Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 104-116.

The interferon (IFN) system forms an essential part of the innate immune response, up-regulating hundreds of IFN-stimulated genes (ISGs) in response to viral infection. A key protein in this response is the human myxovirus resistance protein MxA, an IFN-induced GTPase with broad-spectrum antiviral activity, capable of inhibiting many RNA and DNA viruses. One of the most studied antiviral effects of MxA is the inhibition of influenza A virus replication, yet the molecular mechanism of antiviral activity is still unknown. Influenza A viruses are inhibited by MxA at two distinct stages of viral replication; during viral entry and following primary transcription of viral mRNAs. The antiviral effects of MxA during viral entry are highly dependent on IFN, however activity exerted after primary transcription can occur in the absence of IFN. This study provides evidence that MxA exerts its antiviral activity at these two stages of viral replication through distinct mechanisms, and outlines a potential model of MxA antiviral activity following primary transcription. A potential third antiviral mechanism of MxA is proposed based on the findings that MxA is able to regulate cellular lipid metabolism, thereby potentially affecting virion composition. Mutational analysis of MxA highlights the significance of GTPase activity to the antiviral effects of MxA, while also demonstrating that natural single nucleotide polymorphisms in MxA have the potential to severely impair or prevent antiviral activity. Finally, this thesis shows for the first time that MxA exhibits antiviral activity against influenza B viruses. Overall this thesis provides new information illustrating how MxA provides potent antiviral activity against influenza viruses. Such information is vitally important as understanding the molecular basis of how proteins such as MxA function against many human pathogens is fundamentally important in our efforts to create better long-term treatment options for all viral diseases.

In this study, the production of monoclonal antibodies directed against the activating enzyme for an S6 kinase is examined and described. Evidence is presented for the association of an Mr. 55,000 abd Mr. 95,000 protein with the s6 kinase. These proteins are phosphorylated in the presence of Activating Enzyme. A sequence of regulatory events for insulin-stimulated phosphorylation of ribosomal protein S6 in cells is postulated as follows: insulin activates the receptor tyrosine kinase, which phosphorylates the Mr 116,000 subunit of Activating Enzyme. The Activating Enzyme then activates the S6 kniase by phosphorylation, and phosphorylation of the ribosomal protein s6 is promoted.

Mainstream philosophy of action and mind construes intentional behaviour in terms of causal processes that lead from agent-involving mental states to action. Actions are construed as events, which are actions in virtue of being caused by the right mental antecedents in the right way. Opponents of this standard event-causal approach have criticised the view on various grounds; they argue that it does not account for free will and moral responsibility, that it does not account for action done in the light of reasons, or, even, that it cannot capture the very phenomenon of agency. The thesis defends the standard event-causal approach against challenges of that kind. In the first chapter I consider theories that stipulate an irreducible metaphysical relation between the agent (or the self) and the action. I argue that such theories do not add anything to our understanding of human agency, and that we have, therefore, no reason to share the metaphysically problematic assumptions on which those alternative models are based. In the second chapter I argue for the claim that reason-explanations of actions are causal explanations, and I argue against non-causal alternatives. My main point is that the causal approach is to be preferred, because it provides an integrated account of agency by providing an account of the relation between the causes of movements and reasons for actions. In the third chapter I defend non-reductive physicalism as the most plausible version of the standard event-causal theory. In the fourth and last chapter I argue against the charge that the standard approach cannot account for the agent’s role in the performance of action. Further, I propose the following stance with respect to the problem of free will: we do not have free will, but we have the related ability to govern ourselves—and the best account of self-determination presupposes causation, but not causal determinism.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Germline mutations in the tumor suppressor gene PTEN cause PTEN Hamartoma Tumor Syndrome (PHTS). Pediatric patients with PHTS frequently develop lipomas. Treatment attempts with the mTORC1 inhibitor rapamycin were unable to reverse lipoma growth. Recently, lipomas associated with PIK3CA-related overgrowth syndrome were successfully treated with the novel PI3K inhibitor alpelisib. Here, we tested whether alpelisib has growth-restrictive effects and induces cell death in lipoma cells. We used PTEN-haploinsufficient lipoma cells from three patients and treated them with alpelisib alone or in combination with rapamycin. We tested the effect of alpelisib on viability, proliferation, cell death, induction of senescence, adipocyte differentiation, and signaling at 1–100 M alpelisib. Alpelisib alone or in combination with rapamycin reduced proliferation in a concentrationand time-dependent manner. No cell death but an induction of senescence was detected after alpelisib incubation for 72 h. Alpelisib treatment led to a reduced phosphorylation of AKT, mTOR, and ribosomal protein S6. Rapamycin treatment alone led to increased AKT phosphorylation. This effect could be reversed by combining rapamycin with alpelisib. Alpelisib reduced the size of lipoma spheroids by attenuating adipocyte differentiation. Since alpelisib was well tolerated in first clinical trials, this drug alone or in combination with rapamycin is a potential new treatment option for PHTS-related adipose tissue overgrowth.

