O crescimento da população e os aumentos de consumo e da poluição dos recursos hídricos, tem gerado grande preocupação com a qualidade das águas. A indústria metalúrgica gera efluentes com contaminantes, tais como óleo, graxa e metais pesados. Estas substâncias ou uma combinação de substâncias tóxicas são susceptíveis de causar alterações no sistema endócrino dos organismos vivos, como o câncer, puberdade precoce, infertilidade masculina, feminização de animais silvestres, entre outros.O estudo teve como objetivo apontar o que aconteceu com ratos Wistar que consumiram efluente de indústria metalúrgica antes e depois do processo de tratamento, a fim de analisar a presença de substâncias com disfunção endócrina potencial no plasma sanguíneo. Foram avaliados os níveis de glicose, triglicerídeos, colesterol, estradiol, e as enzimas aspartato aminotransferase (AST) e de alanina aminotransferase (ALT). As amostras do plasma dos ratos que consumiram o efluente tratado mostrou altos níveis de glicose e estradiol. Não houve lesões no fígado de camundongos analisados como também não houve alteração nos resultados das enzimas AST e ALT. Sugere-se que essas alterações foram provocadas pelo nonilfenol, um conhecido desregulador endócrino presente na maioria dos detergentes industriais.Os padrões de emissão de efluentes industriais estipulados no nosso país refere-se a contaminantes físicos, químicos e biológicos que podem ser emitidos para o meio ambiente após a realização de tratamento. A legislação ambiental pertinente em nosso país trata desses limites de emissões, mas não deixa claro no que se refere às substâncias consideradas como desreguladores endócrinos. Esta pesquisa tem como objetivo a obtenção de dados através de revisão da literatura, sobre normas de emissões toleráveis, que não prejudiquem as atividades vitais dos seres vivos. A legislação brasileira cita a proibição da utilização de certas substâncias com potencial de desregulação endócrina, mas sem uma abordagem satisfatória. A União Europeia (UE), os Estados Unidos e o Japão têm metas e regulamentações sobre a não utilização de substâncias com potencial de interferência endócrina. / Population growth and consumption increases and pollution of water resources, has generated great concern about water quality. The metallurgical industry produces effluents with contaminants such as oil, grease, and heavy metals. These substances or a combination of toxic substances are likely to cause changes in the endocrine system of living organisms, such as cancer, early puberty, male infertility, feminization of wild animals, among others. The study aimed to point out what happened to Wistar rats who consumed metallurgical industry effluent before and after the treatment process in order to analyze the presence of substances with potential endocrine dysfunction in blood plasma. We assessed the levels of glucose, triglycerides, cholesterol, estradiol, and the enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Plasma samples from rats that consumed the treated effluent showed high levels of glucose and estradiol. There were no lesions in the liver of mice examined, as there was no change in the results of AST and ALT enzymes. It is suggested that these changes were caused by nonylphenol, a known endocrine disrupter present in most detergents industrials. The emission standards for industrial wastewater stipulated in our country refers to physical contaminants, chemical and biological that may be issued to the middle environment after the completion of treatment. The relevant environmental legislation in our country comes to these emission limits, but does not make clear in relation to substances considered as endocrine disruptors. This research aims to obtain data through review of the literature on standards of tolerable emissions, not impairing the vital activities of living things. Brazilian law cites the ban on the use of certain substances with endocrine disrupting potential, but without a satisfactory approach. The European Union (EU), the United States and Japan have goals and regulations on non-use of substances with endocrine interference potential.

