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<p>The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines.<p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / Exposure to oxidative stress leads to accumulation of reactive oxygen species and this stimulates protective cellular functions as a compensatory response to prevent the spread of apoptotic signal and prevent cell death. The purpose of this dissertation is to understand the importance of PKCγ activation and regulation of the retinal gap junction protein Cx50, and what role PKCγ plays in this neuro-protective effect. Through electron microscopy we were able to show that PKCγ knockout mice retinas had incomplete cellular organization in the outer plexiform layer (OPL) of the retina, the layer of retina where Cx50 plays an important role in retinal cellular synapses. Electroretinograms confirmed that this structural disorganization also led to loss of functional response to light stimuli in PKCγ knockout mice retinas. In vivo exposure to 100% hyperbaric oxygen (HBO) caused significant degradation of the retina in knockout mice compared to control mice. Thicknesses of the inner and nuclear and ganglion cell layers were increased, with complete disruption of OPL in PKCγ KO mice retinas. Damage to the outer segments of the photoreceptor layer and ganglion cell layer was significantly more apparent in the central retinas of HBO-treated knockout mice. Cx50 immunolabeling showed significant reduction to HBO treatment of PKCγ control mice retinas, HBO treatment failed to produce reduction of Cx50 immunolabeling in KO mice retinas. In the R28 retinal cell line, PKCγ enzyme was shown to be activated by phorbol ester (TPA) and hydrogen peroxide. This resulted in translocation to the cellular membrane as confirmed by western blot and confocal microscopy. Suppression of PKCγ by siRNA rendered R28 cells more sensitive to oxidative stress-induced cell apoptosis, the process of apoptosis started earlier, and this resulted in cell death. R28 treatment with phorbol esters and hydrogen peroxide led to reduction in gap junction activity and Cx50 gap junction cell disassembly. This dissertation shows that PKCγ plays an important role in structural organization of retina and has a neuro-protective effect in response to oxidative stress, in part because of its control of Cx50.

Doctor of Philosophy / Department of Biochemistry / Dolores J. Takemoto / The avascular lens tissue depends on the gap junction channels to facilitate intercellular communication for supplying cells deep within the lens with nutrients and removing waste products of cellular metabolism. In the absence of the protein synthesis machinery in the inner lens fiber cells, the proper regulation of gap junction channels becomes extremely important as disturbance of the lens homeostasis can lead to cataract development. Phosphorylation of gap junction subunit connexin proteins has been shown to play an important channel-modulating role in a variety of tissue. Protein kinase C-[Gamma] (PKC[Gamma]) has been implicated in the phosphorylation of connexins in the lens. Here the role of PKC[Gamma] in the regulation of gap junction coupling in the mouse lens has been investigated. We have compared the properties of coupling in lenses from wild type (WT) and PKC[Gamma] knockout (KO) mice. Western blotting, confocal immunofluorescence microscopy, immunoprecipitation, RT-PCR and quantitative real time PCR were used to study gap junction protein and message expression; gap junction coupling conductance and pH gating were measured in intact lenses using impedance studies. PKC[Gamma]was found to regulate the amount and distribution of Cx43 in the lens. Gap junction coupling conductance in the differentiating fibers (DF) of PKC[Gamma] KO lenses was 34% larger than that of WT. In the mature fiber (MF), the effect was much larger with the KO lenses having an 82% increase in coupling over WT. Absence of PKC[Gamma] in the KO mice also caused abnormal persistence of nuclei in the typical nucleus-free region in the DF. These results suggest a major role for PKC[Gamma] in the regulation of gap junction expression and coupling in the normal lens mediated by phosphorylation of the lens connexins. This becomes very vital in the diabetic lenses which contain a depleted amount of PKC[Gamma] and people suffering from spinocerebellar ataxia type-14 (SCA14) who have a mutated inactive form of PKC[Gamma]. Prolonged exposure of lenses to oxidative stress in these patients can lead to cataract formation. In cultured human lens epithelial cells (HLECs), 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the depletion of Cx43 protein level via PKC-mediated phosphorylation of Cx43. At the same time Cx46 protein and message levels were upregulated in response to TPA treatment. So, the PKC activator regulates Cx43 and Cx46 in opposing ways. The possible mitochondria localization of Cx46 reported here could help in finding the non-junctional roles for Cx46.