This thesis aims to improve the understanding of transport and critical layer mixing in the troposphere and stratosphere. A dynamical approach is taken based on potential vorticity which has long been recognised as the essential field inducing the flow and thermodynamic structure of the atmosphere. Within the dynamical framework of critical layer mixing of potential vorticity, three main topics are addressed. First, an idealised model of critical layer mixing in the stratospheric surf zone is examined. The effect of the shear across the critical layer on the critical layer evolution itself is investigated. In particular it is found that at small shear barotropic instability occurs and the mixing efficiency of the critical layer increases due to the instability. The effect of finite deformation length is also considered which extends previous work. Secondly, the dynamical coupling between the stratosphere and troposphere is examined by considering the effect of direct perturbations to stratospheric potential vorticity on the evolution of midlatitude baroclinic instability. Both zonally symmetric and asymmetric perturbations to the stratospheric potential vorticity are considered, the former representative of a strong polar vortex, the latter representative of the stratospheric state following a major sudden warming. A comparison of these perturbations gives some insight into the possible influence of pre or post-sudden warming conditions on the tropospheric evolution. Finally, the influence of the stratospheric potential vorticity distribution on lateral mixing and transport into and out of the tropical pipe, the low latitude ascending branch of the Brewer-Dobson circulation, is investigated. The stratospheric potential vorticity distribution in the tropical stratosphere is found to have a clear pattern according to the phase of the quasi-biennial oscillation (QBO). The extent of the QBO influence is quantified, by analysing trajectories of Lagrangian particles using an online trajectory code recently implemented in the Met Office's Unified Model.

La consommation d'un régime hyper protéique (HP) améliore l'homéostasie glucidique, le gain de poids, l'adiposité, en réduisant le tissus adipeux blanc et la taille des adipocytes. Les adaptations métaboliques dues à l'augmentation de l'apport protéique sont au moins caractérisées, au niveau du foie, par la diminution de la lipogenèse et l'augmentation de la conversion des acides aminés (AA) en glycogène. Cependant, le rôle des acides aminés dans le contrôle de ces adaptations métaboliques et des voies de transduction responsables de la transmission du signal " acides aminés " n'ont pas encore été élucidés. L'objectif de notre étude a été de déterminer l'effet de l'augmentation de l'apport en acides aminés sur la traduction et la protéolyse, et d'identifier les voies de signalisation impliquées dans la détection des acides aminés ainsi que l'acide aminé ou le groupe d'acide aminés responsable de ces effets, en utilisant des approches in vivo et in vitro. Les extraits protéiques ont été analysés par western blots pour examiner l'état de phosphorylation des protéines impliquées dans les voies de signalisation qui participent à la détection des AAs et à la régulation de la traduction, à savoir les voies: " mammalian target of rapamycin " (mTOR), " adenosine monophosphate-activated protein kinase " (AMPK) et " general control non-depressible kinase 2 " (GCN2). Cette étude a montré que l'adaptation à un régime de HP est caractérisée par la stimulation de la traduction dans le foie, au moins au niveau de l'étape d'initiation. Cette activation requiert à la fois la présence de fortes concentrations en AA (au moins la leucine ou des AAs à chaîne branchée) et d'insuline, comme l'indique l'augmentation de la phosphorylation de mTOR, 4E-BP1 et S6 et la diminution de la phosphorylation de l'AMPK et GCN2. L'utilisation de l'AICAR (activateur de l'AMPK) et de la rapamycine (inhibiteur de mTOR) nous a permis de montrer qu'en présence de fortes concentrations en AA et d'insuline, mTOR n'est pas le seul régulateur de 4E-BP1 et de la S6K1 (cibles de mTOR) et que l'AMPK peut également jouer un rôle important dans la régulation de leur état de phosphorylation. En outre, l'augmentation de l'apport protéique provoque une inhibition de la dégradation des protéines dans le foie et une diminution de l'expression des gènes codant les principales protéines du système autophagie et de l'ubiquitine-protéasome. En conséquence, les protéines sont moins ubiquitinées, donc moins dégradées. Les AAs et l'insuline semblent être les principaux régulateurs de la voie de protéolyse ubiquitine-protéasome et les voies mTOR et AMPK seraient les médiateurs des effets acides aminés et de l'insuline. Ces résultats suggèrent que le contrôle des voies cataboliques et anaboliques du métabolisme des protéines sont régulées par les mêmes signaux et font intervenir les mêmes voies de signalisation.

