The uptake and binding of tritiated cortisol, corticosterone, and dexamethasone in cytosols isolated from various regions of non-adrenalectomized (intact) and adrenalectomized sheep brains and pituitaries were studied. [3H]-cortisol and [3H]-corticosterone were bound preferentially by cytosols from the hippocampal and septal regions of the brain, whereas, [3H]-dexamethasone was preferentially bound by cytosol from the pituitary. Cytosols isolated from the brains of adrenalectomized sheep bound significantly more [3H]-corticosterone than did cytosol from the brains of intact animals. Pre-incubation of cytosols from the hippocampus of adrenalectomized animals with non-radioactive testosterone or estradiol did not significantly block the subsequent binding of [3H]-cortisol. Results of pre-incubations with non-radioactive progesterone were non-reproducable with a large standard deviation among replicates. These findings provide evidence that the sites of feedback inhibition are the same in the sheep as described for other mammalian systems, and that in the sheep as in other mammals the principle site of localization of cortisol and corticosterone is in limbic structures of the brain, whereas, the principle site of uptake of dexamethasone is in the pituitary.

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Early growth response 1 (Egr1) is a transcription factor critical for learning and memory in the hippocampus and pituitary cell differentiation. Egr1 has been shown to extend continuation of the long-term potentiation in the hippocampus and is credited for forming long-term memories. The somatotrophs in the pituitary produce growth hormone and are found to be decreased in Egr1KO mice. These animals are also found to be sterile due to a decrease in LHB, which blocks ovulation. All previous studies have evaluated these physiological processes with complete Egr1KO research strains or antisense oligonucleotides, up until now, no data specific to individual type of cells has been generated. In an attempt to focus on the understanding of the functions of Egr1 gene in neural cell lineage, we are using an Egr1cKO Nestin-Cre model. Nestin allows for targeting neuronal lineage specific cells. In Chapter 1, we provide a systemic view of Egr1 gene and Nestin-Cre as a system for generating conditional knockout mouse strains. The Chapter begins with the identification of Egr1 gene and its protein structure, then proceeds to grasp its link to memory with behavior testing. The critical role of Egr1 in the pituitary and what cell populations are affected is also described. The same goes for Nestin-Cre, along with its limitations and understanding how to account for them in a study. The Egr1cKO Nestin-Cre system is the best form to understand neurological cell populations with Egr1 removal. In Chapter 2 and Chapter 3, we employ the Egr1cKO Nestin-Cre mouse model to understand cell-specific knockout of Egr1 in the nervous system by evaluating the hippocampus and pituitary. We explore learning and memory through behavioral tests and ribonucleic acid sequencing (RNA-seq) analysis to understand gene expression changes with Egr1 removal. Females showed higher activity during behavior tests, with more movement in the elevated plus maze and lower freezing times during the contextual fear conditioning. RNA-seq had higher changes in females than males but was not affected by the Nestin-Cre system overall. The same RNA-seq changes in the pituitary gland were present, with females having higher genomic differentiation. Females had growth-specific pathways altered by Nestin-Cre. / Doctor of Philosophy / Genetics has become a very important forerunner in scientific research. One gene that has become important in many different research arenas is Early growth response 1 (Egr1). This particular gene is critical for learning, memory, and cell changes in the pituitary. In Chapter 1, we have analyzed the current research landscape of information on Egr1 in its functions with learning and memory, as well as the pituitary. Most previous studies that have been completed only evaluate this gene by its removal from the entire body. This leaves a large gap in information about how this gene functions with specific cell types. To limit the type of cells from which Egr1 has been removed, we have selected Nestin-Cre, a tool to remove genes from neuronal stem cells. The capabilities and limitations of this tool have also been explained in this chapter, along with how the two together can accomplish a cell-specific knockout of Egr1. In Chapter 2, we have constructed an experiment with behavioral tests for mice, along with RNAseq data from the hippocampus to evaluate what changes have occurred in the Egr1cKO Nestin-Cre model. Female mice are more active in the behavioral test, including the elevated plus maze (EPM) and Contextual fear conditioning (CFC), than male mice. The same holds for differences in the RNAseq data as well. In Chapter 3, the pituitary of Egr1cKO Nestin-Cre mice is the main focus. We evaluated RNAseq data and determined growth rates of transgenic mice. The mice had different growth rates over twelve weeks between the controls and the knockout. The RNAseq data also revealed many differences between males and females. Female mice had specific growth genes effects by the knockout of Egr1

The purpose of this research is, first, to determine by laboratory methods, that species specificity does not exist in closely allied taxonomic animals; second, to determine a unit of activity for the gonadotropic hormone. For this purpose a quantitative method for determining potency is necessary to ascertain the seasonal production of the gonadotropic factor in fish. A further aim in this investigation is to demonstrate that the diversity of the gonadotropic factor, in relation to phylogenetic variations, is not as ineffectual as previously reported.

