBgQEurAsLC3ft2gU1LygouHTpksC+URyXp06diq2KGgMWUUhHPy0hxNzcHAL9X79+jeaXuXPnQk5aY5SVla9evQrpx48fRysQXqYXL17cuHEDDVMcx9XU1ODYeuLECcz2lRpz69YtrD+cVFFREUzmWrVqdePGDcyJPX/w4MEcx1VXV5uYmEDKnDlzsJK4Ct+yZQvHcTweD68gXhSOkqhOnTrBvSmsMQUFBTo6OpA4btw4/G1KSgoKHl4gWmNcXV1hklFRUTFo0CBIlJeXT01N5f6uMW3atDl9+jRcX2woQoi1tTV0tuTkZDzWkiVL4FiJiYmwKFFSUnr9+jVWDFctK1euhBTa9jNnzhwYJ+/cuSMrKwuJv/76K/4cZ04wz0YqKiouX75MH4jjuLVr10JmJycnHAz/cxojzLRp0yCPsMZglD1tBoVppqysLPquv0xj+vTpgyrFcVxMTAzMPeXk5J4/f15cXFxYWPjp06fIyEi4G9XU1ARiCuqkaTUGXKavXr2CwVdUVBRU4fjx43ixOY5LT08X1hg0LPTp04cunL6LAgMDIbHxGoOLqo4dOx49elQgyvzt27dKSkpQ1Xv37n369KmwsLC4uPjFixdwCtLS0gIdF2lyjRHo9IMHD4Z0W1tbSEGNUVNTox3CN2/exPnyzZs3OY47e/YsfFRRURFwHeO+ruHDh3Mc9+nTJ2zerVu3NnCVaXg83oIFC+BXuHiiNYb2QOBKUU5Ojt51izaoUaNGQQqtMQEBAfQRzczMIH3kyJH11aqkpASXj9u3b4fEr9eY27dv49WEJdedO3dg3q2hoQGO8cLCwpKSkitXrkC2rl27wpiAFh4jIyPwksbFxYHBFufydCcMDQ0V7oRSUlLQhYQ1JiIiAu53KSkp2pjMUZaDXr16QQpqjKqqKu1Rv3XrFvYfkExaY9atW4c5z58/D4kCBigPDw9Ix42N6Ojt06dPSUnJx48fCwsLy8rKcKzw8PCAnKgx4AJAcIGFusVx3JQpU/6xGwB8Ph99ot27d0cX9X9OY+Tk5FxcXPpRnD9/HvIIawxeAGtrawiI2rp1K4i5oaEhysaXaczatWvpGuLiWkxMTFdXV09Pr3379vr6+hoaGpjemJ3kzaExHMc5OztDCkzf0Dq/Z88ejuNSUlKENQancgLXns/n4yiMQfGN15hLly7RvgQ9Pb1ly5Zhsz979gzSRUREtLW1oRn19PS0tbXxV/Xt4KE1xtfX99KlS+f+x59//onztcZrjIBPG4enTp06QX9AjenYsSN9sV68eIHG6DNnznDUZbW0tBSIL0K7f8+ePTmOy8jIQEsUHSghAI/HCwoKmjVr1oABAywtLQ0MDPDcMYqU1pi9e/fib3FEVlFRoQV71qxZkBlVitYYmDsjKAOOjo6YmJeXd/DgwXHjxjk5OVlYWOjr66PRD6cjX68xx44dw/5z/fp1jrKetWrVCjpM+/btO3TogNN5RUVF6GP37t2Dc5eQkIAdUehGsrOzg0WYQCfU19cX7oTh4eFcXRpz+vRpSNHW1hbwHeIOrfbt25eXl3OUxnTo0IGegL58+RJts2AdpTWGnjFfv34dEjU0NGhfMvZ2XF7Mnj0bUmRlZeGMoInatm0L6VZWVuB3QI0R2FSHVtalS5di4uTJkyHR29tb+ErdvXt30aJFgwcPtra2NjQ0RIOblZXVf1djGohdFtYYPp+Pkbvi4uL0hnbanEVrDB3d1LDG4NISwL4lDPRLExOTxjydqZk05sCBA5BiYGAQHh4OZp+2bdvCYq5OjUG/wsKFCwXKx0bAboEaQ7s069QYjuNOnTpFm4wJId26dQOzPnpf60NXVzczM7POs6Y15tq1awLf4jDUeI0R8P2sW7cO6wAmR9QYfX192nKVlpbWrl07+sRxkWFnZydQMZQu6NsvX75ERyv6qAQoLS2ljSQC1KkxO3bswJ+jxrRr146OIsPBBUugNYa+ZTjK4Na7d29IefnypYWFRX21akKNQVWWkZEBUxJO0oWBM7W3t4dLVllZ2bVrV/gK7GA438IpxT92Qh0dHbhxhDVmx44dkNKhQweBsOD9+/fDV+rq6vAVaoyenh4dgpyeno6uCzAG0hpDh4fg1FZdXZ3eVDBx4kRIHz16NKQIbPEWQFxcHC142A3QGwRgrEFjNKampmbp0qX1He4/rTENxC4La0xiYiKYHZSVlaGrSUpKGhkZCYTV16cxuLJrjMZcvnwZ0uXk5P7444+IiIhwinv37tHbcRqgmTTm1atXMLOWkpLq1q0btAYOJXVqDA6148ePpwuvqqrCgM7NmzcLZKY1pqysDOKayN81huO4vLy8Q4cO0e5xmHA9fvwYPoqKih49elS4GYXDMZEv2B/TsMZs3LiRLgFNzz169IAZH72OoTc0PHnyBOfvMNFGr7KxsbHA1gcsdsiQIXAtMNbu7NmzdZ4plqaiohIQEHDr1q3IyEiMgmsmjRF4dAI6nCFzVVUVzjMsLS2PHTt29+7d8PBwVJ2m0pgPHz6g36h79+4wGhw6dAhS1NTUgoOD6W4TFhYWFhaGkWAcJVF9+/Z99+4drBgkJCTQ1vTXX39hJzxy5EgDnVBYY3AyRwdxABgL0KNHD6g2vY6hA8CePn2K/QdmS1+vMTNmzIAUa2vryMhI+ozCwsIiIyPRzflZGoMLJgFbGa41paWlV65cef369cjISOy3/3WNqW9vkbDGgHRLSEj89ddf8fHx9+7de/HihfD2q/DwcPihlJQUhnxw1Mk3RmOePn0KQ4OEhISAHfaz2Lp1K5Svq6tLu0+Bjx8/3r9//9GjR/QuDWGENYaj7P4Imhnr1BicqHbp0oX2PCUkJKD3GwOO0VFPu/5CQ0Nxq4SAxgC1tbW459TY2JjH471//76B+OOGaXKN6d+/P3a2iooKnP+iKQk1Rk5ODsMfOI7bvHmzQI/CmEM6rIjjuJqaGvRCg5m7oKAA57AYXADk5+eDjQUH7sOHD+O3eNGbSWNwExjHce/evQOPBflftHp6ejosv6SlpemdIuh++zKN6d+/P51eUFBAx6phLA8E2sGFaGAWAoSFhYHLRF1dfd68edAUdnZ2eK0zMzOxI2HcWp0IawzGI4iJiQl0YGwKnPKjxsjJyd25cwdz4lAmKSkJ4ctfrzFYZteuXRtun8/SGJQumCEheJnoZ8xcunQJEr8HjQEh6dOnDyFERERkwoQJwcHBDx8+DA0NjY6OFtgZm5iYiDE/gwYNyszMfPnyJW2LaIzGlJSUYOy5vb09bc8pLS0NCQmBLZb1kZGRERMTk5iYiE9mU1dXf/DgwfPnz1NSUuB0ysvL4TKLiIgsW7asgdLq1Bjaik0I0dLSwnaoU2PQX0oIWbFiRWlpKZ/Pz8zMRNeOsrIyRFJxlD9TTk7u3LlzhYWFp0+fhtA1ADSGx+Pt37+f3jWCM1ArK6va2tra2los38TEhPZglZeXh4eHQ9xknTS5xsBtlpaWlp+fTwfb4D5TOq5MXV09KCiouLj45s2bKBLW1tYwG3j9+jUavt3c3LKysvh8fmlp6cKFC7GE27dvcxzH5/PR9966devjx4+XlJR8+vTp/PnzBgYGIOqodliTtLQ0FJ5m0hhCyKxZs7Kzs9+8eYM7UURERGBwjI+PhxRpaWncs0I3xZdpTM+ePRMSEhISEiIiInbt2oWBBtBhcKGfl5eHO7Hc3d3pVUtxcXFwcDDdkSoqKnBXEJolad9bdXU1rtKMjIxoySwvL4+IiMB7RFhjCgsL0T5sbW2dkpJSW1tbUVEREBCAbvwjR47Az+m4MnV19YsXLxYXF9+6dQut+lZWVtB/vl5joqOj8TlVixcvhskKdJLs7OygoCA04XyWxvj7+0OilpZWUlJSfn4+rN4wNA63ZObn52Pif1Fj8FaE2W6deVBjbGxsIM9vv/2GgaGIuLi4srLy5MmTUQZ4PJ6joyNmkJOTQ8mBLkhrDOakI1CBkydPYiGamprDhw+fPn36wIED4S6lL4wAubm5+vr6kpKS0tLS2A8IIZKSklJSUvLy8jDzffv2LYYPYmhTneCqnJ4GZmZmos+NEDJ58mT8KiUlBRqK1piamho6AL9Tp06Ojo60T4sONKLDSQkhOJ6i+xo0prS0VEFBoVWrVkOGDPHz85s2bRr6LTBYBU2XhBBlZeWhQ4fOmDFj6NChBgYGIiIiAiGSNPHx8bhsEtaYPXv2wFeysrKftT+mTZs29Fl37NiR3upM/o66ujoG9RIqYJfjuDVr1mC6iopK3759cTAihAwbNgyXrc+fP0dTCSGkffv2OIBCM/r4+MBHNTW1VatWLVmyBM1H5O8ag4NyYzQG56R1+vwBJSUlvLiEkJ9++gmqXVJSglslLC0tf/nll4kTJ9JN8Vkag3sYRUREpKSkpKSkcIAGDA0NBZ6tvm3bNvxWX19/1KhR06ZN+/nnn2ETMR4dEIhTFRcXp7cnc3/vhEpKSgKdEO1Cde6PobfvyMvL29vb4+4uQkjv3r3RTyOwP4YI9R/0gVVXV6MuNkZj0AhB3y90zLqpqemECRMmTZrUt29fuAdx+z12A4w0A3A0oOPKMGCSEKKoqNi2bdtBgwZx1I54GRmZ+fPnr1mzBtfr5O8a85977nKHDh3q0xg8K1zrZGVl4RZI+sYA7O3t8TwfPHhAT7oJIRoaGrt27YItct26dUONQRcCvaEdWbFiBd1FaOg9OgKkpKTU+RMEnm+Rl5eHcTIDBw5soLkwDv2nn36i0+nH2dJ70JKTkzGdfoLFixcv6I3BSNu2bXfu3EmXzOPxcOqEjBs3Di/cb7/9xnFcTU1Nz549hQvs168f7TPfvn07qqkADSzg4uLicCQS9mTs3LkTC8HnJkVGRmIijhGoMX369MEwYjxxevsFaoympqbwHv6xY8fS1s6SkpI6HycsIiIyY8YMgU0/e/bsoWUGUFVVhaHwxYsX9NN9AHd3d7DWwsZbjuP4fD7KWJ0TAklJSfqBaWhbx96FGiMiIuLs7CwQGq6rq0vb/c6fPy9QZ2VlZfSoo+suKSkJM9S3Kh03bpxwQwEaGhrz58+nVyoAj8fDwVEYgf6Au7MBtK7T7Nixo75OiIMszlEIIbh6q62tpffb0gwfPpyOXEWN0dDQEO4/Y8aMwf5TXV2NT9zAHYEc5QaWlZWlQwRxKUzvAs7Ly6vzcQ/k7yqL3QB21yH29vaQjksTjuOKi4vpTaPkf8+sysnJEb7T+/fvD+varl274tiLNvxv/P6Y8PBwb29vT0/PXbt2CTtUgKioqBEjRmCe8vJyEF4xMbHNmze/efMmKSnp6dOnS5YswbUC/Tj3+Pj40aNH9+rVq0+fPrNmzYLdTKtXr/b09Ny+fTse9PDhw56enp6envXF/ERERCxdutTDw6NPnz62trbOzs4zZsy4dOkSLk6Fqays3LBhw7x58xYK4evru3z5cuiXfD7/1KlTnp6eI0aMaMBkxHHc48ePR44c6enpiRu2gbi4uLFjx3p6eq5atYoe/kpLS/38/Dw9PWfNmiVw9xYXF+/fv3/QoEG9evWys7Pr27fv3LlzBSZ9AJ/PDwwMdHJy6tmzp6urKxgEnj175unpOWrUKNxFnJGRsXr1aldXV1tb2z59+gwZMuTo0aPCTwB89uyZv7//8OHDHRwcbG1tHR0dJ0+efPbs2QbeqVNUVLRixQpPT88pU6ZAuBFNUlLSxIkTPX1tvYkAACAASURBVD09V6xYgYf78OHDtGnTPD09fX19sWTUmM2bN2dkZEydOrV37969evWaOHEibeXjKI3p2rVrbm7uvn374PRdXFwOHDggHJzC4/H+/PPPUaNG9enTx87Ork+fPmPGjAkODq7zdKKioqZOnWpvb29ra+vk5LRq1Srok0BmZqavry8cbsCAAX/++SePx1u8eLGnpycdALZ9+3ZPT89hw4bRz5d78+bNlClTPD09lyxZQrfn9evXoW9jmDK9jrlw4UJMTIy3tzfU3NfXV3ijUkRExOjRo+3t7e3s7Hx8fOLj49+/fw9dEXvsx48f586d6+npOW3atPqeDPTkyZP58+cvWLAA7oIFCxYsWbJk586dISEhdISuMLdu3VqwYIG7u3vv3r1tbW379es3b968mzdv0g5FjuNqamoCAgLgZH18fCAQWZh/7ITZ2dnTp0/39PScN2+eQIROSEjIpEmToCl69eo1bNgw4XkPakyXLl1yc3P3799P9x9a9vh8/pYtW+Dep+++V69eTZo0SaBXcxwXFBQEZyfw4L7q6upz587NmDHDzc3Nzs7Ozs5uwIABK1eu/Ouvv3CIu3btmkA3AI4fP+7p6enl5UW7jjiOy8nJmT9/PtzRbm5u+G1RUdGaNWtcXV179uzp7Oy8b9++mpoa6JBbtmzBw505cwYOJxwL+mU077uWaUJDQ/H+p9OLi4vRokI//w6ora2tT8O+gAYefdri4PF4jTwd4VCF+gpsZFM3ssCmQjh2GRxFwjlRY/T09HDcaUxt+Xx+U51+sz7fntYYtPs1pht8855fW1vbtHX44k7I4/HqC4VFjWnfvj1K1L/W2xt/RzeS+mr+L7+C4d/TGDQIwKMXcnJyioqK0tLS5syZA8ZoZWXlFvcqM8a/QH37Y4Spb3/Md0MD+2MYX099+2MYX8O/pzF5eXm4OQNmCt26daOfOi5gR2IwANy3KxA6KMzvv/8OOXV0dL7LMaKsrAwdP+BRYzQhOEfR1tZm892m4t/TGI7jkpOTR40aJeDtl5WVHThwYANP6WD84OA+BoE9mMJcvHgRcnbu3LkxD6NrcZSXl2OcdH0bQhlfTFBQELRtx44dBd5zyPhi/lWNAd68eXPt2rVjx44dOXLkzJkzP+YrURmNJzEx8ejRo6dPnxZ4bZownz59CgoKOnToUH0xuN8BT548OXTo0MWLFxuItmB8Gdh/6OAjxlfyDTSGwWAwGD8ITGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYxn+CrKysgICAVatWXbhwAZ9NGRQU5O/vHxAQ8ObNmyY5Sk5Ozt69e3fu3AlvXK6P3NzcHTt2+Pv7//777//yA0AZLYKcnJy3b99+61q0DL4TjYmNjT158uQvv/yycePGM2fOCD9J/nsiPz//+vXr+/btW7169f79+2/fvi38KP4WB75bqV27dvAYj4qKCnhdECHk6NGjTXKUEydO4APNBN4QQxMcHAzZpKWlG352fVORnJx84cKF7du3r1mzJjAw8Ny5cw2/p/W/QFFRUWho6O3bt+/8nZCQkPv37zf89vEWzf79+zU0NFRVVceNG/cdn2ZT0eI1Jjs7e/z48fhCe0BZWdnPz49+gnd5eXl6ejq+iriFUl5evn79enzPG2JkZHTu3LnmPvr79+/T0tIaeO/O14Aag4+8LS8vx2cMN5XG4CsR6bdnCoMao6Ki0lRLqPqIjIwcMmSIkpKSwDWVk5OztbWFdzn/N6HfSy2AmJhYne80+j7A97tLSUnhK1wZ9dGyNaakpARfBieAhoZGYWEhZKuqqho4cKCKioquri79usmWRVFR0YABA+q7q9u2bSv8LsIm5M8//9TV1VVRUaFfQtyE4GPV8bH85eXl+LbgpnrG8LFjx6DAzp07C7zDiubGjRuQTVVVtVlNIhs2bMA32NeJoqJic4vcF1PfOxyBJ0+efOsKNhczZ86EczQ2NmaPZ/5HWrbGbNq0CS62uLj4smXLwsLCQkNDd+zYYWxs3LVrVxxEeDyehoYG5Gyhb92ora3F17XC2DdhwoRt27atXbvWxcUFXsATHx/ffBXAVyN369atOcr/ATXm119/pQdlDw+P48ePX7x48bfffvPz88M34+I7g/9ruLu7Qw2NjIx27ty5bdu2rVu3bt26NSAgYM+ePd/lc6+Bjx8/BgcHX79+nS1iGkPL1hh857azszOdXlJS8vTpU3AdV1dXZ2Zm4ls39u7dW1BQIPzg7vz8/LS0tOjo6MzMzAZuDx6Pl52dnZGRkZWVVVBQUFBQkJ+fjwsmpLKyMiMjIzo6Oi0trYGxLCsrKy4u7sWLF/W94xa5cuUKDkaGhoZxcXH0t0FBQT4+PgJTKh6P9+bNm+jo6Li4uHfv3tVZ7MePH+nKZ2RkxMfHCz/euLi4GOXcxMTk/fv3eXl5+Lrc6urqnJwcfB/4ixcv4uPjBezUfD7/7du3MTExsbGxdY7aX6Axb9++jY2NffnyZQOviqmsrExLS0tMTIRrii+YaaTGqKioQOEfPnyIjY1NTU0VWMOVlpbm5ubW6dr5+PFjbm6uwEuFkbi4OElJSVSyM2fOCOe5evXq9OnT6RfOA9iY9Z17WVlZbm4uvlQxJycnPj7+zZs3DbxmsaKiAntsI5/ojBpDv6NegE+fPuXl5QlXsqioKCcnp7S0FD7W1NTk5OSgGbaysjI5Ofn58+d1Ls2LiorwBXTFxcUxMTHCNvDs7OyEhAR4t3R9dSssLExNTYUOWefS/MOHD4mJiYmJicKXgKv/1aKlpaUZGRl//fVXcnJynfXn8/l5eXnYZ3g8XnJycmJi4nf5Vj2upWuMra0t9PKePXvWl8fX11dTU1NcXBxytmvXTk9Pr1OnTmA04/P5V65cGT16tI6OjpiYGCFESkrKwMBg2bJlwjfGyZMn+/bt265dO1FRUQUFhQ4dOujp6bVv375jx470a7oPHTrUs2dPsIGIiYl169Ztx44dAkWFhoa6ubnJy8tDrTQ0NGbOnNnAqDd48GDIKSYmFhoa2nCz8Hi8AwcO/PTTTzIyMiIiIqKiovLy8u7u7gLBVDdu3OjcuXOHDh3CwsLS0tJGjhzZunVrMTExNTW1UaNGoa87NjbW0NBQUVERKtCqVav27dtra2uPGDECVHzMmDEaGhre3t4FBQVz5sxp1aqVmJjYpk2b8EDHjh1zdnZu3bo1VKZ169YDBgwICgqiK/NZGnP58mVXV9fWrVsTQkRERHR1dRcvXizceleuXOnZs6eEhISYmJiBgcGBAwcOHz4Ma75GaoyOjs6zZ88WLFgAF11cXNzBweHZs2eQLS0tzdLSUlNT09jYOCYmhi4hPDy8c+fOmpqa2EoCTJ48GScNn+VL+/PPP52dnWVlZQkhoqKi7du3X7ZsGS1yGRkZ3bt319LSCgwMLC4uXr58uYaGhpiYmLy8fN++ff/66y/hMvfv329jY4M91sjIaNeuXf9YE9QYd3f3OjN8/Pixb9++2traurq669atw/SbN2927dpVQ0Nj4sSJkDJz5kx1dXUo5/LlyzY2NuLi4qKiourq6qtXr6YbMDw83MDAQE9P79atW48fPzY0NBQVFdXW1saJ0bNnz0aOHKmiogJ1U1JSGj58+MuXL+mKFRYWLl++vGPHjqKiooQQGRkZe3v78PBwzBAdHT18+HBlZWUoRFlZ2dvbGydqp0+f7ty5s5aW1pw5c+i65ebmrlq1ytzcXEJCghAiLi4O5ygQvrF582YdHR1LS8usrKywsLC+ffuKi4uLiYnp6+uvWLGivklJy6Vla8zo0aPxRp08ebJwCFBtba2JiQmpC3jh1YULF+r8lhAyePBgejK+Zs2a+nISQhYvXgyHQ1utAN7e3th7wsPDYXwUICwsrM7TzMrKUlNTgzy9e/du+LXz5eXlI0aMqLMOIiIiBw8exJxo/ho6dKi+vr5A5t69e8M0E0d/AVq1agUvBgfPvJaWloeHB32+HMfV1NRMmjSpvkYLCAjAyjReY3bs2FFnadbW1vS08dy5czBpoNHW1oZhpZEaIy8vL9wympqaT58+5TguOzsbX7g3ffp0uoSpU6diMwqXX1hY2KFDB8jg7Ozc8AWl2bJlS53nbmdnh1OiJ0+eQKKtra2wt1JZWfnFixdYII/HmzZtWp1l+vj4NPzid9SYYcOG1ZmBx+ONGTMG8sjIyIAtt6SkxNzcHBK3bdsGOS0tLQkhampqs2bNEq7J7NmzsUy0dg4ePFhXVxfzgPDfu3cP1UXgkj148ABKKCsrGzRokHCeoUOHQoYXL15oaWkJZ8BOuGLFCkixsrLCa5eZmYnnJYCKigr9Qhq4PUVFRUeOHCktLS2Qee7cuY3rCy2Glq0x+N5DQFtb29/fX8BIGhAQ8PPPP8O8jxBiYWExdOhQd3f3yMhIjuO2b99OCOnatauvr+/BgwePHj1KD9D4ds6YmBgYsFRVVY8ePRoVFbVgwQJI0dDQmD59enR0NMdxv/32G/xQWVl5x44dUVFRe/fuRXk4ffo0lObm5gYp48ePj4yMvHfv3rp166ytret7s1ZYWBjMvgkhS5cubbhNVq9ejfV3dXX19/efPXu2qqoqVgx9yPv376dbT1dX18PDAx1XhJDff/+d47iXL1+OGDHCyMgIEuXl5d3c3AYNGrR48WI+n8/n80HFsYaamppt2rSBcSEwMBBL69u3r5+fn6+vr7a2NqTIycnhBLORGvPo0SOYJLZq1WrZsmWRkZGnT5/u1q2bQOOUl5djhSUkJNzd3XFABBqpMYCqqurIkSNhHARw5o5jor6+PppYS0pKUJloUUeePXuG+rdv3z5M//Tp07t377IoaPmJiIgAgZSSkvLz84uMjDx58mSXLl2gnFWrVkG26OhoyAbIysq6u7vTM61Zs2ZhmQcPHoREFRWV3bt3R0VF7d69G4fpCxcuNNDTsElNTU33Uhw4cADNPpmZmbgaGDVqFEeF9llaWmLMfe/eveku5ODg4OnpieOviIgIjtFo7cSLq6Oj06ZNm4yMjKKiIlTuESNGhISEBAcHY2CCvb09SObly5fxkl24cOHx48cnTpwYMGDA/PnzBa6pm5vb3bt3IyMjt27damdnd/HiRciwdu1aLBMN8nggCQmJ0aNHr169etSoURjQYW9vjxPWcePG0SdrYWExaNAgGRkZ+CgpKZmQkNBAs7c4WrbGcBw3b9488ndUVFTWrl1LG1irqqowChbGTSQ1NfXUqVMCDpUePXpA5i1btkDK7t27IWXt2rWYDSx1qqqqMN+vrKyEYUhUVPTw4cOY7fDhw/BbcBrxeDx0Dj18+BCzlZeX1xevha+AJYTs3r27gdbIycnBAWLy5MmYfv/+fVRZf39/SKQ1xsHB4fXr1xzHJSQkoMyMHz8eS9izZw8ODXRQOI/Ho8evGTNmfPjw4fXr1/n5+cXFxRhmPWLECPxVTEwMTv/nzJkDiY3UmPHjxwv8kOO4x48fgy1UV1cXpvPnz5+HbCIiIjiI//HHHzjeNV5jLC0tweteWlqKFktZWVlYDYSFhaFaXLlyBX5+69YtGEGUlJQyMzOFyw8JCcHyr127BokfP37s06ePmpqaOoWbmxvUk8/njxw5En4Ci2a8slCBTp06gZeR1hhtbe2IiAgo3MHBARJNTExgvKuoqDAzM4Mee+LECSxz3759kLN///71NRFHaYwwOBxz1IgsIyNz48aN7t27w3Whw7JBYwghEhISW7duBVdHUFAQygwuZWiNUVNTu3nzZnZ2NkwrDx06BOl2dnZYcmlpKei9iIgITCvXr18P2YYPH06fDjqHsDL0WFFVVYV2CGGNuXfvHtZq7969+Cs0FdAXGjQGmD59OloLUJDoacd3QIvXGI7jtm3bpqOjI9DLx44di0N2ZWUlTnBOnTr1jwUOHz5c4GZetWoVpOzfvx+z9evXjxAiLi4Ow010dDR4cRUVFVNTU6uqqiorK2tqah48eAD3vK6uLsykcEbco0eP69ev/+M2rtOnT+N5HTlypIGcuLFDTk5OIB4JDVmDBg2CFNQYHR0dOihgwoQJkO7m5oaJtMbQWkhrTL9+/eh594MHD9DFJfDyWpQKBwcHSGmMxnz8+BFFKzg4mMfjVVZWVlVV5eTkwEJNVFQU5oALFiyAbObm5nSVtm7dCumNt5XRjpaIiAh07MEcv6amxsLCAlLGjRsH2XDjyMCBA+ss/88//8QLisH0GRkZGAWAiIuLw1IvLy8PDTihoaE1NTWVlZXV1dVZWVmwt0ZCQiIlJYWjNEZcXPzGjRt4UFyyaGlpwZLr8ePHMK4pKSm9evUKe2xERARopL6+fgOdEzVGVFRUSkpKUlJSUlKyVatWUlJSaADgOK6wsBBneGgidnJyoovCYX3KlCl0OtxidD9BjZGWlhZw6eEMYPXq1RzHVVZWVlZW8vl8FxcXSIddVgcOHMDKrF+/Xjg2bMiQIdhJTp8+LdxPaI0RSDEyMqINjGVlZdiHcXqKGuPk5ISdk8fjgd4TQmjf1XfA96AxHMe9e/du48aNuD4AcDguKytDjaHna0B1dfXly5cXLVrk7e3t5ORkZWWF/m3UGDTK2djYJCYm5uXlnThxAlYGurq6MBO5dOkS5GnVqpWxsbGZmZmpqamFhUXnzp3xrkhPT+eoXg5YWFgEBgbiNEoY2iRIL6SEwRmogYGBQLAT+pPMzMxgSYEaY2pqSt8YdQ6Ru3btwtrS4w6tMQcOHKCPiNsqtbW1BQLn0IbWoUMHKK0xGpOamooz9C5dupibm5uampqZmRkZGYEBjRACcQ04S5g6dSp93M+NK1NTU6Oj7DIzM9HyiZPNjRs3Qoq6ujosiNFMV9/W0bt37+IFRYd/VVXVihUrhg4d6u3tbWNjA9+2adMmOTmZ47iEhAT8iYGBAZy7ubm5oaEhyt6dO3c4SmOkpKRo1wu6HrW1tUFjMKW+HisrK5uRkVFfK6HGODg4xMTEREdHR0dHP3v2LCYmRsBxfeLECdo3JicnJzDnQI355Zdf6PQlS5ZAeteuXWFxg1ewU6dO9OMtamtr0WSqra1tYWFhamoKTYSRNStXruQ47v3797SPTU1NbfLkybRb/vr164TC0NBw3bp1dASQsMaMHTsWUsANSYOG8UmTJkEKagyGPADoPFu/fn19bd4S+U40BsjJyZk5cyYaOvv16wfpDWhMQUFBA0t+1Bgej4dx0rKysjgvI5TfEr2R9dGtWzewZtTW1m7ZskVga7e9vb1w0DBw9+5dPKkJEyY00AK0kAhY3nA3RseOHeEr1BhjY2P6dp0/fz6kf67G7Nmzhz4iGgo6duwoIHh4aHV1dfiqMRqD3uz6kJOTg3UMXqwFCxbQx/3K/TG5ubk4PAUGBkJiUlISTs+vXr368uVLuFjKysr17a2Jj4+XkpKCn8DAJwC2BmpMZGRkw+euoKCA62nUGHqzPZaJGoPGpfowMjJqoJXwxvH09KwvDxAbG0tvNVVRURFoGdQYgSkU9mc9PT3otLTG0HsMaB9YnYiLi+PeuJiYGLQcYjufP38eSztx4gQdUEAIMTQ0RMEW1pj+/ftDioBscJT9AE1zqDFjxozBbHw+v0+fPpDONOa/jp2dHd4hMGEvKyvD/nfy5Ek6M9qFNDU1N23a9Mcff1y7dg37H2pMZmYmzJLQNUcI0dLS2rJlC6520QegoKBw8uTJq1evXqG4dOlSWloafejU1NT169fjnJEQsnDhwjrPKDs7W11dHfJ07NixAfMFzqkF7kCO45YuXQpfOTo6QspnaQwKhqWlJT1LpTVGwFeEiyoNDQ2BHQa428bKygquUWM0JjExEdvq119/FWjhoKAgiLzgOG7o0KF13vO4tGqkxqirq9MrsMzMTHR30TtacIiZMmUKXgIPD4/6yi8rK8O1TufOnYX3o5w6dQq+RY2Jjo7Gc9+2bZvwueNMvPEag+O1oqLi77//Tpd5+fLly5cvw7K7Pv4xdhkRDnScN28enQE15tdff6XTp0+fDuk2NjZwo9EaQ+9yq6mpwVtp4sSJ165dE2if8PBwerFeU1MTFBTk7u6OC6x27drRk7z379/v3LmTDvTACyqsMQMHDoQUYbnFB88sWbIEUpjGtBhqamrqdKhigIeFhQX0y/Lycux/tDe+qKgI/NsiIiL03hEMt0WN8fX1hWwhISEPHz68ePFiVFSUwCCFfhcBG0XDfPz40dvbGw5nZ2dX38YuT09P7Ot1znwfPHhQW1uLY6iUlNTjx4/pDNiD0Xz0WRqzd+9eSDQyMqpvHSOgMWhzkJCQoA30HGVAGDFiBKQ0RmNyc3NxiG94TwlGkHft2pUeWTBCpJEaIyMjc/fuXUzHSqIDGTh58iSkKygo4GxG2CpLs2zZMrygY8eOFXiqKVpHUWOysrLQhHv58uUGSm68xmC8ooyMjMAEqDGgxjSgphzH3b59G2MycSyWlpam9xGjxtBjdHV1NSoxbvOsT2M4juvbty98Rcc6/yNBQUG4DBXopRzHVVZWoulYT08P2k1YY2bPng0p7du3p5fs79+/x5BOHHmYxrQYkpOTtbS0vL294cHDPB6voqJi//79GIuCwYi1tbX4ZA5vb+/q6uqCgoLy8vKcnBwFBQVCiKioKIbPJyUloSChxoDbUEREZObMmZGRkYmJic+ePUtNTaUfEPnx40ccEwcNGkTv2uXxeFFRUTBYVFZWnjp1ijZz44aPvn370iFbNHfv3qUDUseOHXv79u309PSUlJRz584NGjSodevWGRkZdFyZm5sb7Pzn8/kBAQFobTt79iyU+Vkag+olJycXERFRXV0NE3x6B5KAxuTl5aHBwcHBAXdcHzhwAF0IGEPRGI3h8Xjo1zU0NKSHRT6fHxcXh8/IwjUlIWTZsmXl5eWfPn3y8/PDxMbHlbVv3z44OLimpiYuLg4Dhbt160b/PDc3V2BHhaqqap2bw5EPHz7Qzza1tbW9cOFCSkpKWVlZamrq3LlzIR01pqamxtXVFRLNzc0hCBDPPTY2FneGNl5jCgoKUBGHDh1KR1fW1NQ8fPgQggjqAzXG2dk5MzMz/X+kpaWhKayqqqpXr16Qzd/f/8OHDxhSSO+qQY0hhEyePDknJ6esrGzx4sWYiP2kAY3ZsGEDfCUvLy+gFm/evLl37x7cXPfv36dDIYqLi9FIADF458+fpx/LhLHOnTt3BqepsMbcunULq7p8+XKY1hQXF+MGPklJSQzTZxrTYoiPj4dxU0REREtLy8TEhDbIKisr0yt9jPskhHTp0kVdXf3YsWMcx1lbW0OimZnZoUOH/P39cd5BKI05dOgQGtAJIWJiYrBfXUdHx9fXF0dPDHEmhBgYGEybNm3FihU+Pj5mZmatWrVas2YNx3F5eXmtWrVSVlaeMmVKYGDgokWL8IgQD1Mf6P8EREREFBUVQSMBWDxhaCYhREdHx93dHUOx4a5AOfksjaF9IUpKSh06dLCwsIBVV30aI9AgGhoagwYNwkczEEIsLS1xXGtk7DLtmlJXVx8zZszKlSsnTZrUs2dPWVnZIUOGQLaSkhLaCNmxY0eMB/msPZjY1F26dEHXMaGcMciUKVPon4wcObKBSwlEREQI+OQUFBS0tLToa9qqVSscm+haaWpqjhs3buXKlRMnTrSxsZGRkcEVYeM1hvvf/jCgW7du06dPX758uY+Pj6mpqYSEBOxTrg/cySghIaGkpKT4PxQUFNq1awfxeMePH4c8KioqILoYokk/sYLWGLiy9L3cvn17nLE1oDFZWVm49UpKSsrd3X358uWzZ892cnJSUlLS0dGBTg7bY52dnTds2BAQEICrH21tbTBa6urqtm7d2sfHZ9u2bf7+/ujKRUkQjl3m8/m4RCOEmJiYDB06FGckhBBfX1+sJ9OYFsOnT58cHR1JXRgaGgo8cCUsLEzgAbcwTFy7dk14qy16jHHP7cePHw0MDLD7CueHp3XV1tbiBm9hdu7cyXFcaWkp9loaJyenhh9YxOfz/fz8cJuLAJ6enjDJordxCODh4UFPrtGNr6enR2sMGprQcwOn5uXlRZfWtm1b0BgcDrZv3y5Q5+rqarQECtC/f396Mo5jBz4irLy8HCeY9E7Gbdu2Ccf4AnRARFBQkMCVkpCQWLRoEcRraGhoNPBUuqtXr2KrYuQr4uLiIqxP4eHhuDgjf/fWNMDTp0+dnZ3rPBfAwcGBXl788ssvGEEnwIwZM7BMTKQDr7GFFRQUcHTm8XgTJ06s7+h0pL4w6PSqk3v37hUVFeGqGg28tHPRwsICLAGoMdbW1gK6KysrS++kwagNjOKjuXXrFgb+CWBoaAh+RPQF0sjJyaEFUiAcADAxMcEoZ9zn3717d3THvnjxwtDQUPiHEhISixcvpl2YGPRIP+eNz+fDziFC7WD7PmjBGsNxXFFR0ZEjR4YOHWpubm5sbGxtbT1w4MADBw7UOXzcuHGjf//+ENTo6emJq5z79+97enpaWloaGxs7OTkFBwfn5eV5eXk5OjpC566pqcFnHi9cuDApKenRo0d3796dM2cODiuwygYuX748ZcqUvn37mpmZGRsb9+jRY9SoUSdPnkTXbmxs7IwZM3r06GFsbGxubu7o6Lhnz55GPogwNjbW39/fyckJCre1tZ08efK1a9cE9pyeOnVq8ODBZmZmJiYmpqamP//88/HjxwUMcY8ePRowYICjo6O/vz/91bVr1xwdHR0dHendZNDaixYt6tWrl5GRUY8ePTCKbPXq1Y6Ojv369RMISAVqa2vPnz/v4eFhbm4OlXFxcTlw4IDAc0ri4uIGDRrk6Oi4cOFCOJfa2tply5Y5Ojr+/PPP6MwHHj58OH/+fFdXVwsLC2NjY0tLy6FDh+7evVsg7r03NAAAIABJREFUMO/evXtDhw41NTU1MTEZPHgweFbWrl3r6Oi4aNGiBh4QmZKSMnDgQEdHxzt37pSUlKxevdrKysrIyMja2nrdunV1PgGTz+ejk0lVVfUfH3JKt8+NGzdmzpzZo0cPExMTExMTMzMze3v7WbNm/fHHH8IPb71///68efP69esH5969e3cPD4+9e/fiEbOzs0eNGuXo6Dh69Gg64vbly5fQwgsWLBCIOQwKCpo8efJPP/1kamoKPdbHx+fUqVMNvMaN47g7d+6MHz9+4sSJk/7OhAkTpk2blp+fn5GR4eTk5OjoOH78ePpEzp8/7+zs7OjoOGzYMFBr1JiAgID4+HgvLy+IPPby8rp//z590NjYWLg0S5YsqXPP8qtXr9atWzd48GC4v8zMzFxcXNauXYsu0qKioh07djg7O8PJ2tnZTZ8+nXYOpaenL1682NbW1tjY2NTU1MHBYdOmTfRjikJCQlxcXBwdHXGPNpCfn79t27affvrJxMQEuuXIkSOFny546tQpuL8EPHbbtm2D++j27dsNNHuLo2VrDFJVVVVaWtqY5z5VVVXV+Zat6urq+naoPH78GG4APT09gZ+0a9cOvrp69arwDysqKhrY9cJxXGlp6Re/R+8fC4c8ZWVlTf5WsYaHnvqorKxs8srAVWv4fTbl5eVf/57Q2trakpKSBjpYdXU1DpS4GfOz4PP5ZWVlZWVl+ATrhoE+37Tv8mlMp2oOsOlw+2F5eflXdhU+n19aWtpAY5aXl//j7flldSgrKystLW34aW8/Dt+JxjQrYWFhcAO0adMmKCiouLi4srIyJydn3bp1EDAjLS39n32RFOPf4d69e+goavgxXwxh6tsfw/gOYBrzzxQUFNAOZCMjIxsbG3zVPKG2YTJ+WDCCSFVVVdhPwGgYjARpOOyF0RJhGtMo4uPjBw8eTG/AJISIiora2dlhKDDjh+X9+/foqRZ4yD+jMWBI1YYNG751XRhNDNOYz+Dly5dnzpzZvn371q1b9+7dGxUVxUyuDI7jysvLf//9982bN587d66BkGhGfTx+/Hjr1q379u1rvjdbM74VTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXTGMYDAaD0VwwjWEwGAxGc8E0hsFgMBjNBdMYBoPBYDQXX6gxOZ+qN914t+TP14svpLG/7+Bv2cV0/8uvlvzx7WvC/tgf+/uMvz/SVl3/cCepsGmFoQn5Qo2JSi+a9ntaVHpxVHoR+/sO/v54ljv2aNJDdkHZH/traX877n5Yfy2jaYWhCflCjXmYXrTsYlrTVoXxDckoqJh7NuVb14LBYHw21+PyN17/7jQmKr1oyR9MY74fXmaXzTmTUsv/1vVgMBifyaWYPKYxjP86TGMYjBYK0xhGC4BpDIPRQmEaw2gBMI1hMFooTGMYLQCmMQxGC4VpDKMFwDSGwWihMI1pkbx79y4mJqa6uvpbV+Rf4ofVmLS0tMTERD7/xztzxvfCj6gxfD4/Pj7+5s2b169fj4iISE1NbXGD9eTJkwkhaWnfs47S/KPGfPjw4c6dO8HBwSEhIQkJCfn5+f9i7ZoROzs7NTW1kpKSb10RBuML+RE15s2bN4qKioTCxsbm1q1bX1rJJiA5OTkhIaHx09Xx48cTQpKTk5u1Vv8d/lFjvL296Quqpqb2yy+/fP30/9WrV9HR0bW1tV9ZDlJQUPD48ePGa0bPnj1VVFQ+fvzYVBVgMP5lfkSNSUpKIoQ4OTmFhITcuHFj/fr1kpKSMjIyDx8+/NJ6fi29e/c2MDAoLy9vZP6JEycSQlJSfpSt7/+oMX369JGTk7tw4cLt27d/++03S0tLQoifn99XHtfLy0tRUbGsrOwry0GOHDkiLi4eERHRyPx2dnbq6upFRUVNVQEG41/mR9SYly9fEkKmTp2KKSdPniSEjBs3Tjiz8LjP4/F4PJ5wTpw119bW1jfz5fF4NTU1wvPr7t276+jolJaWCv+kpqZG+HBMYwSwt7fX0tKqqamBj7m5uZqamq1btxZYAdR37QDhph48eLC0tHSd1wULbHxpHMft37+fEBIWFtbI/ExjGC2dH1djJkyYgCmZmZkSEhJ2dnbw0c/Pb+TIkcnJyfb29goKCmvWrIH0pKSk4cOH6+joaGtrDxky5MmTJ1jCnj17XFxc3r17t2TJEl1dXS0trblz5xYUFGCGhw8fDhs2TENDQ0NDQ19ff9WqVTByhYWFde/eXUZGRlJS0sLCwszM7MyZMxzHlZaW7t6929LSUkNDQ01NzcHB4ebNm1ga0xgBHBwcNDQ0Cgv///GuQ4cOJYS8fPkSPj579mzgwIGqqqqqqqo2NjZHjx6lf37p0qUePXpoaWnBhauqqkpNTbWxsVFQUBATE4Prsn37do7jMjIyzMzMQkJCrl+/bmNjo6qq6uLicv/+fSwqOzt72bJlXbp00dDQUFdXHzFiRGJiIsdxPB7Py8tLS0uLEGJgYGBhYTF27FiO4/Lz81etWtWtWze40B4eHrGxsVga0xhGS4dpDMdxXHx8vIiIyIgRI+DjsGHD2rZt2759+y5dugwZMiQgIIDjuNjY2LZt2+rp6W3ZsmXHjh2dO3eWk5N7+vQp/GThwoUSEhJdunRp37790qVL+/fvTwjx8fGBBU1hYWGXLl1MTEz8/f13797t6upKCFm2bBkUO3bsWCUlJTk5uVGjRo0ePTo0NJTjuJMnT8rIyHh7e+/YsWPNmjVKSkoyMjLPnz+HwzGNEUBYY3r27CkhIQHO/5CQEBkZGXV19YULFy5atMjExIQQsnr1ash57949ERERNze306dPr1q1ysHBISsrKzs7e9KkSdra2hISEqNGjfLx8blw4QLHcc+fPyeEmJiYqKioTJw4cc6cOa1atVJSUkLf2PLlyxUVFadOnbpnz55FixaJiop269atpKSEz+evXLnS2tqaEOLq6jp69OiNGzdyHLdx40Z5eflJkybt2bNn6dKlrVq16tChA85OmMYwWjo/rsZMmzYNPhYWFnp4eBBCLl68CCkQtdWrVy80ktTW1rq4uKirq+PN/+HDBxkZGQ8PD/i4atUqQoitrW1eXh6k9O3bV0xM7PXr1xzHVVVVxcbG0ga0Dh066OrqYoqtrW3Hjh3pSr579y41NRU/3rp1ixCybds2+Mg0RgCwlUF78vn83bt3E0K8vLz4fH55eXm3bt2UlZUzMzMhc2VlpZ2dnYSEBKxy5s6dSwjJycmBb/l8PsYZDhs2rE2bNvSBEhISREVF5eTkbty4ASmHDx+mL01KSsqHDx8w/7p16wghd+7cgY8nTpwghDx+/BgzpKWlvXv3Dj9u27aNEHL58mX4yDSG0dL5cTVGQ0PDycnJ0dFRXV2dELJgwQJ0k4wbN44QEhMTgz/Jzs4mhIwdO7asrKywsLCwsLC0tLRXr14qKirgsFm5ciUh5MGDB/iTzZs3E0JwoYPweLzS0lJXV1eMF+Lz+T169NDX1//06VOdFa6oqHj69CkhZOnSpZDCNEaAfv36SUpKOjg4ODo6GhsbE0KsrKxAVMLDwwkhvr6+dH4Y6w8fPsz9z0eyYMEC4YjnIUOGyMnJ0dclISGBEDJr1ixMSUpKEhUVnTlzpnCtSktLQYFOnToFKQcOHCCE3Lt3r86zKCsrO3XqFCFk//79kMI0htHS+XE1RkFBwdjY2M7Obtq0aTgnBcaMGSMqKvrmzZv/LzAqSlRUVEtLq2fPnt27d+/evXuPHj2UlZX19fUhDnXFihUC89MdO3bQKRUVFXv27HF2djY3Nzc0NJSWltbT0wPbTn0aExcXN2vWLBsbGzMzMz09PULIihUr4CumMQK4urqKiIh07drV0tLSy8trz5492Ji///47IeT48eN0/rCwMELIypUrOY4rLS0dPXo0IURFRWXlypX0KqQ+jZk/fz6mvHr1SkZGZsqUKZgSGho6cuRIa2trY2NjNTU1QsjJkyfhqzo1JiIiYsyYMdbW1iYmJjDjYRrD+G74cTWG9scIABqTnp6OKQ8ePCCEeHt73759++bNmzdv3rxx40ZoaGh8fDxkAI2JiorCnwQGBhJCIC6gpKTE1dVVVFR0zJgxW7Zs+e2330xMTDQ1NVFjrK2tO3ToQG+bCA0NVVJS6tSp0/Lly/ft27dx40Z04XBMY4RwcHBQV1evcx9JAxoDHhHg6tWrgwYNIoR06dIFLJwcxw0ZMqRNmzb0dQGNmTdvHqakp6fLyMhgmOLhw4chfmTNmjWHDh0Cu6vAOiY8PBx/furUKSkpKSsrq9WrVx88eHDmzJlMYxjfEz+uxkycOLG+DMIaA7ay8ePH1/eT+jQGbGU3btwQGNFGjBihrKyMGmNra6urq0uPZW5ubuLi4mip//jxIyFkyZIl8JFpjADg8y8uLhb+KiIiQkAVuP/Zx65cuSKQ+ciRI4SQ9evXw8chQ4ZISkrSxTasMZWVldra2mZmZvhtcHCw8Drm9u3b8LGmpqZz586dO3fGqGsQv3379sFHpjGMls6PqzGftY6pra0Fiz+tIgUFBehGblhjzp49SwjZu3cvfBUdHa2mpqalpYVxUIMGDRITE6OPaG9v37p1a8ywevVqZiv7rLgypKSkpGPHjgoKChBDzHFcRkZGx44ddXV1IX9MTAyqyMOHDwkhmzZtgo9gQ4uOjsbSGtaYT58+ycjI2NjYwFelpaUQToLrmDNnzhBCAgMD4WNFRYWSkpKxsTF+9PHxIYQcOHAAUpjGMFo6P6LGwD5/jFQWxtPTkxBCh3VxHBcbG6uhoSEmJubh4bFgwYIhQ4YoKiqiXX7BggWEkMjISMz/66+/EkIePXrEcdyHDx90dXVFRERGjRo1ZswYLS2tdu3aKSkpYZTazp07CSFGRkaurq6HDh3i/jeh7tKly+zZsx0cHDQ1NemhbeTIkfTmj++ef9SY7t27t27dmt6QRBMcHNymTRtZWdnBgwe7u7vLy8srKiri04OcnJzU1dWnT58+c+ZMZWVlNTU1fBDcb7/9RgjR09MbMGDAhg0bOI6Li4sT2MCbmppKCBkzZgx8nDNnDkQYzp0719jYWFtbmxBy5MgR+DYpKQn2Qg0ePHj06NF8Ph9mJ1ZWVnPnzjU3N9fR0SGE7NixA/KbmprKysqyZ8kwWi4/osbk5OS4ubmhyVuYPXv2eHl5CQ9Yr1698vX17dmzp4mJib29/YoVK9Bwf/78+YEDB+JHjuOCg4NdXV1fvXoFH5OSkry9vS0tLfv163f9+vXLly+PHTu2oqICvq2oqFizZo2VlVX37t0xzvXw4cO9evUyNzefMWNGWlrarFmzfvvtN/jqwIEDHh4eGCf93fOPGuPv7z9+/PgGHsYTExMzZcoUKysrMzOzsWPH0vL85MmTMWPGmJqaCn9VU1Ozfft2Gxsbc3Pzc+