Les ultraviolets A (UVA) sont carcinogènes et produisent des espèces réactives de l'oxygène (ERO). Le récepteur à la mélanocortine de type 1 (MC1R) est un récepteur couplé aux protéines G (RCPG) qui est impliqué dans la mélanogénèse et dans l'inflammation cutanée. Certains variants du gène sont associés à un risque accru de mélanomes et de carcinomes cutanés. Le MC1R est exprimé surtout dans les mélanocytes mais son expression peut être induite par les UV in vitro dans les kératinocytes et in vivo dans la peau. Le récepteur MC1R est activé par l'α-MSH. L'objectif de ce travail de thèse a été d'étudier les effets du récepteur MC1R sur le stress oxydatif induit par les UVA dans des lignées kératinocytaires humaines HaCaT exprimant le récepteur MC1R ou son variant non fonctionnel Arg151Cys. Nous avons montré que la production d'ERO intracellulaire induite par les UVA est fortement inhibée dans les cellules HaCaT-MC1R et que cette inhibition est renforcée en présence d'α-MSH. L'inhibition du stress oxydatif induit par les UVA dans les cellules transfectées par le MC1R est en partie dépendante de la phosphorylation de la sous-unité activatrice, NoxA1 de la NADPH oxydase. Le traitement des cellules HaCaT-MC1R par un inhibiteur du récepteur au facteur de croissance épidermique (EGFR) restaure l'habilité de ces cellules à induire un stress oxydatif après irradiation UVA. Ces résultats montrent que l'activité constitutive du récepteur MC1R dans des kératinocytes pourrait inhiber le stress oxydatif induit par les UVA via des mécanismes dépendants de l'AMPc et de l'EGFR. / Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to Melanocortin-1 Receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R151C) non- functional variant (HaCaT-R151C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R151C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by α-MSH treatment of HaCaT-MC1R cells before UVA exposure; (3) after UVA irradiation, NoxA1 phosphorylation was increased i n HaCaT-MC1R compared to HaCaT and HaCaT-R151C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of UVA-ROS production; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive disease that is mainly caused by chronic exposure to high altitude, chronic obstructive lung disease, and obstructive sleep apnea. The increased pulmonary vascular resistance and increased pulmonary arterial pressure result in increased right ventricular afterload, leading to right heart failure and increased morbidity. There are several clinical reports suggesting a link between PH and diabetes, insulin resistance, or obesity; however, it is unclear whether HPH is associated with diabetes as a progressive complication in diabetes. The major goal of this study is to examine the effect of diabetic ''preconditioning'' or priming effect on the progression of HPH and define the molecular mechanisms that explain the link between diabetes and HPH. Our data show that HPH is significantly enhanced in diabetic mice, while endothelium-dependent relaxation in pulmonary arteries is significantly attenuated in chronically hypoxic diabetic mice (DH). In addition, we demonstrate that mouse pulmonary endothelial cells (MPECs) isolated from DH mice exhibit a significant increase in mitochondrial reactive oxygen species (ROS) concentration and decreased SOD2 protein expression. Finally, scavenging mitochondrial ROS by mitoTempol restores endothelium-dependent relaxation in pulmonary arteries that is attenuated in DH mice. These data suggest that excessive mitochondrial ROS production in diabetic MPECs leads to the development of severe HPH in diabetic mice exposed to hypoxia.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Manganese and iron homeostasis play an important role in oxidative stress protection in a variety of organisms. However, the transport and role of these metals in the periodontal pathogen Porphyromonas gingivalis were not well understood. Analysis of the genome of P. gingivalis W83 revealed the presence of two genes encoding homologs of ferrous iron transport protein, FeoB1 and FeoB2. The goal of this study was to determine the role of these two putative transporters in metal transport, their contributions to resistance to oxygen radicals and intracellular survival as well as the regulation and genetic organization of these two loci. Isogenic mutant strains deficient in FeoB1 and FeoB2, respectively, were generated and used in this study. The transport ability for manganese and iron was assessed and compared in feoB1, feoB2 mutant and wild type strains using 55Fe2+ and 54Mn2+. We demonstrated that feoB2 encodes a major manganese transporter, while FeoB1 functions as a major ferrous iron transporter. The roles of P. gingivalis FeoB1 and FeoB2 in oxidative stress defense and intracellular survival in host cells were determined using an oxidative stress survival assay and an in vitro infection assay, respectively. The feoB2 mutant exhibited reduced survival after exposure to H2O2 and to atmospheric oxygen and inside the host cells compared to the wild-type strain and its revertant, while the feoB1 mutant survived as well as the wild type strain under oxidative stress and possessed better capability to adhere to and survive in the host cells. Our results demonstrate that FeoB2 is required for