Type 2 diabetes affects 10% of the United States population, and patients with diabetes have an increased risk for diseases such as atherosclerosis and hypertension. Since all these diseases are associated with insulin resistance, finding therapies to improve insulin sensitivity could be beneficial for many patients. Studies have shown CYP450 metabolites called epoxyeicosatrienoic acids (EETs) positively regulate insulin action. The purpose of this study is to provide further evidence to support endogenous EETs regulation of insulin action in vivo and to determine how EETs regulate insulin action. To study this in mice, endogenous production of EETs was disrupted by deleting a major EET-producing epoxygenase, Cyp2c44.Glucose tolerance tests (GTTs) were performed with global and liver-specific deletions [Cyp2c44(-/-) and hepCyp2c44(-/-)] to assess glucose homeostasis. Cyp2c44(-/-) mice had impaired glucose tolerance, while hepCyp2c44(-/- ) mice had no alteration. This suggests that EETs increase insulin action but shows disruption of liver-produced EETs, where Cyp2c44 is highly expressed, is not sufficient to alter glucose homeostasis. Therefore, production of EETs within other tissues (e.g., skeletal muscle, vascular endothelium, or adipocytes) must be contributing to decreased glucose tolerance in Cyp2c44(-/-) mice. Insulin signaling in skeletal muscle has previously been shown to be impaired in Cyp2c44(-/-) mice. Therefore, we investigated the effect of endogenous EETs on a critical protein in the insulin signaling cascade, AKT, and a downstream effector, FoxO1. In the present studies, insulin-stimulated AKT and FoxO1 phosphorylation were unaltered in Cyp2c44(-/-) mice. Therefore the effect of EETs on insulin signaling in skeletal muscle could either occur at a different downstream AKT effector or within other insulin-stimulated pathways, like the MAP kinase pathway. Complementary studies will help determine the roles of gender, age, dietary modifications and other experimental conditions on these differences in insulin sensitivity.

The norepinephrine transporter (NET) clears norepinephrine (NE) from the synapse after vesicular release. NET is a target of the psychostimulant amphetamine (AMPH). We have recently shown that AMPH alters trafficking of the transporter, causing a net decrease in surface NET in the monoaminergic CAD cell line. In this study we demonstrate, by confocal imaging of immunostained superior cervical ganglion (SCG) neurons, that AMPH causes an increase in NET levels inside terminal boutons. Further, the intracellular compartment in which NET accumulates upon AMPH exposure is not known; such information would inform investigation of the mechanism by which the drug affects NETs cellular distribution. We show that after AMPH treatment, NET co-fractionates with both Rab4 and Rab11, and that AMPH increases co-localization of NET with these endosomal markers as indicated by the intensity correlation quotient (ICQ). Finally, we show that the functions of both GTPases are involved in AMPH-regulated NET trafficking by transfection of dominant negative (DN) constructs followed by cell-surface biotinylation. Our results support the conclusion that AMPH-regulated NET trafficking occurs through endosomes.

Long bone lengthening occurs at the growth plate (GP) by well-regulated chondrocyte proliferation, hypertrophy and terminal matrix deposition. GP chondrocyte (GPC) hypertrophy has been implicated to be the main determinant of bone growth rate; however the mechanism is poorly understood. The work of this thesis examined some of the cellular process that drives the chondrocyte swelling or hypertrophy particularly in a mammalian post natal GPs using living in situ GPC and fixed GP tissues. Confocal scanning microscopy (CLSM) was used to determine living in situ GPC volume and dimension changes in proliferative zone (PZ) through to hypertrophic zone (HZ) chondrocytes of different GPs of various bones. While PZ cells showed similar volumes and dimensions, HZ cells varied in different GPs, even within the same long bone but at opposite ends. However, the hypertrophic cell volume measured at a single post natal age (day 7) was independent of the corresponding bone length. This could reflect a complex interplay between local and systemic factors in different GPs, which occurs throughout the active phase of bone growth. Maintaining GPC morphology was critical in studying GPC hypertrophy using fixed tissues. This work highlighted a problem caused by conventional fixative solutions, which caused up to 44% hypertrophic GPC shrinkage following GP fixation. This artifact appeared to be associated with the hyperosmotic nature of the fixatives used and could be abolished by adjusting the fixative osmolarity close to physiological level (280 mOsm), or could be significantly reduced by bisecting bone tissues prior to tissue fixation. This thesis proposed roles for plasma membrane transporter(s) in mediating GPC hypertrophy. This hypothesis was tested by examining roles of sodium-hydrogen exchanger (NHE) and anion exchanger (AE) in GPC hypertrophy using an ex vivo bone growth inhibition model. Inhibition of bone growth by inhibitors of NHE (EIPA) and AE (DIDS) respectively was shown to be dose-dependent. The histology of bones demonstrated that the late HZ width was significantly reduced in GPs treated with EIPA or DIDS. Although in situ GPC volumes in the PZ and HZ were not notably different in DIDS-treated GP, the cell volumes in both zones were significantly reduced by EIPA treatment. Fluorescence immunohistochemistry revealed distinctive cellular localisations of NHE1 and AE2 in the PZ and early HZ. These results suggest a possible role of AE in mediating GPC volume increase in PZ chondrocytes and those in the early stages of cell hypertrophy, whereas NHE could possibly maintain intracellular pH of GPC throughout all GP zones. This thesis has characterized various changes in volume and dimensions of living in situ GPC from PZ through to HZ of different GPs of postnatal rats. This work emphasized the importance of fixative osmolarity in order to accurately preserve the normal volume/morphology of cells within tissues. Most importantly, this thesis confirmed a potential role of the plasma membrane transporters, AE and NHE in GPC hypertrophy of growing bones.