fOcRz3/v37gQMHnjhxAvPk5+d7enrCzIDjuIqKirVr1/bo0cPKymrVqlWpqane3t70xv4bN244OzubmprCqrSqqmrTpk02NjaWlpbLli1LT0/38fHB/bYrV66cNGlSVVXVZzYYg/Ff4UfUGEaL44d9tj+D0dJhGsNoATCNYTBaKExjGC0ApjEMRguFaQyjBcA0hsFooTCNYbQAmMYwGC0UpjGMFgDTGAajhcI0htECYBrDYLRQvk+NeZhWtPSP1H/Ox2ghvMqvmHuWXVAGo+Vx7Xnehu9SY2adSXvxoYz9fR9/NxMKJh57kZhV+s1rwv7YH/v7rL8DEdnrrr5uUl1oSr5QY9Lzyhf/mTHvbNrcMyns7zv48z2buuB86ryzqd+8JuyP/bG/z/pbejX7/JOcphWGJuQLNQbgcxyfz/7YH/tjf+zv2/01lRo0D1+lMQwGg8FgNADTGAaDwWA0F0xjGAwGg9FcMI1hMBgMRnPBNIbBYDAYzQXTGAaDwWD8H3v3HdfU1f8B/PpYFRcOEI0IKFQFRMGNgrNOqq1Fq1VmUMHViiLVaoH2cSu4cLPCCEtx4AYVF4qAoCIZQBYJO8wQAmSc3x/HJz+qdmlCQL7v1/flixxubk4umA93naMukDEAAADUBTIGAACAukDGAAAAUBfIGAAA+KzQiuqTGXXPOXWNzXJN9wUyBgAAPiOPmNWu4QVrLpS7RHL8rvEyuXWa7Q9kDAAAfCYamuXb4vPJVL5D0BvHYLprJNeZkn/yQQlXKNFUlyBjAADgM1ErkW6i5rmEsxyCcnE5hjDIVMHaqIKY9IpqcXPrdwkyBgAAPh+UJyVr4sudwvKUMeMQlOsUlu8WLdhygZ1Mq2qWtepIzZAxAADw+ZA0yyOflZIpeWRqoWMIvWXSuESwXSK5vyVys3itd5IGMgYAAD43eaXiw3cELuEsl0iuQxDt/w+dBdNco3iOIYyT9wWFla1xkgYyBgAAPk/P2XW/XuWRqQJnSkHLHRrHEAY5mu8emReXUV7ToN6TNJAxAADwOUum13hd5DiFMlvu0DgE5TqH5ZOpfO+L7Ed5NQqFuk7SQMYAAMC/IG6S1zfKPr3ETTJRo6ykpqmourGopulTq7qxqLqRWSrOLapvWcxSMbuiIYVe5X2B9c6FAP87ScNxieAcuC2gFYvVsbkgY0D71iyTN8sUzTL5J5ZUrmiWKSrrm4WiZmH9J5eoWShq5lU2sssb2BWSTyxOhYRdIcktrn8tqH/FF6mknuTXJOdW3qWpoFIY1YkvhdHPy2LTVVBx6WVhT4qP3+WfUEUF3uMfvs3zvcrxucJWSe26zNoal78lLn9LbN4nFl6PRwTDPYLhoYpyj2A4h9De2VnB5RZGdwujv9/+v5M0dNconislPzS1rLS2SbX/Q1WcMQoVFUJIrkD1jTKRSkoiEzXKSmubSmpUUKW1zaW1TRyhpKBcUlDWoJLK4tWl5tc8LVBBpbFqk3OrrmSXX31Z8el17WVFbHpp0KOi4MfFn16hT0pOpwj23+Ltv8nbf5P76bX3OndnAmvnJRXUrkusnZdYP0Xn/RjN3ET91PoxOu/H6DxyKN05hOaikgqlOVMKXKN4rpFcKM2WSzjbJZzlEs5WRbGcw/KdVFihTMcQxocr+E8z5m3ShDLI0YKNMeyEFxVCkcpO0nx8xtzMEZ56VNHyT4YjSYW7r3F2X+fsvvaptec65/dEjveFgm3x+dsuFHxqxed7xee7hzPWhDPWqqbojsE053CWM6VANRXOclFhRbDx/q9KyjWS6xrFc41UUUXxyNRCVZVrVKEK36lLBEdlP1BKgTOlwCmUqcJyDKY7BNEcglVUb//apamo/urDC6od1erYktVxZRcyyzSfMQducz2u1LlFC5RFjua7RvFUVpFcl3C2Cj98ncLyVFiOIXTHYJoKS2WfHcpPEBV/GEFBQeF693/EJ/3f/7Pdjo+qv/obJeRv9mOcKflkKv/Xq7zn7LommcoG0/z4jDl2t9A1qlDTP2yoDl8qzuZ3V+4YTP/4UunHx19/gvzbcg7L/1c7ZA5Bb1pulncWcIlgq+xIVCTHNYpHpvJVVtECt9gSldTq2BK3mCKnUKbDeeXWeOMYTP+EIwdsfBRLBRVCdwymO4fQXENpLn8s11CaRwRjTTj9A7/hQbkOb8eb4XtE5V/OrqhvlKkqXTCVZ4yqdr1V+pd4MO0vPize3+7/9CequnIKZTpT8p3DVFThLPzr+8nFcQxh/P+Hy/k3TqFMMpX/8Ue3ogrJVL5bTLHKKrpIlcfZIznv/DI4hTKdwvI++uC445/8l/64cgrOVc2pnRCaSyhtQxRza1y+Z+zf15a4/HURjP//KziY5hSc6xmb7xmThxfYHJO38xJrz3Xuf69xVFHcg7d4p+4LTqqiAu/xzz0sin5eppKKSS+Pzyjbe53jEsHGW8OZUrA5Nj8pt+rOm39dt99UJr2pzODUZvFELz69uHVZPFFBeQNPKOG2KH5VY1ltUwandtdlllPoe9eVBdNcI3lOocxTKUWFlY0qjBalTzhWdovrcaWOHC3A5RYtIEcL3h64V82xMp6Kzqq9Laew/A8c9QplOoYw3v3P/I+Olanor49gumMIY3UYfUMUc0NU3qfX+kjm1rj8Xy+zd11ifWL9epm1Npyu3D5OoXmbqMwjSYWHb/M+rg7d4h67y1fJ5QPBj4tDnpRQUksuZ1dcziq/nFXxiXUluyI+o4wcSlO+X5cINiW15FlBzZP8f12P82ue5Ne8KapnlNQzSsSfWPTienpJfVF1I75cTSVV2yD9p9fgNslvva50iy5SfqpujGJWiaXKBUSNssZmuUyuUFGp8PNNLeIzytziSvDWcI0q/P0aR9M9+isKhSIpt3JLXME7t2E6BOU6U1hkaqHvVW4mt1Z9Hfj4jLn+uuLUI+Hxu3xcJ+4JTqUIop6VUtNKqWlln14xz8tuvxEm5VaqpJJzKzM5tdm8uqw/Vg5fFP281DWSq/y7dXUY/Rmr5mWhKOu9hVsWp0IiqGpUTVU3ldU2VdY3V4pUUfXSOomsUSqXNH9qNUnl/rd5ZCofbxy36KKT9/gq/OVraxQKtC6C4RSWj98vOaYok6PhuTfaiAf0areYYmXG/BjNlKvtlr22j5pW6hb7dmu4RvJ8r7I13aM/lVUo8rtWSI4qdG4xErNDUK5jKJMcLfgxln39dWWjVL2pDvfHoHR2rVtM0f/2YPLXRTBk8o77/+cdx5ILydGCDpIx9Y0yj5YZEy14WqDGv+/akXu0qpYZs4nKVPcHU1vWLjJGUNV47lGpMyXPNZLX8ggwHq/MJSwvOLWsrK41hvqHjEGp+TVu//sYdQrL94hgqPysV/t1NOkPGRMIGdMhQca01MYzRqZQJL6sWB/DJkcL3jkR4BLOco3k7b7Be80XtVp/IGMgY/4KZIymO9UmQMa01MYz5nJWuVtMsXP4H86+4Kt1NkXn33lT1dy6p7wgYyBj/gpkjKY71SZAxrTUljOmSabYfqHANYrX8ipZMrXQJZQR8ri4olUOjr0DMgYy5q9Axmi6U20CZExLbTljpHKF3xX22xPMwTR839KBW3x6Sb2mugQZAxnzVyBjNN2pNgEypqW2nDEIoTRW7ZooNjla4BrJ+/kS71FejVyjFzFBxkDG/BXIGE13qk2AjGmpjWcMQii/TByfWX6PUVMnkWq6L5AxkDF/CTJG051qEyBjWmr7GdOmQMZAxvyVI3cK3WJL8GBZq2NLT9z9zDNmbTjdOZyN369bbEkqZAxCCKF7tKrVcWV4s7hEcDZEMTpyxkQ9K1kd/3ZrkKmCX6+wNN2jNg0yBjLmrxxJKnSLKXYIpjsE091iSk587vsxa8MZzhTW/95vcWo+ZAxCCN3NrXKLLcWbxTmcvSGqQ+/HRKWVro57uzXIUXyfK7Af81cgY9CTvJrVMcV46DBnCss9nA4Zo8QVSrJ4olf8+lf8+iyeiFcp0XSP1EimQLlF4peF//9+q8QauNazDaqsb37BrcOb5SVfRCsWa/Y0smaV1DRl/W9rZPNE+WUNmu5RmwYZgx4xq90TqvC4mWSqwDWUJmoDJ8oAAOAzABmDymqbk3OrUxjVKYzqe/SqR3k10g78NxoAAKgQZAwAAAB1gYwBAACgLpAxAAAA1AUyBgAAgLpAxgAAAFAXyBgAAADqAhkDAABAXSBjAAAAqAtkDAAAAHWBjAEAAKAukDEAgA8oLi7esGHDvHnzKioqPriASCRydXWdO3cuhUJp5b6BdgQyBgDwB1Kp9Pz58wMGDCAIgiAINvvDY9f7+fnhBX744YdW7iFoRzpixsTHx0dFRX3wW3V1ddevX9+7d6+fn9+lS5fq6upauW/tQkPD5zmYOYPBuHr1amIL165du3HjRnZ2tqa71qq2bNlCEISXl9fXX3/duXNnLpf7/jJ0Ol1LS2vBggU9e/Z0cHBo/U62pidPnvz222/r16/fu3dvZmbmO98ViUTh4eGenp4bNmw4efKkQCDQSCfbrI6VMa9fv/7qq68Ighg8eLBM9u4kMbdu3Ro5ciRBENra2n369PnPf/4THByskX62WTk5OYsXL545c+Zn+R9p+/btxB917tyZIIivvvpK011rVefOnbt79y5CaPXq1QRBfDBjvv76axMTk9TU1K5du65atarV+9hKZDKZi4sLQRBdu3bt1asXQRBaWlqhoaHKBVgs1rhx4zp16jRq1CgzMzOCIIyMjF68eKHBPrc1HShjjh492qNHj7FjxxoYGLyfMW/evOnTp4+RkVFSUlJNTY1YLE5LS8vPz9dUb9uaxsbGgwcP9u7dmyCIbt265ebmarpHqsflcu/cuZOcnHz37t27d++mpKQEBAQQBLFp0yZNd00znJycPpgxN27cIAji+vXrPB6PIIjPOGNEItFXX3116NAhLpfb0NBw+fLlvn379u7dW/k3Ft5EN2/elMlkMpns2rVrBEHY2dkpFDA/yFsdJWOkUunXX3+9Z88eqVQ6ffr0/v37v5MxK1asIAiiox0V+efWrFmjpaUVEBBllQvbAAAgAElEQVSwYMGC7t27MxgMTfeoNXh4eBAEkZWVpemOaMYHM0YikRgbG8+aNQshlJOT83lnjFwul0r/MGPhxo0bCYK4ffs2fmhsbGxkZNRygSFDhowYMeL9wyQdVkfJGIVCoTyLMHnyZB0dnZa/BCUlJb169Vq8eLGGetcOXLhwAQfw0qVLv/jii46QMSwWS0tLa/78+ZruiMZ8MGP27dtHEMTLly8RQtnZ2Z93xrzv119/JQgiKSkJP5w3b94XX3yhPEnz8uVLgiBcXFw01r+2p6NkTEvvZ8zjx48Jgjh16lRhYWFcXFxMTExaWhr8JfJB3377bZcuXTpCxuDTM9euXdN0RzTm/YxhsVja2tq+vr7KhwRBuLq6aqiDGmBra6ulpVVaWoofpqam9unTx9DQ8NatW48fPzYxMbGysuLz+ZrtZJsCGYMQQhcuXCAIYsyYMXp6et27d8fne+fNmwe/K+/rIBlTUVExYMCAkSNHNjU1abovGvN+xri5uf3nP/+JiYlJTk5OSko6e/YsQRCzZ89OTk5Wfux+xm7evEkQxObNm1s20mg0Q0ND/KFhZWUlEok01b22CTIGIYQiIiIIghg3blxMTExBQUFubu4vv/xCEMSKFSvg3N07OkjGnDx5Eu/aarojmvR+xkybNo34E+fPn9dgV1tBaWnp0KFDTUxMqqqqlI1yufzo0aMkEmnBggWTJk3S0tJav359TU2NBvvZ1kDGIIRQTEwMQRDv3DRjaWnZt2/fjvDX2b/SETKmsbFxxIgR/fv3Ly8v13RfNIlMJhME0XIj5OTk3L59Ozk5GV99d+7cOYIgZs2adefOnbKyMg12Vd3EYvGsWbO++OKL1NRUZaNcLl+/fj1BEMeOHUMISSQSX19fgiAWLVokkUg019m2BTIGIYSSkpIIgjhy5EjLxZYsWdK9e/c/u8m5w+oIGXPp0iV8E6KmO6IZHA7n0aNH6enpc+fOJQjiwoULqampH/yJ5+fnEwTh6OjY+p1sTU1NTfb29gRBXLx4sWX78+fPCYJwc3Nr2ejm5kYQxMOHD1u3j21XR8wYa2trHR2dli15eXmdO3e2s7Nr2WhmZmZoaCgWi1u3d21dR8iY6dOnd+3alUajabojmrF58+auXbt27969a9euXbp06datW7du3ezt7d9fMicnp2vXrs7Ozq3fyVYjkUiWL19OEERsbOw730pISCAI4sSJEy0b8VHW5OTkVuxjm9aBMkahUEilUqlUOmnSpP79+zc2NiqvfJfL5TY2NgRB3L9/H7ccP36cIIidO3dqrr9t1Pfff9+lS5fP+MDIkydPCIJYsmSJpjuiMXw+PzU19dmzZ+np6RkZGc+ePUtNTf3g/chNTU0vXrzg8Xit38nW0dDQ4ODgQBDE9u3bKyoquFwuh8NhsVhCoRAhlJub27lzZzMzMxaLhZdns9lmZmba2trFxcUa7Xgb0oEyZtWqVbq6urq6uvgUpa6uro6Ozr59+/B3Hz9+3LdvX4IgbGxsRo0aRRDE119/XV1drdk+tx1paWnBwcFBQUEWFhYEQfz3v/8NDg5+/PixpvulYgqF4ptvviEIAg+mAjq4zMxM/HHRr1+/Xr16de/evXv37lpaWnPmzGlubkYIHT9+vHPnzr17954wYcKECRO0tbX79u37ziG1Dq4DZcyJEydcXFzWrFmzadOmn376ac2aNc7OzgkJCcoFcnJyPD09Z82atXLlypCQEPw7BDB8lJkgiC5duvTs2RN/vWzZMk33S8XkcvmuXbv27Nkjl8s13RegedXV1adPn/b39z9w4MC+ffv279+/f//+3bt3U6lU5W9Idnb2/v373dzcPDw8jhw5wuFwNNrlNqcDZQz4FKWlpdnZ2TktZGdnf5YjYwIAVAgyBgAAgLpAxgAAAFAXyBgAAADqAhkDAABAXSBjAAAAqAtkDAAAAHWBjAEAAKAukDEAAADUBTIGAACAukDGAAAAUBfIGAAAAOoCGQMAAEBdIGMAAACoC2QMAAAAdYGMAQAAoC6QMQAAANQFMgYAAIC6QMYAAABQF8gYAAAA6gIZAwAAQF0gYwAAAKgLZAwAAAB1UW/G8Ksk7PIGtb4EaGVcoeQZp6GyvlnTHQEAtAPqypg6iTQmo3xtVAE5gv2AWa2mVwGtjF/V6BGZtzqubHMc+/abymaZQtM9AgC0aarPGJlccZ9ete0ih0zlO4XluydU+d8pVPmrAI1glIhdIzkuEWzncJZrJM/3KieLV6fpTgEA2i4VZ0yOQLTnBs8lguMSznYKzSNHCzbHsWnFYtW+CtAUuQJdflG+OjyPTOU7hjJco3iOocyT9wWFVY2a7hoAoC1SWcaU1jadf1TsFMIgRxU6htBdo3iuYczwp6VCUZOqXgK0EezyhuN3BU6hTJdwlmMIgxwt8IjKT3hRUd8o1XTXAABtiwoyRtwku/aqcmN0AZnKdwplOgTTXEIZAUmF7ArJp68ctFmZXJFvIs8plOkQlOtMySdT+Tsuc58U1Gq6XwCANuRTMyaDU+ubWEim8p0pBQ5BuQ5BuY4hjA1RzKBHRRFPS4IeFZ9/VNQh6mHRwVu8Pde5eztGHbjJO5rM97nCcgqhOwTTHIJyHYJoLhEclwj24SRBXikcHQUAIPQpGVNY2Xg4qcglnO0ayfnfp8z/KpjmEsF2jeJ1rIrkukRyXDtSOYXl/eHnHpTrGEx3iylyieRGPy9VwEVnAHR4H58xvMrGg3cELhFs10juuxkTRHMJ73gZE8VzjSrsOOUSwXEMpr/zc3eN5DpTCvyTBLnF9ZAxAIBPPVb2nF3re5VHjip0DmfhDxqnUObGKGbEs5L4zLKotNLIZx2iIp6WXs0uv50jvNUB6naO8B696i6t8r/XOE4hTPxzd6YUkKn8Xy5xnrFqVPKrCQD4DKjgnL+kWX7tpXBjdAE5+u05f+cQ+pFkPgvu8P98ldU2BT0uWROR5xhMdwxlkqP566LyLmWV1zfKNN01AEAborJrl8tqm84/KnIOobtGFTqGMFyjeK5hjLDU0rJauHb5s1LfKL/wosI96u1lhK5RPKdQ5qkUQVE1XEYIAHiXiu/BpBXX77/Fd4lgu0SwnUKZbtGCn+LYb4rqVfsqQFOqG6Q+V9huscVOYfn4Vv89N/ivBfDzBQB8mOrHkpErFE8Kardf4pKj+c6UAveEqsO3eSp/FfWRy+Wa7kLbRSuuJ1P5ZCqfHC3YEs++x6iWymBzAQD+lLrGxKxvlCZkCT2oLLdIVlsbE/PIkSOOjo7vt9fW1trb2xsZGfn5+bV6p9qHhibZ6RTBzsSiqLTyKjEMvQwA+BvqHdu/tLapsFL1Z/43bdpkaWlpZmZmZma2bdu2ysrKf/X0bdu2TZ48GSEklUrnz5+/f/9+3O7r62tiYpKcnMxgMFTYWzc3Nw8PDxWuUONEEhgzBgDwj7S/OcoUCsW4cePs7e0vXboUHR09ePDgGTNmNDb+izEZd+7cOWPGDISQVCpdunTp0aNHcfv8+fM9PT0/vYd+fn7Hjx9XPty0adOWLVs+fbUAANDutL+Mkcvl1tbWhw4dwg9v3LhBEERGRoZCoWCxWAqFgslkvn79Wip9+7d2dnZ2ZmYml8tVrkGZMQihsrKy6upqhBCNRhs3bpyLi0tmZiZuEYvFmZmZL168UD6xqqqqqKhIJpO9ePGCz+cjhIqKivAy9fVvz3szGIzx48fPmzcvMzOzqKgIIVRZWVleXq5ciUgkyszMzMzMVD5FJpOxWCyEUGlp6YsXL0pLS9Ww2QAAQAPaZcZMmTJl7969+OHjx4+1tLRoNFpDQ8P48eMdHR2NjIysrKyEQmFFRcWSJUt0dHT09fUHDRp05MgR/JSWGfPVV1/t2rULITR37txu3br17t1bX1//6dOn2dnZVlZW+vr6AwYMmD9/fnFxMUIoIiIC70KRSCQfH5+goCBTU1O8cktLSxqNhhD67rvvtLS0evbsqa+vj4NwzZo19vb2+OWSkpIsLCz09fUHDx5sbm7+5MkThFBtbe3kyZOdnZ0nT55MIpEGDx789OnT1tykAACgJu0yY6ZOnbp9+/bS0tKcnBwzM7OJEyc2NTXV1dUZGhoOHz48JydHLBYrFIrvv//ewsKCzWaLxeLw8HCCIJKTk9EfM2bWrFne3t4IIYlEYmtr6+npKRaLq6urJ0yYcPjwYYSQTCabNm2au7s7Qig+Pp4gCHd395qamsbGxosXL2ZkZOD1TJs2bdWqVXg9dnZ2rq6uYrG4ubkZIUQmk5csWYIQKi4uHjhw4ObNm8VicV1d3bJly4yNjevq6hobG4cPHz5y5MisrKza2lpbW9sFCxa0/oYFAACVa38ZgxCaM2dOr169Bg0a1K9fv0WLFrHZbIRQRUWFiYlJXFwcXqa4uFhXV/fy5cvKZ82cORPHyTv7MT///LNyge3btyOEUlJSevfu7eXl9csvv+zatWvq1KlmZmYIISqVOmLECOUxLoRQdnb2L7/8snPnzokTJy5fvhw3fvPNNziTsNWrV+P9mIiIiEGDBonFb8ckLigoGDhwYFpamlwuNzU1pVAouP3UqVNmZmYyGdwwDwBo99pfxsjlchsbG09Pz4KCAnwaA6uoqBg+fHhCQgJ+yGAwhgwZ0vKg05IlS9auXYv+PGNmzJiBQyg2NrZHjx6LFi1asmTJkiVLHBwc8HG2iIiIUaNG1da+nSLlwIEDJBIJL2NsbKy8Hnrx4sUfzJjjx4+PGzdOef+NQCAwMDC4c+cOQsjc3DwiIgK3BwUFDR06tKkJxkcAALR77TJjpkyZorzgWAlnTHx8PH4oFApJJFJISAh+KJVKDQwM/P390T/ImPv372tra5eVlb3zEhQKxdzcHF8RUFlZ2b9/f7xChJCHhwc+IIYQWrhwoZubm/JZyoy5dOmSjo5OSUkJbk9KSurVq1dBQUFzc7O5ublyP+bcuXPGxsaQMQCAz0C7zBhLS0t8or6l8vJyEolEpVKVLVu2bOnevXtQUFBCQsLixYuNjIzwhV5eXl4TJ07Ey0yZMmXz5s3464kTJ/70008IoaamptmzZ48ePTouLi4hIWH79u34WuTg4GAjI6OqqiqEUENDw+jRo+fNm5eQkLBr166uXbsqj5WtW7du4MCBFy5cwNekOTg4LFy4ECFUVVVlbm5uY2OTkJAQHh4+aNAgFxcXhFBtba2RkVFQUBB++smTJ/X09CBjAACfgXaZMW5ubsHBwe+0V1dXL1269O7du8qW5ubmvXv3mpqampqarly5Unlg7fTp08qbIjds2BAYGIi/9vDwOHHiBP5aKBSuW7cOP3fJkiVv3rxBCF2/fn358uV1dXV4mZycnPnz55uamjo5Of3222++vr64nc/nL1q0yNTUFK95z549yl0lPp/v4OCAV7t79+6GhgaEUH19/fLly69fv46XuXTp0pIlS/D1AgAA0K61v4wBAADQXkDGAAAAUBfIGAAAAOoCGQMAAEBdIGMAAACoC2QMAAAAdYGMAQAAoC7tOGNevnz55MmTnJwcTXcEAADAh7XLjJFKpVu3bu3fv7+RkZG2travr69yELCWFAoFj8fLy8t7/7sKhSI/P18gEOCHJSUl6pi1RSAQCIVCla8WAADai3aZMQkJCV26dElJSWlubj558mS3bt1aDo6pJBaLJ0yYQBCE8u59paNHjxIEMXfuXPxw3Lhxs2bN+vSOCYVC5ajMTU1NytFiAACgY2qXGRMQEDBkyBC8d5KQkNC3b188vP87RCLRpEmTzM3NR48eLRKJlO11dXUTJkwwNTWdO3cuXsmFCxeuXbv26R2bOXPm2bNn8ddyuTw8PDwlJeXTVwsAAO1Uu8wYJpPZv3//4ODglJQUHR2d33777YOLiUSicePG+fn5mZiYREVFKdvDw8Otra19fHxmzJiBp2lJT09PTU3F3xWLxQEBAX5+focPHz5y5AgeZDMtLe3BgwdZWVl+fn54xpfg4GA/P7///ve/dDodISSRSI4dO6arqztr1iw/P7+srCyE0KNHj/AX2I0bN/z8/H7//Xc8+hlCqKqqKjY2VigUUiiU33///eHDh2rZXgAAoCHtMmMQQlu3biUIYsKECXjaFT6fHx8f/85YxSKRyMLCIi4uzsvLy9bWFseJTCaztbU9fvz4qVOnbGxs8H6Mvb09nnqyurp6zpw5lpaWCxcu7N69O0EQfn5+CCFfX189PT3cHhcX5+7uPnnyZDs7Oxsbm4EDB6anp0skEjKZrK2tPWLEiIULFyYlJSGErK2t16xZgxCSSqUeHh4kEmnhwoXTpk3r0aPHxYsXEUJ5eXkDBgwYPXr0ggULbG1tO3fufPv27VbekgAAoD7tMmMSExMtLS0tLCy+/PJLPJtLZGRkz549a2pqWi4mEolGjRoVExNDo9G0tLRevnyJELp//76urm5VVdWRI0emTp2KM8bFxQWPzB8XF9e/f3+pVIoQolKpxsbGeIH9+/f37t0bJwdCKC8vT/kqkyZNwjMC4K/xLADYvHnzPD09cYe7deuWlpaG2zds2DB8+PDm5maBQKCtrb1+/XrcPnPmzBUrVqhhg6mSVPaByytAK5PLFZruAgD/SPvLGDab3adPnxMnTojFYisrq7Fjx0okkt27d9vY2CgUf/iPhzMGT1M2fvx4PE/MypUrf/jhB4TQvn373s8YGo02bNiwI0eO3Lp1a/HixRMmTJBIJAghX1/f2bNnK9csFou9vb3Hjh07fvz4fv36/frrr7h93LhxLSdPmzdv3pYtWxBC27Zts7W1Vbanp6fr6+vzeDyBQDB06NDnz5/jdm9v72nTpqljo6lEbpHo4G2+zzX+y8L6v18aqA2jROybyN17k5dVKPr7pQHQqPaXMRERET169MATHguFwilTpowfP97IyOj06dPvLIkz5vz58wihM2fOGBsbp6amGhoaPnjwACG0d+/e9zMmKyvL2tp67NixFhYWK1eu5PF4eFV+fn7Tp0/HGVZbWzt16tRp06ZRKBQqlWppaamcHmbs2LGHDh1SdkCZMV5eXvPnz1e2P336VE9Pj8vlFhUVGRkZKa8L+Pnnn6dMmaKGbfapyuqaQlNLXcPzyVS+e0LVrssfuIoPtJqT9wUel2vJ1EKn0LzTD4r5VY2a7hEAf6r9ZcyDBw8IgoiOjsYPaTQaQRAEQdy5c+edJVtmTFlZmZGRkYGBwezZs/GJmQ9mzKFDhyZMmMBkMvPy8rhcrnJVLTMmJyeHIAgcVAihCRMmbNu2DX9tYWHRcoJO5bGy0NDQ3r17K1f4448/jhgxorm5mcPhtMwYb2/vqVOnqnJjfbJGqfxmTtWmWDaZyncKzXMJZ7tFC+Iz3p2FGrSmFEa1czjLNZLnFMokU/nrqAWXsoT1jVJN9wuAD2h/GSOXy9evX9+rV69Vq1Y5OTmNGzfuhx9+WLt2ra6u7sGDB1suWVdXRyKRlDfHbNy4kSCIyMhI/HDXrl3m5ubvnPMvKCjQ1dXV09PT19cfNGjQ3Llz8akXb2/vMWPG4IwRiURTpkwxNzd3cnKysbHp3LmzMmM8PDz69Onj5OSEz9xMnjwZn/OvqamZNWuWsbGxk5OTnZ1d3759L1++jBBiMBi9evVSnubZsGGDubm52rfgP5bOqfv1KpdM5TtTCpzD8slUvvdFzpP8GgTnAjTtBbfO91qha1Th2x9NtGDHJd5TVp2m+wXAu9pfxmAJCQnu7u7u7u5XrlzBLRQKJSAgoOUyTU1Nx48fz8jIwA+ZTObu3burqqrwwwcPHpw7dw7HxsWLF2NiYhBCZ8+eXbBgAZ/Pr66uFgqF48aNW7lyJULo3r17QUFByvM9QqHw119/dXd3T0hIuHbt2q1bt3B7bW3t77//7u7unp6ejhAKCwu7ceMG/pZMJjty5Ii7u/uGDRtoNJpyPYcPH1be3HPr1q1z586pY3P9WwXl4qPJfGdKvmsk1yGY5hzK2HaRfetNlab7Bf7gYV7Nzis8lzCmQzDNJYLjEsE5nCSgl4g13S8A/l97zRg1mTVrFt6hQQiVl5ebmZkpL/rqICrrm6OelZLDGGQq3zGE4RCU6xhCJ4fRQx4XP8qrfsCsvkurSqZVQmm27tKrUuhVT/KqqWmlayOYb39SwXRyFN85lBH8uKistunvf9gAqB9kzB9cu3Zt+PDhQ4YMGTp06ODBg+3t7UtKSjTdqVYiapTdfF25JZ5FpvKdwvIcgnJblks42y26yC2mGKotVZFLBMchmNbyJ+UUyiRHCzZG51/OqqhpgJM0QMMgY94lFAofPHiQkpLy7NkzTfelVRWUNXjG5q29KHSmFDgEvWnxsfXGMYThGsl1jeJBtbmK5DmFMt/5eTmH5a+5ULGemkcvhuNmQMMgY8D/EzXKErIq1lPz3aIFjqEM/JnlGMJwj2Dsv8Hdc53zWyJUG6rd1zkHb3HXR709VoZ/WGQq3z2yICa9vFoMOzFA8yBjwLuKqhvPPSpxpuS7RvEcgmmOIYyN0ayErMqGZpmmuwb+QCqT36FVb45jO4YwHIJprpFcZ0rB8XtFPKFE010D4C3IGPBhr/mi3Tf4rlE8Z0qBUyjTlVroncB5mFfzzmAKQFPS2LW7rnDJUYVOYXnOlAJXauGvV3kZXLjzH7QtkDHgT0lliqTcKs94tlu0wCkszyWc7RrF23uDx6+EP5M1qbS2KSCJ7xLOcongOIUyydH8DdH5114JJc0wlBxocyBjwN8QiprDn5a6hTPJ1ELHEMbaC8IDt7h//zSgNiGPi90TqpxCma5RPKcQ+vlHxUXVMJwMaKMgY8A/wigRH7jFcwlnrYkvP50i0HR3OrT4jLLVcWUuEZz/XuO+5sPBMdCmQcaAf0qBUDqn7j6jVtQIJ/81qaFZ9jCv5hmrTgoj/IM2DzIGAACAukDGAAAAUBfIGAAAAOoCGQPaFjw9KL4LRyaTcbncsjKYrgaA9goyBqiXQqEQiURVVVWVlZWVlZVNTX8YD7ipqam+/v9nbvby8urbt+/EiRPr6+sbGhqWLFmio6ODp49rBfX19c3Nzf98ealUWl9fDzelAvAXIGOAeolEounTpw8cOJBEIpFIpFGjRlEoFOV3f/75Z2NjY7FYjBB68uRJnz59kpOTS0tL5XL5+fPnSSQSjUarqKhQU9+kUqkyIcrKyvT09Pz9/f9ieblcLpX+/yBgJ06c0NHRKSoqUlP3APgMQMYA9aqurjY2Nt63bx+dTqfT6fv27evRo8ePP/6Iv3vp0iUfHx+8cxMTEzNixAjlE7dt26acy0dNZs6cee/ePfx1bW2tt7f33bt3/2L5xMTEuXPnKh/ev39/27ZtNTU1au0kAO0aZAxQr+rq6pEjR8bGxipb4uPjCYK4c+cOQqi0tDQ7Oxsh9OrVK0dHR319/eDg4LS0tEePHs2YMcPKyopCoeTm5iKEKisrw8PDKRQKh8PB6xEKhY8ePaqtrY2MjFTOV81msykUSkREhHLCUx6Pl5aWJpFIYmNjqVRqZWUlQqiuri44OLhXr15r166lUCjFxcUIoZcvXyp3SgoLCykUCoVCefDgAW55+fKlk5NT3759z58/j6c3ra6uzszMVB5ea2hoiIqKolAoKSkpyjebmZlZUFBQXFxMoVASExPVsYUBaMsgY4B64YyJiIhQtigUCmNjYw8PD4RQQECAsbExPjI2ZMgQLS2tMWPG7N+/f+fOnTo6Otra2paWlvHx8a9fvx41apSFhcWYMWP09fVv376NEEpNTR00aJCtre2IESPIZDJC6OrVq4aGhlZWViNGjDAzM8vJyUEIUSgUfX39OXPmTJo0SUdHZ8KECXV1dQKBYMqUKV27djUyMrK0tHz+/LlcLjcxMTl48CBC6O7du+bm5lZWVlZWVgMGDNi/f79CoTh//ryBgUG3bt3GjBnj5OSEEIqPj9fV1cX59OrVqzFjxgwbNszS0lJXV3fTpk34qJqLi8uYMWNmzpxpaWnZpUuXTZs2yeUwqhjoQCBjgHq9nzEIoa+//nrJkiUIoZMnT44ZM6axsREhFBQUZGFhoVxm48aNixcvxl/PmDHDz88Pf+3r6ztu3DiZTPb69esvvvjCxcUFn1MpLy9vucP0/fff29vbI4QSEhK++OKLQ4cOIYRYLJa2tjbuTFNTk7GxcUJCAl5eIpFYWVkdO3YMIcThcPDeFUIoPDxcT08P7/2Eh4ebmpoqe5iQkGBsbFxRUaFQKGxsbBYsWIDfyP379zt16nTlyhWE0Lp16/r37//w4UOEUFhYWO/evVksluq2LgBtHWQMUK8PZszMmTOXLl2K/pgxp0+fNjc3V154tm7dOjs7O4RQQUGBrq7urFmz7Ozs7OzsJkyY0KtXL7FY/PLlS0NDw4KCArz8nTt3evTo8fXXX9vZ2S1atGj48OEjR45ECEVHR5ubm0skb8eKnjRpEo6rurq6YcOGxcTE4PaWGYMQotFoS5cutbOzs7a2xkGCEAoKChoxYoRyVThjamtri4qKdHV1W57LmTNnjpeXF0LI1dV11apVuJHD4QwYMCA1NVWlGxiANg0yBqjX+xlTW1vbv3//vXv3IoQCAwOVGXPq1Clzc3P8NULIw8MDZ0xWVlafPn3c3d19fX19fHz27t0bEREhk8nS09MNDQ3fvHmDl4+Li9PW1vb29vbx8fHx8Tlw4MDly5cRQlFRUWZmZsrTM5MnT96xYwfuxrBhw+Li4nB7y4y5ePGijo7OunXrfH19V61apcyYc+fOmZqaKnuIM6auro7NZuvr66enpyvf44IFCzZu3IgQIpPJK1euxI1cLldHR0d5ggeAjgAyBqgXzhj8cY/9+OOPWlpaeP/jn2RMaWmprq5ufHz8O2t++vSpoaEhPumCEEpPT+/Vq5cycpQiIyPNzMzwwS6E0KRJk3bu3IkQqq2tJZFIkZGRuDMBF38AACAASURBVL2hocHKyur48eMIoUWLFs2aNQu3x8bGGhgY4Iw5e/bskCFDlGvGGVNVVdXQ0KCvr3/48GHcXlZW1qdPn7CwMIQQmUz+4YcfcDubzdbR0cHHzQDoICBjgHpVV1cPHjzYzs7Oz8/Pz89v3rx5enp6+FwFQujw4cOGhob46FNAQMDgwYOVR6IcHR1tbW3x13v37tXS0vL09PTz83Nzc8O7CKmpqb1793758qXytchk8qBBg3bt2uXn5/f999/v378fIRQSEkIikYRCIV5m5MiRnp6eCCG5XG5jYzNq1Cg/Pz86nd7c3Dxs2LADBw4ghA4ePNizZ08fHx9vb+9+/foNGDCgvLwcIfTw4cNOnTqtXr365MmTCKG4uLj+/fuXlJTgp3zxxRe4h5aWljY2NnV1dQihZcuWLVq0CL90QUFBp06d/vryaAA+M5AxQL0kEskvv/zyzTffzJ8/f/78+Tt27Gh502JSUtIvv/yCL8FKTk7++eeflTc5hoSEKPcMEELR0dELFiyYP38+mUzG+0D5+fk//fQTn89XLiOTyY4fP45faOvWrXgQmidPnvz8888i0dt5Vnx9fZW7RHQ6ffny5fPnz8c7Qzt37sRXrDU3N+P1ODg43Lp1y8/PD98Eo1AogoKC7Ozstm7dihDKzMzcsmWL8v6Y+Ph43MPff/8d31WKEDp37hwOJIRQeXm5h4cHnU5X7RYGoC2DjAEAAKAukDEAAADUBTIGAACAukDGAAAAUBfIGAAAAOoCGQMAAEBdIGMAAACoC2QMaA0SiSQsLOzMmTM3b97UdF8AAK0HMgaonUAgsLW1NTQ0nDhxYs+ePbdv3/5n8xMrFIpr165duHChww7qJZFI7t+/j+cLAOAzABkD1M7Pz09fX7+2thYh5O/vr6Ojoxw9rKWSkpIFCxYMGTLEzMyMRCKdOHFC3R0rKSl5/fr1nwWeRjAYDIIg8LBpAHwGIGOA2nl7e48aNQp/7eXlZWZmVl9f//5i27dvHzRoEI6i/Pz858+fq7tjZ86cMTc3V/er/CsNDQ0PHz7EY6AB8BmAjAFql5OTo6Ojc+7cuQMHDvTp0wfPsvy+devWDR8+vGVLUVHRxo0blTPE1NfXe3t7Z2Rk5OTkHD169OXLl87OzitWrLh//75yAQqFYm9vb29vv2fPHuXQZ8ePH3/16lVMTIy9vf2PP/6IYywyMnL06NG9e/desmSJr68vXpLP53t4eNjb269Zs4bNZit7EhUVZW9vv2zZsrVr165atSotLQ23nzlzxt7efvny5ZmZmbjlzp07586de/bs2ffff79y5UrlXGcIITab7eLiYm9vv2/fPuUMza9evfrhhx/s7e3xTMwSieTo0aMvXrxACCkUipCQEHt7+9WrV7cclg2AdgQyBrSG7777jiCI2bNn5+bmIoRSU1MDAgLeWSY1NbVv377Tpk1LSUnBLXV1dXp6engASoTQnTt3CIIoKChITEzs0qWLpaWlt7f3/PnzlUP6U6lUa2vr7du3b9++/csvv3R1dcXHwWxsbExNTRctWrR161YjI6OJEyfW19ffuHFj5syZurq6np6ep0+fRghlZ2cbGhriIS/nzZtHIpFwbwMDA0kk0tatW1evXk0QxPTp0/EYmuvXr7ewsNi+fTuZTNbT08PnkPz9/bt27Tp58mQvL6/JkycPHjwYn1zJzs42MTFxcnLavn27paXl8uXLFQoFi8UyMTEhk8leXl5kMlkqlVZXV+vp6Z0/fx4hdOzYMQMDg59//nnVqlVnzpxphR8TACoHGQPUbseOHRMnTpw7d66pqSkeY9/Hx2fo0KHvL/n48eOpU6d26tRp8eLFhYWFCKE9e/YYGBg0NDQghNasWYPHyb9y5Yq2tjYeJF8mk5mYmPj7+yOElHNoIoTOnj1rZGSEZ6OxsbFZuHChXC5HCGVnZ3fv3j05ORkhFBQUNHbsWOVTFi9ePHfuXOXDSZMmrVmzBiE0ZcqULVu24MYJEyb8/vvvCKGMjIzBgwfjEEIILV269Ntvv0UIBQYG6urq4swrKyvT1dW9cOECQmj58uVOTk544czMzH79+r169SolJaV79+7KM/wKhaKysnLkyJF4VpulS5cq+9PyrQHQjkDGAPVKSkrq3Lnzixcv5HL5uHHjxowZU15evmLFCjKZ/GdPSUxMHDBgwOzZs5ubm5XzejU0NCinRo6JiTE3N1cO1z9t2rRt27YhhBobG/fu3TtkyJAhQ4bo6uqOGTMGfzTb2toeOnQILyyVSseOHYvH2z969Ojo0aPxITWJRDJq1KiWJ9t9fHxmzpyJEDpy5MjgwYMPHTq0c+dObW1tPDVASEhIt27dhg0bNmTIEAMDgz59+uD8CwgImDJlivK1Ro4cee7cOYTQ+PHj9fT0DAwMcPc6d+6ckpLS2Ni4atWq/v37e3p6crlchJBQKBw5cmR4eDhCKC0tzdTU1NLSMiwsrE1dmADAPwcZA9TLz8/PwMAAf11bW/vVV18ZGxv/7bT2kZGRBEHweDyE0KJFi9zc3C5duqTcoYmNjR05ciSeNwwhZGtrizNm27Ztenp6d+7cycrKOnDggJmZGZ7xzMbGBk8+hhBqaGgwMTEJCgpCCAUEBFhaWuKP76amJktLy5YZs3Hjxjlz5iCEvLy8vvrqq2+++ebbb79VzmJ5+vRpfX39Z8+eZWRkZGRk0Ol03B9/f/+JEyfibGtubh4xYgQ+zGVmZrZ58+aXL19mZGRkZWWx2WzlHDMPHjyYMGGCqalpWVlZfX29MmMQQpWVlWfOnNHT01PuSAHQvkDGAPW6fv06QRBRUVH44aVLlwiCIAgCH61q6eHDh8rrzdzc3IYNG4ankrx06ZKhoeG4ceOUn7PvZ8zPP/+MEJo+ffo333yDG9etWzdixAh8rGz69OlWVlZ4bjQKhdK5c2cajYYQOnHihJ6ennLmzR9++MHKygp/9PN4PB0dHbz3M3nyZG9v75SUlJSUlIyMDLxwRkZGz549r1+/jh9WVlbiRGyZMU1NTSNGjMD7TCtWrFBO64kQotPpcrmcxWLhy7gLCgoIgnj69Cne9YmIiEAIvXr1Ci/s7e1tbGwMuzKgPYKMAeolk8m8vLz69u1rbW1tbW1tbm6+f/9+Hx+fvn377tu3r+WSdnZ2xsbG1tbWFhYWOjo6ly5dwu319fXm5uYEQSinVY6IiBg4cGBpaSl+OGbMmPXr1yOEIiMj+/XrZ21tPXnyZENDw5EjR+L8WLBggbm5+aRJk6ytrXv27IlPqCCEsrOzdXR0zMzM8HkXDodjaWk5bNgwa2trAwODZcuW4czbt29fp06dTE1NTU1N9fX13d3d8Wr37t3bv39/5fsKCwtDCO3Zs2fkyJHKjNHT08NBxePxxo4dO3ToUGtr63Hjxi1YsKC5uTkyMhK/5S+//PK7774Ti8U1NTUDBw4MDQ1FCH3//fejR4+2trYeMmQInPMH7RRkDGgNjx49CgwMDAwM5HA4uOX27dtJSUktl6mvrw8NDQ0MDDx16hQ+4a/k7u4+duxYmUyGH3K53MTEROX+x507d/DFvgihtLS0wMDAuLi4srKypKQkfK7F1tb28OHDT58+DQwMfGf/KTs7+/Tp08rdkfr6+qCgoMDAwMuXL+OWsrKyiRMn4guLEUI5OTmdO3dW9hyvMzAwUDkwAY1Gu3XrFr6+QC6XX716lcFg4G+JxeLg4ODAwMCzZ89WVVXhBS5evBgYGBgREYHfXWNjY2JiIovFQggVFxefOnUqMDDw8ePHH7fZAdA4yBjQ1tXU1BgbGx87duyj1zB58mQ/P7+Pey6dTicI4vz583w+n8/n+/r66ujo0On0j+4MAB0KZAxo6y5evKivr4//tP848+bNU57z/7dkMhk+vT9o0KBBgwZNmjQJhvUE4J+DjAFtXXV1dUVFxaesoaqq6oOj1/xzlZWVAoFAIBDAfSoA/CuQMQAAANQFMgYAAIC6QMYAAABQF8gYAAAA6gIZAwAAQF0gYwAAAKgLZAwAAAB1gYwBAACgLpAxAAAA1AUyBgAAgLpAxgAAAFAXyBgAPge8Sgmz5JPGZAOfvWaZIvJZyeG7ZY/zalptxjvIGADat2qxNCqtbHVEgTOFdY9WpenugLYrnVPnFlO8OrbEJZy9/yaPVtwaf5RAxgDQXsnlivuMGq8LHDJV4BSWv/Zi5bG7hX//NNBRFZQ3rA5nukUXOYUyXamFLqHM0CclpbXqHUocMgaAdim3WLzvFt8lkusSznYKyydHC7wTuMxSsab7Bdo0erF4/61Clwi2SwTbKTSPHC3YFMO69kooaZap6RUhYwBoZ4qrG88+KHIOZbpG8RxDGK5RvLVR+THp5bXiZk13DbQPGZy6364XulDyHIJpzpQCMrXQJ5GXzqlTx2tBxgDQbjQ0y6+9Eq6LyiNHC5xCmY7BNJcwZuD9Il6lRNNdUz2FQlHfJGtokmuymuW1DdLimibNVklNU1F1I6NUTCuupxeLP7GYpQ1coSSTK9pzg+cclucQlOsQTHON5DpTCo7eK+IIVfy7BBkDQPuQya3zucolU/nOlAKHoFyHoFzHEIZHBOPkPcH5R8Un7wkCVVEn7wn23+D6JXJ9r7I1W78kFGyNy/eK12Rti8/3jM3ziGS4RzA8IjVXEQz3CIZjcK7D+Vz8o//0IofSV4fRHVu0OIbQydH8tRH5Ec/KiqobVfV7CxkDQFtXWNnon1TkHM5yjeQ6BNPe+bBwCWe5RnJVWrw2UKp9R59ULm2kIjgqLNdIrjMl/73soeELz+IyylT12wsZA0Bbx6ts9E/GGcN7J2Mcg+mukVxytECF5RZT5BZTrOGKLXGN5DqGMBxDmRosp9A8hyCaWxjdLQz/q7FaTaFviGJuouZtpDI/vTZFM7fF52+IYji2+HVyDme5RvF+u16YzROp8O4ZyBgA2ofsQtHv1wtdo3gu4ay3ARPK/DEmn5pWevVlRXxGWczzUpXU9VfCZFqlZusurfpRXvULbl0mV6TByuDUveKL2BUSdnkDu0KiqWKVS7hCSZVYWi2WVombP6WqxdL6Rll9oyyDU7vzEtspNM8hKNcplEmm8jdFF9zMqWySylX7ewsZA0C70SSV335T6RnPJkcLnELzHIJpZEqef5Igr6xB010D7UZtgzT6edmaiDxnSoFjMM01qtApmB70qLhMPTfKQMYA0M4IRc3hT8vIFCaZyncMYbhGcp1CmWceFAmqPsOry4BqpTCqPOPZZKrAKTTPJYLtGsnde7PwTZEab/iHjAGgXWKWiv2T+M6UApwx5GjB+mhWOqdW0/0CbRe9ROwUlucayXUKyyNHC7YlcO4zamRy9Y5cBhkDQDuWml+zI4FFjip0Dme5X6red4Or6R6BtoteInYKYayOLVkTmR+dUVHbIG2FF4WMAaB9a2iSXXtV+WMsa30sN4VRrenugLZLoVA8ZFZT04UF5a13Ag8yBoDPgVDULKhS2X1zAKgKZAwAAAB1gYwBAACgLpAxAAAA1AUyBgAAgLpAxgAAAFAXyBgAAADqAhkDAFCxqKiosWPHWlpaLl26lMfjabo7GpCbm2tra5uamqrpjmgeZAwAQJXOnj3bo0ePw4cPUygUFxeXsLAwdb8ilUr18fFR96v8K69evRo7duyjR4803RHNg4wBAKjS3Llz7ezsWrbU1dVxOBzlQ4VCwePxRCJRXV2dUChECDGZzLy8vJZPqa+vp9PpdDq9ru7tJPNyubyoqAghJBaL6XR6aWkpbi8uLl65cqWpqSmdTscLYAUFBXQ6XSAQvL9aJpPJZrOZTGZDw9vb3aurq+l0ekFBgXJJoVCIX5pOp7NYrHfeI5PJpNPpIpGoZSODwaDT6TKZDCEkk8lKSkoaG9/eFVtbW0un0/l8/j/Yfp8byBgAgCp5eXn16dPnyZMnypbs7Ow+ffo8fvwYP0xLS+vXrx+TyQwLC5s2bdrq1asNDAy0tLQ2btyoUCgQQomJiePHjyeRSCQSycLCIi0tDSEkEonmzJnz+++/z5w5k0Qi6enpXblyBSHk6enZs2fPrl27kkikdevWIYTq6+s3