Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Capillary perfusion in skeletal muscle is highly coordinated both spatially and temporally with the level of muscle fiber activity. Increased oxygen demand in working muscle is met by increasing capillary perfusion. Blood flow to skeletal muscle increases proportionally with exertion during exercise, and returns to resting levels almost immediately after cessation of activity. Matching blood flow with fiber activity is the result of multiple mechanisms working in combination to dilate and constrict smooth muscle in the arteriolar walls. In this study, theoretical models were used to examine the regulation of capillary perfusion based on arteriolar vasodilation in response to local metabolic changes in the muscle tissue. The level of tissue oxygenation in working muscle was predicted for mechanisms of blood flow regulation based on vascular sensing of decreased tissue PO₂ and of increased interstitial [K⁺]. Two hypothetical mechanisms for vascular sensing of PO₂ and interstitial [K⁺] were considered: direct sensing by arterioles, and sensing by capillaries with stimulation of feeding arterioles via conducted responses. Grouping of capillaries into functional units called microvascular units (MVUs) increased the predicted fraction of capillaries necessarily perfused to maintain adequate oxygenation relative to control by individual capillaries. Control by arteriolar sensing of oxygen and K⁺ resulted in poor control of tissue oxygenation at high levels of muscle activation. At higher levels of muscle activity, the terminal feeding arteriole for a MVU typically flows through a region perfused by another MVU such that the metabolic conditions near the arteriole is not sufficient to trigger vasodilation. Control of microvascular unit perfusion by capillary sensing of oxygen and K⁺ resulted in adequate tissue oxygenation over the full range of activation, without a significant increase in the number of perfused MVUs relative to control by individual capillaries. Oxygen consumption in active muscle fibers does not reach a maximal rate until 15 to 25 seconds after initial recruitment, but increased blood flow to MVUs is observed within 5 seconds after initial recruitment. A simulation of the time course of the response mechanisms suggests that vascular sensing of tissue PO₂ does not fully trigger MVU perfusion until 15 seconds after initial recruitment. Increased interstitial [K⁺] can trigger MVU perfusion within 2 seconds after initial recruitment. During the initial 10 seconds of muscle activation, increased perfusion in the absence of an increased rate of oxygen consumption may function to minimize the build up of interstitial [K⁺], which is important in maintaining fiber excitability and avoiding early onset of fatigue. MVU perfusion triggered by capillary sensing of local metabolic conditions resulted in a lower mean [K⁺] across the muscle tissue over the range of activity levels than mechanisms based on arteriolar sensing.

I begin by exploring the distinction between the physiologic, quantitative, and qualitative conceptions of futility. I argue that if medical futility is going to be a useful and appropriate normative tool in the medical lexicon, it should not duplicate and confuse judgments which we already have the tools to make. Hence, I distinguish qualitative futility from the concepts of distributive justice, rationing, harm, and insufficient benefit. Lastly, I consider the argument that providing qualitative futility violates professional integrity. Next I consider the claim that futility judgments are a form of unjustified paternalism. I also explore the relationship of physician imposition of values and the ideas of individual patient well-being, and self-determination. I consider an argument put forth by Thomlinson and Brody that futility judgments actually support autonomy, concluding that their argument must be restricted to individuals whose choices can be shown to be inconsistent with their values and aims. Lastly, I provide a comparison of futility judgments and the ordinary/extraordinary distinction which shows futility to be normatively vague and clinically dangerous. Because of the potential for misuse and confusion, I compare futility and rationing judgments. I argue that rationing decisions are necessary but should be explicit rather than disguised as futility. The consequences of failing to adequately distinguish these two are unfairness to individual patients, and harm to the doctor-patient relationship and societal trust of medicine. I detail a number of models of the physician patient relationship and attempt to determine two things: (1) whether these allow for physician authority to withhold qualitatively futile care, and (2) how well these models can answer this question in the absence of an account of the goals of medicine. I conclude that various accounts offer little specific guidance about the physician's right to withhold qualitatively futile treatment. Finally, in chapter seven I attempt to ground the debate about medical futility in the larger context of a debate about the appropriate ends and goals of medicine, arguing that such limits require an extended social dialogue.