Molecular dynamics (MD) simulation, which employs an empirical potential energy function to describe the interactions between the atoms in a system, is used to investigate atomistic motions of proteins. However, the timescale of many biological processes exceeds the reach of standard MD due to computational limitations. To circumvent these limitations, steered molecular dynamics (SMD), which applies external forces to the simulated system, can be used.Dynamical properties of the gonococcal type IV pilus (GC-T4P) from the bacteria Neisseria gonorrhoeae are first considered. T4 pili are long, filamentous proteins constructed from a subunit (pilin) found to emanate from the surface of pathogenic bacteria. They can withstand large forces (~100 pN), and are implicated in infection. SMD simulations are performed to study the response of the filament to an applied force. Our simulations reveal that stability of the pilus likely results from hydrophobic contacts between pilin domains buried within the filament core. Along the filament surface, gaps are formed between pilin globular head domains. These gaps reveal an amino acid sequence that was also observed to become exposed in the experimentally stretched filament. We propose two other regions initially hidden in the native filament that might become exposed upon stretching.The multidrug resistance transporter EmrD, found in the inner membrane of Escherichia coli is also the target of our studies. EmrD removes harmful drugs from the bacterial cell. We use MD to explore equilibrium dynamics of the protein, and MD/SMD to study drug interactions and transport along its central cavity. Motions supporting a previously proposed lateral diffusion pathway for substrate from the cytoplasmic membrane leaflet into the central cavity were observed. Additionally, interactions of a few specific residues with CCCP have been identified.Finally, we describe network analysis as an approach for analyzing conformational sampling by MD simulations. We demonstrate for several model systems that networks can be used to visualize both the dominant conformational substates of a trajectory and the connectivity between them. Specifically, we compare the results of various clustering algorithms to the network layouts and show how information from both methods can be combined.