bNigo6NDIpEGDRp06NAh/LpZWVljx44lkUh9+/bt1KnT4MGD09PTEUI3btwwMzMjkUi6urpeXl5SqRQh9MsvvyxcuHDVqlUkEql79+67d+/GK6mvr1+zZs2AAQNIJNLo0aMfPnyIEKqurnZ2dh4wYMDAgQO3b9+OEOJyuV9++eXdu3cRQqmpqVZWViQSaejQoQ8ePGidn0LbARkDAFClsrKyefPmEQSxbNmyrKwshFBzc7O1tfWqVavwAt7e3uPHj0cIBQUFEQSxZcsWoVAYGxv7n//8B39kX79+PSEhAS/8ww8/TJs2DSHU2Nhobm5uYmJy8+bNysrK1atXa2trV1RU1NfX//jjjzY2NpWVlXjHwtPTU19fPyMjo7KyMjY2tlOnTjiNbG1tnZ2da2pqmEzmgAEDTp06JZVK8/PzjY2NL168iBDicrlGRkZnz55FCO3atatTp04HDhyorKw8cuSIlpYWg8FACG3btm3GjBnV1dUIIR8fn1GjRonFYiqVqqOjU11dXVlZmZiYiBBis9mDBw/GGTNr1iwHBweZTPb8+XOclx0KZAwAQMXkcjmVSh09enTPnj1xWpw9e3bQoEEikUgul1tYWJw4cQIhdPz48dGjR+OnyGSyESNGnDlzBj8sLy8/ePDggQMHFi1aNHXqVISQRCIxNzc/ffo0XoDH4w0ePDg5ORkhtGPHjlmzZuH2urq6oUOHBgYGKjuzePHiNWvWIIRmz569detWhJBQKOzbt29QUBBC6NSpUyQSKSAg4MCBA/7+/l9++aW9vT1CyMvLa+bMmcrOkEikpKQkhUJhbm6+YsWKQ4cOHThw4KeffiIIgsFgPHnyREdHx8fHp7b27dwKBQUFhoaGOGPWrVtnYWFx6dIl9W3wtgwyBgCgFlKpdNGiRUZGRs3NzdXV1fr6+levXn306BGJRCorK0MIHTt2bOLEic3NzQghuVxuZmYWEBCAELpw4YK+vv706dPnzp07YsQInB8SiWTMmDERERF45UKh0NjYOD4+HiHk7e2tzIOSkhJjY+Nr164pu7FmzZrvvvsOIbRv374uXboMHTpUV1d35cqV+FSQj49Pv379FixYMHfu3Llz5y5duhRfpLB161blWaWqqioSiXTt2rWGhgYDA4Nx48bhhe3s7MhkMj7lk5CQMHnyZLx7hBBis9mGhoZJSUkIoaamph07dowcOdLa2jo3N7cVtnybAhkDAFCXAwcO9O/fXywWI4TIZPLXX3/t5ua2dOlS/N1jx46NHz8enxiXSqWmpqZ4/8PCwsLV1RUvExAQMGnSJIRQY2OjmZnZnj17cHt6enqvXr0yMjIQQlu2bMHH0xBCYrF4+PDhynMwCCFLS8sdO3Y0NjZaWVmFhIS8ePHi5cuXyu8eOXLE2Nj4/Z5v3bp1wYIF+GuhUEgikRITE2UymZGR0blz5/7s/R49erRHjx4CgaCkpESZMVhzc/PMmTPnz5//77Zg+wcZAwBQmfr6+mXLloWFhSUnJ4eFhfXp02fbtm34W0+ePOnevXu3bt3u3buHWw4fPmxubq7MGENDQ39/f4TQvHnzJkyYcPfu3bNnz/br18/a2hoh1NTUZGVl1bdv32PHjiUnJ48ePdrW1hZfxBUQENC1a9eLFy/iyPHz8+vWrRuFQklOTvbw8NDW1sanUpYsWTJixIgZM2bMmDHj+++/x1eR4WNuK1asSE5OTk5O9vT0xPtAmzZtUu4bVVRUaGtr432m3bt39+7dOzQ0NDk5mUql/vTTTwqF4uzZs0ePHr13796uXbtIJJJQKOTz+bq6unfv3pVKpVu3bk1MTHz48KGlpaUyOzsOyBgAgMpIpVJ8VsPExGTYsGH79u1TXh8sl8snT55sZGSkvKI3MjJy+fLlTU1NCCGZTLZ48eLw8HCEEIvFWrx4sYmJydy5c319fT08PBBCDQ0NEyZM2LZt27fffmtiYmJvb19cXIzXIxQKly9fbmxsvHnzZvxCBw4cGDZsGF7DixcvEEKNjY2rVq1auXJlcHBwcHCwg4PDl19+ideQm5s7f/58ExMTExMTR0dHfKXyoUOH8OsihKqrq2fOnInP/cjl8kOHDhkbG5uYmIwePRofu3v48KGVlZWJicnkyZPxfZd8Pn/OnDn4DL+fnx9enkwm4wN0HQpkDACglUydOvWXX375uOfW19dbWFhERkZ+3NMfPHigra2tvKPlwYMH3bp1a3nQDKgJZAwAoDVcuXKFIIhXr1593NNFItGQIUPwBWkfIS8vz9jYeMqUKWQymUwmGxkZrV69Gh9qA2oFGQMAaA2JiYkHDx786I/1pqamo0ePPn/+/KM7UFxcvGXLFpwxFy5c+Oj1gH8FMgYAAIC6QMYAAABQF8gYAAAA6gIZAwAAQF0gYwAAavHq1auXL19WVlZquiNAkyBjAAAqxuFw5syZo6urSyKRTExMUlJSPrhYU1NTUVHRB29LbGxsLCoqemeCltZUV1dXUVGhqVf/nEDGAABUzM3NzdjYmM/n19bW2trazp49+4OLpaam9uzZc/DgwXisFyW5XL548WItLa1jx479xatIpdLa2lq5XK6SPkskkpaRtnr1aiMjIzwGAfgUkDEAABWzsbHBw+kjhBwdHZUD77/j3r17gwcPNjAw2LRpU8v2+/fvGxgYGBkZKUfA/CA6nT5q1Cg8lcun27dvn3KwToTQjRs3Tp8+DTdpfjoNZIxcLr9x44aaph2Vy+U3b97k8XjvfysuLs7f3185RWsb0QrTFmVlZf3ZwQoA1OHo0aN6enpsNtvf3793797379//4GJ4aEsfHx8DA4Py8nJl+4oVK9asWfPNN9/89ttvuKWoqCggIMDf3z88PBzPlclkMjds2NCtW7cdO3acPXu2ubmZz+dTqVTl3Mz379/Hk06WlpbevHmzrKzs6NGjeBKXp0+f+vv7+/v7K2elvHXrlo2NDR6U8/r16wghDofz+PFj/FoIITabfeTIEX9//5bv5fbt22w2OyMjw9/fXzmpGniHujKGwWCYmZmdP3/+/W81NTWNHj368uXL/3xtxcXFKSkpeA7UvyaVSq2srOLi4t5pP3PmTK9evaZNm6baEYpCQkLMzc3xjBFKcXFxZmZmeDa9v+Xu7u7m5vYRL/3o0aOWc6T/BS8vr4ULF37wWzQazdXV1cTExNzcHE8FiBCSy+VHjx7FQwTu2LFDIpEghKRS6YEDB0xMTIyNjX/77Tc85wdCqLCw0MnJaeXKlR/xFsBnzNjYuG/fvnPnzsWTLmdmZj579uydZZKTk01MTHJycoYPH378+HHcyGKxDAwMMjMz7ezs/Pz8EEIMBmPMmDFTp06dNm2agYHBsmXLxGJxSkrKmDH/1965BzV1bX88nSloioEJgRi1IqQ8RAKEl3LyKgHUQR5GfGCM0ajFNoFgQUVAeSnlIT44CFEGrHUk4gjYYkVA2hEItFXboghqtVLqiK3WB/JSQbLuH3vumVy58/v5h8ztY3/+OqwTds5kzjnftddaey8Pc3NzX19fmUz27NmzqqoqGo128+ZNNM6CBQvCwsIAwGAwMJlMb29vb2/vrVu3Hjx40NXVVSwWi8ViFouFHt6srKwZM2ZYWVmJxWIkbJmZmU5OTmgeU1lZyWaz/fz8xGIxk8mktlwLCAjg8XgikUgsFr/99ts7d+6c+N/1r8dEaUxycjKDwVi8eDHlCJgikUiQs/CanDlzxsXF5TUDr1KpdHzLuaVLl1I+0RskLi6OTqeLxWJqc1mj0bh06VI6nR4TE/M6IyQmJr4SKHhNfHx8ysrKXueTqampK1as+K+nysvLMzMz6+rqUlJSLC0tOzs7AUCn01lbW1dXV586dYrFYqWlpQFAfn4+m82uqak5efKklZVVXl4eAFy8eDEsLEwmk0kkkjcVFsf8DcjKyvL09JwyZYpGo0GW8PBwquUXRWNjo52dXV9f37Zt2zw8PJBx06ZNKH8jlUqRxvT19V27dg2d7erqsrCw+P777wGgvb19+vTpqN0ZAFRWVlpbW6NdkwFg6dKlyPW5dOmSmZkZ6oAJAN3d3VQyPzU1lcfjoeOUlJTg4GDq2vLy8nx9fQHgyZMn06ZNo3Tl5MmTZmZm6AKCg4Pd3d17e3sBYNeuXXZ2dtQsCkMxIRrT39/v7+9fUVHh6+uLdroGgIGBgejoaIIg9u3bJ5FI6uvrAeDcuXPBwcEEQSQmJiJ/uba2liTJQ4cOEQTxwQcfjIyMdHR0uLq6WlhYzJ07Fzk7Fy5cCAwMJAhi9+7dSMOGhoY+/PBDgiD27t0bEBBQU1Njej1ZWVksFsvR0VGpVL58+fLAgQMlJSXh4eFJSUkA8OTJE5VKRRBERETErVu3AODBgwfp6ekVFRVSqTQsLOzOnTvV1dUEQaxfv76vr890ZK1Wu3jxYolEQqlaW1tbaGjounXrPvroI2Q5ffq0UCgkCCI7O5uaihUXFwsEgvj4eJVKlZyc3NPTEx0d/fjxY3R2x44dr4QXjh07RhCEQCBA2pyQkMBgMLhcbnh4OLXDOQC8ePFCr9cLhUKBQLB3717khf0fGmOKm5vbiRMnAEAikVAtb8vKyng83sDAgFAopFoQFhQUeHp6jo2NdXd3//zzz/X19VKpFEeuMYhjx45NmjTpxo0bLS0tNBpt165dAEAQRGZm5iufbGxsnDlz5sOHD7u6uhgMxqVLl0ZGRhwcHCoqKgBAIpEgjQGA7u7uJUuWCAQCX19fKyurH3/8EQAMBgOHw0HPLABUVlYymczxGtPW1mZvb9/T00N9b3V1NXpGHB0dRSIRMpo2VwYTjWlubra1taViBi9fvnR3d0dRCqlUSnmuzc3NhwtP/AAAC05JREFUbDZ7glIAf2kmRGPq6uqCgoLGxsYUCgVy0o1Go0Kh8PLyIkkyJCTknXfeMRgMY2NjUVFReXl5JSUlDg4OqampAKDT6SwsLJRKJUmSjo6OsbGxDx482Lhx47vvvpubm2swGDo7O0UiUWlpqV6v9/f3R29DlUrl6elJkmRoaCidTjdtPwcA586d8/LyioyMPHr06MjIiEwmo9PpiYmJZ8+effHixfz58wMDA0mSVCqVLi4u9+/ff/TokY2NDZfL3bdvX2Bg4KxZs4KCgkiStLe3p5QDoVar1Wp1dnb2smXLkEWlUuXk5KSmpkZHRwPA+fPnmUzmjh07SJK0s7NLSUkBAL1eb21tvWfPnvj4eDqdnpaWNjw87ODggITqxo0bs2fPvn79OvUtSO30en1JSQmfz//uu+9Onz7N5XKjoqLKysqoFuIA0NPTEx4eXlxcXFRUZGNjg7pxvI7GdHZ2crnc7u7u4eFhLpfb0tKC7K2trbNnz75y5YqTkxPy3QCgoaHB0dGRctkqKyuxxmAoIiIi5s+fj46/+OILBoMRGRnp4OBgeksjkMag97JUKtVoNOXl5c7OzsjdpDSmtbWVw+Go1WqSJNPS0mxtbVFLGNS2ubu7G41WXV1ta2tL5WLlcvnKlSsBoK2tbdasWdRMKD09ncViZWdnHzhwYNmyZQRBIHtCQoJp/RulMV999ZWdnR3lyY2MjMyZM6eoqAgAAgMD0RONLsba2vo1w9f/KCZEY2JiYlAYp6Ghwc/Pb3R09N69e25ubigXMjQ05ObmdvbsWfThkZGR4eHh+Pj4yMhIACgqKhIKhejUmTNnUBvUlpYWPz8/ZExPT3/vvfdiYmK0Wu2cOXMWL17c19fH4/HQG/D58+fu7u6vzGMAYM2aNaWlpeg4JCQE9TICgNraWldXV6pmkc/nl5aWPnv2jMvlom593377LYvFQk6QTqd7//33TYdVq9UbNmzo7u5GlZr9/f1z5869c+fOtm3bNm7cCAAymQzNlgDg888/d3JyGhoaWr58+SeffIKMSqUSyXBMTMyqVasAgCRJ1H6cIjQ01N/fX6vVajQaS0vLnJwcAECq819//+fPnw8NDclkMtSC8HU0JiIiIj4+HgDu3r07a9YsSk5++OGH2bNnNzY2Ojs7U0+pwWBwcHD47bff0J9YYzCmaLVaNptNrXrZvXs3jUbjcrnj64CRxiBV0Ov1lpaWbDabqiWjNCYpKWnmzJnIeOHCBWtra9Tv0mAwTJkyBbWzBICWlhYrKyvUO+DevXsODg7r1q2D/9SYsbExFxcXKo6dnJzs7e2NjuPj4318fKhrozTml19+YTAYx48fR/bz58+bm5tfvHgR/lNjmpqaqBcFxpQ3rzEPHjywt7dftGiRRqNRKBTm5uZXrlzp7Oz09fWlAk2BgYFIYwoLC+3s7DgcDpPJ3LBhA7JQL8QbN264u7sPDw/X1tZ6eXkhx3nt2rV8Pl+r1cbGxiYlJdXV1XV0dPj4+FDLiRcsWDA+HxMVFUXV2i9atIjqQlFaWkp17UYfy8/PHxwc9Pb2RnPwb775xsPDA3XuO3LkSGhoqOmwarVaoVAAwMKFCz/99FO9Xi+TyQAgLi4ONdGTSqXULuLXr1/n8/m9vb1BQUFUOiolJUWr1QKAwWDw9PTs6+uTyWSmtRJGo5HP54eGhsbGxsbGxu7YsaO9vd1oNIpEovElFc+fP09OTuZwOBwOx9LSEinZ/6sxarU6KCgICe2TJ08cHR2bm5vRqaamJmdn566uLhcXF/RgA0BtbS2Xyx0cHER/Yo3BmPLrr7+6urryeDyNRqNSqVxcXFAc29vbm6rjQtTW1lpYWCDfv7+/n8Vi0Wg0Ktzk4eGxdetWAGhoaJg0aZJcLo+JibG3t6fRaOhW7O3tnTp1qlgsTkhIePjw4dOnT728vJydnTUajVQqZbFYyGlrampiMBhXr15Fw3788cc2NjYajSYqKsrCwsLLywvF248fP06j0dasWYOqADIyMhwdHVGWccuWLQwGY+PGjRqNZurUqVR/TB8fH8pbbWxspNFoVKQOQ/HmNaaysnLatGkqlUoulyuVSjc3t5iYmPv37zs7O3d0dADA4OCgq6trc3PzzZs32Wx2a2vr0NBQUlISuiGKiorEYjEaqqamhsfjjY6O1tTU8Pl8ZNy8eXN4eLjpN96+fdvJyam9vR0Anj17xuPxxs9jXtEY1DYcAKqqqpycnKjXpY+Pj16vHxgY4PP5XV1dANDa2urm5obev2VlZa/kLdVqNYr5Hj161MfHZ968eaikTavVohtx4cKFVLawqqqKz+cPDAyEhYWhuQgAKBQKyqsKDg5OS0uTSCSveENSqTQ3N9fUYjQa582bh0Jhppw6dWrGjBm3b9/u7++Xy+XIyTLVmPElGGlpaSEhIZR9dHTU29sbpfQBIDMzUygUDgwMeHp6ovgAACQmJkokEupf6uvrFyxYABjMv+nv79++fbtcLl+3bh2aEw8ODsbHx1PRC8RPP/2Unp5OpSH1er3posvCwsK6ujp03NDQsHr1aqVSaTAYSJKkHpCWlhaVShUbG4sixr29vZs2bZLL5W1tbV9++SVqmtnd3Z2RkUEtWhgdHS0oKJDL5WlpaU1NTcXFxUhIxsbGdDqdQqFA4fevv/56z549VCVLRUUFatWMckWIoqIiylm8detWcnIy3jhnPG9YY4xGY3Bw8ObNmylLdXU1k8l8/PjxqlWrhEIhSZLh4eFmZmYtLS09PT0zZsxIT08/dOgQh8NBC6A+++yzyZMnq1QqlMBARU2XL1+2sbFJTk5ua2u7fPkyk8mMjY0lSTI6Oho5HVFRUf7+/iRJymQyMzOz8UVrYWFh1HtTIpFQ8/G+vj5fX1+UbomMjPTz8xsaGrp37x6Xy0WK2NTUNHPmTDSFKi4ulkgkpsOuXbs2IiICAP744w8GgzF9+nR0r2/YsAFJZlVVlYWFRWpqakFBwbRp09AjdPjwYVtbW5IkExIS6HQ65Qrt37//rbfeQkE2U0pKSqZMmbJz506SJJcvX44Wu6xYsUIgEBw+fNi0lKW+vp7NZpMkuWfPHnNzcxSm27JlC5p+nT171snJyXTx0L59+2g0WnJy8sGDB0mSRIUGOp2OTqfn5OTk5uYymUz0UOXn51tYWOTn52dlZTGZTKTid+/eLSwsVCgUHA5n//79VBYHg8FgEG9YYwYHB9VqNRVUAYDHjx+vXr26o6Pj6dOnSqWSIIijR49mZ2cj76ayslIkEoWFhWVnZyO10Ol0ISEh2dnZBEFs376dKsTKzs4WCoUoKnrhwgVUjbZ+/XpUOPjo0aO1a9cSBHHkyJGcnJzxqxrz8vKoyU1GRoZpMO3333+Xy+UEQahUKrQQ7OHDh3FxcXfu3AGAa9euaTQalIQ8d+7cKwXQOp0uPz8fHefm5lKefklJCRWOO3HiBCpiOXz4MLK8fPkyLy+PIIi0tLTS0tIjR44g+9WrVydPnvyKrwcARqOxvLxcIBAQBJGRkYFk7PLly4GBgStXrjTdAMNoNBYWFgoEglWrVu3cuRPJQ3l5OdLXtra2JUuWUHkUACgoKAgJCQkICCAIAtXpUdcvEAgEAgH1QxmNRpIkBQKBSCSqra1Fxvb2dpFIFBAQsHDhQoFAcODAAcBgMBgT/nR7yeTn54+vo/+HUFlZ6enpSYUOMBgM5q/On05jysvL4+Li/tdX8b9Bq9VSMwkMBoP5G/Cn05h/MnipPAaD+ZuBNQaDwWAwEwXWGAwGg8FMFFhjMBgMBjNRYI3BYDAYzESBNQaDwWAwEwXWGAwGg8FMFFhjMBgMBjNRYI3BYDAYzESBNQaDwWAwE8W/AFbfItbSZ003AAAAAElFTkSuQmCC" />   During the very early prenatal period, genes corresponding to general cellular function are prominent (Mody et al., 2001).  These are followed in time by genes regulating neuronal differentiation and migration in the mid to late gestational period. From late gestation (gestational day 15) until birth almost all the cells in the CA fields switch from a highly active proliferation state to a postmitotic state, and then undergo differentiation and migration. Expression of proliferative genes involved in cell cycle progression are highly expressed at gestational day 16, then subsequently are silent immediately after birth when genes directing neuronal growth switch on. The pyramidal neurons of the CA fields in the hippocampus proper develop in advance of the granule cells that comprise the principal cells of the dentate gyrus. As such, the genes controlling the distinct phases of neurodevelopment are expressed at different times in these two hippocampal subregions (Altman and Bayer, 1990a; b). In both subregions, however, many phenotypic changes within the hippocampal neuron occur in the period immediately after birth (postnatal day 1 to 7). Almost all neurons show extensive growth and differentiation during the first postnatal week. These cellular changes are marked by rapid cytoskeletal changes, production of cell adhesion molecules, and extracellular matrix formation. The gene families involved in these processes include actins, tubulins, and chaperonin proteins essential for promoting correct protein folding of cytoskeletal components. Cell adhesion and extracellular matrix proteins are also upregulated during this period as these genes are critical for differentiation and synaptogenesis. During late postnatal hippocampal development (postnatal day 16-30), hippocampal circuits become more active and exhibit increased synaptic plasticity. Many genes upregulated during this phase of development are involved in synaptic function and include genes regulating vesicle associated proteins and calcium-mediated transmitter release, neurotrophins, and neurotransmitter receptors. Efficient energy utilization is essential during this period of increased synaptic activity, events mirrored by an upregulation of enzymes involved in glucose and oxidative metabolism.