Master of Science / Department of Biochemistry / Dolores J. Takemoto / Spinocerebellar ataxia type 14 (SCA14) is a rare, autosomal dominant neurodegenerative disease caused by mutations in the gene encoding for protein kinase Cγ (PKCγ). These mutations affect the translocation and activation of the protein and are particularly damaging to the Purkinje cells of the cerebellum. This translocation and activation leads to the down regulation of gap junction activity by direct phosphorylation on the C-terminal tail of connexin proteins. This process is necessary in terminating the propagation of apoptotic signaling and is disrupted by SCA14-type mutations. Gap junctions allow the passive diffusion of small molecules from one adjoining cell to another. Gap junctions function as electrical synapses in neuronal tissue and are formed from connexin proteins. The connexin family of proteins contains approximately 20 members, each of which is expressed in a tissue dependent manner. One of the dominant connexin proteins expressed in Purkinje cells is connexin 57 (Cx57). Here, I have tested if Cx57 is regulated by PKCγ. This thesis shows that activation of PKC and PKCγ caused internalization of Cx57 gap junction plaques in HT-22 cell culture. PKC and PKCγ activation led to the phosphorylation of Cx57 primarily on serine residues. Furthermore, the expression of SCA14-type PKCγ led to increased sensitivity to oxidative stress, resulting decreased cell viability.

Dans notre laboratoire, des études antérieures ont montré que la stimulation du récepteur AT[indice inférieur 2] par l'angiotensine II menait à l'activation des p42/p44[indice supérieur MAPK] (GENDRON et al., 1999) par une voie impliquant les protéines Rapt et B-Raf chez les cellules NG108-15 (GENDRON et al., 2003a) et que cette voie était nécessaire à l'élongation neuritique. Parallèlement à l'activation de la voie MAPK, il a été montré que le R-AT[indice inférieur 2] active la voie NOS/GCs/GMPc (GENDRON et al., 2002). Puisqu'il est connu que les protéines kinases C (PKC) participent à la différenciation neuronale dans certains types cellulaires, le but de mon travail a été de déterminer si les PKC pouvaient participer aux mécanismes initiaux menant à l'activation des voies précédemment décrites. Nos résultats indiquent que les isoformes [alpha], [epsilon], [iota] et [zéta] sont exprimées dans cette lignée cellulaire et que l'inhibiteur de PKC[alpha], le Gö6976, induit une élongation neuritique importante en plus d'une diminution de la prolifération. Lors de traitements courts avec l'Ang II, nos données indiquent qu'il y a translocation de PKC[alpha] de la membrane plasmique à la fraction soluble, entraînant ainsi son inactivation. De plus, le traitement des cellules avec le Gö6976 provoque l'inhibition de p21[indice supérieur ras] mais active Rap1 de façon similaire à l'Ang II. Par contre, l'inhibition de PKC[alpha] n'a pas d'effet sur la capacité de l'Ang II à activer les p42/p44 MAPK . L'ensemble de ces résultats indique que PKC[alpha] serait en amont de la cascade de signalisation des p42/p44[indice supérieur MAPK]. Ainsi, l'inhibition de PKC[alpha] mène à la diminution de l'activité de p21 ras et de la prolifération cellulaire, ce qui pourrait contribuer à favoriser l'activation de la voie menant à p42/p44[indice supérieur MAPK] et l'élongation neuritique. Les isoformes PKC[epsilon], et PKC[zéta] seraient impliquées dans des processus différents de PKC[alpha] puisque l'utilisation d'inhibiteurs pharmacologiques sélectifs, soit le Gö6983 (inhibe PKC[alpha] et PKC[zéta] ou le GF109203X (inhibe PKC[alpha] et PKC[epsilon]), a montré qu'ils induisent l'élongation neuritique de façon similaire à l'Ang II ou l'inhibiteur de PKC[alpha]. En conditions basales, PKC[zéta] est associée à l'actine tandis que l'activation du récepteur AT[indice inférieur 2] cause sa relocalisation dans le cytosol. Il pourrait s'agir d'un mécanisme de régulation de la polymérisation de l'actine. Pour sa part, l'isoforme [epsilon] semble impliquée dans la production de GMPc induite par le R-AT[indice inférieur 2]. De plus, la différenciation des cellules avec un traitement de 3 jours avec l'Ang II augmente l'association de PKC[epsilon] avec les microtubules. Cette isoforme pourrait être utile à la régulation de la ramification neuritique des cellules NG108-15. Ces résultats indiquent que les PKC sont impliquées dans la physiologie des cellules NG108-15, chacune ayant un rôle précis et distinct à jouer.

