Traumatic brain injury (TBI) is a major cause of hospitalization and death. To mitigate these human costs, the search for effective drugs to treat TBI continues. Even mild injuries can lead to long-term deficits in memory and cognition. Predicting which patients will have long lasting memory issues following mild TBI is challenging. Our group has previously shown that in vitro models of TBI result in cell death, decreased long-term potentiation (LTP), and glial activation. In this thesis, we used chemical and electrical treatments to modulate the outcome following injury to inform future therapies. In the first aim of this thesis, we evaluated the efficacy of a novel neurosteroid, NTS-105, to reduce post-traumatic pathobiology in an in vitro model of moderate TBI that utilizes an organotypic hippocampal slice culture. Treatment with NTS-105 starting an hour after injury decreased post-traumatic cell death in a dose-dependent manner, with 10 nM NTS-105 being most effective. Post-traumatic administration of 10 nM NTS-105 also prevented deficits in LTP without adversely affecting neuronal activity in naïve cultures. Our results suggest that the pleiotropic pharmacology (affinity for the androgen, mineralocorticoid, and progesterone receptors) of NTS-105 may be a promising strategy for the effective treatment of TBI. In the second aim, we evaluated the mechanisms of NTS-105 in an in vitro model of mild blast TBI, a model in which NTS-105 is known to preserve LTP. Treatment with NTS-105 starting an hour after injury reduced a marker of microglial activation and increased expression of the GluR1 subunit of the AMPAR, which is a postsynaptic protein associated with LTP. NTS-105 is known to inhibit activation of the androgen receptor and the mineralocorticoid receptor, partially activate the progesterone B receptor and not activate the glucocorticoid receptor. NTS-105 treatment did not alter the expression of any of the oxosteroid receptors (progesterone, androgen, mineralocorticoid, and glucocorticoid). In order to demonstrate the benefits of mineralocorticoid antagonism following TBI, we administered eplerenone immediately after injury. Eplerenone treatment preserved LTP, but did increase spike magnitude at high concentrations. In the third aim, organotypic hippocampal slice cultures were biaxially stretched to model a mild TBI and serial electrophysiological recordings were collected. In this in vitro model, stretchable microelectrode arrays were embedded within the culture substrate to both deform the adhered culture and record neural signals, which are indicators of neuronal health and network connectivity. Multiple spontaneous and evoked recordings were obtained while maintaining sterility to study and modulate the electrophysiological response to injury. Bursting activity increased 2 hours after injury but returned to baseline by 24 hours. However, 24 hours after injury, both LTP and long-term depression (LTD) were impaired. In another experiment, LTP was induced multiple times, both 24 hours before and 24 hours after injury, to study how the state of the pre-injury network affected electrophysiological outcome after injury. We provide preliminary evidence that induction of LTP before injury to increase synaptic strength was detrimental to neuronal plasticity (LTP) after injury. This thesis has expanded upon the understanding of TBI injury mechanisms and hormone receptor modulators following TBI. Future studies will continue to examine NTS-105 and study the benefits of androgen receptor antagonism. Future studies will also continue to use the stretchable microelectrode arrays and our induction paradigm to test if induction of LTD, a weakening of synaptic strength, could increase resiliency to injury.