protection of the bacterium from oxidative stress and for intracellular survival of P. gingivalis in host cells. However, FeoB1 is dispensable for both processes. Quantitative RT-PCR analysis revealed that expression of feoB2 in P. gingivalis is induced by oxidative stress. However, expression of feoB1 increased 2-fold upon exposure to lower growth temperature. Both observed inductions were specific and not detected under other stress conditions. We have also showed in this study that feoB2 is the second gene transcribed in an operon that is composed of a total of five genes and feoB1 is only co-transcribed with one downstream gene encoding a hypothetical protein. Notably, we also identified tandem repeats with potential to form stable stem-loop RNA secondary structure within the feoB2 and feoB1 transcripts.To our knowledge, this study has demonstrated the first connection among metal homeostasis, oxidative stress resistance and response to host cells in the periodontal pathogen, P. gingivalis.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in models of cardiovascular disease. We previously suggested sphingosine-1-phosphate (S1P) activates volume-sensitive Cl- current (ICl,swell) by ROS-dependent signaling. S1P and its analog, FTY720 (fingolimod), primarily act via G-protein coupled receptors (S1PR; S1PR1-3 in heart), but several intracellular S1P ligands are known. We investigated how these agents regulate ICl,swell. ICl,swell was elicited by bath S1P (500 nM), FTY720 (S1PR1,3 agonist; 10 μM), and SEW2871 (S1PR1 agonist; 10 μM) and was fully inhibited by DCPIB, a specific blocker. These data suggested role of S1PR in activation of ICl,swell. Surprisingly, neither CAY10444 (S1PR3 antagonist; 10 μM) nor VPC23019 (S1PR1,3 antagonist; 13 μM) blocked FTY720-induced ICl,swell. Also, gallein a pan Gbeta-gamma inhibitor, failed to block the S1P-induced current. Moreover, 100 nM FTY720 applied via the pipette evoked a larger, faster activating current than 10 μM bath FTY720. Similarly, 500 nM S1P gave larger, faster activating ICl,swell when added to the pipette than when added in the bath. In contrast to FTY720, bath S1P-induced ICl,swell was blocked by CAY10444, but a 3-fold higher concentration failed to eliminate the response to pipette S1P, and VPC23019 failed to suppress bath and pipette S1P-induced currents. Taken together, inconsistencies in the responses to S1PR agents and the greater sensitivity to pipette than bath S1P and FTY720 support the notion that intracellular ligands rather than sarcolemmal S1PR activated ICl,swell. Next we tested if S1P and FTY720, like osmotic swelling, require both NADPH oxidase and mitochondrial ROS production to evoke ICl,swell. S1P- and FTY720-induced ICl,swell were blocked by rotenone but were insensitive to gp91ds-tat, suggesting only mitochondrial ROS production was needed. One possibility is that S1P and FTY720 elicit ICl,swell by binding to mitochondrial prohibitin-2, an S1P ligand whose knockdown augments mitochondrial ROS productions. These data suggest ICl,swell may be activated by S1P accumulation in ischemia-reperfusion and CHF. Understanding S1P-signaling that elicits ICl,swell may provide insight into electrophysiological mechanisms of cardiac pathology and help identify novel targets for therapy.

Title of Dissertation: FUNCTION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 7(IGFBP7) IN HEPATOCELLULAR CARCINOMA By Dong Chen. Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring the development of innovative and effective targeted therapies. The oncogene Astrocyte Elevated Gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates Insulin-like Growth Factor Binding Protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarrays by real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at the IGFBP7 locus. Stable IGFBP7- overexpressing clones were established in the background of AEG-1- overexpressing human HCC cells and were analyzed for in vitro proliferation, senescence, in vivo tumorigenesis and angiogenesis. HCC cell lines infected with an adenovirus expressing IGFBP7 (Ad.IGFBP7) were analyzed by using in vitro cell cycle, apoptosis, in vivo tumorigenesis assays. Results: IGFBP7 expression is significantly downregulated in both human HCC patients’ samples and cell lines compared to normal liver and hepatocytes. IGFBP7 expression was also found to inversely correlate with the stages and grade of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence. When injected into nude mice, in vivo growth was profoundly suppressed, potentially as a result of inhibition of both angiogenesis and IGF1R activation by IGFBP7. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing Reactive Oxygen Species (ROS) and activating a DNA damage response. N-acetylcysteine could neutralize ROS and rescue the cells from apoptosis. In early phase after Ad.IGFBP7 infection, activation of cell cycle control proteins like Rb, p53, ATM, ATR, CHK1 and CHK2 were identified and G2/M cell cycle arrest was recorded by FACS. Ad.IGFBP7 infection resulted in the activation of p38 MAPK, and a p38 MAPK inhibitor SB 203580 could block the apoptotic process. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intra-hepatic metastasis. In a nude mouse subcutaneous model, xenografts from human HCC cells were established in both flanks and only left- side tumors received intratumoral injection of Ad.IGFBP7. Ad.IGFBP7 markedly inhibit growth of both left-sided injected tumors and right-sided un- injected tumors by profound suppression of angiogenesis. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Targeted overexpression of IGFBP7 may be a potential novel and effective therapy for HCC.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Dans la recherche de thérapie antitumorale de plus en plus innovante, nous avons évalué un traitement local basé sur l’utilisation de plasma froid. Le plasma froid (dans ce cas, <40°C) est un gaz ionisé par un apport d’énergie. Il contient des charges (électrons, ions), des radicaux libres et des molécules excitées. Il peut être généré à l’extrémité de cathéter permettant un traitement locorégional comme le traitement de dysplasie ou encore de tumeurs non résécables. Une activité antitumorale importante du plasma a été mise en évidence in vitro sur diverses lignées tumorales (colorectale, pulmonaire, pancréatique et cérébrale). Par ailleurs les cellules tumorales sont plus sensibles au plasma que les cellules normales. Les ROS générés sont à l’origine des principaux mécanismes d’action du plasma. Ils induisent de nombreux dommages à l’ADN, suivi d’un arrêt du cycle cellulaire conduisant à l’apoptose des cellules. Les études de tolérance ont mis en évidence l’innocuité de faibles doses de plasma sur le tissu traité permettant de définir les doses de plasmas utilisable dans le cadre de traitements antitumoraux. En utilisant des tumeurs xénogreffées en sous cutané et l’imagerie de bioluminescence, une activité antitumorale du plasma froid a été mise en évidence pour la première fois in vivo avec une augmentation de la survie des souris traitées d’environ 60%. Le traitement induit un arrêt de la prolifération tumoral avec une induction d’apoptose dans l’ensemble de la tumeur sans augmenter la surface de nécrose. L’effet antitumoral a également été démontré en utilisant le plasma gun sur un modèle de tumeurs colique et pancréatique en situation orthotopique chez la souris avec une augmentation de la survie (115%) accompagné d’une diminution de la métastasie. Ces résultats obtenues dans une démarche de recherche translationnelle montrent l’intérêt potentiel du plasma comme nouvel agent antitumoral. / In the context of new innovated antitumor treatment discovery, we evaluated the efficacy of a new local treatment based on non-thermal plasma (NTP). NTP is a cold (in this case, <40°C) ionized gas (a ir or noble gas) thanks to an electric discharge. It contains free charges (electrons, ions), free radicals and excited molecules. It can be generated at the end of a catheter allowing a local treatment that is compatible with usual endoscopes for dysplasia or non resecable tumors. We showed that NTP has a significant antitumor effect in vitro on various cell lines including colorectal, pancreatic, lung and brain tumor cells. The major action mechanisms of NTP was linked to a high rate generation of ROS in the vicinity of tumors cells and others plasma components have a minor implications. These ROS induce lethal DNA damages leading to a multiphase cell cycle arrest and finally to apoptosis. In vivo, a good tolerance of plasma treatment was highlighted and NTP treatment parameter was defined. Using subcutaneous xenografts and bioluminescence imaging, we showed a major antitumor effect of plasma in vivo with a 60% increase of mice life span. NTP treatments of tumor induce a tumor cell cycle arrest with a significant apoptosis induction in the whole tumor without increase of necrotic area. This in vivo antitumor effect was also observed with an in situ treatment using plasma gun of colorectal and pancreatic orthotopic tumor xenografts. A significant increase of mice lifespan (115%) was obtained together with a metastasis decrease. These results obtained in translational research showed the potential antitumor activity of NTP as a new type of treatment for cancer treatment.

Aim: To determine the effect of diet-induced steatosis in the development of mitochondrial dysfunction in the liver and hepatic sensitivity to the partial ischemia. Methods: Male Wistar rats (361 ± 8.8 g) were fed standard (SD) or high-fat diet (HFD). Partial ischemia was induced by short-term clamp (20 min) of vein porta two days before the end of the experiment. Results: Ten-week HFD administration lead due to increased ketogenesis the altered glucose tolerance elevated serum NEFA. We demonstrated the inhibitory effect of HFD on the respiratory capacity of mitochondria in vitro. HFD negatively affected the activity of antioxidant systems and stimulated the formation of lipoperoxides. Partial ischemia had no efect on the mitochondrial oxidative capacity but significantly elevated the oxidative stress. Conclusion: HFD administration lead to the development of fatty liver that was still not accompanied by biochemical markers of liver injury. Nevertheless, we proved the impairment of to mitochondrial respiratory capacity, signs of structural damage of mitochondria and the increased sensitivity to oxidative damage of the liver. Subject words: biochemistry, physiology Keywords: mitochondria, HFD, ischemia, respiratory chain, antioxidant systém, ROS