Growth hormone (GH) is a vital pituitary hormone controlling somatic cell growth and development. GH has a multitude of effects on the body: deficiency can lead to dwarfism while excess can cause conditions such as gigantism. Human patients with mutations in the transcription factor Pit-1 show decreased GH and prolactin levels. However, Pit-1 is known to control multiple pituitary hormones, so what other factors lead to the specificity of transcriptional regulation of the GH gene via its promoter? In order to study this, our lab has been analyzing rat pituitary cell lines to understand the role of ZNF292 (formerly called Zn-16), a selective GH transcription regulator with 16 zinc fingers that bind to the GH promoter DNA. This work has used rat MtT/S cells that are unique in that they exclusively express GH. MtT/S cells were procured from Riken Cell Bank in Japan, cultured, and examined for GH hormone and RNA expression. Results confirmed that the MtT/S cells are terminally differentiated as somatotrophs. To understand the role of ZNF292 in transcription of the GH gene, recent rat genomic data was analyzed to determine the positions of 7 exons upstream from the large exon 8 that contains the important zinc finger-encoding portions of the protein. First, MtT/S RNA was reverse transcribed into DNA, then PCR amplification was performed using primer pairs encompassing various sections of the exon 1 – 7 region. Specific PCR products were found with distinct products ranging in size from 130 to 960, all of which agreed with the predicted sizes of these exons. It had previously been theorized that ZNF292 contained a single large exon; however, these results show that splicing of the primary transcript does occur in this upstream region. Characterizing this exonic region was performed because it has been shown that ZNF292 produces circular RNA (circRNA) of unknown function in human endothelial cells and in certain types of cancer. CircRNAs are thought to be created by the “back-splicing” of exons, so that rather than a linear transcript, the ends are circularized for a portion of the transcript. Having determined the sequence and organization of these upstream exons, we are now testing primer sets that will demonstrate productive amplification only from circRNAs. Further, we are removing linear RNAs using RNAse R treatment to selectively enhance circRNA presence in the reactions. Finally, data from RNA-Seq analysis of the MtT/S cells will be scrutinized to determine if additional exon/intron boundaries or alternative splice sites exist in this upstream 7 exon region. The study of circular RNAs could be very important in understanding its role in acting as a microRNA sponge or RNA-binding protein sponge during their regulation of downstream gene transcription. Also, analysis of this mechanism shows potential as a clinical tool in cancer treatment because ZNF292 functions as a tumor suppressor in colorectal and chronic lymphatic leukemia.

Small cell variant of medullary thyroid carcinoma is an extremely rare histologic entity with a paucity of data. As such, there is a lack of clinical experience regarding this disease. In this case, a 52-year-old patient with small cell variant medullary thyroid carcinoma was experiencing intractable nausea, vomiting, and diarrhea. The initial workup was extensive yet unrevealing. He was refractory to all treatments. On further laboratory analysis, the calcitonin was substantially high and the thyroid stimulating hormone level was mildly elevated. This case is presented to highlight a possible treatment for this rare cancer through thyroxine suppression therapy. This case is presented due to the lack of literature available on small cell medullary thyroid carcinoma and also to discuss a possible direct relationship between thyroid stimulating hormone and calcitonin levels in this disease population.

Exposure to certain stressors during a sensitive period around puberty can lead to enduring effects on an animal’s response to estradiol. In estradiol-influenced behaviors, such as sexual receptivity, hippocampal-dependent learning and memory, depression-like behavior, and anxiety-like behaviors, exposure to a peripubertal stressor such as shipping stress or an injection of lipopolysaccharide (LPS) can eliminate or even reverse the normal response to estradiol. In addition to regulating these behaviors, estradiol play a role in the regulation of body weight. While some of the previous studies touched on short-term effects on body weight, no systemic long-term study of the effects of a peripubertal stressor on body weight, particularly without interruption by ovariectomy, have been undertaken. This paper introduces a hypothesis that proposes that increased adiposity following exposure to a peripubertal stressor leads to the changes to estrogen-dependent behaviors through altered levels of estrogens and changes to estrogen receptors. The first chapter examines body weight data collected during studies with other aims, and then proposes an experiment to test whether either of two peripubertal stressors results in increased weight gain and body weight. The following chapter proposes further experiments designed to determine the proximate mechanisms leading to weight gain following peripubertal stressors and the role of diet on weight gain. The final chapter proposes experiments to test the effects of adiposity on peripheral levels of testosterone, aromatase, estradiol, and estrone; central levels of estradiol and estrone; and estrogen receptors in the brain.