This dissertation is focused on the application of Markov State Models on protein folding and designing of small drug-like molecules, as well as application of computational tools on the study of biological processes. The central focus of protein folding is to understand how proteins obtain their unique three-dimensional structure from their aminoacid sequences. The function of protein critically depends on its three- dimensional structure; hence, any internal (such as mutations) or external (such as high temperature) perturbation that obstructs three-dimensional structure of a protein will also interfere with its function. Many diseases are associated with inability of protein to form its unique structure. For example, sickle cell anemia is caused by a single mutation that changes glutamic acid to valine. Molecular dynamics (MD) simulations could be utilized to study protein folding and effects of perturbations on protein energy landscape; however, due to its inherent atomic resolution, MD simulations usually provide enormous amount of data even for small proteins. A thorough analysis and extraction of desired information from MD provided data could be extremely challenging and is well beyond human comprehension. Markov state models (MSMs) are proved to be apt for the analysis of large scale random processes and equilibrium conditions, hence it could be applied for protein folding studies. MSMs can be used to obtain long timescale information from short timescale simulations. In other words, the combination of many short simulations and MSMs is a powerful technique to study the folding mechanism of many proteins, even the ones with folding times over millisecond. This dissertation is centered on the use of MSMs and MD simulation in understanding protein folding and biological processes and is constructed as the following. The first chapter provides a brief introduction into MD simulation and the different techniques that could be used to facilitate simulations. Protein folding and its challenges are also discussed in chapter one. Finally, chapter one ends with describing MSMs and technical aspects of building them for protein folding studies. Chapter two is focused on using MD simulations and MSMs to design small protein like molecules to prevent biofilm propagation by disrupting its lifecycle. The biofilm lifecycle and strategy for its interruption is described first. Then, the designed molecules and their conformational sampling by MD simulations are explained. Next, the application of MSMs in obtaining and comparing equilibrium population of all designs are discussed. At the end of chapter two, the molecular descriptions of best designs are explained. Chapter three is focused on the effects of mutations on the energy landscape of a sixteen residue protein from c-terminal hairpin of protein G, GB1. Three mutations, tz4, tz5, and tz6 are discussed, and their folding rates and folding mechanisms are compared with wild-type GB1 using MSMs built from a significantly large MD simulation data set (aggregating over 9 millisecond). Finally, chapter four is focused on the application of MD simulations on understanding the selectivity of Na,K-ATPase, a biologically critical protein that transports sodium ions outside and potassium ions inside against their concentration gradient in almost all eukaryotic cells. Multiple MD approaches, including metadynamics and free energy perturbation methods are used to describe the origins of selectivity for Na,K-ATPase. / Chemistry

Yes / In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport. / This work was supported by Biotechnology and Biological Sciences Research Council grant BBS/B/14418 (Membrane Protein Structure Initiative).

No description available.

Membrane proteins play an integral role in the control of ion transport across the cell membrane in biological systems. However, due to experimental constraints, structural and functional data available for these proteins is limited, especially considering their importance. In this study, two membrane proteins which transport nitrate and nitrate into the model filamentous ascomycete Aspergillus nidulans were investigated. Work on the twelve trans-membrane domain nitrate transport protein NrtA is well established. As a member of the major facilitator super family (MFS) the role of signature sequences characteristic of this family have previously been studied. Here, a series of point mutations were made to facilitate an understanding of key residues in the nitrate binding domain, the first nitrate signature motif and residues of the unique fungal central-loop domain. Using an expanded alignment package, the proposed secondary structure of NrtA was enhanced and used as a starting point for mutagenesis. Alanine scanning mutagenesis showed that glycine residues in the conserved nitrate nitrite porter (NNP) motif were critical for NrtA function. Two asparagines in the NNP were investigated; N160 and N168. N168 was found to be critical for NrtA function as all mutants were devoid of growth on nitrate solid agar medium though they expressed in the membrane to varying degrees. The nitrate binding site has been studied previously, revealing the interaction of conserved arginine residues with the anion as it traverses the bilayer. Though it was thought that mutations of residue T83 to a small, charge neutral, amino acid would substitute for no alteration to enzyme kinetics in mutant T83S was found when using ¹³NO₃⁻. Another major part of this thesis examined NitA which is part of a distinct nitrite transport family to NrtA (the Formate Nitrite Transporters, FNT). A mutagenesis approach targeted NitA residues conserved amongst homologous proteins. Residues in position D88 in an alignment of homologues were conserved in terms of charge. Mutagenesis of D88 revealed that maintaining charge at this position was essential for NitA function, likely due to a role in salt-bridge formation during conformational changes. Mutations to asparagine, glutamine, serine and valine showed reduced growth on agar though the protein was expressed to approximately wild-type levels. Nitrite uptake assays using a ¹³NO₂⁻ tracer were performed on D88N, D88E and D88Q and all showed wild-type Km and Vmax. Finally, the role of conserved asparagine residues found throughout NitA was investigated by mutagenesis. Expression studies revealed that mutants created in N122 and N246, changed to aspartic acid, lysine, glutamine and serine were generally not present in the membrane and thus did not grow on nitrite agar. However, mutations in N173 (in Tm 4) and N214 (in Tm 5), which are conserved in > 95 % of NitA homologues, showed varying degrees of growth and expression. Both of these residues are located in FNT signature motifs, so it is likely that they are involved with conformational changes or protein dynamics.

