L’insuffisance rénale chronique (IRC) est associée à une diminution de la clairance métabolique des médicaments résultant en partie de l’inhibition des cytochromes P450 (CYP450) et des enzymes de phase II, notamment la N-acétyltransférase 2 (NAT2), tel que démontré chez le rat. Nous avons précédemment démontré le rôle de l'hormone parathyroïdienne (PTH) dans la diminution des CYP450 hépatiques chez le rat souffrant d’IRC. Toutefois, l’étude des mécanismes sous-jacents pouvant être facilitée par l’utilisation de souris transgéniques, l’objectif de cette étude consiste à confirmer ces résultats dans un modèle murin. D’abord, afin de valider ce modèle expérimental, une IRC a été induite par néphrectomie subtotale 3/4 chez des souris C57BL/6, puis l’expression protéique et génique des CYP450 et de la Nat2 hépatiques a été étudiée. Les résultats indiquent que l’IRC induit effectivement une diminution d’expression de ces enzymes dans un modèle murin. Ensuite, des souris mutantes pour le gène codant la PTH (PTH-/-) et les souris correspondantes de type sauvage (PTH+/+) ont été néphrectomisées, puis l’expression protéique et génique des CYP450 hépatiques a été analysée. Si la PTH est responsable de la diminution du CYP450 en situation d’IRC, les souris PTH-/- atteintes d’IRC ne devraient présenter aucune baisse d’expression. Les résultats obtenus pour les souris PTH-/- ne peuvent être interprétés, puisque chez les souris PTH+/+ atteintes d'IRC, le CYP450 hépatique est inchangé par rapport aux souris PTH+/+ témoins. Des expériences supplémentaires seront requises afin de déterminer si la régulation à la baisse du CYP450 précédemment observée est contrecarrée par l’absence de PTH. / Chronic renal failure (CRF) is associated with a decrease in the metabolic clearance of drugs, which is partly due to a reduced expression of cytochrome P450 (CYP450) and phase II enzymes, namely N-acetyltransferase 2 (NAT2). This phenomenon has been shown in the rat. We have previously demonstrated the role of parathyroid hormone (PTH) in CYP450 down-regulation in rats with CRF. However, the study of mechanisms underlying the down-regulation of CYP450 by PTH should be confirmed with the use of knockout mice. The aim of this study was, therefore, to confirm these results in a murine model. Firstly, to validate this experimental model, CRF was produced in C57BL/6 mice using the 3/4 subtotal nephrectomy. Protein and mRNA levels of hepatic CYP450 and Nat2 were then analyzed. The results showed that CRF down-regulates these enzymes, as previously observed in the rat. Finally, PTH-null mice (PTH-/-) and their corresponding wild type (PTH+/+) were nephrectomized in order to analyze protein and mRNA expression of hepatic CYP450. If PTH is responsible for the decrease of CYP450 in the presence of CRF, then PTH-/- mice with CRF should not show any reduction in CYP450 expression compared to controls. The results concerning the PTH-/- mice could not be interpreted because PTH+/+ mice with CRF did not show any significant difference of CYP450 expression when compared to PTH+/+ controls. Thus, additional experiments must be conducted in order to determine the role of PTH in CYP450 down-regulation in CRF mice.