Measurement of genomic profiles in developing brain use methods that are well established and accepted in the published literature.  Microarray studies with expression profile analyses have been conducted in cortex and hippocampus of humans (Zhang et al., 2002), non-human primates, and rodent brains of various ages (Mody et al., 2001; Royland et al., 2008; Dong et al., 2015). More commonly, quantitative rtPCR or in situ hybridization have been used to probe individual gene transcripts (Dowling et al., 2000, Morte et al., 2010) or their protein products (Alvarez-Dolado et al., 1994; Gilbert et al., 2007). Recently RNA-Seq technology was applied to T3-treated primary mouse cortical cells and gene targets enriched in astrocytes and neurons to identify TH-responsive genes (Gil-Ibanez et al, 2015).

Gene expression in the developing brain in general is analogous across most mammalian species (Kempermann, 2012). Most of the empirical data on gene expression in hippocampus is from rat, mouse and human studies.

UBERON:0000955

UBERON brain

High Female High Male

High

During brain development

High High Moderate

Altman J, Bayer SA. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J Comp Neurol. 1990a Nov 15;301(3):365-81. Altman J, Bayer SA. Prolonged sojourn of developing pyramidal cells in the intermediate zone of the hippocampus and their settling in the stratum pyramidale. J Comp Neurol. 1990b Nov 15;301(3):343-64. Alvarez-Dolado M, Ruiz M, Del Rio JA, Alcantara S, Burgaya F, Sheldon M, Nakajima K, Bernal J, Howell BW, Curran T, Soriano E, Munoz A (1999) Thyroid hormone regulates reelin and dab1 expression during brain development. J Neurosci 19:6979-6993. Bernal J. (2105)  Thyroid Hormones in Brain Development and Function. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.. Dong H, You SH, Williams A, Wade MG, Yauk CL, Thomas Zoeller R (2015) Transient Maternal Hypothyroxinemia Potentiates the Transcriptional Response to Exogenous Thyroid Hormone in the Fetal Cerebral Cortex Before the Onset of Fetal Thyroid Function: A Messenger and MicroRNA Profiling Study. Cereb Cortex 25:1735-1745. Dowling AL, Zoeller RT. 2000. Thyroid hormone of maternal origin regulates the expression of RC3/neurogranin mRNA in the fetal rat brain. Brain Res: Molec Brain Res.  82:126-132. Gilbert ME, Sui L, Walker MJ, Anderson W, Thomas S, Smoller SN, Schon JP, Phani S, Goodman JH (2007) Thyroid hormone insufficiency during brain development reduces parvalbumin immunoreactivity and inhibitory function in the hippocampus. Endocrinology 148:92-102. Gil-Ibanez P, Garcia-Garcia F, Dopazo J, Bernal J, Morte B. 2015. Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types. Cerebral cortex. Nov 2. Kempermann G.  New neurons for 'survival of the fittest'.  Nat Rev Neurosci. 2012 Oct;13(10):727-36. Mody M, Cao Y, Cui Z, Tay KY, Shyong A, Shimizu E, Pham K, Schultz P, Welsh D, Tsien JZ. Genome-wide gene expression profiles of the developing mouse hippocampus. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8862-7. Morte B, Ceballos A, Diez D, Grijota-Martinez C, Dumitrescu AM, Di Cosmo C, Galton VA, Refetoff S, Bernal J.  Thyroid hormone-regulated mouse cerebral cortex genes are differentially dependent on the source of the hormone: a study in monocarboxylate transporter-8- and deiodinase-2-deficient mice. Endocrinology. 2010. 151:2381-2387. Royland JE, Parker JS, Gilbert ME. A genomic analysis of subclinical hypothyroidism in hippocampus and neocortex of the developing rat brain. J Neuroendocrinol. 2008 Dec;20(12):1319-38. Zhang Y, Mei P, Lou R, Zhang MQ, Wu G, Qiang B, Zhang Z, Shen Y. Gene expression profiling in developing human hippocampus. J Neurosci Res. 2002 Oct 15;70(2):200-8. AOPWiki 2016-11-29T18:41:26

2018-08-11T09:26:56

Hippocampal anatomy, Altered

Tissue

The hippocampus is a major brain region located in the medial temporal lobe in humans and other mammals (West, 1990). Developmentally it is derived from neuronal and glial cells in the neural tube and differentiates in the proencephalon and telencephalon.  The hippocampus is a cortical structure, but only contains 3-layers, distinct from the 6-layered neocortical structures. For this reason, it is known as archicortex or paleocortex meaning old cortex. Within humans, the structure is identified as early as fetal week 13 and matures rapidly until 2 to 3 years of age (Kier et al 1997), with continuing slow growth thereafter until adult ages (Utsunomiya et al., 1999).  In rodents, the hippocampus begins to form in midgestation, with the CA fields forming in advance of the dentate gyrus. Dentate gyrus forms in late gestation with most of its development occurring in the first 2-3 postnatal weeks (Altman and Bayer, 1990a; 1990b). The structure of the hippocampus has been divided into regions that include CA1 through CA4 and the dentate gyrus. The principal cell bodies of the CA field are pyramidal neurons, those of the dentate gyrus are granule cells. The dentate gyrus forms later in development than the CA fields of the hippocampus. These regions are generally found in all mammalian hippocampi. The major input pathway to the hippocampus is from the layer 2 neurons of the entorhinal cortex to the dentate gyrus via the perforant path forming the first connection of the trisynaptic loop of the hippocampal circuit. Direct afferents from the dentate gyrus (mossy fibers) then synapse on CA3 pyramidal cells which in turn send their axons (Schaeffer Collaterals) to CA1 neurons to complete the trisynaptic circuit (Figure 1). From the CA fields information then passes through the subiculum entering the fiber pathways of the alveus, fimbria, and fornix and it routed to other areas of the brain (Amaral and Lavenex, 2006). Through the interconnectivity within the hippocampus and its connections to amygdala, septum and cortex, the hippocampus plays a pivotal role in several learning and memory processes, including spatial behaviors. The primary input pathway to the CA regions of the hippocampus is from the septum by way of the fornix and direct input from the amygdala. Reciprocal outputs from the hippocampus back to these regions and beyond also exist.   Trisynaptic Hippocampal Circuitry <img alt="" 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" />

Data in support of this key event have been collected using a wide variety of standard biochemical, histological and anatomical methods (e.g., morphometrics, immunohistochemical staining, in situ hybridization and imaging procedures). Many of methods applied to reveal anatomical abnormalities are routine neurohistopathology procedures similar to those recommended in EPA and OECD developmental neurotoxicity guidelines (US EPA, 1998; OCED, 2007). Subtle cytoarchitectural features depend on more specialized birth dating procedures and staining techniques. It is essential to consider the timing of events during development for detection to occur, as well as the timing for detection (Hevner, 2007; Garman et al., 2001; Zgraggen et al., 2012). Similar techniques used in rodent stydies have been applied to postmortem tissue in humans.  In humans, structural neuroimaging techniques are used to assess hippocampal volume with an analysis technique known as voxel-based morphometry (VBM). Volume of brain regions is measured by drawing regions of interest (ROIs) on images from brain scans obtained from magnetic resonance imaging (MRI) or positron emission tomography (PET) scans and calculating the volume enclosed. (Mechelli et al., 2005). Similar imaging techniques can be applied in rodent models (Powell et al., 2009; Hasegawa et al., 2010; Pirko et al., 2005; Pirko and Johnson, 2008).

The hippocampus is generally similar in structure function across most mammalian species (West, 1990). The vast majority of information on the structure of the hippocampus is from mice, rats and primates including humans.

UBERON:0000955

UBERON brain

High Male High Female

High

During brain development

High High High

Altman J, Bayer SA. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J Comp Neurol. 1990a Nov 15;301(3):365-81. Altman J, Bayer SA. Prolonged sojourn of developing pyramidal cells in the intermediate zone of the hippocampus and their settling in the stratum pyramidale. J Comp Neurol. 1990b Nov 15;301(3):343-64. Amaral D, Lavenex P (2006). "Ch 3. Hippocampal Neuroanatomy". In Andersen P, Morris R, Amaral D, Bliss T, O'Keefe J. The Hippocampus Book. Oxford University Press. ISBN 978-0-19-510027-3. Garman RH, Fix AS, Jortner BS, Jensen KF, Hardisty JF, Claudio L, Ferenc S. Methods to identify and characterize developmental neurotoxicity for human health risk assessment. II: neuropathology. Environ Health Perspect. 2001 Mar;109 Suppl 1:93-100. Hasegawa M, Kida I, Wada H.  A volumetric analysis of the brain and hippocampus of rats rendered perinatal hypothyroid. Neurosci Lett. 2010 Aug 2;479(3):240-4. Hevner RF. Layer-specific markers as probes for neuron type identity in human neocortex and malformations of cortical development. J Neuropathol Exp Neurol. 2007 66(2):101-9. Kier, EL, Kim, JH, Fulbright, K, Bronen, RA. Embryology of the human fetal hippocampus: MR imaging, anatomy, and histology. AJNR Am J Neuroradiol: 1997, 18(3);525-32. Mechelli A, Price C, Friston K, Ashburner J (2005) Voxel-Based Morphometry of the Human Brain: Methods and Applications. Curr Med Imaging Rev 1:105-113. OECD. 2007. OECD guidelines for the testing of chemicals/ section 4: Health effects. Test no. 426: Developmental neurotoxicity study. http://www.oecd.org/dataoecd/20/52/37622194. Pirko I, Fricke ST, Johnson AJ, Rodriguez M, Macura SI. Magnetic resonance imaging, microscopy, and spectroscopy of the central nervous system in experimental animals. NeuroRx. 2005 Apr;2(2):250-64. Pirko I, Johnson AJ. Neuroimaging of demyelination and remyelination models. Curr Top Microbiol Immunol. 2008; 318:241-66. Powell MH, Nguyen HV, Gilbert M, Parekh M, Colon-Perez LM, Mareci TH, Montie E. Magnetic resonance imaging and volumetric analysis: novel tools to study the effects of thyroid hormone disruption on white matter development. Neurotoxicology. 2012 Oct;33(5):1322-9. U.S.EPA. 1998. Health effects guidelines OPPTS 870.6300 developmental neurotoxicity study. EPA Document 712-C-98-239.Office of Prevention Pesticides and Toxic Substances. Utsunomiya, H., K Takano, M Okazaki, A Mitsudome Development of the temporal lobe in infants and children: analysis by MR-based volumetry. AJNR Am J Neuroradiol: 1999, 20(4);717-23. West MJ (1990). "Stereological studies of the hippocampus: a comparison of the hippocampal subdivisions of diverse species including hedgehogs, laboratory rodents, wild mice and men". Progress in Brain Research. Progress in Brain Research 83: 13–36. Zgraggen E, Boitard M, Roman I, Kanemitsu M, Potter G, Salmon P, Vutskits L, Dayer AG, Kiss JZ. Early postnatal migration and development of layer II pyramidal neurons in the rodent cingulate/retrosplenial cortex. Cereb Cortex. 2012 Jan;22(1):144-57.    AOPWiki 2016-11-29T18:41:26

2022-05-20T05:45:05

Hippocampal Physiology, Altered

Tissue

The hippocampus functions as a highly integrated and organized communication and information processing network with millions of interconnections among its constitutive neurons. Neurons in the hippocampus and throughout the brain transmit and receive information largely through chemical transmission across the synaptic cleft, the space where the specialized ending of the presynaptic axon terminus of the transmitting neuron meets the specialized postsynaptic region of the neuron that is receiving that information (Kandell et al., 2012). During development (see KE: Hippocampal anatomy, Altered), as neurons reach their final destination and extend axonal processes, early patterns of electrical synaptic activity emerge in the hippocampus. These are large fields of axonal innervation of broad synaptic target sites that are replaced by more elaborate but highly targeted and refined axonal projections brought about by activity-dependent synaptic pruning and synapse elimination.  This is a classic case of the interaction between physiological and anatomical development, where anatomy develops first, and can be ‘reshaped’ by physiological function (Kutsarova et al., 2017). In the rat, excitatory processes are fully mature in area CA1 of hippocampus within 2 weeks of birth with inhibitory processes lagging begin by several weeks (Muller et al., 1989; Michelson and Lothman, 1988; Harris and Teyler, 1984). In hippocampal slices, inhibitory function in areaCA1s is first seen on postnatal day 5 and increases in strength at postnatal day 12 through 15.  In vivo studies fail to detect inhibition until postnatal day 18 with steady increase thereafter to adult levels by postnatal day 28. Synaptic plasticity in the form of long-term potentiation (LTP) is absent in the very young animal, only emerging about postnatal day 14, appearing to require the stability of both excitatory and inhibitory function to be established (Muller et al., 1989; Bekenstein and Lothman, 1991). These features of the maturation of hippocampal physiology are paralleled in dentate gyrus, but as with anatomical indices in the rat, the development of these physiological parameters lag behind the CA1 by about 1 week.