The glucose toxicity has been recognized over the last several years as a factor contributing to both impaired insulin secretion and insulin resistance in patients with diabetes. However, the molecular mechanisms that underlie the changes in glucose transport activity induced by hyperglycemia have not been fully understood. The purpose of the present investigation is to determine if acute hyperglycemia affects an activation of glucose transport and also if hyperglycemic-induced change in insulinstimulated glucose transport is mediated via a PKC-dependent signaling system. Animals were anesthetized, and the soleus (SOL) muscles were isolated and clamped at their resting length. After a 10 minute recovery period the muscles were transferred to preincubation vials containing KHB supplemented with 4 or 16 mmol of glucose and 16 mmol/1 mannitol with or without insulin and/or inhibitors for 30 minutes. Following an incubation series to prepare the muscle, the muscle was incubated in radioactive 3-0- [3H] methylglucose and [14C] mannitol for 10 min. in the presence/absence of insulin and inhibitors, and the amount of glucose transport was measured. A total of 100µU/ml insulin with 4 mM glucose led to increase glucose transport by 155%, whereas the same amount of insulin with 16 MM glucose led to 80% increment in glucose transport. Also, 16 mM glucose in the absence of insulin induced an increase of glucose uptake by apporoximately 50% compared with 4 MM glucose. However, the addition of insulin reduced that difference to 5.3%. The conventional PKC inhibitor GF 109203X in the muscle incubated with 16 MM glucose led to a decrease in insulin-stimulated glucose transport (1l%), whereas the inhibitor with 4 mM glucose induced a decrease in insulin-stimulated glucose transport (24%). These findings suggest that glucose can directly regulate glucose transport activity by a mechanism that possibly involves a facilitated GLUT1 transporter activity. In addition to the mass action of glucose, the hyperglycemic-induced increase in insulin stimulated glucose transport may be partially mediated via a PKC-dependent signaling system. / School of Physical Education