[ES] Se han preparado varios materiales metal orgánicos de circonio MOF-808 para evaluar sus propiedades catalíticas en reacciones tipo Meerwein-Ponndorf-Verley (MPV) para la reducción de compuestos carbonílicos. En particular, se han sintetizado compuestos tipo MOF-808 modificados en los que una pequeña fracción de los ligandos trimesato presentes en el MOF original se ha reemplazado por ligandos dicarboxilato, como una estrategia para inducir la creación controlada de defectos estructurales. Los ligandos utilizados han sido: isoftalato (MOF-808-IPA), 3,5-piridindicarboxilato (MOF-808-Pydc), 5- aminoisoftalato (MOF-808-NH2) y 5-hidroxiisoftalato (MOF-808-OH). Todos los materiales obtenidos presentan una elevada cristalinidad y son isoreticulares respecto al MOF-808 original. Se ha evaluado la actividad catalítica del MOF-808 original y de los materiales modificados en reacciones tipo MPV, utilizando ciclohexanona como compuesto modelo. Todos los materiales presentan una elevada actividad catalítica, superior a la del tereftalato de circonio UiO-66 utilizado como referencia. Esta mayor actividad catalítica se corresponde a un mayor número de iones Zr4+ con insaturación coordinativa presentes en el MOF-808 con respecto al UiO-66. Además, los materiales MOF-808-IPA y MOF-808-Pydc presentan una mayor actividad que el MOF-808 original, lo que se debe a la presencia de centros activos menos congestionados estéricamente debido a la introducción de los ligandos dicarboxilato. Una ventaja adicional de compuestos MOF-808 con respecto al UiO-66 es su sistema de poros más grande, lo que permite la conversión de moléculas de mayor tamaño. Para evaluar esta característica, se ha utilizado un compuesto de gran tamaño, la estrona, capaz de penetrar en los poros del MOF-808 pero no en el UiO-66. En consecuencia, el MOF-808 es capaz de convertir por completo la estrona de forma selectiva a estradiol, mientras que el UiO-66 apenas presenta actividad. Además, cuando se usa el MOF-808 como catalizador, se produce una cantidad considerable del isómero 17alfa-estradiol, difícil de obtener por otros medios, por lo que las propiedades de diastereoselectividad del MOF-808 en reacciones MPV resultan de gran interés preparativo. Con el fin de estudiar en mayor detalle la diastereoselectividad de reacciones MPV catalizadas por MOF-808, se ha estudiado la reducción de ciclohexanonas substituidas: 3-metilciclohexanona (3MeCH), 2-metilciclohexanona (2MeCH) y 2-fenil-ciclohexanona (2PhCH). En función del alcohol utilizado como reductor y de la posición del grupo substituyente en la ciclohexanona, el MOF-808 favorece selectivamente la formación de uno u otro isómero, con una diastereoselectividad variable: 82%, 61% y 94%, respectivamente para 3MeCH, 2MeCH y 2PhCH. Es posible racionalizar estos resultados considerando la formación preferencial de uno u otro estado de transición en el espacio confinado disponible dentro de los poros del MOF. Las características energéticas del proceso se han analizado mediante el uso combinado de estudios cinéticos y cálculos teóricos. Finalmente, en vista las interesantes propiedades del MOF-808 como catalizador para reacciones MPV, se ha extendido con éxito el uso de este material a la preparación de compuestos hidroxiesteroides de difícil obtención y de interés farmacológico mediante la reducción quimio-, regioy diastereoselectiva del correspondiente oxoesteroide. De esta forma, se han conseguido obtener en un solo paso de reacción y con una elevada selectividad los siguientes compuestos: 17alfa-estradiol, 5alfa- androstan-3beta,17alfa-diol y epitestosterona, lo que demuestra el potencial del MOF-808 como catalizador para la síntesis de compuestos de alto valor añadido. / [CA] S'han preparat varis materials metall orgànics de zirconi MOF-808 per avaluar les seves propietats catalítiques en reaccions tipus Meerwein-Ponndorf-Verley (MPV) per a la reducció de composts carbonílics. En particular, s'han sintetitzat composts tipus MOF-808 modificats en els que una petita fracció dels lligands trimesat presents en el MOF original s'han reemplaçat per lligands dicarboxilats, com una estratègia per induir la creació controlada de defectes estructurals. Els lligands utilitzats han sigut: isoftalat (MOF-808-IPA), 3,5-piridindicarboxilat (MOF-808-Pydc), 5-aminoisoftlatat (MOF-808- NH2) i 5-hidroxiisoftalat (MOF-808-OH). Tots els materials preparats presenten una elevada cristal·linitat i són isoreticular respecte al MOF-808 original. S'ha avaluat l'activitat catalític del MOF-808 original i dels materials modificats en reaccions tipus MPV, utilitzant ciclohexanona com a compost model. Tots els materials presenten una elevada activitat catalítica, superior a la del tereftalat de zirconi UiO-66 utilitzat com a referència. Aquesta major activitat catalítica es correspon a un major nombre d'ions Zr4+ amb insaturació coordinativa presents en el MOF-808 respecte a l'UiO-66. A més, els materials MOF-808-IPA i MOF-808-Pydc presenten una major activitat que el MOF-808 original, el que és debut a la presència de centres actius menys congestionats estèricament debut a la introducció