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13553号 / 論医博第2282号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齋藤 潤, 教授 万代 昌紀, 教授 長尾 美紀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>The risk of fracture is higher in adults with type 2 diabetes compared to controls without type 2 diabetes, despite normal or higher than normal bone mineral density (BMD). In addition to BMD, bone strength depends on other factors such as structural and material <em>bone qualities</em>, which are not accounted for in BMD measurements. The objective was to determine whether structural and material <em>bone qualities</em> are different in adults with type 2 diabetes compared to controls without type 2 diabetes. First, a cross-sectional study was undertaken using MRI to investigate distal radius trabecular bone microarchitecture, a structural <em>bone quality.</em> In women with type 2 diabetes, trabecular bone holes were larger compared to controls, which is important because greater trabecular bone hole size is related to reduced bone strength. Next, a two year prospective study was conducted with the participants involved in the cross-sectional study to determine whether changes in trabecular bone microarchitecture are different in women with type 2 diabetes compared to controls. There was a greater increase in the number of trabecular bone holes in women with type 2 diabetes compared to controls, which provides early evidence of trabecularization of cortical bone in women with type 2 diabetes. In the third study, quantitative backscattered electron imaging (qBEI) was used to derive bone mineralization density distribution (BMDD) outcomes for bone samples from adults with and without type 2 diabetes to compare material <em>bone quality. </em>There was evidence of elevated bone calcium concentration and reduced mineralization heterogeneity in bone samples from adults with type 2 diabetes compared to controls, which may contribute to bone brittleness. In summary, differences in structural and material <em>bone qualities </em>identified in this body of work provide explanations for elevated fracture risk in adults with type 2 diabetes.</p> / Doctor of Science (PhD)

Prostate and breast cancer are the leading causes of cancer-related death in men and women, respectively, and metastasis is the primary factor underlying the high mortality rates.1 Snail transcription factor is an important molecule that drives prostate and breast cancer metastasis through the process of epithelial mesenchymal transition (EMT). Proteolytic enzymes that promote invasion and metastasis such as the lysosomal cysteine protease cathepsin L (Cat L) have been shown to degrade E-cadherin, promoting the epithelial mesenchymal transition (EMT).2 It has also been shown that silencing Cat L can inhibit transforming growth factor-beta (TGF-β)-mediated EMT by suppressing Snail transcription factor.3 Several recent studies have highlighted an additional unexpected localization and site of action for Cat L within the nucleus in breast, colon and prostate cancer.4 Natural products have been shown to be efficacious in prevention and possible treatment of cancer.5 Specifically, we have been studying Muscadine Grape Skin Extract (MSKE) as a possible candidate to inhibit Snail signaling. MSKE has previously been shown to promote prostate cancer apoptosis.6 We hypothesized that Snail promotes nuclear localization of Cat L, which promotes EMT associated with increased migration and invasion, and that antagonizing Snail-Cat L signaling would lead to mesenchymal epithelial transition (MET). We showed for the first time that MSKE promotes apoptosis through induction of endoplasmic reticulum stress response and autophagy. Additionally, MSKE could inhibit Snail-mediated EMT via scavenging reactive oxygen species. Moreover, Snail could promote nuclear localization of Cat L, which then promoted cleavage of CDP/Cux, increased Snail transcription and decreased E-cadherin transcription by direct promoter binding of cleaved CDP/Cux, leading to EMT associated with increased migration and invasion. Interestingly, Z-FY-CHO, a small molecule specific inhibitor of Cat L, as well as MSKE could antagonize this signaling by promoting nuclear to cytoplasmic re-localization of Cat L. Therefore, we have dissected novel mechanisms of action of Snail and how it can be antagonized by MSKE natural product.