O objetivo desse trabalho foi estudar os mecanismos que alteram a produção de melatonina na glândula pineal durante o processo de caquexia associada ao câncer e o papel de citocinas neste contexto. Os resultados mostraram um aumento da produção de melatonina no grupo inoculado com o Tumor de Walker 256 (GT) junto com uma maior atividade e expressão gênica da enzima AA-NAT, principal enzima reguladora da síntese de melatonina. O estudo in vitro mostrou que a glândula do GT produziu menos melatonina que a glândula do grupo controle (GC) após estimulação com noradrenalina (NOR). Além disso, o TNF-<font face=\"symbol\">a foi capaz de modular a síntese de melatonina em cultura, promovendo um efeito estimulatório 2h após o inicio da estimulação com NOR, e um inibitório 4h após essa estimulação. Dessa forma, os resultados indicam que produtos na circulação do rato do GT estão envolvidos na modulação encontrada in vivo e que o TNF<font face=\"symbol\">a- é um forte candidato a participar dessa modulação promovida pelo estabelecimento da síndrome da caquexia sobre a produção de melatonina na glândula pineal. / The purpose of this work was to investigate the mechanisms that modify melatonin synthesis in pineal gland during cancer related cachexia and the involvement of cytokines in this context. The results showed an increment of melatonin production in the group inoculated with Walker 256 Tumor (GT) together with a higher activity and gene expression of AA-NAT enzyme, main enzyme that regulates melatonin synthesis. The in vitro study showed that glands from GT produced less melatonin than glands of the control group (GC) after noradrenalin (NOR) stimulation. Besides, TNF-<font face=\"symbol\">a was capable to modulate melatonin synthesis in pineal gland culture, promoting a stimulatory effect 2 hours after NOR stimulation and an inhibitory effect 4 hours after this stimulation. Therefore, the results indicate that products from tumor bearing rat\'s circulation are probably involved in the modulation found in vivo and that TNF-<font face=\"symbol\">a is a strong candidate to participate in cachexia-related modulation of melatonin production in pineal gland.

L’insuffisance rénale chronique (IRC) est associée à une diminution de la clairance métabolique des médicaments résultant en partie de l’inhibition des cytochromes P450 (CYP450) et des enzymes de phase II, notamment la N-acétyltransférase 2 (NAT2), tel que démontré chez le rat. Nous avons précédemment démontré le rôle de l'hormone parathyroïdienne (PTH) dans la diminution des CYP450 hépatiques chez le rat souffrant d’IRC. Toutefois, l’étude des mécanismes sous-jacents pouvant être facilitée par l’utilisation de souris transgéniques, l’objectif de cette étude consiste à confirmer ces résultats dans un modèle murin. D’abord, afin de valider ce modèle expérimental, une IRC a été induite par néphrectomie subtotale 3/4 chez des souris C57BL/6, puis l’expression protéique et génique des CYP450 et de la Nat2 hépatiques a été étudiée. Les résultats indiquent que l’IRC induit effectivement une diminution d’expression de ces enzymes dans un modèle murin. Ensuite, des souris mutantes pour le gène codant la PTH (PTH-/-) et les souris correspondantes de type sauvage (PTH+/+) ont été néphrectomisées, puis l’expression protéique et génique des CYP450 hépatiques a été analysée. Si la PTH est responsable de la diminution du CYP450 en situation d’IRC, les souris PTH-/- atteintes d’IRC ne devraient présenter aucune baisse d’expression. Les résultats obtenus pour les souris PTH-/- ne peuvent être interprétés, puisque chez les souris PTH+/+ atteintes d'IRC, le CYP450 hépatique est inchangé par rapport aux souris PTH+/+ témoins. Des expériences supplémentaires seront requises afin de déterminer si la régulation à la baisse du CYP450 précédemment observée est contrecarrée par l’absence de PTH. / Chronic renal failure (CRF) is associated with a decrease in the metabolic clearance of drugs, which is partly due to a reduced expression of cytochrome P450 (CYP450) and phase II enzymes, namely N-acetyltransferase 2 (NAT2). This phenomenon has been shown in the rat. We have previously demonstrated the role of parathyroid hormone (PTH) in CYP450 down-regulation in rats with CRF. However, the study of mechanisms underlying the down-regulation of CYP450 by PTH should be confirmed with the use of knockout mice. The aim of this study was, therefore, to confirm these results in a murine model. Firstly, to validate this experimental model, CRF was produced in C57BL/6 mice using the 3/4 subtotal nephrectomy. Protein and mRNA levels of hepatic CYP450 and Nat2 were then analyzed. The results showed that CRF down-regulates these enzymes, as previously observed in the rat. Finally, PTH-null mice (PTH-/-) and their corresponding wild type (PTH+/+) were nephrectomized in order to analyze protein and mRNA expression of hepatic CYP450. If PTH is responsible for the decrease of CYP450 in the presence of CRF, then PTH-/- mice with CRF should not show any reduction in CYP450 expression compared to controls. The results concerning the PTH-/- mice could not be interpreted because PTH+/+ mice with CRF did not show any significant difference of CYP450 expression when compared to PTH+/+ controls. Thus, additional experiments must be conducted in order to determine the role of PTH in CYP450 down-regulation in CRF mice.