dels lligands dicarboxilat. Un avantatge addicional dels MOF-808 respecte a l'UiO-66 és el seu sistema de porus més gran, que permet la conversió de molècules de major tamany. Per avaluar aquesta característica, s'ha utilitzat un compost de gran taman, l'estrona, capaç de penetrar en els porus del MOF-808 però no en els de l'UiO-66. En conseqüència, el MOF-808 és capaç de convertir completament l'estrona de forma sel·lectiva a l'estradiol, mentre que l'UiO-66 gairebé no presenta activitat catalítica. A més, quan s'usa el MOF- 808 com a catalitzador, es produeix una quantitat considerable de l'isòmer 17alfa-estradiol, difícil d'obtenir per altre medis, de manera que les propietats de diastereoselectivitat del MOF-808 en reaccions MPV resulten de gran interès preparatiu. Per tal d'estudiar en major detall la diastereoselectivitat de reaccions MPV catalitzades per MOF-808, s'ha estudiat la reducció de ciclohexanones substituïdes: 3-metilciclohexanona (3MeCH), 2- metilciclohexanona (2MeCH) i 2-fenil-ciclohexanona (2PhCH). En funció de l'alcohol usat com a reductor i de la posició del grup substituent en la ciclohexanona, el MOF-808 afavoreix selectivament la formació d'un o de l'altre isòmer, amb una diastereoselectivitat variable: 82%, 61% y 94%, respectivament per a 3MeCH, 2MeCH y 2PhCH. És possible racionalitzar aquest resultats considerant la formació preferent d'un o l'altre estat de transició en l'espai confinat disponible dins dels porus del MOF. Les característiques energètiques del procés s'han analitzat mitjançant l'ús combinat d'estudis cinètics i càlculs teòrics. Finalment, en vista de les interessants propietats del MOF-808 com a catalitzador per a reaccions MPV, s'ha estès amb èxit l'ús d'aquest material a la preparació de composts hidroxiesteroids de difícil obtenció i d'interès farmacològic mitjançant la reducció quimio-, regio- i diastereoselectiva del corresponent oxoesteroid. D'aquesta manera, s'ha aconseguit obtenir en un únic pas de reacció i amb una elevada selectivitat els següents composts: 17alfa-estradiol, 5alfa-androstan-3beta,17alfa-diol i epitestosterona, el que demostra el potencial del MOF-808 com a catalitzador per a la síntesi de composts d'alt valor afegit. / [EN] Various zirconium-containing MOF-808 compounds have been prepared as potential catalysts for the Meerwein-Ponndorf-Verley (MPV) reduction of carbonyl compounds. Modified MOF-808 have been synthethized in which a small fraction of the trimesate ligands present in pristine MOF-808 has been replaced by dicarboxylate ligands, as a strategy to induce a controlled creation of defects. The linkers used are: isophthalate (MOF-808-IPA), 3,5-pyridinedicarboxylate (MOF-808-Pydc), 5-aminoisophthalate (MOF-808-NH2) and 5-hydroxyisophthalate (MOF-808-OH). All these compounds are highly crystalline and isoreticular with pristine MOF-808. The catalytic activity of pristine and defect engineered MOF-808 has been evaluated for MPV reactions, using cyclohexanone as model substrate. All the materials show a higher catalytic activity than that of zirconium terephthalate UiO-66 used as reference. This higher activity is attributed to the higher amount of coordinatively unsaturated Zr4+ ions in MOF-808 than in UiO-66. Moreover, MOF- 808-IPA and MOF-808-Pydc are more active than pristine MOF-808, which is due to the creation of less sterically crowded sites due to the introduction of defective dicarboyxlate linkers. A further advantage of MOF-808 over UiO-66 is the presence of a wider pore system, which allows converting bulkier substrates. To evaluate this characteristic, a bulky ketone has been used; estrone, which can enter the pores of MOF-808 but not those of UiO-66. Accordingly, MOF-808 can fully convert estrone selectively to estradiol, while UiO-66 shows barely any catalytic activity. Interestingly, when MOF- 808 is used as catalysts, a noticeable amount of the isomer 17alpha-estradiol is produced, which is difficult to obtain by other means. Therefore, the diastereoselective properties of MOF-808 for MPV reactions are interesting from the preparative point of view. In order to investigate in more detail the diastereoselective properties of MOF-808 for MPV reactions, various substituted cyclohexanones have been considered: 3-methylcyclohexanone (3MeCH), 2- methylcyclohexanone (2MeCH) and 2-phenylcyclohexanone (2PhCH). Depending on the alcohol used as reducing agent and the position of the substituent in the cyclohexanone molecule, MOF-808 selectively favors the formation of one isomer or the other with a different diastereoselectivity: 82%, 61% and 94%, respectively for 3MeCH, 2MeCH y 2PhCH. These results can be rationalized by considering the preferential formation of a given transition state in the confined space available inside the MOF pores. The energetic characteristics of the process have been analyzed by a combined use of kinetic studies and theoretical calculations. Finally, in view of the interesting properties of MOF-808 as catalyst for MPV reactions, this material has been successfully applied to the preparation of a number of challenging hydroxysteroid compounds with