Thioredoxin is a small ubiquitous oxido-reductase found in all species. The highly conserved active site, which facilitates thioredoxins redox activity, contains two redox active cysteine residues. Thioredoxin has numerous protein substrates to which it donates H+ ions and it can also function as a free radical scavenger. Through these activities thioredoxin is able to influence the redox state of not only its protein targets, but also the entire cellular environment. Thioredoxin has been implicated in many biological functions, and one mechanism by which it influences these functions is through interactions with a number of transcription factors including NF-kappa-B and p53. Thioredoxin also has numerous extracellular biological roles. It has been shown that thioredoxin is actively secreted from a number of normal and transformed cell lines including fibroblasts and activated B and T cells. This study investigates the role of thioredoxin in embryonic implantation and cancer cell metastasis, two physiological functions which rely on the same basic processes. Thioredoxin expression has previously been shown to be increased in many cancers. However it has not yet been established whether this increase is a causative or a side effect of the cancerous phenotype. Similarly thioredoxin expression has previously been shown to be increased during different phases of the oestrus cycle and pregnancy. This thesis describes the role of thioredoxin in embryonic implantation using a marmoset model. A thioredoxin cDNA was isolated and subsequently sequenced. Preliminary antibody experiments indicated that the anti human thioredoxin monoclonal antibodies available in our laboratory would recognise marmoset thioredoxin. Subsequently immunocytochemistry using anti human thioredoxin antibodies was carried out on sectioned marmoset uterus and embryonic tissue. The results indicated that thioredoxin is expressed by cells at the embryonic-maternal interface of early implantation sites. Further studies demonstrated that thioredoxin is also expressed and secreted by cultured blastocysts in vitro. This thesis also describes the role of thioredoxin in cancer cell metastasis. Results of this study indicate that thioredoxin is actively involved in facilitating the invasive phenotype of breast cancer cells. The two cell lines utilised were MCF-7, a well differentiated, relatively non-invasive breast cancer cell line; and MDA-MB-231, a poorly differentiated, highly invasive breast cancer cell line. The cell lines were transfected with thioredoxin sense, antisense and 1SS (encodes thioredoxin with both active cysteine residues mutated to serine residues and is thus redox inactive) constructs. The results demonstrate that when endogenous thioredoxin levels are increased, i.e. transfected with a sense thioredoxin construct, the invasive breast cancer cell line MDA-MB-231 becomes more invasive, conversely when endogenous levels are decreased, i.e. transfected with antisense or 1SS constructs, the invasive capacity of these cells decreases. However, when the endogenous level of thioredoxin was manipulated in the relatively non-invasive cell line MCF-7 very little effect was observed. Results also indicate that thioredoxin has the ability to act as a chemoattractant for actively invading breast cancer cells. Both of these functions appear to be dependent on thioredoxin's redox activity. Additional studies described in this thesis have shown that thioredoxin is involved in the regulation of Sp1 in vitro. Sp1 is a transcription factor known to regulate the transcription of a number of genes whose products are intimately involved in the invasive phenotype. The results in this study suggest that Sp1 DNA binding is regulated by thioredoxin such that when reduced by the enzyme its binding to DNA is facilitated. Results also indicate that Sp1 may regulate the transcription of thioredoxin by binding to Sp1 sites within the thioredoxin promoter.

Le facteur de transcription Sp1 régule la transcription de nombreux gènes à partir des sites riches en GC. Mon projet a été d'étudier le rôle de Sp1 dans la régulation de l'apoptose et du cycle cellulaire, qui est encore mal défini à l'heure actuelle. Nous avons montré que la surexpression de Sp1 induit un ralentissement du cycle cellulaire en phase G1 et l'apoptose. L'étude du transcriptome a permis d'identifier les mécanismes qui pourraient être à l'origine du ralentissement du cycle cellulaire : la répression de la cycline D2 et l'induction de p18 et cycline G2. Concernant l'induction de l'apoptose, les mécanismes mis en jeu sont spécifiques du type cellulaire et sont différents de ceux décrits jusqu'à ce jour. De façon inattendue, nos résultats montrent que l'ensemble de ces perturbations cellulaires requièrent la liaison de Sp1 à l'ADN mais pourrait être indépendant de son activité transcriptionnelle.