Germline mutations in the genes, BRCA1 and BRCA2 have been implicated in hereditary breast cancer (HBC) and hereditary breast and ovarian cancers (HBOC). A large-scale multi-center genetic linkage analysis revealed that while linkage to BRCA1 and BRCA2 was evident in the majority of HBOC families, the large proportion of HBC families remained unaccounted for by either of these genes. These findings supported the hypothesis that other novel breast cancer susceptibility genes could account for the unexplained clustering of female breast cancer cases in the HBC families. Previous studies have identified common BRCA1 and BRCA2 mutations in the French Canadian (FC) population. The objective of this thesis was to establish the contribution of BRCA1 and BRCA2 mutations to the HBC and HBOC families in the FC population of Quebec. Mutations were identified in 96 of 224 (43%) families that met our inclusion criteria of at least three verified cases of breast cancer diagnosed below 65 years of age or ovarian cancer. An additional 13 novel mutations were added to the FC mutation spectrum including the BRCA2:3398delAAAAG mutation which was identified in eight independent families. Haplotype sharing of closely related alleles in the carriers of this novel BRCA2 mutation suggested that the mutation was introduced by a common ancestor. Although the FC population was more heterogeneous than originally expected five common mutations accounted for 84% of the mutation carrier families. The BRCA1/BRCA2 phenotype was further defined to include metachronous bilateral breast cancer. In addition, 38 mutation-negative families were identified that were phenotypically indiscernible from the BRCA / Les mutations germinales dans les gènes BRCA1 et BRCA2 ont été impliquées dans la prédisposition aux syndromes du Cancer Héréditaire du Sein (CHS) et des Cancers Héréditaires du Sein et de l'Ovaire (CHSO). Une grande étude multi-centres a révélé que bien que la ségrégation associée à BRCA1 et BRCA2 soit évidente dans la majorité des familles avec le CHSO, une grande proportion des familles avec le CHS ne sont pas visiblement associées à ces gènes. Cette observation supporte l'hypothèse que des gènes de prédisposition au cancer du sein inconnus pourraient être responsables de l'excès de cancer du sein féminin dans ces familles CHS. Des études précédentes ont identifié des mutations communes dans BRCA1 et BRCA2 dans la population canadienne française (CF). L'objectif de cette thèse portait à déterminer la contribution des mutations dans BRCA1 et BRCA2 chez les familles avec le CHS et le CHSO dans la population canadienne française du Québec. Des mutations furent identifiées dans 96 des 224 (43%) familles rencontrant nos critères d'inclusion, soit au moins 3 cas vérifiés de cancer du sein (diagnostiqués avant l'âge de 65 ans) ou de cancer de l'ovaire dans la famille. Treize mutations additionnelles furent ajoutées à la gamme de mutations CF, dont la mutation BRCA2:3398delAAAAG identifiée dans 8 familles indépendantes. Les porteurs partagent un haplotype d'allèles très similaires ce qui suggère que la mutation fut introduite dans la population par un ancêtre commun à tous les porteurs. En dépit de l'observation que la population CF semble plus hétérogène qu'originalement anticipé, 5 mutations communes sont responsab

The hepatitis C virus (HCV) is a positive-strand RNA virus that belongs to the Flaviviridae family. HCV expresses the non-structural protein, NS5B, an RNA-dependent RNA polymerase required for genome replication. Here, we describe the allosteric properties of both the GTP-specific binding site (G-site) of the HCV polymerase and VIR-1327, a benzimidazole-derivative non-nucleoside inhibitor (NNI). Using biochemical assays, we identified certain G-site residues that affect NS5B activity, including serine-29, proline-495 and valine-499. We also demonstrated that VIR-1327 is a potent inhibitor with a resistance profile consisting of P495A and V499A mutants, but is non-competitive with regards to GTP. Moreover, biophysical results suggest that the mechanism of action of VIR-1327 is to prevent the formation of a stable enzyme-template complex. Further analysis of the G-site and NNIs will not only provide a better understanding of HCV replication, but also validate different allosteric sites as virus-specific targets for the development of anti-HCV therapeutics. / Le virus de l’hépatite C (VHC) est un virus d’ARN de polarité positive qui appartient à la famille Flaviviridae. Le VHC exprime la protéine non-structurelle NS5B, une ARN polymérase dépendante de l’ARN indispensable à la réplication du génome virale. Nous décrivons ici les propriétés allostériques du site GTP-spécifique (site-G) du VHC polymérase, ainsi que les propriétés allostériques de VIR-1327, un inhibiteur non-nucléoside (INN) qui est dérivé du benzimidazole. En utilisant des tests biochimiques, nous avons identifié certains résidus du site-G qui influencent l’activité du NS5B, incluant sérine-29, proline-495 et valine-499. Nous avons également démontré que VIR-1327 est un puissant inhibiteur avec un profil de résistance comprenant les mutants P495A et V499A, mais qu’il est non-compétitif avec le GTP. De plus, des résultats biophysiques suggèrent que le méchanisme d’action de VIR-1327 est de prévenir la formation d’un complexe enzyme-matrice stable. D’autres analyses du site-G et des INN vont non seulement fournir une meilleure compréhension de la réplication du VHC, mais aussi valider différents sites allostériques comme étant des cibles virus-spécifiques pour le développement de thérapeutiques contre le VHC.