pharmacologic interest through a chemo-, regio- and diastereoselective reduction of the corresponding oxosteroid. In this way, it has been possible to prepare in one single reaction step the following compounds: 17alpha-estradiol, 5alpha-androstan-3beta,17alpha-diol and epitestosterone. This demonstrates the high potential of MOF-808 as a catalysts for the synthesis of high added value compounds. / I want to thank the European Union’s Horizon 2020 research and innovation program for a contract under the Marie Sklodowska-Curie grant agreement No. 641887 (Project acronym: DEFNET). / Mautschke, H. (2019). Zirconium Metal Organic Frameworks as Heterogeneous Catalysts for Meerwein-Ponndorf-Verley Reactions [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/130203

Withanolides are a class of ergostane natural products found in plants of family Solanaceae. Plants of this family are used in traditional medicine in Asia and South America. Recently, a series of 17β-hydroxy withanolides were identified from high-throughput screens as inhibitors of androgen-induced changes in gene expression of prostate cancer cells. Therefore, these compounds may have important applications as new therapies against prostate cancer. We have devised a synthetic route to members of this family and their analogues which allows stereoselective introduction of C14, C17 and C20 hydroxyl groups in separate steps. This will allow preparation of differentially hydroxylated analogues so as to identify which contributes to the potency and thus gain a better understanding of the SAR of this class of bioactive molecules. As part of this we have shown that the stereochemical outcome of the epoxidation of Δ 14-15 cholestanes with m-CPBA is controlled by the steric bulk of a C17 substituent. When the C17 is in the β configuration, the epoxide is formed on the α face, whereas if the C17 is trigonal (flat) or the substituent is in the α configuration, the epoxide is formed on the β face. The presence of a hydroxyl substituent at C20 does not influence the stereochemical outcome of the epoxidation. We have successfully introduced aldehyde functionality to the lateral side chain 14 hydroxyl compound. This aldehyde compound is a key intermediate from which many of the withanolides can be made. We have also investigated the introduction of a hydroxyl at the C18 as an entry into the physachenolides. Finally, we have carried out an assessment of the potency of the synthesised compounds against hormone-insensitive prostate cancer cell line, PC-3.

Steroid hormones belong to a group of compounds known as endocrine disruptors. They are hydrophobic compounds and are categorized as natural and synthetic estrogens. Some common household products have been implicated as estrogen mimics. Exposure effects of these compounds are felt by human and wildlife, such reproductive alterations in fish and frogs. They mainly introduced into the environment through veterinary medicines administration to animals and the discharges from wastewater treatment plants (WWTPs). In this study, a new alternative analytical procedure that is simple, rapid and fast for the determination and quantification of five steroidal hormones: estriol (E3), beta estradiol (β-E2), alpha estradiol (α-E2), testosterone (T), progesterone (P) and bisphenol A (BPA) using the High pressure liquid chromatography coupled to a charged aerosol detector (HPLC-CAD). These compounds were studied because of their strong endocrine-disrupting effects in the environment. Under optimum conditions, a linear graph was obtained with correlation coefficient (R2) ranging from 0.9952 - 0.9996. The proposed method was applied to the analysis of water samples from a wastewater plant and the results obtained were satisfactory. The limits of detection (LOD) for the target analytes in wastewater influent was between 0.0002 – 0.0004 μg/L and the limit of quantification (LOQ) was 0.001 μg/L respectively for each of the analytes. Enrichment factors of 148- 258, and extraction efficiency 84- 102% were obtained for the target analytes; relative standard deviations (% RSD) for m = 6 were between 2.8 and 7.6%. The concentration of the EDCs in environment sample was between 0.2 - 2.3 μg/L. / Chemistry / M. Sc. (Chemistry)

Les acyl stéryl glycosides (ASGs) appartiennent à une famille de glycolipides qui possèdent un caractère amphiphile particulier dû à la présence de deux parties hydrophobes, un stéroïde et une chaine grasse. Dans le cadre de nos études des propriétés d’auto-organisation des glycoamphiphiles, ce travail est dédié à l’étude de deux ASGs, α-CAG et BbGL 1, composés naturels présents respectivement dans les membranes des bactéries Helicobacter pylori et Borrelia burgdorferi., présentant des structures similaires mais des activités biologiques différentes. Notre travail a consisté à déterminer les paramètres structuraux qui gouvernent leurs propriétés d’auto-assemblage. Deux séries de 6-O-acyl cholestéryl glycosides (glucosides et galactosides) variant dans leur configuration anomérique et la longueur et le niveau d’insaturation de leur chaine grasse ont été synthétisées