O objetivo desse trabalho foi estudar os mecanismos que alteram a produção de melatonina na glândula pineal durante o processo de caquexia associada ao câncer e o papel de citocinas neste contexto. Os resultados mostraram um aumento da produção de melatonina no grupo inoculado com o Tumor de Walker 256 (GT) junto com uma maior atividade e expressão gênica da enzima AA-NAT, principal enzima reguladora da síntese de melatonina. O estudo in vitro mostrou que a glândula do GT produziu menos melatonina que a glândula do grupo controle (GC) após estimulação com noradrenalina (NOR). Além disso, o TNF-<font face=\"symbol\">a foi capaz de modular a síntese de melatonina em cultura, promovendo um efeito estimulatório 2h após o inicio da estimulação com NOR, e um inibitório 4h após essa estimulação. Dessa forma, os resultados indicam que produtos na circulação do rato do GT estão envolvidos na modulação encontrada in vivo e que o TNF<font face=\"symbol\">a- é um forte candidato a participar dessa modulação promovida pelo estabelecimento da síndrome da caquexia sobre a produção de melatonina na glândula pineal. / The purpose of this work was to investigate the mechanisms that modify melatonin synthesis in pineal gland during cancer related cachexia and the involvement of cytokines in this context. The results showed an increment of melatonin production in the group inoculated with Walker 256 Tumor (GT) together with a higher activity and gene expression of AA-NAT enzyme, main enzyme that regulates melatonin synthesis. The in vitro study showed that glands from GT produced less melatonin than glands of the control group (GC) after noradrenalin (NOR) stimulation. Besides, TNF-<font face=\"symbol\">a was capable to modulate melatonin synthesis in pineal gland culture, promoting a stimulatory effect 2 hours after NOR stimulation and an inhibitory effect 4 hours after this stimulation. Therefore, the results indicate that products from tumor bearing rat\'s circulation are probably involved in the modulation found in vivo and that TNF-<font face=\"symbol\">a is a strong candidate to participate in cachexia-related modulation of melatonin production in pineal gland.

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii, a single-celled eukaryotic pathogen, has infected one-third of the world’s population and is the causative agent of toxoplasmosis. The disease primarily affects immunocompromised individuals such as AIDS, cancer, and transplant patients. The parasites can infect any nucleated cell in warm-blooded vertebrates, but because they preferentially target CNS, heart, and ocular tissue, manifestations of infection often include encephalitis, myocarditis, and a host of neurological and ocular disorders. Toxoplasma can also be transmitted congenitally by a mother who becomes infected for the first time during pregnancy, which may result in spontaneous abortion or birth defects in the child. Unfortunately, the therapy currently available for treating toxoplasmosis exhibits serious side effects and can cause severe allergic reactions. Therefore, there is a desperate need to identify novel drug targets for developing more effective, less toxic treatments. The regulation of proteins via lysine acetylation, a reversible post-translational modification, has previously been validated as a promising avenue for drug development. Lysine acetyltransferases (KATs) are responsible for the acetylation of hundreds of proteins throughout prokaryotic and eukaryotic cells. In Toxoplasma, we identified a KAT that exhibits homology to Elongator protein 3 (TgElp3), the catalytic component of a transcriptional elongation complex. TgElp3 contains the highly conserved radical S-adenosylmethionine and KAT domains but also possesses a unique C-terminal transmembrane domain (TMD). Interestingly, we found that the TMD anchors TgElp3 in the outer mitochondrial membrane (OMM) such that the catalytic domains are oriented towards the cytosol. Our results uncovered the first tail-anchored mitochondrial KAT reported for any species to date. We also discovered a shortened form of Elp3 present in mouse mitochondria, suggesting that Elp3 functions beyond transcriptional elongation across eukaryotes. Furthermore, we established that TgElp3 is essential for parasite viability and that its OMM localization is important for its function, highlighting its value as a potential target for future drug development.