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Peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1 alpha (PGC-1α) is a transcriptional co-activator responsible for mitochondrial biogenesis and oxidative metabolism. Many isoforms of PGC-1α have been described in the literature, most of which are shown to function similarly to the canonical PGC-1α protein. Recently, however, a novel isoform of PGC-1α was identified, PGC-1α4. It was shown to have a different, yet complementary function to canonical PGC-1α (PGC-1α1) in muscle. It is also expressed in other metabolically active tissues; however, it is unknown whether it has additional distinct tissue-specific functions. Furthermore, PGC-1α plays an important role in controlling metabolism in pancreatic β-cells and expression of the co-activator is decreased in diabetic islets; however, the role of PGC-1α isoforms in diabetes is unknown. Our objective is to determine whether PGC-1α4 has a unique function in β-cells and whether it plays a role in the pathogenesis of diabetes. We show that stimulation with forskolin, exendin-4 and a cytokine cocktail of TNFalpha, IL-1beta and IFNgamma, induced specific PGC-1α isoforms in β-cells. Following over-expression of these isoforms in INS-1 cells, PGC-1α4 prevented the cleavage of caspase-3 in response to cytokines, suggesting that the novel isoform is uniquely anti-apoptotic. To assess whether PGC-1α isoforms play a role in β-cell survival in vivo, mice with a β-cell specific PGC-1α knockout of all isoforms were subjected to low-dose streptozotocin (STZ) treatment to induce β-cell apoptosis. Unexpectedly, knockout mice were protected from STZ induced hyperglycemia. However, there was no difference in percentage of cleaved caspase-3 positive cells in control versus knockout mice, suggesting no difference in apoptosis. Therefore, PGC-1α4 could be a novel factor important for β-cell survival and over-expression of this unique isoform may protect against the pathogenesis of diabetes. / Peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1 alpha (PGC-1α) est un co-activateur transcriptionnel responsable de la biogenèse mitochondriale et du métabolisme oxydatif. De nombreux isoformes de PGC-1α ont été décrits dans la littérature, dont la plupart ont été démontrés comme fonctionnant de manière similaire à la protéine PGC-1α canonique. Récemment, cependant, un nouvel isoforme de PGC-1α a été identifié, PGC-1α4, possédant une fonction différente mais complémentaire de la protéine canonique (PGC-1α1) dans le muscle. PGC-1α4 est également exprimé dans d'autres tissus métaboliquement actifs, mais il n'est pas connu s'il démontre de nouvelles fonctions dépendantes du tissu. PGC-1α joue un rôle important dans la regulation métabolique des cellules β du pancreas; de plus l'expression du co-activateur est dérégulée dans les îlots de Langerhans de patients diabétiques. Notre objectif est de déterminer si PGC-1α4 a une fonction unique dans les cellules β du pancréas et s'il joue un rôle dans la pathogenèse du diabète. Nous montrons que la stimulation avec la forskoline, l'exendine-4 et un cocktail de cytokines, TNFalpha, IL-1beta et IFNgamma, induit les isoformes de PGC-1α dans les cellules β. Suite à la surexpression des isoformes dans les cellules INS-1, PGC-1α4 empêche le clivage de la caspase-3 en réponse à des cytokines, suggérant qu'uniquement le nouvel isoforme est anti-apoptotique. Pour déterminer si les isoformes de PGC-1α jouent un rôle dans la survie des cellules β in vivo, les souris déficientes de tous les isoforms de PGC-1α, spécifiquequement dans les cellules β, ont été soumises à une faible dose de streptozotocine (STZ) provoquant l'apoptose des cellules β. De façon inattendue, les souris déficientes en PGC-1α ont été protégées contre l'hyperglycémie induite par la STZ. Cependant, il n'y avait pas de différence du pourcentage de cellules en apoptose chez les souris témoins par rapport aux souris déficientes. Donc, PGC-1α4 pourrait être un nouveau facteur important pour la survie des cellules β du pancreas et la surexpression de cette isoform unique, peut protéger contre la pathogenèse du diabète.