et leur capacité à former des cristaux liquides et à promouvoir une ségrégation lipidique dans des monocouches de Langmuir modèles de membrane ont été étudiées. Les relations structure-propriétés établies montrent que la longueur de la chaine grasse est le paramètre le plus discriminant dans le comportement d’auto-assemblage dans les deux types d’expériences. Pour les cristaux liquides thermotropes, l’autre facteur discriminant est la configuration anomérique, deux phases colonnaires successives rectangulaires puis hexagonales étant observées pour les séries α alors qu’une seule a été observée en séries β Changer de sucre n’induit pas de différence significative dans le comportement LC. Concernant la formation de domaines lipidiques, les modifications de la configuration (α/β) et du sucre influencent significativement leur temps d’apparition, apportant pour la première fois une définition claire des paramètres structuraux et physicochimiques qui gouvernent le comportement de l’α-CAG et ses analogues, en lien avec les données commues sur l’augmentation de pathogénicité d’Helicobacter pylori. Ce travail de thèse donne une illustration de l’importance de la structure des carbohydrates dans les processus biologiques et du concept de glycoamphiphilie. / Acyl steryl glycosides (ASGs) are a family of glycolipids which exhibit a peculiar amphiphilic character based on the presence of two hydrophobic appendages, one steroid moiety and one fatty alkyl chain, attached on a polar carbohydrate backbone. In the frame of our studies on the self-organisation properties of carbohydrate-based amphiphiles, this thesis is an investigation of the behavior of ASGs, in particular α-CAG and BbGL 1, two natural compounds found in bacterial membranes, Helicobacter pylori, Borrelia burgdorferi repectively, who exhibit close structures but different bioactivity. Our work has aimed at determining the key structural parameters governing their self-organization behavior. Two series of acyl cholesteryl glycosides (glucosides or galactosides) have been synthesized, with variations in the anomeric configuration, the 6-O-acyl chain length and level of unsaturation, and investigated with respect to their ability to form liquid crystalline mesophases, and to drive lipid domain segregation in Langmuir monolayers as model membranes. Structure-properties relationships have been established, indicating that the fatty chain length showed the most remarkable influence on the self-organization behavior, in LC and model membrane experiments. For the LC mesophases, the other important parameter is the anomeric configuration, two successive columnar phases, rectangular then hexagonal, being observed for the α-anomers, whereas only one was found for the β-anomers. No significant changes were observed when comparing glucosides and galactosides. With respect the formation of domains, configuration modifications at both C-1 (α or β) and C-4 (gluco or galacto) influenced significantly the domains appearance time, giving the first, clear physicochemical proof of the structural influential factors in the behavior of α-CAG and analogues, in the context of the known increased pathogenicity of Helicobacter pylori. Overall, this thesis provides a nice illustration of the subtlety and the importance of carbohydrate structure in biological processes, and of the concept of glycoamphiphilicity.

Steroid hormones belong to a group of compounds known as endocrine disruptors. They are hydrophobic compounds and are categorized as natural and synthetic estrogens. Some common household products have been implicated as estrogen mimics. Exposure effects of these compounds are felt by human and wildlife, such reproductive alterations in fish and frogs. They mainly introduced into the environment through veterinary medicines administration to animals and the discharges from wastewater treatment plants (WWTPs). In this study, a new alternative analytical procedure that is simple, rapid and fast for the determination and quantification of five steroidal hormones: estriol (E3), beta estradiol (β-E2), alpha estradiol (α-E2), testosterone (T), progesterone (P) and bisphenol A (BPA) using the High pressure liquid chromatography coupled to a charged aerosol detector (HPLC-CAD). These compounds were studied because of their strong endocrine-disrupting effects in the environment. Under optimum conditions, a linear graph was obtained with correlation coefficient (R2) ranging from 0.9952 - 0.9996. The proposed method was applied to the analysis of water samples from a wastewater plant and the results obtained were satisfactory. The limits of detection (LOD) for the target analytes in wastewater influent was between 0.0002 – 0.0004 μg/L and the limit of quantification (LOQ) was 0.001 μg/L respectively for each of the analytes. Enrichment factors of 148- 258, and extraction efficiency 84- 102% were obtained for the target analytes; relative standard deviations (% RSD) for m = 6 were between 2.8 and 7.6%. The concentration of the EDCs in environment sample was between 0.2 - 2.3 μg/L. / Chemistry / M. Sc. (Chemistry)

No description available.