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

A insuficiência cardíaca é uma síndrome clínica de mau prognóstico caracterizada por disfunção cardíaca associada à intolerância aos esforços, retenção de fluído e redução da longevidade. Dentre as serina/treonina quinases associadas às alterações funcionais e estruturais cardíacas observadas na progressão da insuficiência cardíaca, a família das proteínas quinase C (PKC) composta por 12 diferentes isoformas parece modular a contratilidade miocárdica e o remodelamento cardíaco. No presente estudo, caracterizamos o fenótipo cardíaco e o perfil de ativação das diferentes isoformas de PKC na progressão da insuficiência cardíaca de etiologia isquêmica em ratos. Além disso, estudamos o efeito da inibição sustentada da isoforma PKC&#946;II sobre a sobrevida, o remodelamento cardíaco e a função ventricular em modelo de insuficiência cardíaca de etiologia isquêmica. Conseguinte, identificamos possíveis substratos cardíacos da PKC&#946;II envolvidos na progressão da insuficiência cardíaca. Para isso, avaliamos os efeitos agudo e crônico da inibição da PKC&#946;II sobre o transiente de cálcio e a contratilidade de cardiomiócito isolados de ratos adultos com insuficiência cardíaca. Por fim, testamos as inibições específicas das PKC&#946;II e PKC&#946;I na progressão da hipertrofia cardíaca compensada para a insuficiência cardíaca em modelo animal de hipertensão arterial sustentada. Nossos resultados sugerem que a inibição sustentada da PKC&#946;II reverte o quadro de insuficiência cardíaca, melhorando a função ventricular, o remodelamento cardíaco e a sobrevida dos diferentes modelos de insuficiência cardíaca estudados, constituindose em uma estratégia terapêutica celular promissora / Heart failure is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality worldwide. Protein kinase C isozymes emerge as important potential therapeutic targets in chronic cardiovascular disease. However, individual PKC isozymes play different roles in the pathogenesis of cardiac diseases. Here, we characterized the cardiac phenotype as well as the different PKC isozyme activation profile during myocardial-induced heart failure progression in rat. Furthermore, we evaluated the role of selective PKC&#946; II inhibition on survival, left ventricle remodeling and cardiac function in myocardial-induced heart failure. Moreover, we identified the cardiac PKC&#946;II substrates related to heart failure. Finally, PKC&#946;II and PKC&#946;I specific inhibitors were chronically delivered to hypertensive-induced heart failure rats and the cardiac phenotype was evaluated. Our data suggest that 6-wks of PKC&#946;II inhibition, but not PKC&#946;I, improved animal survival by restoring cardiac function and promoting cardiac anti-remodeling effect in both myocardial infarctioninduced heart failure and hypertensive-induced heart failure rats. The improved cardiac function and anti-remodeling effect of PKC&#946;II inhibition seems to be associated with increased contractility of cardiac myocytes, improved miofilaments/Ca2+ sensitivity and decreased cardiac inflammatory response. Altogether, the results provide evidence for beneficial effects of PKC&#946;II specific intracellular inhibition on cardiac function and remodeling, which may be a promising cellular therapy for heart failure treatment

Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the frontline of defence within the innate immune system and are among the first cells to arrive at the site of inflammation. Effective phagocytosis and killing of invading pathogens by neutrophils is of significant importance for successful resistance to infectious diseases. An important complication in diabetes mellitus is an increased sensitivity to infections and increased tissue damage, leading to many secondary diseases. This may in part be explained by an impaired function of neutrophil granulocytes. Since the exact mechanisms underlying defective neutrophil function in diabetes mellitus are not fully understood, the aim of the present study was to investigate the effects of elevated glucose and insulin concentrations on phagocytosis of opsonized yeast and on production of reactive oxygen metabolites (ROS) in normal human neutrophils. Elevated D-glucose concentrations (15-25 mM) inhibited the phagocytosis of C3bi- or IgG-opsonized yeast particles, which was neither an osmotic effect nor an effect due to reduced binding of opsonized yeast particles to the neutrophils. Inhibition of protein kinase C (PKC) by GF109203X or Go6976 could completely reverse the inhibitory effect of 25 mM D-glucose on phagocytosis. Diacylglycerol (DAG) dose-dependently inhibited phagocytosis and suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect. Elevated concentrations of insulin (80-160 μU/ml) also inhibited neutrophil phagocytosis, an effect shown in part to be due to a delayed phagocytosis process. Insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. The inhibition of phagocytosis by either glucose or insulin resulted in an expected reduction of intracellular respiratory burst. However, the extracellular release of ROS during phagocytosis was increased by insulin, but inhibited by glucose. The ability of insulin to enhance ROS production was found to be F-actin dependent. Data suggests that glucose inhibited intracellular respiratory burst activation by interfering with intracellular signaling downstream of PKC activation, whereas extracellular release of ROS was inhibited by glucose upstream of PKC signaling. Taken together these results suggest that both hyperglycemia and hyperinsulinemia inhibit complement receptor and Fc receptor-mediated phagocytosis in human neutrophils. Insulin, but not glucose, also induced an enhanced extracellular release of ROS during phagocytosis. The combination of reduced phagocytosis and alterations in ROS production may possibly explain both the increased sensitivity to infections and tissue damage seen in type 2 diabetes.