Thesis (Ph. D.)--University of Wisconsin--Madison, 1987. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 152-157).

La mitochondrie et le réticulum endoplasmique (RE) forment un réseau dans les cellules qui contrôle la fonction et le destin cellulaire. La mitochondrie de la cellule ß pancréatique joue un rôle central dans la sécrétion d’insuline en réponse au glucose de par sa capacité à produire de l’ATP. Le RE lui prend en charge la mise en conformation de l’insuline et joue le rôle de stock calcique. Ces 2 organites se rejoignent au niveau de points de contact appelés Mitochondria Associated endoplasmic reticulum Membranes (MAMs,). Les MAMs sont le siège d’échanges calciques et lipidiques entre les 2 organites. Les altérations de la mitochondrie et du RE ont été montrées comme des facteurs contribuant au développement du diabète de type 2. L’implication des MAMs n’a cependant jamais été étudiée dans la cellule ß.La glucotoxicité provoquée par une exposition chronique à des concentrations élevées de glucose, est un facteur clé de la dysfonction ß pancréatique au cours du diabète de type 2. J’ai pu démontrer que la glucotoxicité augmentait la fission mitochondriale et le nombre de MAMs dans les cellules bêta humaines et INS-1E mais que ces MAMs présentaient des défauts d’échanges calciques, pouvant ainsi contribuer au défaut de la sécrétion d’insuline. J’ai ensuite modulé les MAMs soit via une stimulation aigue au glucose soit par l’utilisation d’un siRNA qui rompt partiellement les contacts entre le RE et la mitochondrie ou par l’utilisation d’un linker qui artificiellement force ces contacts. La stimulation aigue au glucose augmente les MAMs et le transfert de calcium du RE vers la mitochondrie alors que la rupture des contacts diminue la secretion d’insuline. Enfin le linker en forçant les rapprochements RE-mitochondrie mime les effets de la glucotoxicité.Ce travail, constitue la première étude structurelle et fonctionnelle des MAMs dans la cellule ß pancréatique, éclairant leur rôle dans la dysfonction ß pancréatique lors du développement du diabète de type 2 / Mitochondria and endoplasmic reticulum (ER) form a network in cells that control cellular function and fate. Mitochondria play a central role in insulin secretion in ß cell by its ability to product ATP. ER takes in charge of insulin folding and is the major cell calcium store. Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), a multiprotein complex implicated in calcium transfer and lipid exchange . Alterations of mitochondria and ER have been shown to contribute to metabolic disorder such as type 2 diabetes. MAMS were recently implicated in the regulation of glucose homeostasis But the role of MAMs in ß cells is still largely unknown and their implication in glucotoxicity-associated ß cell dysfunction remains to be defined.Here, I report that acute glucose stimulation stimulated ER-mitochondria interactions and calcium (Ca2+) exchange in INS-1E cells, whereas disruption of MAMs altered glucose-stimulated insulin secretion (GSIS). Conversely, chronic incubations with high glucose of either INS-1E cells or human pancreatic islets altered GSIS, and concomitantly reduced ER Ca2+ store, increased mitochondrial Ca2+ and reduced ATP-stimulated ER-mitochondria Ca2+ exchanges, despite an increase of organelle interactions. Furthermore, glucotoxicity-induced perturbations of Ca2+ signalling are associated with ER stress, altered mitochondrial respiration and mitochondria fragmentation, and these organelle stresses may participate to increased organelle tethering, as a protective mechanism. Lastly, sustained induction of ER-mitochondria interactions using a linker induced mitochondrial fission and altered GSIS.Therefore, dynamic organelle coupling participates to GSIS in ? cells and over-time disruption of organelle Ca2+ exchange might be a novel mechanism contributing to glucotoxicity-induced ß cell dysfunction in type 2 diabetes