Le récepteur de la progestérone (PR) est un facteur de transcription hormono-régulé qui joue un rôle crucial dans la coordination de tous les aspects de la fonction de reproduction chez la femme. Pour activer ses gènes cibles, PR recrute de façon dynamique, séquentielle et combinatoire différents partenaires moléculaires : les corégulateurs transcriptionnels. Les coactivateurs de la famille p160 (Steroid Receptor Coactivators : SRC-1, -2, -3), dont l’expression est augmentée dans certains cancers hormono-dépendants, sont les partenaires privilégiés de PR. Au cours de ce travail, nous avons étudié les mécanismes d’interaction entre le récepteur de la progestérone et ses corégulateurs ainsi que leurs conséquences fonctionnelles sur l’activité de PR. Nous avons ainsi pu mettre en évidence l’importance de la dégradation des complexes PR/SRC-1 par le protéasome sous l’effet du ligand agoniste de PR, et le caractère nécessaire de cette régulation négative pour l’activation de la transcription des gènes cibles de PR. Nous avons également identifié un candidat possiblement impliqué dans la dégradation des complexes PR/coactivateurs p160 : le corégulateur transcriptionnel Jab1. En effet, il a été décrit comme un coactivateur des complexes PR-SRC-1 au laboratoire, et nous avons pu observer que, hors du cadre de l’activation par l’hormone, Jab 1 régule les niveaux d’expression de SRC-1 et SRC-2. En revanche, ce corégulateur reste sans effet sur SRC-3. Enfin, nous avons mis au point les conditions expérimentales de l’étude de la dynamique des interactions entre PR et ses corégulateurs par la techninique de FRET.Les évidences croissantes de l’implication de PR et de ses cofacteurs (SRC-1, SRC-3, Jab1) dans le développement et les métastases des cancers du sein font de la compréhension de leurs mécanismes d’action un élément important dans la recherche de nouvelles thérapies. La détermination du rôle exact des corégulateurs de PR dans ces processus permettra une éventuelle redéfinition des cibles pharmacologiques dans le traitement de ces maladies, qui représentent un véritable enjeu de santé publique. / The progesterone receptor (PR) is a ligand-activated transcription factor playing a crucial role in female reproduction. To regulate gene expression, PR recruits several coregulators to target gene promoters, in a cyclic and combinatorial manner. Among these coregulators, PR recruits most notably members of the p160 family coactivators (Steroid Receptor Coactivators SRC-1, -2 and -3) which have recently been implicated in several hormono-dependent cancers.Here, we studied the mechanisms of interaction between PR and its coregulators as well as their functional consequences on PR transcriptional activity. We have demonstrated that PR activity is paradoxically coupled to the agonist ligand-dependent down-regulation of PR/SRC-1 complexes. Two degradation motifs found in SRC-1 were identified as signals involved in this proteasome- and ubiquitin-mediated process. We also identified a putative candidate implicated in the degradation of these complexes, namely the transcriptional coregulator Jab1. Indeed, Jab1 has previously been described in our laboratory as a coactivator of PR/SRC-1 complexes. We observed that it can specifically regulate SRC-1 and SRC-2 expression in absence of hormone. Finally, we optimized the experimental conditions of FRET experiments to get new insights on the dynamic interactions between PR and its coregulators. Collectively our findings are consistent with the emerging role of proteasome-mediated proteolysis in the gene-regulating process. Understanding PR mechanisms of action is an important step in the development of new therapies, due to growing evidences of PR and its coregulators implication in breast carcinogenesis and metastasis. Deciphering precisely the role of PR coregulators in these processes will permit to define new pharmacological targets for the treatment of these diseases, which represent a serious public health problem.