Background: Persistent stress contributes to the onset and maintenance of problem gambling (PG), increasing risks for physiological disturbances. However, minimal research examines acute stress effects and relationships with PG features such as impulsivity or gambling urges. Purpose: The current study examines multiple facets of acute subjective and physiological stress responses in PG. Stress effects on gambling urges and relationships with impulsivity are also explored. Methods: A PG (n=21) and healthy control (HC; n=21) group were exposed to acute psychosocial stress. Saliva samples were collected while participants completed self-report measures of mood and gambling urges. Gambling urges, salivary cortisol (sC), salivary alpha-amylase (sAA), and subjective stress reactivity and recovery were compared within and between groups from baseline up to an hour following stress. Relationships between trait impulsivity, gambling urges, and all aspects of the acute stress response were examined. Results: The PG group showed blunted sC reactivity but reported heightened mood disturbances compared to HCs, while sAA levels did not differ between groups. Within PG and HC groups, sAA had a more rapid acute stress onset than sC, but between-marker differences were pronounced in PG. Self-reported gambling urges were high among PG participants but remained relatively unchanged following acute stress. Impulsivity was positively associated with gambling urges, however, it was not correlated with the acute stress response. Conclusions: This study provides a novel and wide-ranging assessment of the acute stress response in PG, for which research is currently limited. These results indicate that high stress in PG contributes to multi-faceted alterations of the acute stress response relative to HCs. Self-reported gambling urges are elevated and associated with greater trait impulsivity in PG but are unaffected by acute stress exposure. Our findings provide insight into acute stress processing dysfunction in PG and have implications for potential harms, such as increased suicide risk. / Thesis / Master of Science (MSc) / Chronic or continuous life stress can increase the risk of developing and sustaining gambling problems. The current study assessed the effects of a 20-minute stressful task on mood, physiological reactions, the desire to gamble, and their relationships with impulsivity in healthy individuals compared to those with problem gambling (PG). Results showed that after the stressor, the PG group reported more negative mood, but physiological reactions were reduced when compared to healthy participants. In individuals with PG, a greater desire to gamble was correlated with being more impulsive. Overall, our findings show that the long-term stress typically experienced by individuals with PG can affect their mental and physiological reactions to temporary stressors. Additionally, more impulsive individuals show a greater desire to gamble, which may contribute to problematic gambling behaviours. Future research should assess the life impacts of chronic stress in PG, such as the risk of gambling relapse and suicide.

Adenomas hipofisários são tumores benignos, de crescimento lento, originados no interior da sela túrcica e constituem de 10% a 15% dos tumores intracranianos, Os adenomas clinicamente não funcionantes (ACNF), correspondem aproximadamente um terço dos adenomas em geral. Por não apresentarem síndrome clínica hormonal são geralmente diagnosticados devido a sintomas neurológicos ou oftalmológicos, como macroadenomas, com grandes dimensões, invasão de estruturas circunvizinhas e hipopituitarismo. A cirurgia é o tratamento de escolha para estes tumores e apesar de ser eficaz na resolução do quadro compressivo, a possibilidade de cura cirúrgica é reduzida principalmente em tumores invasivos. Seu acompanhamento pós-operatório é efetuado por exame de imagem, preferencialmente ressonância magnética, devido à indisponibilidade de marcadores séricos. Nesta pesquisa avaliou-se a relação da expressão dos genes relacionados à apoptose, Bcl-2, Bax e Caspase-3 e sua relação com o comportamento dos ACNF. Na Divisão de Neurocirurgia Funcional do Instituto de Psiquiatria do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo foram operados 119 doentes com tumores hipofisários, de 28/05/08 à 07/04/09, 50 deles com ACNF, 30 deles foram estudados. A ressonância magnética da região selar pré-operatória possibilitou a medida dos três maiores diâmetros do tumor, ou seja, antero-posterior (AP), crânio-caudal (CC), látero-lateral (LL) e avaliar a invasão do seios cavernoso e esfenoidal. O tamanho dos tumores foi avaliado pela soma dos três diâmetros, pelo maior diâmetro e pelo cálculo do volume, efetuado pela fórmula AP x CC x LL x 0,5. No intraoperatório foram avaliados, a consistência e invasão tumoral. A análise histológica por hematoxilina-eosina, foi efetuada em todos os tumores, assim como a análise imuno-histoquímica (IHQ) dos hormônios hipofisários, Ki-67, p53 e Bcl-2. Foi realizada a análise molecular dos genes Bcl-2, Bax e Caspase-3 por RT-PCR. Dados demográficos: 17 do sexo masculino, 13 do sexo feminino, mediana da idade foi de 54,5 anos e mediana da duração dos sintomas de 31 meses. Todos apresentavam macroadenoma, 87% deles com perda