La NADH:ubiquinone oxydoréductase (complexe I) est le plus gros complexe enzymatique du système mitochondrial d'oxydation phosphorylante (43 sous-unités chez l'homme). Très peu de données sont disponibles concernant les mécanismes régulant l'expression de ces protéines. <br />Cette étude a été initiée par l'étude des promoteurs de deux gènes du complexe I mitochondrial humain. Les résultats montrent que le gène NDUFS8 qui code pour la sous-unité 23 kDa (TYKY) est transcrit sous le contrôle des facteurs de transcription YY1 et Sp1 tandis que gène NDUFS7 codant pour la sous-unité 20 kDa (PSST) est régulé par NRF-1 et Sp1. <br />Dans la deuxième partie de ce travail, une méthode d'analyse du protéome mitochondrial humain par électrophorèse bidimensionnelle a été développée. Le but est d'aborder de manière globale et sans a priori l'expression des protéines du complexe I : déterminer qui est régulé et comment en réponse à un stimulus déterminé? <br />Des cartes de référence ont été développées à partir de mitochondries extraites de placenta humain en utilisant deux types de gradient de pH : l'un est adapté aux protéines acides et neutres, l'autre aux protéines basiques. Sur ces cartes, 85 protéines mitochondriales ont été identifiées par spectrométrie de masse dont 17 sous-unités du complexe I. Cette technique d'analyse protéomique a ensuite été utilisée pour étudier la régulation de l'expression des protéines mitochondriales par le fer. Sur le plan technique, les premiers résultats sont encourageants : les gels d'électrophorèse bidimensionnelle préparés avec des mitochondries extraites de cellules en culture sont de bonne qualité et des variations reproductibles de l'expression de sous-unités du complexe I et d'autres protéines mitochondriales ont pu être détectées. Sur le plan fondamental, les données obtenues sont préliminaires. Il sera nécessaire de réaliser de nouvelles expériences pour confirmer les premières observations et analyser la cinétique des variations détectées.

There is an unmet medical need for the efficient treatment of neuropathic pain, a condition that affects approximately 10% of the population worldwide. Current therapies need to be improved due to the associated side effects and lack of response in many patients. Moreover, neuropathic pain causes great suffering to patients and puts an economical burden on society. The work presented in this thesis addresses SP1-7, (Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), a major metabolite of the pronociceptive neuropeptide Substance P (SP). SP is released in the spinal cord following a noxious stimulus and binds to the NK1 receptor. In contrast to SP, the degradation fragment SP1-7 is antinociceptive through binding to specific binding sites distinct from the NK1 receptor. The aim of this thesis was to investigate the impact of SP1-7 on neuropathic pain. To understand how SP1-7 exerts its effect, a series of N-truncated forms of the heptapeptide were biologically evaluated. A set of small high-affinity ligands was evaluated in animal models of neuropathic pain. To confirm a clinical relevance the levels of SP1-7 in human neuropathic pain were assessed incerebrospinal fluid (CSF) collected from neuropathic pain patients. The results showed that SP1-7 could alleviate thermal as well as mechanical hypersensitivity in three different animal models of neuropathic pain. C-terminal amidation was connected with increased efficacy. N-terminal truncation of SP1-7 indicated a necessity of five amino acids in order to retain biological effect. One small high-affinity ligand showed a significant anti-allodynic effect. CSF levels of SP1-7 in neuropathic pain patients were lower compared to controls. Taken together, these findings demonstrate that the formation of SP1-7 may be attenuated in neuropathic pain. C-terminal amidation and a majority of its amino acids are necessary for stability and permeability. Clearly, SP1-7 and SP1-7 mimetics with high affinity to the SP1-7 binding site ameliorate neuropathic pain-like behaviors in animal models of neuropathic pain. Overall, the findings presented in this thesis contribute to new knowledge regarding the role of SP1-7 and related analogues and fragments in neuropathic pain. In a future perspective, this could be essential for the development of efficient strategies for managing patients with neuropathic pain.