To investigate how protein folding is affected by sequence connectivity five topological variants of the ribosomal protein S6 were constructed through circular permutation.  In these constructs, the chain connectivity (i.e. the order of secondary-structure elements) is changed without changing the native-state topology.  The effects of the permutations on the folding process were then characterised by φ-value analysis, which estimates the extent of contact formations in the transition-state ensemble.  The results show that the folding nuclei of the wild-type and permutant proteins comprises a common motif of one α-helix docking against two β-sheets, i.e. the minimal structure for folding.  However, this motif is recruited in different parts of the S6 structure depending on the permutation, either in the α1 or α2 half of the protein.  This minimal structure is not unique for S6 but can also be seen in other proteins.  As an effect of the dual nucleation possibilities, the transition-state changes describe a competition between two parallel pathways, which both include the central β-stand 1.  This strand constitutes thus a structural overlap between the two competing nuclei.  As similar overlap between competing nuclei is also seen in other proteins, I hypothesise that the coupling of several small nuclei into extended ‘super nuclei’ represents a general principle for propagating folding cooperativity across large structural distances.  Moreover, I demonstrate by NMR analysis that the existence of multiple folding nuclei renders the H/D-exchange kinetics independent of the folding pathway. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper IV: Manuscript

N-heterocyclic carbenes (NHCs) have become a very popular class of ligands, which has found uses in numerous catalytic applications. The use of such compounds in combination with phosphorus-based ligands within metal complexes has enabled the design of very active yet robust catalytic systems. The following chapters will describe the design of novel well-defined palladium- and ruthenium-based pre-catalysts featuring a NHC and a phosphorus-based ligand, referred at as mixed ligand systems. Such species were employed in catalysis where their properties appeared highly beneficial, uses at low catalysts loading and under harsh conditions were then envisioned. The preparation of a series of well-defined palladium mixed NHC/phosphine species is presented in chapter 2. Their catalytic activity in the aqueous Suzuki-Miyaura reaction of aryl chlorides and boronic acids, using low catalyst loadings, is described. The observation of catalytic activity of the latter systems in the hydration of nitriles prompted us to further investigate this reactivity. This reaction appeared to be operative in the absence of palladium species and could be performed under base-catalysed conditions, which was studied in detail and depicted in chapter 3. The combination of a NHC and a phosphite ligand in ruthenium olefin metathesis pre-catalysts has been underexplored. Preliminary results showed that such species could be readily prepared and presented an unusual geometry and a high catalytic activity. Variations in phosphite-containing ruthenium olefin metathesis pre-catalysts are presented. Chapter 4 describes the investigation of various Schrock carbene moieties in such architectures, as well as their implications in structure and catalysis. Chapter 5 depicts attempts to design olefin metathesis Z-selective pre-catalysts by inserting a chelating NHC moiety within phosphite-containing ruthenium species. This dissertation concludes on the potential of such mixed species in catalysis, and armed with the new knowledge provided by this work, proposes potential developments of such chemistry in the design of always more robust and active catalytic systems.