Among Human Immunodeficiency viruses (HIV), type-1 (HIV-1) is the most common worldwide and has the highest virulence and infectivity. Though the virus only infects a few cell types, the infection affects almost every organ system causing multiple comorbidities. One of the comorbidities is HIV-associated Neurocognitive Disorders (HAND). Interorganelle communication regulates many cellular functions including calcium exchange, lipid exchange, intracellular trafficking, and mitochondrial biogenesis. Interestingly, all these processes have been implicated in HAND suggesting that dysregulation of interorganelle communication plays a role in the progression of HAND. Using neuronal cell cultures, I show that mitochondrial-associated ER membranes (MAM)-associated protein and MAM-tethering protein expression and interactions are altered in the presence of the HIV-1 protein Tat. I also show, PTPIP51 and VAPB, two MAM-tethering proteins, expression is altered in the MAM fraction but not the whole cell fraction, indicating a localization problem. Phosphorylation of PTPIP51 has been shown to regulate the subcellular localization and I show that tyrosine phosphorylation is upregulated with Tat. In addition, I show that PTPIP51 binding with VAPB can be rescued with the addition of kinase inhibitors blocking PTPIP51 phosphorylation suggesting that Tat is altering the phosphorylation of PTPIP51 affecting its subcellular localization and binding to VAPB. Furthermore, I show that ER-Golgi communication is altered in the presence of Tat where there is an increase in the interactions between YIF1A and VAPB, two ER-Golgi tethering proteins. The altered iv interactions between MAM and ER-Golgi tethering proteins in the presence of Tat lead to the disruption of cellular pathways associated with dysfunctional interorganelle communication that can lead to neuronal dysfunction and can contribute to HAND. / Biomedical Sciences

Le réticulum endoplasmique et la mitochondrie sont deux organites majeurs impliqués dans la régulation du métabolisme glucido-lipidique. Ces structures interagissent au niveau de points de contact étroits appelés Mitochondria-Associated Endoplasmique Reticulum Membranes (MAMs). Les MAMs sont une zone de communication et d’échanges, de lipides et de calcium entre autre, indispensables à l’activité des deux organites et au maintien de l’homéostasie cellulaire. Des connexions physiques sont assurées par l’interaction de protéines complémentaires, comme le canal anionique voltage-dépendant (VDAC)-1, la protéine chaperonne (Grp)-75 et le récepteur de l'inositol 1,4,5-triphosphate (IP3R)-1, constituant un complexe impliqué dans le transfert de calcium. D’autres acteurs comme les mitofusines 1 et 2 (MFN1/2) assurent également un rapprochement entre les deux organites et semblent jouer un rôle dans les échanges des lipides. Récemment, les MAMs sont apparues comme un nouveau carrefour de la signalisation de l’insuline dans le foie. Le monoxyde d’azote (NO) participe également au contrôle de la réponse à l’insuline hépatique et a une action spécifique sur la mitochondrie. Mes travaux de thèse ont montré que le NO à des concentrations physiologiques module les interactions entre le RE et la mitochondrie dans le foie et que son action sur les MAMs implique la voie de signalisation sGC/cGMP/PKG. J’ai également démontré que la modulation des MAMs par le NO semble jouer un rôle clé dans la régulation de la voie de signalisation à l’insuline (projet1). Par ailleurs, j’ai exploré l’importance des MAMs dans la régulation du métabolisme lipidique. Pour cela, j’ai modulé l’expression protéique de Grp75 et Mfn2 sur un modèle d’hépatocarcinome humain (Huh7). Mes résultats ont montré qu’une surexpression des deux protéines améliore les MAMs et la β-oxydation mitochondriale mais conduit à une accumulation intracellulaire de lipides. Ceci serait dû à un défaut de sécrétion des lipides dans les lipoprotéines VLDL et pourrait impliquer l’apparition d’un stress mitochondrial et une altération des échanges de phospholipides entre les deux organites (projet 2). Par conséquence mon travail confirme le rôle physiologique des MAMs et éclaire les mécanismes d’actions de cette plateforme cellulaire dans la régulation du métabolisme glucido-lipidique hépatique. A plus long terme ces connaissances participeront peut-être à l’identification de potentielles cibles thérapeutiques afin de prévenir la stéatose et la résistance à l’insuline hépatiques et leurs complications / The endoplasmic reticulum and mitochondria are two major organelles involved in the regulation of glucose and lipid metabolism. These structures interact at close contact points called Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs). MAMs constitute an area of communication and exchange, of lipids and calcium among others, essential for the activity of both organelles and the maintenance of cellular homeostasis. Physical connections are ensured by the interaction of complementary proteins, such as the voltage-dependent anionic channel (VDAC)-1, the chaperone protein (Grp)-75 and the inositol 1,4,5-triphosphate receptor (IP3R)-1, constituting a complex involved in calcium transfer. Other actors such as mitofusins 1 and 2 (MFN1/2) also connect the two organelles and are involved in lipid exchanges. Recently, MAMs have emerged as a new carrefour for insulin signaling in the liver. Nitric oxide (NO) also helps control the response to hepatic insulin and has a specific action on mitochondria. My thesis work showed that NO at physiological concentrations modulates the interactions between RE and mitochondria in the liver and that its action on MAMs involves the sGC/cGMP/PKG signalling pathway. I also demonstrated that NO modulation of MAMs plays a key role in regulating the insulin signaling pathway (project 1). In addition, I explored the importance of MAMs in the regulation of lipid metabolism. For that purpose, protein expression of Grp75 and Mfn2 was modulated in a human hepatocarcinoma model (Huh7). Results showed that overexpression of both proteins improves MAMs and mitochondrial β-oxidation but leads to intracellular lipid accumulation. This could be due to a defect in lipid secretion in VLDL lipoproteins and could imply the appearance of mitochondrial stress and an alteration of phospholipid exchanges between the two organelles (project 2). Consequently, my work confirms the physiological role of MAMs and sheds light on the mechanisms of action of this cellular platform in the regulation of glucose and lipid metabolism in the liver. In the longer term, this knowledge may contribute to the identification of potential therapeutic targets to prevent steatosis and hepatic insulin resistance and their complications

