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

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

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

18 effects of TZDs are probably due to the remodeling of adipose tissue and increased adiponectin secretion. SUMMARY Studying the pathogenesis of insulin resistance and the role of PKC in insulin resistance In HHTg rats, elevated serum triglycerides and FFA were associated with the ectopic accumulation of triglycerides in tissues and reduced insulin sensitivity of peripheral tissues. Impaired glucose utilization in the peripheral tissues was associated with the reduced activity of GS in skeletal muscle. Decreased GS activity and glucose utilization in peripheral tissues indicate a possible defect in insulin signal transduction. In line with this, our results show that skeletal muscle IR was associated with the increased activation and translocation of PKC θ. Nutritionally induced obesity of HHTg rats resulted, in many cases, in the further deterioration of metabolic abnormalities associated with IR. We found that PKC θ, in particular, could contribute to the metabolic abnormalities associated with IR and obesity. The age-related increase in IR and deterioration of some parametres of carbohydrate and lipid metabolism, were not associated, in HHTg rats, with obesity but with increased serum levels of triglycerides and FFA. The age-related worsening of IR in HHTg rats was accompanied by increased...

Orientador: Ricardo da Fonseca / Resumo: Em fêmeas bovinas a liberação de prostaglandina F2α (PGF2α) endometrial pode ser induzida in vivo pelo estradiol (E2), entretanto, seu papel ainda não foi esclarecido. Em estudos prévios realizados pelo grupo, o tratamento com E2 in vivo duas horas antes das vacas serem abatidas no 17º dia do ciclo estral, associado à adição de cálcio ionóforo (CI) in vitro em explantes endometriais, exacerbou a síntese de PGF2α quando comparado ao grupo de vacas não tratadas com E2 e CI. O mesmo foi observado em células endometriais bovinas (BEND) expostas ao E2 e CI simultaneamente. Considerando que as concentrações plasmáticas de PGF2α aumentam a partir de 3,5 horas após da aplicação do E2 in vivo, possivelmente tais ações envolvam a síntese de proteínas. Hipotetizou-se que o E2 estimule a expressão e/ou a atividade das enzimas dependentes de cálcio, especificamente a proteína quinase C (PKC) e fosfolipase A2 citosólica (cPLA2). Assim, objetivou-se analisar o efeito dos inibidores de Proteína G, PKC e PLA2 na síntese de PGF2α induzida pelo 17β-estradiol e CI em células endometriais bovinas BEND (Experimento 1), e avaliar as proteínas PKC e PLA2 e ERK 1/2, pela técnica de Western Blotting, em explantes endometriais obtidos de fêmeas bovinas abatidas 2 horas após a administração de 17β-estradiol no 17o dia do ciclo estral (Experimento 2). No Experimento 1, células endometriais bovinas (BEND) foram submetidas a ausência ou presença de um inibidor de proteína G - 10µM de Guanosine 5’- {B-thio}... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