visual, 53% com cefaléia, 17% com outras alterações neurológicas e um assintomático diagnosticado incidentalmente. Avaliação hormonal, disponível em 26 doentes, confirmou deficiência em 92% deles, com mais de dois eixos acometidos em 50% dos casos. A mediana do volume dos tumores foi de 11,6 cm3, do maior diâmetro de 3,8cm e da soma dos três diâmetros de 8,6cm, observou estreita correlação significativa estatisticamente entre as três medidas. Quarenta porcento dos tumores eram gigantes (diâmetro maior ou igual a 4 cm). Consistência amolecida e invasão tumoral foram observadas em 87% e 67% dos tumores, respectivamente. Todos doentes foram operados pela via transesfenoidal, exceto um operado por craniotomia pterional. Complicações cirúrgicas ocorreram em cinco pacientes, três com fistula liquórica, dois com meningite e dois óbitos. A análise histológica confirmou o diagnóstico de adenoma hipofisário em todos os casos. A IHQ foi negativa para todos hormônios em 18 e positiva em 12 tumores (TSH, FSH, LH, GH ou ACTH). A IHQ para proteína P-53 foi negativa em todos os casos. A IHQ para KI-67 revelou ausência da proteína em 11, positividade em menos de 3% das células em 15 e em mais de 3% em 4 tumores. A IHQ para Bcl-2 foi positiva em apenas três pacientes. A análise molecular dos genes Bcl-2, Bax e Caspase-3 revelou expressão muito inferior nos tumores em relação à observada para um pool de hipófise normal. Observou-se correlação positiva estatisticamente significante entre os três genes porém não foi observada correlação entre os níveis destes três genes e nenhum fator de prognóstico tumoral estudado, quais sejam, idade, positividade para hormônios na IHQ, tamanho ou invasão tumoral / Pituitary adenomas are benign, slow-growing tumors that arise in the sella turcica and account for 10% to 15% of all intracranial tumors. Non-functioning pituitary adenomas (NFPA) account for around one third of all pituitary adenomas. NFPA do not clinically present as hormonal syndromes and are generally diagnosed as macroadenomas due to marked neurological and ophthalmologic symptoms and invasion of surrounding structures, beside hypopituitarism. Surgery is the gold standard to treat these tumors. It effectively relieves compressive symptoms but cure is uncommon. Despite benign in nature, NFPA usually show aggressive behavior. There are no hormonal markers and the follow-up usually is made only by magnetic resonance imaging. Apoptosis-related genes, Bcl-2, Bax, and caspase-3, were here studied in NFPA to assess their role as potential markers of tumor behavior. Out of 119 patients with pituitary adenomas treated by surgery, 30 patients (17 men, 13 women, median age 54.5 years old) harboring NFPA who underwent surgery in the Department of Functional Neurosurgery at Hospital das Clínicas Psychiatric Institute, University of S. Paulo Medical School, from August 2008 to July 2009, were studied. Information on gender, age, pituitary function, symptoms and their length was collected. Tumor dimensions were measured using magnetic resonance imaging of the sella turcica. The tumor volume was calculated by the following equation: anterior-posterior diameter x cranial-caudal diameter x lateral-lateral diameter x 0.5. Intra-operative information such as tumor invasion and consistence was recorded. Histological examination by hematoxylin-eosin staining and immunohistochemistry analysis of pituitary hormones, Ki-67, p53, and Bcl-2 were performed. The molecular analysis of Bcl-2, Bax, and caspase-3 genes was performed by real-time polymerase chain reaction (RT-PCR) in all tumor specimens collected during surgery and compared to a poll of normal pituitary gland. All patients had macroadenomas diagnosed due to visual loss (87%), headache (53%) and other neurological symptoms (17%) and one case was incidentally found. Hormonal deficits were seen in 92% of 26 cases; more than two axes were involved in half of these patients. There was found good correlation between tumor volume, largest diameter and the sum of the 3 diameters, and tumor volume was used to assess the correlations with other parameters. The median volume was 11.6 cm3. Giant tumors (4 cm) were diagnosed in 40% of the patients. Soft tumors and tumor invasion were observed in 87% and 67% of cases, respectively. A transsphenoidal approach was used in all patients, except one who had pterional craniotomy. Five patients presented post-operative complications: three had CSF leakage, two meningitis and two died. The histological examination confirmed pituitary adenoma in all cases, 18 of them were null cell and 12 showed a positive immunohistochemistry analysis for one or more hormones, mainly TSH. Immunohistochemistry analysis results for p-53 was negative in all cases; for Ki-67 was negative in 11, positive in less than 3% of the cells in 15 and positive in more than 3% of the cells in 4 cases; and for Bcl-2 was positive only in three patients. Bcl-2, Bax and caspase-3 molecular analysis revealed very low expression compared to normal pituitary values. There was found a positive correlation between these three genes but no correlation between them and age, tumor volume or invasion. The Bcl-2, Bax, and caspase-3 gene analysis by RT-PCR in NFPA did not evidence their potential as markers of tumor behavior