Orientadores: Luis Otávio Zanatta Sarian, Sophie Françoise Mauricette Derchain / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-07T13:36:45Z (GMT). No. of bitstreams: 1 Toledo_MariaCarolinaSzymanski_M.pdf: 838964 bytes, checksum: 3c546db1a656989db9f433b93287b2d2 (MD5) Previous issue date: 2012 / Resumo: Introdução: o câncer de ovário é comumente diagnosticado em estágios avançados, sendo atualmente a neoplasia ginecológica de maior letalidade. As pesquisas têm direcionado esforços na tentativa de descobrir novos fatores prognósticos e métodos terapêuticos. Muitos trabalhos estudam a expressão dos receptores de esteróides (dentre eles, estrógeno (RE), progesterona (RP) e andrógeno (RA)) e HER2 (receptor estimulador de crescimento epitelial - subtipo 2) como fatores prognósticos e associados à resposta terapêutica, apresentando; entretanto, resultados conflitantes. Até onde conhecemos, não há estudos desta natureza no Brasil. Objetivo: Correlacionar à expressão dos RE, RP, RA e HER2 aos fatores clínico patológicos, ao intervalo livre de doença e à sobrevida nos cânceres epiteliais de ovário. Material e métodos: Este é um estudo observacional de coorte retrospectiva. Foram incluídas 152 mulheres com tumores epiteliais malignos, selecionados através dos prontuários no período de 1993 a 2008, no Centro de Atenção Integral à Saúde da Mulher (CAISM) da Universidade Estadual de Campinas - UNICAMP, São Paulo, Brasil. A avaliação da expressão dos RE [subtipos alfa (RE?) e beta (RE?)], RP, RA e HER2 foram realizadas por imunoistoquímica através da construção de microarranjos de tecidos (TMA). Inicialmente, foi realizada análise uni variada da expressão dos receptores acima quanto à idade, estado menopausal, índice de massa corpórea (IMC), tipo histológico, grau histológico, estadiamento inicial de acordo com a classificação proposta pela FIGO e presença de doença residual pós-tratamento cirúrgico. Em seguida, as pacientes foram divididas em dois grupos: ausência da expressão de RE?, RP e HER2 (triplo negativo) e positividade para pelo menos um desses receptores (não triplo negativo); e, avaliadas em relação aos critérios clínicos e epidemiológicos acima. Foram, então, realizadas análises multivariadas dos padrões de expressão de RE ? e ?, RP, RA, HER2 e triplo negativo em relação a estes mesmos critérios. Por fim, análise de sobrevida multivariada foi realizada utilizando-se todos os padrões de expressão e os fatores clínicos epidemiológicos estudados. Resultados: Nas análises multivariadas, mostraram-se significativas as seguintes associações: do receptor de estrógeno alfa (ER?) com tumores de grau histológico menos diferenciado (p=0.01); do receptor de progesterona (RP) à obesidade (p= 0.01); do receptor de andrógeno (RA) com tumores do subtipo seroso (p= 0.01); do receptor de HER2 com tumores dos graus histológicos II-III (p=0.02); do subgrupo triplo negativo com tumores de grau histológico menos diferenciado (II-III) (p<0.01). Não houve associação do RE? com nenhum dos fatores estudados. Quanto à análise multivariada de sobrevida livre de doença e sobrevida global; dentre os padrões de expressão de receptores, apenas o RE? esteve associado com melhor sobrevida livre de doença (RR=0.39; 95%CI 0.17 -0.90). Conclusões: A expressão do RE? esteve mais significativamente associada a fatores clínicos de pior prognóstico. O RP esteve associado à obesidade. O RA esteve significativamente mais presente nos tumores serosos. A expressão do HER2 e a presença de tumores triplo negativo foram maiores em tumores menos diferenciados. Paradoxalmente; o RE? foi o único receptor a apresentar associação com maior sobrevida livre de doença apesar de sua relação significativa com fatores reconhecidos de pior evolução clínica. Não houve diferença estatística significativa na análise multivariada de sobrevida total e sobrevida livre de doença quanto ao grupo de tumores triplo negativo / Abstract: Introduction: Ovarian cancer is commonly diagnosed in advanced stages and currently is the most lethal gynecological malignancy. Surveys have focused efforts in an attempt to discover new prognostic and therapeutic methods. A plenty of studies investigates the expression of steroid receptors (among them, estrogen (ER), progesterone (PR) and androgen AR)) and HER2 (epidermal growth receptor stimulator - subtype 2) as prognostic factors and associated to therapeutic response, presenting, however, conflicting results. As far as we know, there are no studies of this nature in Brazil. Objective: The aim of this study was to correlate the expression of ER (subtypes ? (ER ?) and ? (ER ?), PR, AR and HER2 to clinical pathological factors, the disease-free survival and overall survival of epithelial ovarian cancers. Methods: This is a retrospective observational cohort study. The study included 152 women with