The nuclear lamina consists of a mesh-like network of lamin proteins anchored to the inner nuclear membrane by interactions with integral membrane proteins such as emerin. Emerin binding to lamin A/C is one of the interactions that connect the inner nuclear membrane to the lamina. Infection by herpesviruses results in changes in the organization of the nuclear lamina, perhaps in order to facilitate envelopment of capsids at the inner nuclear membrane. In HSV-1 infected cells, alterations to the lamin proteins have been shown to involve pUL34, pUL31, and pUS3 proteins, which are also required for normal nuclear envelopment. We tested hypotheses about the mechanism and significance of lamina disruption. This thesis presents the following data. Infection of multiple cell types induced emerin hyperphosphorylation that was dependent on the presence of pUL34 and kinase active pUS3 proteins. The pUL34-dependent component was also sensitive to Rottlerin treatment suggesting that cellular kinases sensitive to Rottlerin were involved in emerin modification. LAP2 (another lamin associated protein) was de-modified (perhaps de-phosphorylated) in a pUS3 and pUL34 independent manner. Emerin was not required for growth of HSV-1. Hyperphosphorylation of emerin was required for its disassociation from the lamina. PKC family members have been implicated in the disruption of the nuclear lamina during herpesvirus nuclear egress. To test the hypothesis that PKC activity was required for viral replication, PKC activity was blocked with PKC inhibitors and dominate negative PKC constructs. Chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta did not inhibit viral growth. In addition to lamin associated proteins, lamin localization is also disrupted during herpesvirus infections. Emerin and lamin A/C are binding partners and the localization of both proteins is disrupted by pUS3 and cellular kinase mediated phosphorylation. To test the hypothesis that HSV-1-induced lamin A/C disruption is mediated via a mechanism similar to emerin's, pUS3 and Rottlerin Sensitive Kinases were inhibited and lamin A/C localization was observed. Unlike emerin, HSV-1-induced disruption of lamin A/C was not altered by Rottlerin Sensitive Kinase inhibition suggesting that HSV has multiple mechanisms for disruption of the lamina. Phosphorylation of lamina components, by Rottlerin Sensitive Kinases, may be a required event prior to primary envelopment. To test this hypothesis, growth of HSV-1 was tested in Rottlerin treated infected cells. Although the inhibitor Rottlerin, did reduce viral growth, it was also was also associated with severe depression of viral late-gene expression. TEM analysis suggested that Rottlerin Sensitive Kinases(s) were required for: (i) nuclear egress and (ii) capsid accumulation or formation supporting the hypothesis that the capsids were made in the presence of Rottlerin were unable to leave the nucleus. pUS3 is a multi-functional protein in alpha-herpesviruses. It has been implicated in lamina disruption, protecting the infected cell from apoptosis, and de-envelopment at the outer nuclear membrane. In BT-549 cells, a breast cancer cell line with low PKC delta expression, the hypothesis was tested that in the absence of cellular lamina disrupting kinases, an US3-null virus would be blocked at the lamina disruption step. In BT-549 cells, the US3-null (vRR1202) virus was 10-fold decreased above the typical 10-fold decrease, compared to WT virus, to produce a 100-fold decrease in infectious PFU yet apoptosis was not increased. Lamin A/C disruption occurred via similar mechanism in both breast cancer cell lines: BT-549 and MCF-7. Interestingly, in the BT-549 cells, emerin was extensively hyperphosphorylated in an US3-null infection, yet was not redistributed along the NE. These data support a model that one or more specific residue(s) must be phosphorylated for emerin disconnection from lamina.

The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells. / Doctor of Philosophy (Ph.D.)