In animals, synaptic function in the hippocampus has been examined with imaging techniques, but more routinely, electrical field potentials recorded in two subregions of the hippocampus, area CA1 and dentate gyrus, have been assessed in vivo or in vitro from slices taken from naive or exposed animals. Field potentials reflect the summed synaptic response of a population of neurons following direct stimulation of input pathways across a monosynaptic connection. Changes in response amplitude due to chemical perturbations and other stressors (e.g., iodine deficiency, thyroidectomy, gene knockouts) is evidence of altered synaptic function. This can be measured in vitro, in vivo, or in hippocampal slices taken from treated animals (Gilbert and Burdette, 1995). The most common physiological measurements used to assess function of the hippocampus are excitatory synaptic transmission, inhibitory synaptic transmission, and synaptic plasticity in the form of long-term potentiation (LTP). Excitatory Synaptic Transmission: Two measures, the excitatory postsynaptic potential (EPSP) and the population spike are derived from the compound field potential at increasing stimulus strengths. The function described by the relationship of current strength (input, I) and evoked response (output, O), the I-O curve is the measure of excitatory synaptic transmission (Gilbert and Burdette, 1995). Inhibitory Synaptic Transmission: Pairs of stimulus pulses delivered in close temporal proximity is used to probe the integrity of inhibitory synaptic transmission. The response evoked by the second pulse of the pair at brief intervals (<30 msec) arrives during the activation of feedback inhibitory loops in the hippocampus. An alteration in the degree of suppression to the 2nd pulse of the pair reflects altered inhibitory synaptic function (Gilbert and Burdette, 1995). Long Term Potentiation (LTP): LTP is widely accepted to be a major component of the cellular processes that underlie learning and memory (Malenka and Bear, 2004; Bramham and Messaoudi, 2005). LTP represents, at the synapse and molecular level, the coincident firing of large numbers of neurons that are engaged during a learning event. The persistence of LTP emulates the duration of the memory. Synaptic plasticity in the form of LTP is assessed by delivering trains of high frequency stimulation to induce a prolonged augmentation of synaptic response. Probe stimuli at midrange stimulus strengths are delivered before and after application of LTP-inducing trains. The degree of increase in EPSP and PS amplitude to the probe stimulus after train application, and the duration of the induced synaptic enhancement are metrics of LTP. Additionally, contrasting I-O functions of excitatory synaptic transmission before and after (hours to days) LTP is induced is also a common measure of LTP maintanence (Bramham and Messaoudi, 2005; Kandell et al., 2012; Malenka and Bear, 2004). Synaptic function in the human hippocampus has been assessed using electroencephalography (EEG) and functional neuroimaging techniques (Clapp et al., 2012). EEG is a measure of electrical activity over many brain regions but primarily from the cortex using small flat metal discs (electrodes) placed over the surface of the skull. It is a readily available test that provides evidence of how the brain functions over time. Functional magnetic resonance imaging or functional MRI (fMRI) uses MRI technology to measure brain activity by detecting associated changes in blood flow. This technique relies on the fact that cerebral blood flow and neuronal activation are coupled. Positron emission tomography (PET) is a functional imaging technique that detects pairs of gamma rays emitted indirectly by a radionuclide (tracer) injected into the body (Tietze, 2012; McCarthy, 1995). Like fMRI, PET scans indirectly measure blood flow to different parts of the brain – the higher the blood flow, the greater the activation (McCarthy, 1995). These techniques have been widely applied in clinical and research settings to assess learning and memory in humans and can provide information targeted to hippocampal functionality (McCarthy, 1995; Smith and Jonides, 1997; Willoughby et al., 2014; Wheeler et al., 2015; Gilbert et al., 1998). Assays of this type are fit for purpose, have been well accepted in the literature, and are reproducible across laboratories. The assay directly measures the key event of altered neurophysiological function.

The majority of evidence for this key event come from work in rodent species (i.e., rat, mouse). There is a moderate amount of evidence from other species, including humans (Clapp et al., 2012).

UBERON:0000955

UBERON brain

High Female High Male

High

During brain development

Moderate High High

Bekenstein JW, Lothman EW. An in vivo study of the ontogeny of long-term potentiation (LTP) in the CA1 region and in the dentate gyrus of the rat hippocampal formation. Brain Res Dev Brain Res. 1991 Nov 19;63(1-2):245- Bramham CR, Messaoudi E (2005) BDNF function in adult synaptic plasticity: the synaptic consolidation hypothesis. Prog Neurobiol 76:99-125. Clapp WC, Hamm JP, Kirk IJ, Teyler TJ. Translating long-term potentiation from animals to humans: a novel method for noninvasive assessment of cortical plasticity. Biol Psychiatry. 2012 Mar 15;71(6):496-502. Gilbert, M.E. and Burdette, L.J. (1995). Hippocampal Field Potentials: A Model System to Characterize Neurotoxicity. In Neurotoxicology: Approaches and Methods. L.W Chang and W. Slikker (Eds). Academic Press:New York, 183-204. Gilbert ME, Mack CM. Chronic lead exposure accelerates decay of long-term potentiation in rat dentate gyrus in vivo. Brain Res. 1998 Apr 6;789(1):139-49. Harris KM, Teyler TJ. Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol. 1984. 346:27-48. Kandell, E., Schwartz, J., Siegelbaum, A. and Hudspeth, A.J.  (2012) Principles of Neural Science, 5th Edition.  Elsevier, North Holland. Kutsarova E, Munz M, Ruthazer ES.  Rules for Shaping Neural Connections in the Developing Brain.  Front Neural Circuits. 2017 Jan 10;10:111. doi: 10.3389/fncir.2016.00111. Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5-21. McCarthy, G. (1995) Review: Functional Neuroimaging and Memory. The Neuroscientist, 1:155-163. Michelson HB, Lothman EW. An in vivo electrophysiological study of the ontogeny of excitatory and inhibitory processes in the rat hippocampus. Brain Res Dev Brain Res. 1989 May 1;47(1):113-22. Muller D, Oliver M, Lynch G. Developmental changes in synaptic properties in hippocampus of neonatal rats. Brain Res Dev Brain Res. 1989 Sep 1;49(1):105-14. Smith, E and Jonides, J. (1997). Working Memory: A View from Neuroimaging. Cognitive Psychology, 33:5-42. Tietze, KJ. (2012). Review of Laboratory and Diagnostic Tests- Positron Emission Tomography. In Clinical Sills for Pharmacists, 3rd Edition, pp 86-122.  Wheeler SM, McLelland VC, Sheard E, McAndrews MP, Rovet JF (2015) Hippocampal Functioning and Verbal Associative Memory in Adolescents with Congenital Hypothyroidism. Front Endocrinol (Lausanne) 6:163. Willoughby KA, McAndrews MP, Rovet JF (2014) Effects of maternal hypothyroidism on offspring hippocampus and memory. Thyroid 24:576-584. AOPWiki 2016-11-29T18:41:26

2018-08-11T09:41:37

Cognitive Function, Decreased

Individual

Learning and memory depend upon the coordinated action of different brain regions and neurotransmitter systems constituting functionally integrated neural networks (D’Hooge and DeDeyn, 2001). Among the many brain areas engaged in the acquisition of, or retrieval of, a learned event, the hippocampal-based memory systems have received the most study. The main learning areas and pathways are similar in rodents and primates, including man (Eichenbaum, 2000; Stanton and Spear, 1990; Squire, 2004). In humans, the hippocampus is involved in recollection of an event’s rich spatial-temporal contexts and distinguished from simple semantic memory which is memory of a list of facts (Burgess et al., 2000). Hemispheric specialization has occurred in humans, with the left hippocampus specializing in verbal and narrative memories (i.e., context-dependent episodic or autobiographical memory) and the right hippocampus, more prominently engaged in visuo-spatial memory (i.e., memory for locations within an environment). The hippocampus is particularly critical for the formation of episodic memory, and autobiographical memory tasks have been developed to specifically probe these functions (Eichenbaun, 2000; Willoughby et al., 2014). In rodents, there is obviously no verbal component in hippocampal memory, but reliance on the hippocampus for spatial, temporal and contextual memory function has been well documented. Spatial memory deficits and fear-based context learning paradigms engage the hippocampus, amygdala, and prefrontal cortex (Eichenbaum, 2000; Shors et al., 2001; Samuels et al., 2011; Vorhees and Williams, 2014; D’Hooge and DeDeyn, 2001; Lynch, 2004; O’Keefe and Nadal, 1978). These tasks are impaired in animals with hippocampal dysfunction (O’Keefe and Nadal, 1978; Morris and Frey, 1987; Gilbert et al., 2016).

In rodents, a variety of tests of learning and memory have been used to probe the integrity of hippocampal function. These include tests of spatial learning like the radial arm maze (RAM), the Barnes maze, and most commonly, the Morris water maze (MWM). Test of novelty such as novel object recognition, and fear based context learning are also sensitive to hippocampal disruption. Finally, trace fear conditioning which incorporates a temporal component upon traditional amygdala-based fear learning engages the hippocampus. A brief description of these tasks follows. 1) RAM, Barnes, MWM are examples of spatial tasks in which animals are required to learn: the location of a food reward (RAM); an escape hole to enter a preferred dark tunnel from a brightly lit open field area (Barnes maze); or a hidden platform submerged below the surface of the water in a large tank of water (MWM) (Vorhees and Williams, 2014). 2) Novel Object recognition. This is a simpler task that can be used to probe recognition memory. Two objects are presented to animal in an open field on trial 1, and these are explored. On trial 2, one object is replaced with a novel object and time spent interacting with the novel object is taken evidence of memory retention (i.e., I have seen one of these objects before, but not this one. Cohen and Stackman, 2015). 3) Contextual Fear conditioning is a hippocampal based learning task in which animals are placed in a novel environment and allowed to explore for several minutes before delivery of an aversive stimulus, typically a mild foot shock. Upon reintroduction to this same environment in the future (typically 24-48 hours after original training), animals will limit their exploration, the context of this chamber being associated with an aversive event. The degree of suppression of activity after training is taken as evidence of retention, i.e., memory (Curzon et al., 2009). 4) Trace fear conditioning. Standard fear conditioning paradigms require animals to make an association between a neutral conditioning stimulus (CS, a light or a tone) and an aversive stimulus (US, a footshock). The unconditioned response (CR) that is elicited upon delivery of the footshock US is freezing behavior. With repetition of CS/US delivery, the previously neutral stimulus comes to elicit the freezing response. This type of learning is dependent on the amygdala, a brain region associated with, but distinct from the hippocampus. Introducing a brief delay between presentation of the neutral CS and the aversive US, a trace period, requires the engagement of the amygdala and the hippocampus (Shors et al., 2004). Most methods are well established in the published literature and many have been engaged to evaluate the effects of developmental thyroid disruption. The US EPA and OECD Developmental Neurotoxicity (DNT) Guidelines (OCSPP 870.6300 or OECD 426) both require testing of learning and memory (USEPA, 1998; OECD, 2007). These DNT Guidelines have been deemed valid to identify developmental neurotoxicity and adverse neurodevelopmental outcomes (Makris et al., 2009). A variety of standardized learning and memory tests have been developed for human neuropsychological testing. These include episodic autobiographical memory, word pair recognition memory; object location recognition memory. Some components of these tests have been incorporated in general tests of adult intelligence (IQ) such as the WAIS and the Wechsler. Modifications have been made and norms developed for incorporating of tests of learning and memory in children. Examples of some of these tests include: 1) Rey Osterieth Complex Figure (RCFT) which probes a variety of functions including as visuospatial abilities, memory, attention, planning, and working memory (Shin et al., 2006). 2) Children’s Auditory Verbal Learning Test (CAVLT) is a free recall of presented word lists that yields measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak 1995; Talley, 1986).  3) Continuous Visual Memory Test (CVMT) measures visual learning and memory. It is a free recall of presented pictures/objects rather than words but that yields similar measures of Immediate Memory Span, Level of Learning, Immediate Recall, Delayed Recall, Recognition Accuracy, and Total Intrusions. (Lezak, 1984; 1994). 4) Story Recall from Wechsler Memory Scale (WMS) Logical Memory Test Battery, a standardized neurospychological test designed to measure memory functions (Lezak, 1994; Talley, 1986). 5) Autobiographical memory (AM) is the recollection of specific personal events in a multifaceted higher order cognitive process. It includes episodic memory- remembering of past events specific in time and place, in contrast to semantic autobiographical memory is the recollection of personal facts, traits, and general knowledge. Episodic AM is associated with greater activation of the hippocampus and a later and more gradual developmental trajectory. Absence of episodic memory in early life (infantile amnesia) is thought to reflect immature hippocampal function (Herold et al., 2015; Fivush, 2015). 6) Staged Autobiographical Memory Task. In this version of the AM test, children participate in a staged event involving a tour of the hospital, perform a series of tasks (counting footprints in the hall, identifying objects in wall display, buy lunch, watched a video). It is designed to contain unique event happenings, place, time, visual/sensory/perceptual details. Four to five months later, interviews are conducted using Children’s Autobiographical Interview and scored according to standardized scheme (Willoughby et al., 2014).

Basic forms of learning behavior such as habituation have been found in many taxa from worms to humans (Alexander, 1990). More complex cognitive processes such as executive function likely reside only in higher mammalian species such as non-human primates and humans.

High Male High Female

High

During brain development

High High High

Alexander RD (1990) Epigenetic rules and Darwinian algorithms: The adaptive study of learning and development. Ethology and Sociobiology 11:241-303. Bellinger DC (2012) A strategy for comparing the contributions of environmental chemicals and other risk factors to neurodevelopment of children. Environ Health Perspect 120:501-507. Burgess N (2002) The hippocampus, space, and viewpoints in episodic memory. Q J Exp Psychol A 55:1057-1080. Cohen, SJ and Stackman, RW. (2015). Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav. Brain Res. 285: 105-1176. Curzon P, Rustay NR, Browman KE. Cued and Contextual Fear Conditioning for Rodents. In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009 D'Hooge R, De Deyn PP (2001) Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 36:60-90. Eichenbaum H (2000) A cortical-hippocampal system for declarative memory. Nat Rev Neurosci 1:41-50. Fivush R. The development of autobiographical memory. Annu Rev Psychol. 2011. 62:559-82. Gilbert ME, Sanchez-Huerta K, Wood C (2016) Mild Thyroid Hormone Insufficiency During Development Compromises Activity-Dependent Neuroplasticity in the Hippocampus of Adult Male Rats. Endocrinology 157:774-787. Gilbert ME, Sui L (2006) Dose-dependent reductions in spatial learning and synaptic function in the dentate gyrus of adult rats following developmental thyroid hormone insufficiency. Brain Res 1069:10-22. Herold, C, Lässer, MM, Schmid, LA, Seidl, U, Kong, L, Fellhauer, I, Thomann, PA, Essig, M and Schröder, J. (2015). Neuropsychology, Autobiographical Memory, and Hippocampal Volume in “Younger” and “Older” Patients with Chronic Schizophrenia. Front. Psychiatry, 6: 53. Lezak MD (1984) Neuropsychological assessment in behavioral toxicology--developing techniques and interpretative issues. Scand J Work Environ Health 10 Suppl 1:25-29. Lezak MD (1994) Domains of behavior from a neuropsychological perspective: the whole story. Nebr Symp Motiv 41:23-55. Lynch, M.A. (2004). Long-Term Potentiation and Memory. Physiological Reviews. 84:87-136. Makris SL, Raffaele K, Allen S, Bowers WJ, Hass U, Alleva E, Calamandrei G, Sheets L, Amcoff P, Delrue N, Crofton KM. A retrospective performance assessment of the developmental neurotoxicity study in support of OECD test guideline 426. Environ Health Perspect. 2009 Jan;117(1):17-25. Morris RG, Frey U. Hippocampal synaptic plasticity: role in spatial learning or the automaticrecording of attended experience? Philos Trans R Soc Lond B Biol Sci. 1997 Oct 29;352(1360):1489-503. Review O’Keefe, J. and Nadel, L. (1978). The Hippocampus as a Cognitive Map. Oxford: Oxford University Press. OECD. 2007. OECD guidelines for the testing of chemicals/ section 4: Health effects. Test no. 426: Developmental neurotoxicity study.  www.Oecd.Org/dataoecd/20/52/37622194.Pdf [accessed May 21, 2012]. Samuels BA, Hen R (2011) Neurogenesis and affective disorders. Eur J Neurosci 33:1152-1159. Shin, MS, Park, SY, Park, SR, Oeol, SH and Kwon, JS. (2006). Clinical and empirical appliations fo the Rey-Osterrieth complex figure test. Nature Protocols, 1: 892-899. Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372-376.Squire LR (2004) Memory systems of the brain: a brief history and current perspective. Neurobiol Learn Mem 82:171-177. Stanton ME, Spear LP (1990) Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol Teratol 12:261-267. Talley, JL. (1986). Memory in learning disabled children: Digit span and eh Rey Auditory verbal learning test. Archives of Clinical Neuropsychology, Elseiver. U.S.EPA. 1998. Health effects guidelines OPPTS 870.6300 developmental neurotoxicity study. EPA Document 712-C-98-239.Office of Prevention Pesticides and Toxic Substances. Vorhees CV, Williams MT (2014) Assessing spatial learning and memory in rodents. ILAR J 55:310-332. Willoughby KA, McAndrews MP, Rovet JF. Accuracy of episodic autobiographical memory in children with early thyroid hormone deficiency using a staged event. Dev Cogn Neurosci. 2014. 9:1-11. AOPWiki 2016-11-29T18:41:24

2018-08-09T11:55:05

<upstream-id>a656c566-b6f8-44d8-8b3e-fea0dba211ec</upstream-id> <downstream-id>a9e86e77-0eb7-44b0-9914-4b27e8166094</downstream-id>

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#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42d424bed0> AOPWiki 2019-06-08T17:42:22

2019-06-08T17:42:22

<upstream-id>a656c566-b6f8-44d8-8b3e-fea0dba211ec</upstream-id> <downstream-id>c5a5e923-ee29-4fb4-86f8-dca2926838e5</downstream-id>

Not Specified

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b43018a1f78> AOPWiki 2019-06-08T17:48:51

2019-06-08T17:48:51

<upstream-id>a9e86e77-0eb7-44b0-9914-4b27e8166094</upstream-id> <downstream-id>4055d62f-cb95-449b-b06a-a3fcde4c7e83</downstream-id> The basic biological processes that link gene regulation in the structural formation and function of all organs of the body are similar throughout the developing organism.  In the developing brain, genes encode proteins critical for developmental events intrinsic to structural development (e.g., neurogenesis, neuronal migration, synaptogenesis, myelination). The development of the hippocampus is no exception to this general rule of biology.

The overall weight of evidence is moderate for a direct linkage between perturbation of the expression of genes in brain (and in hippocampus specifically) and neuroanatomical abnormalities.  It is widely acknowledged that the development of the structure of the hippocampus is under the control of hippocampal gene expression. However, while an extensive body of literature exists linking some genes to hippocampal structure, there is no complete compendium on the total number of genes involved, nor direct causative links between the myriad of genes and the intricate development (both timing and location) of the majority of hippocampal structure.

The biological plausibility of this KER is rated as strong. It is well established that gene regulation controls brain development.  This also applies to the development of the hippocampus, where nuclear thyroid receptors that regulate gene transcription, directly or indirectly via transcription factor regulation, to control translation.

Empirical support for this KER is rated as strong. The number of publications in this area is extensive. A few examples are: Strange et al. (2014); Takei et al. (2016); and Shin et al. (2015). Work supporting the relationship includes use of a variety of animal models (i.e., nutritional deficiencies, chromosome abnormalities, gene deletions, knock out animals, toxicant exposures and developmental hormonal imbalance) (e.g., Frotscher, 2010; Castren and Castren, 2014; Spilker et al., 2016; Skucas et al., 2011; Lessman et al., 2011). Mutant mouse lines generated for genes involved in human cortical malformations such as doublecortin, reelin, Lis1 and Tuba1a also show gross disorganization within the hippocampus (Khalaf-Nazzal et al., 2013). Collectively, data from these studies clearly support the link between alterations in hippocampal gene expression and structural changes in hippocampal volume, cell number, and/or cytoarchitecture. A direct linkage between some specific gene targets and structural change in the hippocampus has been demonstrated using knock out and mutant mouse models (e.g., Grant et al., 1992; Lee et al., 2000; Frotscher, 2010; Castren and Castren, 2014; Spilker et al., 2016; Skucas et al., 2011; Lessman et al., 2011; Khalaf-Nazzal et al., 2013). Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). It is a well-recognized fact that there are critical developmental windows for disruption of TR-regulated genes and subsequent formation of the anatomy of the hippocampus. This has been demonstrated in multiple studies. Many of the gene-anatomy relationships critical to brain development only exist during development, or exist only to a very limited extent in the adult brain.  For example, genes controlling neuronal proliferation and migration are critically essential in hippocampal development, and their disruption results in abnormal hippocampal anatomy. Whereas, in the adult brain the genes are largely without effect as these processes are completed in the early neonatal period. In support of this, a limited number of studies have defined critical periods for the interaction of some genes and resulting neuroanatomical organization of the hippocampus (Lee et al., 2015; Favaro et al., 2009; Lee et al., 2000).  In addition, there are some ‘rescue’ experiments for a select number of genes (eg., Lee et al., 2015; Spilker et al., 2016). Several examples are described below: In the Jacob/Nsfm knockout model, hippocampal dysplasia is seen in hippocampal areas CA1 and CA3, characterized by reduced complexity of the synapto-dendritic cytoarchitecture, shorter dendrites and fewer branches (Spilker et al., 2016). Simplified dendritic trees and reduced synaptogenesis were also observed in hippocampal primary neurons cultured from these knock out mice relative to cultures from wild type mice. The protein product of Jacob/Nsfm regulates activity-dependent brain-derived neurotrophic factor (Bdnf) transcription. Lower BDNF levels were seen in area CA1 of knock out mice on postnatal day 10. The dysplasia seen in hippocampal neuronal cultures from knock mice could be reversed by BDNF supplementation if administered in early (2-4 days in vitro) but not later (15 days in vitro) in development. Neuroregluin-2 (Nrg2) contributes to synaptogenesis of the granule cell layer of the hippocampus. In hippocampal slice cultures, inducible microRNA targeting strategies have demonstrated early suppression of Nrg2 (4 days in vitro) but not late suppression (7 days in vitro) reduced synaptogenesis of inhibitory neurons. On the other hand, late treatment impaired the dendritic outgrowth of excitatory synaptic connections.  These effects could be eliminated with overexpression of Nrg2 (Lee et al., 2015). Many of the gene-regulated processes involved in hippocampal development are also present in the developing cortex. In models of prenatal hypothyroidism, altered expression patterns of many genes involved in neuronal migration and apoptosis are associated with disruptions in hippocampal organization and cytoarchitecture of the cerebral cortex (Pathak et al, 2011; Mohan et al., 2012; Lui et al., 2010). Structural changes in hippocampus and cerebral cortex are dependent on time of exposure (Auso et al., 2003; Berbel et al., 2010; Pathak et al., 2011) and can be reversed with TH supplementation (Mohan et al., 2012; Pathak et al., 2011; Berbel et al., 2010). Dose-Response Evidence: Dose-response data is lacking for this KER.  Papers that utilize knock-out and mutant models do not provide ‘dose-response’ information for gene-anatomy relationships. Studies in which genes and anatomy were reported following developmental hypothyroidism were single high-dose studies that focused on varying the developmental window of exposure, but not necessarily the dose.

There are no inconsistencies in this KER, but there are some uncertainties. Few studies exist that report both gene expression changes and structural changes in the hippocampus in same study to provide direct causative evidence for this KER. Lacking also is the specific suite of genes that are altered in the hippocampus at particular developmental times that are causal to the structural defects reported. For future research, it is critical to generate data in which the upstream KE is modulated in a ‘dose-response’ manner to better support the causative relationship. Significant data gaps also exist for basic fetal hippocampal development.

There are no data on the quantitative linkages between gene expression changes and altered hippocampal anatomy.