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La chromatine est essentielle au maintien de l’intégrité du génome, mais, ironiquement, constitue l’obstacle principal à la transcription des gènes. Plusieurs mécanismes ont été développés par la cellule pour pallier ce problème, dont l’acétylation des histones composant les nucléosomes. Cette acétylation, catalysée par des histones acétyl transférases (HATs), permet de réduire la force de l’interaction entre les nucléosomes et l’ADN, ce qui permet à la machinerie transcriptionnelle de faire son travail. Toutefois, on ne peut laisser la chromatine dans cet état permissif sans conséquence néfaste. Les histone déacétylases (HDACs) catalysent le clivage du groupement acétyle pour permettre à la chromatine de retrouver une conformation compacte. Cette thèse se penche sur la caractérisation de la fonction et du mécanisme de recrutement des complexes HDACs Rpd3S et Set3C. Le complexe Rpd3S est recruté aux régions transcrites par une interaction avec le domaine C-terminal hyperphosphorylé de Rpb1, une sous-unité de l’ARN polymérase II. Toutefois, le facteur d’élongation DSIF joue un rôle dans la régulation de cette association en limitant le recrutement de Rpd3S aux régions transcrites. L’activité HDAC de Rpd3S, quant à elle, dépend de la méthylation du résidu H3K36 par l’histone méthyltransférase Set2. La fonction du complexe Set3C n’est pas clairement définie. Ce complexe est recruté à la plupart de ses cibles par l’interaction entre le domaine PHD de Set3 et le résidu H3K4 di- ou triméthylé. Un mécanisme indépendant de cette méthylation, possiblement le même que pour Rpd3S, régit toutefois l’association de Set3C aux régions codantes des gènes les plus transcrits. La majorité de ces résultats ont été obtenus par la technique d’immunoprécipitation de la chromatine couplée aux biopuces (ChIP-chip). Le protocole technique et le design expérimental de ce type d’expérience fera aussi l’objet d’une discussion approfondie. / Chromatin is essential for the maintenance of genomic integrity but, ironically, is also the main barrier to gene transcription. Many mechanisms, such as histone acetylation, have evolved to overcome this problem. Histone acetylation, catalyzed by histone acetyltransferases (HATs), weakens the internucleosomal and nucleosome-DNA interactions, thus permitting the transcriptional machinery access to its template. However, this permissive chromatin state also allows for opportunistic DNA binding events. Histone deacetylases (HDACs) help restore a compact chromatin structure by catalyzing the removal of acetyl moieties from histones. This thesis focuses on the characterization of the function and of the recruitment mechanism of HDAC complexes Rpd3S and Set3C. The Rpd3S complex is recruited to actively transcribed coding regions through interactions with the hyperphosphorylated C-terminal domain of Rpb1, a subunit of RNA polymerase II, with the DSIF elongation factor playing a role in limiting this recruitment. However, the HDAC activity of Rpd3S depends on H3K36 methylation, which is catalyzed by the Set2 histone methyltransferase. The Set3C complex’ function is still not clearly defined. It is recruited to most of its targets through the interaction between the Set3 PHD domain and di- or trimethylated H3K4. However, Set3C recruitment to genes displaying high RNA polymerase II occupancy is independent of H3K4 methylation. The mechanism by which Set3C is recruited to this gene subset is under investigation. These results have mostly been obtained through chromatin immunoprecipitation coupled to tiling microarrays (ChIP-chip). The protocol and experimental design challenges inherent to this technique will also be discussed in depth.