Introduction: Therapeutic agents like doxorubicin, an anthracycline antibiotic drug, are widely used in cancer chemotherapy. The use of doxorubicin is limited however by an increased risk of cardiac damage as a side effect, and an increased cancer cell drug resistance mediated by efflux drug transporters. Strategies are needed to protect the heart and still allow the benefits of drug treatment. “Basic” fibroblast growth factor-2 (FGF-2) is a multi-functional protein. It is angiogenic and cardioprotective against ischemia-reperfusion injury. FGF-2 can also regulate cancer cell drug resistance or sensitivity, however, so far, there is no evidence that FGF-2 protects against doxorubicin-induced cardiac damage through effects on efflux drug transporter levels or function. Aims: To investigate whether: (1) FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage; and if so whether (2) an effect on efflux drug transporters might contribute to this cardioprotection by FGF-2. Methods: Neonatal rat cardiac myocyte cultures were treated with doxorubicin in the absence or presence of pre-treatment with FGF-2. To assess cell damage: (i) culture medium was tested for lactate dehydrogenase (LDH) activity as an indication of plasma membrane disruption; (ii) cells were stained with fluorescent apoptosis and necrosis biomarkers as well as (iii) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and acridine orange to assess DNA fragmentation or compaction. The role of FGF receptor (FGFR) or protein kinase C (PKC) was addressed through use of inhibitors including SU5402, or chelerythrine as well as bisindomaleimide. Multidrug resistance gene 1a and 1b (MDR1a, 1b), multidrug resistance gene 2 (MDR2) and multidrug resistance-related protein 1 (MRP1) gene expression, as well as the function of MDRs and MRPs protein products were assessed by real-time reverse transcriptase-polymerase chain reaction (qPCR), as well as retention/extrusion of (fluorescent) doxorubicin/calcein in cardiac myocytes, respectively. Efflux drug transporter inhibitors, including 20 µM cyclosporine A (CsA), 2 µM verapamil and 1 µM Tariquidar (XR9576) were used to asssess for a direct effect of FGF-2 on transporter function. Fluorescence-activated cell sorting (FACS) was used to measure fluorescent doxorubicin/calcein levels inside treated cardiac myocytes. Results: Doxorubicin increased the incidence of programmed cell death, DNA damage, and lysosome and LDH activity, while decreasing cell number at 24 hours. FGF-2 prevented the detrimental effects of doxorubicin. In turn, the protective effects of FGF-2 were blocked in the presence of FGFR or PKC inhibitors. FGF-2 treatment significantly increased MDR1a, MDR1b, MDR2, MRP1 RNA levels by qPCR, and protein levels as assessed by function, and specifically extrusion of doxorubicin/calcein, in the presence of doxorubicin when compared to doxorubicin treatment alone. Furthermore, inhibition of efflux drug transporters with CsA and Tariquidar (XR9576) significantly reduced the ability of FGF-2 to protect against doxorubicin-induced damage; the beneficial effect of FGF-2 was completely blocked by pretreatment with verapamil. Conclusion(s): These data indicate for the first time that exogenous FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage, and implicate PKC and regulation of efflux transporter protein levels and/or function in the mechanism.

Introduction: Therapeutic agents like doxorubicin, an anthracycline antibiotic drug, are widely used in cancer chemotherapy. The use of doxorubicin is limited however by an increased risk of cardiac damage as a side effect, and an increased cancer cell drug resistance mediated by efflux drug transporters. Strategies are needed to protect the heart and still allow the benefits of drug treatment. “Basic” fibroblast growth factor-2 (FGF-2) is a multi-functional protein. It is angiogenic and cardioprotective against ischemia-reperfusion injury. FGF-2 can also regulate cancer cell drug resistance or sensitivity, however, so far, there is no evidence that FGF-2 protects against doxorubicin-induced cardiac damage through effects on efflux drug transporter levels or function. Aims: To investigate whether: (1) FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage; and if so whether (2) an effect on efflux drug transporters might contribute to this cardioprotection by FGF-2. Methods: Neonatal rat cardiac myocyte cultures were treated with doxorubicin in the absence or presence of pre-treatment with FGF-2. To assess cell damage: (i) culture medium was tested for lactate dehydrogenase (LDH) activity as an indication of plasma membrane disruption; (ii) cells were stained with fluorescent apoptosis and necrosis biomarkers as well as (iii) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and acridine orange to assess DNA fragmentation or compaction. The role of FGF receptor (FGFR) or protein kinase C (PKC) was addressed through use of inhibitors including SU5402, or chelerythrine as well as bisindomaleimide. Multidrug resistance gene 1a and 1b (MDR1a, 1b), multidrug resistance gene 2 (MDR2) and multidrug resistance-related protein 1 (MRP1) gene expression, as well as the function of MDRs and MRPs protein products were assessed by real-time reverse transcriptase-polymerase chain reaction (qPCR), as well as retention/extrusion of (fluorescent) doxorubicin/calcein in cardiac myocytes, respectively. Efflux drug transporter inhibitors, including 20 µM cyclosporine A (CsA), 2 µM verapamil and 1 µM Tariquidar (XR9576) were used to asssess for a direct effect of FGF-2 on transporter function. Fluorescence-activated cell sorting (FACS) was used to measure fluorescent doxorubicin/calcein levels inside treated cardiac myocytes. Results: Doxorubicin increased the incidence of programmed cell death, DNA damage, and lysosome and LDH activity, while decreasing cell number at 24 hours. FGF-2 prevented the detrimental effects of doxorubicin. In turn, the protective effects of FGF-2 were blocked in the presence of FGFR or PKC inhibitors. FGF-2 treatment significantly increased MDR1a, MDR1b, MDR2, MRP1 RNA levels by qPCR, and protein levels as assessed by function, and specifically extrusion of doxorubicin/calcein, in the presence of doxorubicin when compared to doxorubicin treatment alone. Furthermore, inhibition of efflux drug transporters with CsA and Tariquidar (XR9576) significantly reduced the ability of FGF-2 to protect against doxorubicin-induced damage; the beneficial effect of FGF-2 was completely blocked by pretreatment with verapamil. Conclusion(s): These data indicate for the first time that exogenous FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage, and implicate PKC and regulation of efflux transporter protein levels and/or function in the mechanism.