malignant epithelial tumors, selected through the records from 1993 to 2008, in the Centro de Atenção Integral à Saúde da Mulher (CAISM) at the State University of Campinas - UNICAMP, São Paulo, Brazil. The expression of ER (? and ?), PR, AR and HER2 was evaluated by immuno histochemistry through tissue microarray (TMA) technique. Initially, univariate analyses were performed, evaluating the expression of each receptor mentioned above to age, menopausal status, body mass index (BMI), histological type, histological grade, initial staging as preconized by FIGO staging of ovarian tumors and presence of residual disease after surgical treatment. Then, the patients were divided into two groups: absence of the expression of ER?, PR and HER2 (triple negative) and positive for at least one of these receptors (not triple negative), and evaluated in relation to the clinical and epidemiological criteria mentioned above. Multivariate analyzes were performed with ER ?, ER?, PR, AR, HER2 and triple negative towards these same criteria. At last, multivariate survival analyses were conducted using all the patterns of receptors' expression, epidemiological and clinical factors studied. Results: In multivariate analyzes, there were the following significant associations: of the estrogen receptor alpha (ER?) with less differentiated histological grade tumors (p = 0.01); of the progesterone receptor (PR) to obesity (p = 0.01); of the androgen receptor (AR) with the serous tumors (p = 0.01); of the HER2 receptor with tumors of histological grades II-III ( p = 0.02); of the triple negative subgroup with less differentiated histological grade tumors (II-III) (p <0.01). There was no association of the ER? with any of the factors studied. In the multivariate analysis of disease-free survival and overall survival; considering the patterns of receptors' expression, only the ER? expression was associated with better disease-free survival (RR= 0.39, 95% CI 0.17 to 0.90). Conclusions: The expression of ER? was more significantly associated with clinical factors of poor prognosis. The PR was associated with obesity. The AR was significantly more prevalent in serous tumors. The HER2 expression and the presence of triple negative epithelial ovarian cancer tumors were higher in less differentiated tumors. Paradoxically, the ER? was the only receptor which showed association with better disease-free survival despite its significant relationship with recognized factors of worse clinical outcome. There was no statistically significant difference in multivariate analysis of overall survival and disease-free survival regarding to the triple negative group / Mestrado / Oncologia Ginecológica e Mamária / Mestra em Ciências da Saúde

Glucocorticoids (GCs) have been widely used as a therapeutic agent for diverse inflammatory ocular diseases. However, a high percentage of patients undergoing this treatment develop high intraocular pressure (IOP), which if left unsupervised may lead to glaucoma. It is believed that the IOP elevation in response to GC treatment has a genetic determinant. In order to test this hypothesis, we analyzed in 52 patients the presence of single nucleotide polymorphisms (SNPs) in the glucocorticoid receptor gene (GR), the principal mediator of GCs uptake by the cells. We studied six GR SNPs previously reported to be associated with sensitivity and resistance to GCs: GluArg22/23GluLys (codon 22-23), Asn363Ser (codon 363), IVS2+646C>G (intron 2/BclI), IVS3-46G>C (intron 3), IVS4-16G>T (intron 4), Asn766Asn (Codon 766). Nevertheless, the results of this preliminary study did not show any specific correlation between SNPs in the GR gene and IOP elevation. Therefore, we proceeded to perform a whole genome SNP screen with the DNA samples of these patients to search for possible target genes responsible for the elevated IOP after GC treatment. As a result, we identified forty-eight SNPs in thirty-three genes that correlate with the high IOP response. The gene showing the strongest association is a poorly known G-protein coupled receptor. In addition, four SNPs hit a single transporter gene. Other candidate genes identified are a translation elongation factor, an F-box protein, an oxysterol binding protein, and a solute carrier family gene. These results support our hypothesis that IOP elevation following GC treatment is a genetically determined response. GCs are a common treatment for innumerable medical conditions; we believe that a genetic association between GC treatment and its physiological response may be important for improving treatment management and drug development for retinal diseases as well as for other medical ailments. However, further studies need to be performed to analyze in depth the association between the candidate genes identified in this study and the steroid response.