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmia, caused by genetic defects in sarcoplasmic reticulum (SR) Ca-release channel RyR2, or its accessory proteins. Our previous studies demonstrated that CPVT mitochondria can absorb RyR2-mediated aberrant Ca release (ACR) and behave as an efficient Ca buffer which is critical in mitigating harmful consequences of ACR. In this study, we test the hypothesis that modulating mitochondrial phosphate (Pi) transport or the tethering between SR-mitochondria, known as Mitochondria-associated-membrane (MAMs), impacts arrhythmogenesis in CPVT. We found that inhibiting mitochondrial Pi carrier (PiC) exacerbated cellular arrhythmias whereas overexpressing PiC in CPVT alleviated both cellular and in vivo arrhythmias. In parallel, disrupting MAMs exacerbated arrhythmogenesis in CPVT, but promoting MAMs by overexpressing mitofusin2 tethering protein reduced cellular arrhythmias. Our study provided both pharmacological and genetic evidence that directing more Ca to mitochondria by enhancing mitochondrial Pi transport or targeting MAMs could be promising therapeutic strategies to reduce CPVT arrhythmia.

The theme of this thesis is to analyze the impact of policy interventions such as financing human development (HD), tax reform and conditional cash transfer programmes, under the framework of growth and sustainable development. These policy instruments are evaluated through the application of both partial and general equilibrium models, and the last paper concentrates on developing regional social accounting matrices (SAMs) as a core database for spatial general equilibrium modelling. Essay 1: Trade-offs in Achieving Human Development Goals for Bangladesh investigates the benefits and costs associated with alternative investment financing options for achieving HD goals by applying the MAMS (Maquette for Millennium Development Goals Studies) model. We find that full achievement of these goals would have led to a GDP loss that would have been significantly larger in the domestic borrowing scenario than in the tax scenario. The tax-financing alternative is thus the better option for financing large development programs. In terms of public spending composition, we find that, under some circumstances, a trade-off arises between overall Millennium Development Goal (MDG) progress and poverty reduction. Essay 2: Welfare impact of broadening VAT by exempting Small-Scale food markets: The case of Bangladesh analyses the welfare impacts of different VAT reforms. A general and uniform VAT on all commodities is preferred as it is more efficient and less administratively costly. However, due to equity concerns, food is normally exempted from VAT. On the other hand, exemptions on food mean that an implicit subsidy is provided to high-income households. Hence, we analyze a broad-based VAT regime with a high threshold that excludes small-scale operators (where the low-income households buy their products most, including food) and the simulation result shows that welfare improves for the low-income households. Essay 3: Effect of Girls’ Secondary School Stipend on Completed Schooling and Age at Marriage: Evidence from Bangladesh estimates the effect of a conditional cash transfer programme on education and age at marriage. We apply both difference in differences (DiD) and regression discontinuity methods to evaluate the impact of the policy instrument. Our estimation results show that the girls in the treatment group who were exposed to the programme had a higher average number of completed years of schooling and also delayed their first marriage compared to the girls in the control group. We also show that the DiD approach might produce a biased result as it does not consider the convergence effect. Essay 4: Estimation of Multiregional Social Accounting Matrices using Transport Data proposes a methodology for estimating multiregional SAMs from a national SAM by applying the cross-entropy method. The methodology makes possible the construction of regional SAMs that are consistent with official regional accounts and minimize deviations from transport data.

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