Les ostéoclastes (OCs) sont responsables de la résorption de l’os normal. La différenciation, la maturation et la survie de ces cellules sont principalement stimulées par RANKL. Ces phénomènes se produisent dans les suites de l’interaction de RANKL avec son récepteur RANK situé à la surface de l’OC, via la transduction de signaux intracellulaires conduisant à l’activation de différentes cascades de signalisation. Les principaux facteurs de transcription induits par cette signalisation sont NF-?B et NFATc1, qui modulent l’expression de gènes d’importance majeure pour le bon fonctionnement des OCs. Dans la maladie de Paget, caractérisée par une augmentation multifocale du remodelage osseux avec hyperrésorption osseuse, les OCs ont une morphologie anormale, ils sont résistants à l’apoptose et leur activité est augmentée. Des mutations du gène codant pour la protéine p62 (séquestosome 1 ) ont été décrites dans cette maladie, identifiant p62 comme un facteur important de la signalisation de l’OC. Des travaux de notre laboratoire ont montré une augmentation de l’activation de NF-?B induite par RANKL dans les OCs pagétiques, avec même une activité constitutive de NF-?B en présence de la mutation p62 p.P392L. Notre étude a évalué l’expression et l’activation de NFATc1, et le rôle de PKC[C zêta] dans la signalisation NFATc1, cette kinase appartenant au complexe de signalisation TRAF6/p62 induit par RANKL. En culture primaire, des monocytes de sang périphérique de patients pagétiques et de contrôles (tous génotypés pour la mutation p62) ont été différenciés en OCs matures en présence de RANKL et M-CSF. Nous avons évalué l’effet de RANKL sur l’activation de NFATc1 (translocation nucléaire évaluée par immunofluorescence), en présence ou non d'un inhibiteur de PKC[C zêta], ainsi que l’expression protéique (immunobuvardage) et ARNm (RTPCR quantitative). Nous avons observé que RANKL stimulait l’activation de NFATcl dans les OCs pagétiques, porteurs ou non de la mutation p62 p.P392L, et que PKC[C zêta] intervenait dans cette signalisation. Dans un modèle de différenciation OC à partir de monocytes foetaux, nous avons par ailleurs mis au point l’étude de l’activation de NFATc1 par essai luciférase, et les conditions optimales pour évaluer son expression protéique et génique. Nos résultats démontrent pour la première fois dans des OCs humains que RANKL stimule l’activation et l’expression de NFATc1, et que l’activation de NFATc1 est augmentée dans les OCs pagétiques. De manière tout à fait originale, nous montrons également que la PKC[C zêta] contribue à l’activation de NFATc1. [symboles non conformes]