High Male High Female

Moderate

During brain development

Moderate High

The majority of data in support of this KER is from rodent models.  The evolutionary conservation of thyroid receptors (Holzer et al., 2017) coupled with their role in TR regulated gene transcription in neurodevelopment, suggests that this KER may also be applicable to other species.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b430114ba80> AOPWiki 2016-11-29T18:41:35

2018-08-11T19:05:29

<upstream-id>4055d62f-cb95-449b-b06a-a3fcde4c7e83</upstream-id> <downstream-id>1a569fdf-14a4-4824-904c-753881a7f6d0</downstream-id> The hippocampus is a highly integrated and organized communication and information processing network with millions of interconnections among its constitutive neurons (see Andersen et al, 2006). The neuronal spine is the primary site of action for synaptic interface between neurons. Although difficult to measure due to their small size, large number and variable shapes, changes in the frequency and structure of dendritic spines of hippocampal neurons has dramatic effects on synaptic physiology and plasticity (Harris et al., 1992). Anatomical integrity at a more macro-level is also essential for physiological function. The connectivity of axons emanating from one set of cells that synapse on the dendrites of the receiving cells must be intact for effective communication between neurons to be possible. Synaptogenesis is a critical step for neurons to be integrated into neural networks during development. Changes in the placement of cells within the network due to delays or alterations in neuronal migration, the absence of a full proliferation of dendritic arbors and spine upon which synaptic contacts are made, and the lagging of transmission of electrical impulses due to insufficient myelination will independently and cumulatively impair synaptic function.

The weight of evidence supporting the relationship between structural abnormalities in brain induced and altered synaptic function is moderate. There is no doubt that altered structure can lead to altered function. Many examples from knock out models, genetic mutations, prenatal alcohol, nutritional deficits demonstrate a correlative link between altered structure and impaired synaptic function within the hippocampus (Gil-Mohapel et al., 2010; Berman and Hannigan, 2000; Grant et al., 1992; Palop et al., 2010; Ieraci and Herrera, 2007). However, the scientific understanding of the causative and quantitative relationship between the two KEs is incomplete.

The biological plausibility of alterations in hippocampal structure having an impact on synaptic function and plasticity in brain is strong. Because synaptic transmission in the hippocampus relies on the integrity of contacts and the reliability of electrical and chemical transmission between pre- and post-synaptic neurons, it is well accepted that interference on the anatomical levels will very much impact the functional output on the neurophysiological level (Knowles, 1992; Schultz and Engelhardt, 2014).

Empirical support for this KER is rated as moderate. Numerous examples of a direct linkage between hippocampal anatomy and hippocampal physiology are evident in knock out or transgenic mouse models (eg., Lessman et al., 2011). Other data is derived from nutritional deficiencies, alcohol exposure, and hippocampal slice culture models (Berman and Hannigan, 2000; Ieraci and Herrera, 2007; Gilbert et al., 2016). Although several examples are evident to demonstrate direct linkages between alterations in hippocampal anatomy and disruptions in hippocampal physiology, there is not a mechanism, anatomical insult, or signature pattern of synaptic impairment that accompanies each of these treatments. Below are a few examples where direct linkages have been reported and they serve to bear witness to a direct relationship between altered hippocampal anatomy and altered hippocampal physiology. Fyn is a tyrosine kinase gene involved in synaptic plasticity. Mutations of this gene lead to a lack of expression during development and result in an increase in the number of neurons in the dentate gyrus and CA subfields of the hippocampus. Fyn mutant mice also exhibited impairments in long term potentiation in hippocampal CA1 whereas two other forms of short-term plasticity remained intact (Grant et al., 1992). Neuroregluin-2 (NRG2) is a growth factor and is highly expressed in the hippocampal dentate where it contributes to synaptogenesis of newborn granule cells. In hippocampal slice cultures, inducible microRNA targeting strategies have demonstrated suppression of NRG2 reduced synaptogenesis of inhibitory neurons and impaired dendritic outgrowth and maturation of glutamatergic synapses. These anatomical alterations were accompanied by reductions in the amplitude of excitatory synaptic currents. The magnitude of the impairment was dependent on the timing of the infection and could be eliminated with overexpression of NRG2 in this in vitro model (Lee et al., 2015). Brain-derived neurotrophic factor (BDNF) activation of CREB-activated gene expression plays a documented role in hippocampal synapogenesis, dendrite formation, and synaptic plasticity in the developing and adult nervous system (Lessmann et al., 2011; Panja and Bramham, 2014). Jacob is a protein that translocates to the nucleus upon activation of BDNF-dependent pathways and is involved in both neuronal plasticity and neurodegeneration. Hippocampal neurons in culture derived from Jacob/Nsmf knockout mice exhibit shorter neurite length, reduced branching, and a few synaptic contacts. This effect was specific to hippocampal neurons, as cortical cells derived from the same animals did not display these abnormalities. In vivo, these animals exhibited a reduction of dendritic complexity of CA1 neurons, lower number of branches, decreased spine density.  Deficits in synaptic plasticity in the form of LTP accompanied these structural impairments (Spilker et al., 2016). In Alzheimer’s Disease, amyloid-b protein accumulates in the hippocampus and leads to the formation of amyloid plaques, neuritic dystrophy and aberrant sprouting of axon terminals of the hippocampus. In a developmental germ-line knockout mouse model, high levels of amyloid-b induced aberrant neuronal network excitability and altered innervation of inhibitory interneurons.  Deficits in hippocampal plasticity were seen in the dentate gyrus without change in basal levels of synaptic transmission. In contrast, in area CA1, synaptic transmission was impaired while measures of synaptic plasticity remained intact (Palop et al., 2007). Other evidence for a direct linkage between hippocampal anatomy and hippocampal physiology comes from the area of adult neurogenesis. The neurogenesis process refers to the acquisition of new neurons on the hippocampus of the adult brain and is associated with enhanced hippocampal synaptic function and learning ability (Deng et al., 2010). Manipulations such as caloric restriction, exercise and hormones can enhance neurogenesis and increase synaptic transmission and plasticity (Kapoor et al., 2015; Trivino-Paredes et al., 2016; Deng et al., 2010). A reciprocal relationship also exists whereby increases in hippocampal neural activity serves to increase neurogenesis (Bruel-Jungerman et al., 2007, Bruel-Jungerman et al., 2009, Kameda et al., 2012). Manipulations that decrease hippocampal neurogenesis including exposure to antidepressants, hormone disruption, stress, and alcohol are associated with impaired synaptic function (Herrera et al., 2003; Saxe et al., 2006; Gilbert et al., 2016; Montero-Pedrazuela et al., 2006; Gil-Mohapel et al., 2006; Sofroniew et al., 2006). Temporal Evidence: The temporal nature of this KER is developmental (Seed et al., 2005). This has been demonstrated in multiple studies. A few examples detailed above defined critical periods for the manipulation that alters the structural development of the hippocampus that persists to adulthood to disrupt the synaptic physiology measured in the hippocampus in adulthood (Lee et al., 2015; Grant et al., 1992).  A more limited number of ‘rescue’ experiments have been reported.  Lee et al (2015), using an in vitro model, demonstrated impaired synaptogenesis that was dependent on the timing of the infection and could be eliminated with overexpression of NRG2. In Spliker et al (2016), BDNF application rescued the morphological deficits in hippocampal pyramidal neurons from Jacob/Nsmf mice. Dose-Response Evidence: Dose-response data is lacking for this KER. For future research, it is critical to generate data in which the upstream KE is modulated in a ‘dose-response’ manner to better support the causative relationship.

There are no inconsistencies in this KER, but there are uncertainties. Although several examples are evident to demonstrate direct linkages between alterations in hippocampal anatomy and disruptions in hippocampal physiology, there is not a common cellular mechanism, anatomical insult, or signature pattern of synaptic impairment that defines a common anatomically driven physiological phenotype.  In addition, it is also known that there is an interaction between physiological and anatomical development, where anatomy develops first, and can be ‘reshaped’ by the ongoing maturation of physiological function (e.g., Kutsarova et al., 2017)

Information does not exist to develop quantitative relationships between the KEs in this KER. Papers that utilize knock-out and mutant models have not provided ‘dose-response’ information for anatomy-physiology relationships.

High Male High Female

High

During brain development

Moderate Moderate Not Specified

The majority of data in support of this KER is from rodent models. The evolutionary conservation of hippocampal anatomy in mammals, birds, and reptiles (see Hevner, 2016; Streidter, 2015) suggests, with some uncertainty, that this KER is also applicable to multiple species.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00007b42eb17b048> AOPWiki 2016-11-29T18:41:35

2018-08-11T19:21:12

<upstream-id>1a569fdf-14a4-4824-904c-753881a7f6d0</upstream-id> <downstream-id>c5a5e923-ee29-4fb4-86f8-dca2926838e5</downstream-id> It is a well-accepted assertion that hippocampal synaptic integrity and plasticity are essential for spatial information processing in animals and spatial and episodic memory in humans (Burgess, 2002; Martin et al., 2000; Sweatt, 2016). A large number of studies with a variety of techniques and approaches have linked hippocampal functional deficits to decreased spatial ability, context learning, and fear learning. Study of human disease states and conditions where hippocampal function is impaired (i.e., brain trauma, Alzheimer’s disease, temporal lobe epilepsy, Down’s Syndrome), and imaging studies of hippocampal activation during memory challenge, makes itirrefutable that the hippocampus is essential for specific types of cognition abilities. Decades of animal research has reinforced this assertion. There are many forms of synaptic plasticity and numerous ways in which physiological function of neural circuits can be assessed. Similarly, there are many forms of learning and memory and multiple tasks and specifics associated with these tasks that vary from laboratory to laboratory. An emerging field of computational cognitive neuroscience lies at the intersection of   computational neuroscience, machine learning and neural network theory. These computational and theoretical frameworks support the participation of the hippocampal synaptic transmission and plasticity in learning and memory in animals and humans (for review see: Ashby and Helie, 2012).

The weight of evidence for proper hippocampal function and episodic memory in humans and the animal analogue, spatial and fear-based context learning, is strong. Seminal studies over the past 60 years firmly established the cellular basis of behavior with synaptic plasticity (LTP and LTD). And recent work has provided details on the local hippocampal circuitry needed for memory formation and behavioral change (Sweatt, 2016). In humans, virtual reality experiments in large-scale spatial contexts demonstrate the convergence of spatial memory performance in normal patients with fMRI of the hippocampus clearly demonstrating the essentiality of hippocampal function to spatial learning (Burgess, 2002). This assertion is consistent with a wealth of animal data on hippocampal learning and memory. In rodent models, functional impairment of the hippocampus assessed using electrophysiological techniques is correlated with deficits in spatial memory typically assessed using mazes, and memory for context often assessed in fear-based learning paradigms (O’Keefe and Nadel, 1978; Clark et al., 2000; Squire, 2004; Eichenbaum, 2000; Panjo and Bramham, 2014).

The biological plausibility of the KER is rated as strong. It is well accepted that the normal hippocampal function is critical for the acquisition and memory of context and spatially mediated tasks in rodents and humans (Sweatt, 2016).

Empirical support for this KER is strong. The requisite of hippocampal integrity to optimal visuo-spatial context learning (i.e., episodic memory) in humans and spatial learning in rodents is well documented. In vivo recording in conscious behaving animals has demonstrated activity-dependent neural changes taking place in the hippocampus during spatial learning (Gruart and Delgado-Garcia, 2007). Impairments in hippocampal function induced by drugs, chemicals, lesions, mutant or knock out models that cause changes in synaptic transmission, plasticity, and hippocampal network activity, are coincident with deficits in spatial and context-based fear learning (O’Keefe and Nadel, 1978; Bannerman et al., 2014; Lynch, 2004; Verret et al., 2012). Similarly, treatments found to enhance or facilitate hippocampal synaptic transmission and plasticity are associated with improved learning and memory (Deng et al., 2010; Novkovic et al., 2015; Andrade et al., 2015; Trivino-Paredes et al., 2016).  For example, n-methyl-d-aspartate (NMDA)-mediated glutamatergic synaptic transmission is essential for the induction of hippocampal synaptic plasticity in the form of LTP. Blockade of this form of plasticity by selective NMDA-receptors blockers impairs LTP and hippocampal tests of learning and memory (reviewed in Sweatt, 2016). Perturbation of hippocampal plasticity and impaired spatial learning have been reported in adult offspring following prenatal ethanol exposure (An and Zhang, 2015).  The fyn mutant mouse (fyn is a tyrosine kinase pathway) displays impairments in hippocampal synaptic transmission and plasticity, as well asspatial learning deficits (Grant et al., 1992). Brain-derived neurotrophic factor (BDNF) knock out animals exhibit synaptic plasticity deficits and learning impairments (Aarse et al., 2016; Panja and Bramham, 2014). In the Jacob/Nfsm model which also exhibits pronounced alterations in BDNF-mediated signaling, hippocampal synaptic transmission and plasticity impairments were accompanied by deficits in contextual fear conditioning and novel location recognition tasks (Spilker et al., 2016).  Finally, in rodent models of developmental TH insufficiency, impairments in hippocampal synaptic transmission and plasticity are coincident with deficits in learning tasks that require the hippocampus (Opazo et al., 2008; Gilbert and Sui, 2006, Gilbert, 2011, Gilbert et al., 2016). In humans, hippocampal physiology assessed using neuroimaging reveals activation of hippocampus upon engagement in spatial learning and episodic memory providing a direct linkage of these two specific KEs (Burgess, 2002). In fMRI studies of congenitally hypothyroid children, or children born to women with altered thyroid function during pregnancy, changes in hippocampal activity patterns during memory encoding and retention were observed and associated with memory impairments (Wheeler et al., 2012; 2015; Willoughby et al., 2013; 2014). Temporal Evidence:  The temporal nature of this KER is developmental (Seed et al., 2005). This has been demonstrated in multiple studies.  It is well-recognized that there are critical developmental windows for disruption of the functional development of the hippocampus and the integrity of this structure is essential for later development of spatial ability, context learning, and fear learning. A wealth of studies have shown correlation between hippocampal LTP and spatial learning performance, as well as the role of glutamatergic synaptic transmission and BDNF-mediated signaling pathways in these processes (Bramham, 2007; Andero et al., 2014; Morris et al., 1986; Sweatt, 2016; Migaud et al., 1998). Although studies on reversibility are rare, deficits in hippocampal synaptic transmission and plasticity in slices from BDNF knockout animals can be rescued with recombinant BDNF (Patterson et al., 1996). Dose-Response Evidence: Limited dose-response information is available.  Studies have investigated dose-dependency of both electrophysiological and behavioral impairments in animals suffering from developmental TH insufficiency (e.g., Gilbert and Sui, 2006; Gilbert, 2011; Gilbert et al., 2016).

There are no inconsistencies in this KER, but there are some uncertainties. It is a widely-held assertion that synaptic transmission and plasticity in the hippocampus underlie spatial learning (Martin et al., 2000; Gruart and Delgado-Garcia, 2007; Bramham, 2007). However, the causative relationship of which specific alterations in synaptic function are associated with specific cognitive deficits is difficult to ascertain given the many forms of learning and memory, and the complexity of synaptic interactions in even the simplest brain circuit.

Information does not exist to develop quantitative relationships between the KEs in this KER.

High Male High Female

High

During brain development

High High High

The majority of data in support of this KER is from rodent models. The evolutionary conservation of the role of the hippocampus in spatial cognitive functions suggests, with some uncertainty, that this KER is also applicable to other mammalian species.

#<Reference::ActiveRecord_Associations_CollectionProxy:0x00005eb89f2a0c90> AOPWiki 2016-11-29T18:41:35

2018-08-11T19:24:44

Thyroid Receptor Antagonism and Subsequent Adverse Neurodevelopmental Outcomes in Mammals

TR Antagonism and DNT

Evgeniia Kazymova

Kevin M. Crofton, R3Fellows LLC, Durham, NC USA <croftonwork@outlook.com> <orcid.org/0000-0003-1749-9971>

BY-SA

Under Development

1.84

2.0

This AOP describes one adverse outcome that may result from the antagoism of thyroid receptors (TR) durng brain. Chemical or other stressor antagnoism of TR, the molecular-initiating event (MIE), results in abnormal gene transcription.  TR mediated genes are essential for normal mammalian brain development, both prenatally and postnatally, and abnormal modulation of the these genes during critical developmental windows for brain development can resutl in atypical  neuroanatomical and neurophysiocal development, with subsequent adverse effects on teh developmetn of cognitive functions.  Therefore, chemicals or other stressors that interfere with TR mediated genes have the potential to cause  result in adverse neurodevelopmental effects in offspring. Herein, we discuss the implications of developmental TR antagonism for hippocampal anatomy, function, and ultimately neural function controlled by the hippocampus. The biochemistry of normal  TR function is well known across species. The hippocampus is known to be critically involved in cognitive, emotional, and memory function. The adverse consequences of TR antagnoism depends the severity, developmental timing, and impacts on different TR isoforms, indicating that exposure to TR antagonoists may produce different effects at different developmental windows of exposure. The overall weight of evidence for this AOP is strong. Gaps in our understanding include the roles of TR isoforms and the relationship of TH-dependent gene expression in the complexities of brain development. Although quantitative information at all levels of KERs is limited a number of applications of this AOP have been identified.   Note that this AOP links the MIE, TR antagonism through a new KER to an existing KE in the AOP42.  So all of the KEs, KERs and AO subsequent to the new MIE and KE already exist in the AOPWIki and have been reviewed by EAGMST.

A prime example of impairments in cognitive function as the adverse outcome for regulatory action is developmental lead exposure and IQ function in children (Bellinger, 2012). In addition, testing for the impact of chemical expsoures on cognitive function, often including spatially-mediated behaviors, is an intergral part of both EPA and OECD developmental neurotoxicity guidelines (USEPA, 1998; OECD, 2007).

adjacent

Low

Moderate adjacent

Low

Moderate adjacent

Low

Moderate adjacent

Moderate

High non-adjacent

Low

Moderate

High Male High Female

High

During brain development

High High

<ul> <li>Chemicals: This AOP may be restricted to a limited number of chemical structures that antagonize thyroid receptors.  Recent work from high-throughput testing of the Tox21 library of pharmaceutial and environmental chemcials suggests that only a 3 of the 8,305 unique structures tested were confirmed as antagonists (Paul-Friedman et al., 2019).  This is likely due to the very restrictive ligand-binding pocket for TRs (<a href="#_ENREF_60" title="Meerts, 2001 #811">Meerts et al. 2001</a>).</li> <li>Sex: This AOP applies to males and females. Disruption of thyroid receptor mediated gene expression during fetal and early postnatal develop, as well as the subsequent adverse impacts on nervous system development are similar in both sexes. There are no compelling data to suggest sex differences in susceptibility.</li> <li>Life stages: The relevant life stages for this AOP are fetal and early postnatal ages during critical windows of nervous system development where thyroid receptor gene expresion guides normal development of the brain. Many studies have demonstrated clear windows of developmental susceptibility, with different brain regions showing distinct ontogenetic profiles for TH driven brain development.  It should be noted that the majority of evidence for this statement is derived from stressor expsoures that alter TH levels in tissues rather than perturbations of TR action. This AOP does not apply to adult life states.</li> <li>Taxonomic: Based on the majority of the available evidence the taxonomic applicability domains of this AOP is mammals. Most evidence for this AOP has been gathered primarily from laboratory rodents and humans. However, there are supporting data from amphibians demonstrating the importance of TR mediatived gene expresion for development of the brain.  Due to the conserved nature of TR transcriptional activity, this AOP is likely to be applicable to other classes of vertebrates where thyroid hormones drive development of the nervous system (e.g., birds, fish, reptiles). However, species-specific differences in development and compensatory endocrine responses may influence the outcomes, particularly from a quantitative standpoint.</li> </ul>

It is widely accepted that each of the key events is essential. <ul> <li>Molecular Initiating Event: The molecular initiating event, i.e. antagonism fo TRs, is the essential event that initiates this AOP, as supported by in vitro and in vivo evidence. TRs control and mediate a wide varity of genes during development.  A number of studies have demonstrated that mutations in TRA1 and TRa2 result in clear alteration in gene expression in the developing brain. </li> <li>Hippocampal Gene Expression, Altered: It is well established specific genomic pathways underlie the progression of a number of neurodevelopmental processes in the hippocampus. There is some evidence from ex vivo studies that administration of growth factors will reverse the hippocampal dysplasia seen in Jacob/Nsfm knockout mice (Spilker et al., 2016). Less is known about the impact of hormone replacement on TH-responsive gene expression and the qualitative and quantitative relationships between altered TH-dependent gene expression in this brain region and altered hippocampal cytoarchitectural anatomy.</li> <li>Hippocampal anatomy, altered: It is well accepted that normal hippocampal anatomy is critical for hippocampal physiological function, and that alterations in anatomy lead to altered neuronal activity in the hippocampus (Lee et al., 2015; Grant et al., 1992; Spilker et al., 2016).</li> <li>Hippocampal physiology, altered: It is a well-accepted assertion that hippocampal synaptic integrity and neuronal plasticity are essential for spatial information processing in animals and spatial and episodic memory in humans. However, other brain regions also can influence these complex behaviors. Limited data from studies in BDNF knockout animals demonstrate that deficits in hippocampal synaptic transmission and plasticity, and downstream behaviors can be rescued with recombinant BDNF (Aarse et al., 2016; Andero et al., 2014).</li> <li>Cognitive function, decreased: It is a well-known fact that TH are critical for normal nervous system development (Williams et al., 2008). And this includes development of the hippocampus which plays a major role in spatial, temporal, and contextual memory. Indeed, most developed countries check for childhood hypothyroidism at birth to immediately begin replacement therapy. This has been shown to alleviate most adverse impacts of hypothyroidism in congenitally hypothyroid children (Derksen-Lubsen and Verkerk 1996; Zoeller and Rovet, 2004). The essentiality of the relationship between decreased TH levels and this adverse outcome is well accepted. Decreased cognitive function specific to the hippocampal region are particularly associated with decrements in memory and learning domains of cognition.</li> </ul>

Assessment of quantitative understanding of the AOP:  Currently, there are no data that allow a quantiative assement of the impact of modulation of TR mediated gene expression and changes in gene expression in the brain.  The majority of the evidence for the KER linking the MIE to altered gene expression in the brain is derived from TR knockout models in mice and mutations in both mice and humans.  For the rest of the KERs in this AOP, there is a limited amount of data from dose-response studies (mostly from studies on TPO inhition - see AOP42)  demonstrating increasing impact with increasing chemical dose for all the KEs, and the direct and indirect KERs. At present, the overall quantitative understanding of the AOP is insufficient to directly link a measure of chemical potency as a TR antagonist to a quantitative prediction of effect on cognitive function (e.g., IQ in humans, learning deficits in rodents). Empirical information on dose-response relationships for the MIE and intermediate KEs, currently unavailable, would inform a computational, predictive model for TR antagonist mediated gene changes that resutl in adverse congnitive development.

Applications of the AOP for chemical induced changes in cognitive development may be limited by the very restrictive ligand-binding pockets for TRs (<a href="#_ENREF_60" title="Meerts, 2001 #811">Meerts et al. 2001</a>).

AOPWiki 2019-06-08T17:12:01

2023-09-25T16:27:00