La thèse porte sur le développement de stratégies non conventionnelles de peptides contraints et la modulation des structures secondaires pour augmenter la reconnaissance biologique de ces peptides.Les peptides jouent un rôle important dans de nombreux processus et sont donc d’un intérêt grandissant pour l’industrie pharmaceutique. Cependant, leur utilisation comme médicament reste limitée à cause de leur flexibilité conformationnelle, leur sensibilité aux protéases et leur faible biodisponibilité et pharmacodynamie. Dans ce contexte, la caractérisation des interactions ligands-récepteurs est cruciale pour comprendre les processus de reconnaissance et le design d’agonistes sélectifs, potentiels nouveaux médicaments. Ainsi, le développement d’outils portant des modifications structurales présente un intérêt grandissant pour trouver de nouveaux médicaments. Ces changements structuraux permettent d’affiner les conformations privilégiées et donc de comprendre la spécificité d’interactions par rapport à des sous-types de récepteurs ayant des propriétés physicochimiques et pharmacologiques particulières.Dans la première partie de ce travail, une stratégie optimisée pour la synthèse de fragments N-terminaux (1-34) de la séquence PTHrP.PTHrP(1-34)NH2 est décrite : H-Ala1-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp10-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg20-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu30-Ile-His-Thr-Ala-NH2. D’un point de vue synthètique, la formation de clusters dus à la présence de plusieurs résidus arginine, l’encombrement stérique, la longueur de la séquence et la présence de résidus hydrophobes dans la partie 19-28 du PTHrP rendent la synthèse difficile et donnent un enjeu important à la synthèse. C’est pourquoi, nous avons focalisé nos efforts sur l’optimisation du protocole opératoire. En particulier, nous avons montré l’intérêt d’utiliser une activation sous microondes pour la synthèse, avantages en termes de rendement et de pureté du peptide. La synthèse de PTHrP(1-34) a été optimisée à la fois sous activation par la temperature et sous microondes dans des conditions identiques. Les micoondes ont aussi été utilisé pour la synthèse de PTHrP(1-34)NH2. L’élongation de la chaine peptidique a été suivie par l’analyse UPLC-ESI-MS des fragments obtenus après micro clivage assisté par micro ondes. Cette stratégie nopus a permis , à travers la caractérisation des séquences délétées, d’identifier les points critiques de la synthèse, nécessitant les microondes. Dans la seconde partie de la thèse, ont été entreprises le design et la synthèse d’une série de cyclopeptides (i-to-i+5) 1,4- et 4,1-disubstitués pontés par un triazole, analogues de MTII. MTII, Ac-Nle4-c[Asp5-D-Phe7-Lys10]αMSH4-10-NH2 est un super agoniste agissant d’une manière non sélective des récepteurs de la mélanocortine MC1R, MC3R, MC4s, et MC5s. Ce peptide est caractérisé par un lien lactame entre les résidus Asp5 et Lys 10 stabilisant une structure β-turn, cruciale pour l’activité.Cependant MTII n’est pas sélectif des différents sous-type des récepteurs de la mélanocortine. L’importance particulière du système mélanocortine souligne le besoin de trouvé des analogues plus sélectifs, présentant une meilleure pharmacocinétique et une meilleure biodisponibilité. L’introduction du triazole [1,2,3] utilise dans nos analogues vise à stabiliser une conformation , à la place du lien lactame de MTII. La diversité est apportée par une variation de la position du triazole de i à i+5. Il est obtenu par une cycloaddition alcyne/azoture catalysée par le Cu(I) (CuAAC), générant très sélectivement l’adduit 1,4. Les analogues clickés de MTII présentant une position différente du triazole ont été synthétisé en phase solide et en solution. Les études conformationnelles et biologiques ont été conduites pour identifier l’analogue présentant la meilleure conformation β-turn conduisant à la meilleure activité biologique. / PhD Thesis of Chiara TESTANon conventional synthetic strategies of stapled peptides: modulation of secondary structures to optimise biological recognitionPeptides play an important role in many biologically relevant processes and are of outstanding interest in pharmaceutical research. However their use as drugs is limited by their conformational flexibility, instability to proteases, poor oral bioavailability, and pharmacodynamics. In this contest the characterization of ligand-receptor interactions is crucial to understand the biological processes and to design potent and selective agonist, which could be used as new drug candidates.Therefore, the development of an expansive toolbox of structural modifications that can be used to fine tune the predominant conformations to achieve modulation of specificity toward receptor subtypes, physicochemical and pharmacological properties continues to be of great interest in the development of peptide-based drugs.In the first part of the work it is described an optimized strategy for the synthesis of the N-terminal fragments (1-34) of PTHrP.PTHrP(1-34)NH2 sequence is: H-Ala1-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp10-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg20-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu30-Ile-His-Thr-Ala-NH2. Considering the presence of clusters of arginines, sterically hindered and hydrophobic amino acid residues in the 19-28 sequence of PTHrP, and the considerable length of the peptide, the synthesis of PTHrP(1-34)NH2 is quite challenging. Therefore we focused our effort in the optimization of a synthetic protocol for this peptide. In particular, we showed the advantages that the use of microwaves have, in obtaining the best results in terms of yield and purity of the final peptide. The synthesis of PTHrP(1-34) was performed following the conventional RT and the MW-assisted SPPS protocol. In both cases the synthesis was performed using the same instrument, the same excess of reagents and molar ratios.Microwaves have also been used to monitor the synthesis of the peptide PTHrP(1-34)NH2. In fact, during the elongation