Coffin-Lowry Syndrome (CLS), an X-linked form of intellectual disability, is caused by mutations of the RPS6KA3 gene encoding the growth factor regulated kinase RSK2. To understand the consequences of RSK2 deficiency in the hippocampus we performed a comparison of the hippocampal gene expression profiles from Rsk2-KO and WT mice. It revealed differentialexpression of 100 genes, encoding proteins acting in various biological pathways. We further analyzed the consequences of deregulation of one of these genes, Gria2 encoding GluR2, a subunit of the glutamate AMPAR. An abnormal two-fold increased expression of GluR2 was found in the hippocampus of Rsk2-KO mice. Electrophysiology studies showed a reduction of basal AMPAR and NMDAR mediated transmission, in the hippocampus of Rsk2-KO mice. Activity of ERK1/2 was also abnormally increased in the adult hippocampus of Rsk2-KO mice. P-Sp1 level was also significantly higher in RSK2 deficient cells. Together, my results suggested that over expression of GluR2 in RSK2 deficient cells, is caused by increased Sp1 transcriptional activity on the Gria2 gene, which, itself, is the result of ERK1/2 increased signaling.

Introduction: Genetic rearrangements in Ewing sarcoma, prostate, and leukemia cells result in activation of oncogenic ETS transcription factor fusions. Mithramycin (MTM) has been identified as an inhibitor of EWS-FLI1 transcription factor, a gene fusion product responsible for oncogenesis in Ewing sarcoma. Despite preclinical success, a phase I/II clinical trial testing MTM therapy in refractory Ewing sarcoma was terminated. Liver and blood toxicities resulted in dose de-escalation and sub-therapeutic exposures. However, the promise of selectively targeting oncogenic ETS transcription factors like EWS-FLI1 prompted us to undertake the discovery of more selective, less toxic analogues of MTM. MTM is a potent inhibitor of ubiquitous SP1 transcription factor, likely inducing non-specific toxicity. In collaboration with two medicinal chemistry groups, two semi-synthetic efforts were implemented to develop novel analogues of MTM. The first effort utilized the biosynthetic product mithramycin SA (MTMSA) to modify C3-side chain. The second effort utilized an oxime linker directly formed on MTM’s C3-side chain (MTM-oxime; MTMox). Here I present the pharmacological assessment of over 75 novel MTM analogues towards selectively targeting oncogenic ETS transcription factors, like EWS-FLI1, over ubiquitous transcription factors, like SP1. Methods: Novel MTM analogues were evaluated for selective cytotoxicity against ETS fusion-dependent cell lines. Selectively cytotoxic analogues were evaluated for inhibitory effects on several gene promoters in TC-32 reporter cell lines, a Ewing sarcoma cell line dependent on EWS-FLI1, transfected with luciferase reporter vector. Cloned reporter vectors incorporated NR0B1 (EWS-FLI1 binding), β-actin (SP1 binding) and CMV (non-specific) gene promoters. Furthermore, gene (mRNA) and protein expression changes of EWS-FLI1 and SP1, as well as regulated target genes, namely NR0B1, VEGFA and BCL-2 were evaluated with MTM analogue treatments. The MTM analogues with most selective activity in vitro were administered to mice by intravenous bolus dose for pharmacokinetic analysis. The MTM analogues with highest systemic exposure from each semi-synthetic effort, namely MTMSA-Trp-A10 and MTMox-24, were further evaluated. Metabolic stabilities in whole blood, plasma, and tumor cell matrices, and across multiple species were compared with MTM. Moreover, intrinsic hepatic clearances were estimated using mouse liver microsomes. Tumor and liver distributions were estimated in tumor bearing mice. Additionally, the effect of organic anionic transporter polypeptides (OATP) on distribution of MTM was investigated. Maximum tolerated doses were evaluated for lead MTM analogues, having both selective activities in vitro and high systemic exposure, compared to MTM. Complete blood cell counts and plasma alanine aminotransferase activity were measured to evaluate dose-dependent blood and liver toxicities, respectively. ETS fusion-dependent and non-dependent cell lines were implanted subcutaneously into immunocompromised mice for efficacy studies. Average tumor volumes and survival were tracked for mice receiving treatment, compared to MTM and vehicle treatment. Results: Evaluation of MTM analogues from both semi-synthetic efforts revealed that conjugation of MTM C3-side chain with tryptophan (Trp) and/or phenylalanine (Phe) improved selective cytotoxicity against ETS fusion-dependent cell lines. This was highlighted by MTMSA-Trp-A2 (also refer to as MTMSA-Phe-Trp) and MTMSA-Trp-A10 (also refer to as MTMSA-Trp-Trp), with selective indices of 19.1 and 15.6, respectively, compared to MTM (1.5). Similarly, MTMox-23 (also refer to as MTMox-Phe-Trp) and MTMox-20 (also refer to