of the peptide chain were analyzed by UPLC-ESI-MS intermediate fragments obtained through micro-cleavages assisted by microwaves. This strategy has allowed us, through the characterization of sequences of deletion, to understand what are the critical points of the synthesis that may require the use of microwaves.In the second part of the thesis we reported the design and the synthesis of a series of (i-to-i+5) 1,4- and 4,1-disubstituted [1,2,3]triazole-bridged cyclopeptides, derived from the scaffold of MTII. MTII, Ac-Nle4-c[Asp5-D-Phe7-Lys10]αMSH4-10-NH2, is a potent long acting non-selective super-agonist of melanocortin receptors MC1R, MC3R, MC4s, and MC5s. This peptide is characterized by lactam bridge between residues Asp5 and Lys10 stabilizing a type-II β-turn structure, that is critical for its bioactivity. Nevertheless, MTII is not selective for the different subtype of melanocortin receptors. The particular importance of melanocortin system, underscores the unmet need for highly selective, pharmacokinetically diverse, and bioavailable agonists and antagonists analogs. The introduction of [1,2,3]triazole was aimed to stabilize a β-turn conformation replacing the lactam bridge of MTII with an i-to-i+5 side chain-to-side chain cyclization via CuI-catalyzed azido-to-alkyne 1,3-dipolar cycloaddition (CuAAC) generating 1,4- or 4,1-disubstituted [1,2,3]triazolyl-containing ring structures. Clicked MT-II analogs presenting different permutations of bridges, containing 4 or 5 methylenes, and a triazolyl moiety were synthesized by a combination of solid phase assembly of the peptide and in solution CuAAC. Conformational and biological studies were performed on the peptides synthesized to identify the location and direction of the [1,2,3]triazolyl in the bridge that best reproduce the β-turn conformation leading to highly potent and sel

The circadian rhythm is a rhythm which permeates all aspects of biological life and follows the hours of the sun. The pace of the rhythm is controlled by a collection of neurons in the hypothalamus, called the suprachiasmatic nucleus (SCN), whose signals affect rhythms throughout the body as can be seen in aspects of life from behavior down to oscillations of proteins in the cells. A disruption of this rhythm such as what happens during jet lag, where the rhythm of the SCN is out of synch with the rhythm of the rest of the body, is something that can have adverse effects on mental and physical health. To realign the SCN and the rhythm of the body, different methods and be implemented. This thesis investigated the effectiveness of glucocorticoids on re-aligning the rhythms of the body following a disruption through a meta-analysis and a qualitative review. The meta-analysis and review incorporated experiments from six articles investigating the hours of circadian rhythm shifts in the mouse model, after administering glucocorticoids. What was found was that the individual experiments presented results with high effect sizes; however, the direction of said effects was not uniform as the rhythms shifted in different directions. The lack of uniform direction caused no significant combined effect size to be found by this meta-analysis (MES=0.11 ± 0.06), showing that a statistical analysis based on hours shifted could not find a significant combined effect. The qualitative review, however, indicates that the administration of glucocorticoids shows an effect in re-entraining the rhythm of the peripheral parts of the body to that of the environmental cues and the SCN. Though no significant statistical effect was found in this analysis, the effect of glucocorticoids should not be discounted and could still prove a promising treatment for circadian disruptions, such as jet lag.

Introduction: Rates of childhood-onset type 1 diabetes (T1D) are steadily increasing among American youth, yet Black Americans are more likely to suffer from serious T1D-related complications caused by poor glycemic control. The aim of this thesis is to determine the external factors that are causing discrepancies in the development, diagnosis, treatment, and long-term management of T1D in Black youth. Methods: Epidemiological studies were compiled from the American Diabetes Association, Center for Disease Control (CDC), International Diabetes Foundation (IDF), Kaiser Family Foundation (KFF), and the Claremont Colleges Library network to identify the sociocultural aspects that negatively affect long-term glycemic control in Black youth. Results: Studies indicate that Black youth with T1D are more likely to face disadvantages in treatment regimen which are attributed to insurance coverage, socioeconomic status, education level, and implicit bias. Most studies demonstrate that these factors result in poor glycemic control, which subsequently leads to severe dysglycemia-related complications later in life. Conclusion and Discussion: Many Black youth who suffer from T1D receive insufficient healthcare, which is often exacerbated by a lack of social and economic resources. As a result, they may not have the means to maintain consistent, healthy glycemic levels. System-level changes are necessary to change the morbidity and mortality of T1D in Black youth. Future research should include the analysis of other racial minority groups in order to uncover additional institutional disparities.