as MTMox-Trp) had selectivity indices of 4.6 and 4.5, respectively. These selectively cytotoxic MTM analogues inhibited EWS-FLI1-mediated transcription 10-fold more effectively than both non-specific CMV-mediated and SP1-mediated (via β-actin promoter) transcription in TC-32 reporter cell lines. Moreover, gene (mRNA) and protein expression of EWS-FLI1 and regulated gene, NR0B1, were inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Similarly, SP1 and target genes, VEGFA and BCL-2, gene (mRNA) and protein expressions were also inhibited with MTM analogue treatment (GI50, 6-hour) in TC-32 cells. Conjugation of Trp and/or Phe to C3-side chain of MTM increased systemic exposure in vivo. Most impressively, the addition of two Trp residues, namely MTMSA-Trp-A10 and MTMox-24 (also refer to as MTMox-Trp-Trp), resulted in systemic exposure increases of 218- and 42-fold, respectively, after intravenous (IV) bolus dose. Metabolically, tryptophan/phenylalanine conjugated MTM analogues are liable to esterase activity on carboxy-methyl functional group. Very rapid de-methylation in biological matrix was observed with MTMox-24, compared to MTMSA-Trp-A10, suggesting a regiospecific effect. However, esterase activity was limited to rodent matrices and demethylation occurred at significantly diminished rates in non-human primate and human plasma. MTM analogues were not susceptible to p450-mediated metabolism, with negligible loss in mouse liver microsome assay compared to verapamil control. MTM (1mg/kg) and MTMox-24 (6mg/kg) were detected in subcutaneously implanted (flank) LL2 tumors and liver homogenates after IV bolus dose. Interestingly, MTMSA-Trp-A10 (2mg/kg) was not. Despite a 3-fold increase in systemic exposure with rifampin oral pretreatment, an OATP inhibitor, exposure of MTM was unaffected in Oatp knockout mouse model. Exposure of MTM in liver tissue was 8.4-fold higher compared to tumor tissue with low tissue clearance. This agrees with the lack of metabolism observed in liver microsomes and may provide a mechanism for clinically observed liver toxicity. MTMSATrp-A10 had a single maximum tolerated dose (MTD) of 0.75mg/kg, compared to 1mg/kg for MTM, administered by IV bolus. In contrast, MTM-oxime analogues (MTMox-20, -23, -24 and -25) had single maximum tolerated doses of 20 – 25mg/kg. These increased tolerances are the result of additive differences in whole blood stability, cytotoxicity and systemic exposure. At a dose of 0.75mg/kg, administered every 3 days, MTMSA-Trp-A10 did not result in an efficacious result in tumor xenograft studies. These studies remain under further investigation, but the result may indicate high plasma protein binding of MTMSA-Trp-A10 and lack of free fraction available within tumor. The most selective MTM-oxime analogue in vitro, MTMox-23, significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0025, day 16, one-way ANOVA multiple comparisons test) compared to MTM (p=0.1174, day 16) and extending survival for 17 days out of 48 days on study (p=0.0003, Log Rank (Mantel-Cox) single comparison test) with treatment at MTD every 3 days, compared to vehicle. Additionally, the MTM-oxime analogue with highest systemic exposure, MTMox-24, also significantly inhibited TC-32 (EWS-FLI1+) tumor xenograft growth (p=0.0003, day 21, one-way ANOVA multiple comparisons test) compared to MTM (p=0.032, day 21) and extending survival for 12 days out of 37 days on study (p=0.0004, Log Rank (Mantel-Cox) single comparison test) with treatment, compared to vehicle. Conclusion: These studies in whole highlight the importance of exposure (pharmacokinetics; PK), toxicity and efficacy (pharmacodynamics; PD) relationships. The cytotoxicity and high systemic exposure of MTMSA-Trp-A10 directly contributes to its lower tolerated dose. However, despite a similar tolerated dose to MTM, systemic exposure remains 163-fold higher at the MTD. High systemic exposure may be attributed to high plasma protein binding, but also reduces the exposure of free MTMSA-Trp-A10 within the tumor tissue, which drives the efficacious response. In contrast, the less cytotoxic and rapidly de-methylated MTM-oxime analogues allow for 25-fold higher tolerances in mice. This unique metabolism and clearance may prevent exposures required to induced systemic blood and liver toxicities induced by MTM. Moreover, at these highly tolerated doses, the initial systemic exposure at MTD is highest among analogues tested, which resulted in an efficacious response with MTMox-23 and MTMox-24 treatment in tumor xenograft models. It remains to be determined if these PK/PD relationships can be reproduced in additional animal models, including human, without inducing toxicity. Nonetheless, these initial studies in mice demonstrate that a more selective, more tolerated analogue of MTM has potential for clinical success in treating ETS fusion-dependent tumors.

Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...

L’infection par le rétrovirus complexe T-lymphotrope HTLV-1 (Human T-cell Leukemia Virus type 1), premier rétrovirus humain découvert, touche de 10 à 20 millions de personnes à travers le monde dans des régions endémiques et induit des désordres lymphoprolifératifs de cellules T. Seulement 5 % des personnes infectées développent, après une longue phase asymptomatique, une maladie dont une forme agressive et rapidement mortelle de leucémie nommée ATLL (Adult T-cell Leukaemia/Lymphoma).<p>L’infection par le virus HTLV-1 se caractérise par l’absence de virémie due à la latence du virus dans la majorité des cellules infectées suite à la répression transcriptionnelle de l’expression virale in vivo. Cette latence favorise très probablement le développement tumoral en permettant aux cellules infectées d’échapper à la réponse immunitaire médiée par l’hôte infecté. <p>Au cours de ce travail, nous avons tout d’abord identifié deux nouveaux sites de liaison pour le facteur de transcription Sp1, localisés dans la région R du promoteur LTR du HTLV-1. Nous les avons caractérisés physiquement par des expériences de retard de migration sur gel et avons mis en évidence la liaison de Sp1 au niveau de ces deux sites. Nous avons ensuite déterminé l’affinité de Sp1 pour les différents sites du promoteur du HTLV-1 et avons montré que les sites Sp11 (localisé dans la région U3) et Sp15 (localisé dans la région U5) sont les plus forts. Nous avons étudié l’impact de mutations de tous les sites Sp1 du LTRHTLV-1 sur son activité promotrice en conditions basales et transactivées par Tax dans le contexte d’un vecteur rapporteur épisomal. Nous avons mis en évidence que les sites Sp1 de la région R du LTRHTLV-1 agissent comme répresseurs de la transcription du LTR5’ mais n’ont aucun effet sur l’activité promotrice du LTR3’.<p>Dans une seconde partie de notre travail, nous avons étudié l’implication du cofacteur CTIP2 dans la répression transcriptionnelle du HTLV-1 et avons mis en évidence sa capacité à réprimer la transactivation médiée par Tax des promoteurs (LTR5’ et LTR3’) du HTLV-1 en cellules T-lymphoïdes Jurkat. Nous avons également montré par des expériences d’immunoprécipitation de chromatine, le recrutement de CTIP2 aux promoteurs du HTLV-1 dans une lignée infectée de manière latente par le rétrovirus et son absence dans une lignée productive. A l’inverse, Sp1 est présent dans les deux types de lignées. De plus, nous avons exclus l’implication des sites Sp1 du LTRHTLV-1 dans le recrutement du cofacteur étant donné que CTIP2 réprime toujours la transactivation Tax-dépendante du LTRHTLV-1 muté au niveau de tous les sites Sp1. <p>Finalement, nous avons étudié l’importance des modifications post-traductionnelles de CTIP2 dans son activité de cofacteur transcriptionnel. Nous avons montré que CTIP2 était acétylé par les HATs CBP et p300 et avons identifié 5 sites majeurs d’acétylation. Nous avons mis en évidence l’importance de l’acétylation de la lysine 604 de CTIP2 dans son activité répressive de la transactivation Tax-dépendante des LTRs du HTLV-1.<p>L’ensemble de ces résultats suggère un rôle du corépresseur CTIP2 dans la régulation transcriptionnelle du promoteur du HTLV-1 ainsi qu’un rôle répresseur des sites Sp1 de la région R du LTRHTLV-1 dans la transcription des gènes viraux au départ du LTR5’. La poursuite de ce travail devrait contribuer à une meilleure compréhension des mécanismes moléculaires génétiques et épigénétiques impliqués dans la latence et la réactivation transcriptionnelles des promoteurs du HTLV-1.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...