A cana-de-açúcar é uma cultura agrícola de grande importância econômica para o Brasil, e a expansão de seu cultivo para solos marginais requer uma maior utilização de fertilizantes à base de nitrogênio (N). Na maioria dos países produtores, a adubação nitrogenada se baseia em altas doses de aplicação, enquanto, no Brasil, o seu uso é relativamente baixo devido, em parte, ao processo de fixação biológica de nitrogênio (FBN) pela ação de bactérias diazotróficas. Além da FBN, as plantas adquirem fontes de N, como amônio e nitrato, por meio de transportadores de membranas localizados nas raízes. Há evidências que a associação com microrganismos pode favorecer as plantas por meio da regulação dos genes de transportadores de N. Desta forma, este trabalho teve como objetivo caracterizar o transporte de amônio e nitrato, avaliando a expressão gênica dos principais transportadores de N em cana-de-açúcar cultivada in vitro sob o efeito da associação com bactérias diazotróficas. Também foi descrita a comunidade bacteriana de plântulas in vitro, bem como o efeito da fertilização com N e da inoculação com bactérias diazotróficas em plantas maduras. Plântulas de \'SP70- 1143\' e \'Chunee\', que contrastam para FBN, foram empregadas em ensaios in vitro sob diversas concentrações e fontes de N em associação ou não com uma estirpe de Gluconacetobacter diazotrophicus ou um mistura de bactérias diazotróficas (G. diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense e Burkholderia tropica). A caracterização do transporte de N por meio de ensaios de absorção de nitrato e amônio marcados (15N) revelou que a interação entre cana-de-açúcar x G. diazotrophicus induziu a expressão do gene do transportador de nitrato ScNRT2.1, o que levou a uma tendência no aumento no influxo de nitrato, assim como dos genes de transportadores de amônio ScAMT1.1 e ScAMT1.3, resultando em maiores influxos de amônio apenas para a cultivar \'SP70- 1143\'. Já a associação da cana-de-açúcar com a mistura de bactérias diazotróficas revelou que somente houve indução transcricional de ScAMT1.1, o que resultou na maior absorção de amônio em \'SP70-1143\'. Por sua vez, quando analisada a interação in vitro por 30 dias, a presença da bactéria, apesar de transiente, possivelmente favoreceu a expressão dos genes de transportadores de nitrato ScNRT1.1 e ScNRT2.1, e do transportador de amônio ScAMT1.1, resultando no maior acúmulo de 15N-nitrato de amônio nas plantas de \'SP70-1143\'. Foi detectada uma comunidade bacteriana associada a plântulas micropropagadas, a qual é distinta entre os genótipos \'SP70-1143\' e \'Chunee\' e se altera com a inoculação com G. diazotrophicus. Para as plantas cultivadas em campo, a comunidade bacteriana existente foi alterada pela fertilização de N, mas não pela inoculação com diazotróficas. Portanto, a inoculação com bactérias diazotróficas parece induzir a expressão dos principais genes transportadores de amônio e nitrato em plântulas do genótipo \'SP70-1143\' resultando na maior absorção de fontes inorgânicas de N. / Sugarcane has a large economic importance to Brazil, and it\'s the expansion of cultivation to marginal soils requires a larger application of nitrogen fertilizers (N) to maintain yield. In most producing countries, N fertilization is based on high application rates, whereas in Brazil N fertilization is relatively low, possibly due in part, to the process of biological nitrogen fixation (BNF). In addition, plants acquire inorganic N sources from the soil by membrane transporters that may be regulated by association with microorganisms. This study aimed to characterize the ammonium and nitrate transport evaluating the gene expression profile of the major transporters grown in vitro in association with diazotrophic bacteria. It was also described the bacterial community in micropropagated plants, as well as the effect of N fertilization or inoculation with nitrogen fixing bacteria in mature plants. \'SP70-1143\' and \'Chunee\' which contrasted to BNF, were used in in vitro experiments in several concentrations and N source, in association or not with a strain of Gluconacetobacter diazotrophicus or a bacteria mixture (G. diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense and Burkholderia tropica). The characterization of the N transport by uptake assays with 15N-labeled ammonium and nitrate, revealed that the interaction between sugarcane x G. diazotrophicus induced, the nitrate transporter gene ScNRT2.1 expression, which lead to trend to increase nitrate influx, as well as the ammonium transporter genes ScAMT1.1 and ScAMT1.3, resulting in higher ammonium influx in \'SP70-1143\'. Sugarcane associated with the bacterial mixture revealed a transcriptional induction of ScAMT1.1 resulting in larger ammonium acquisition in \'SP70-1143\'. Further, the presence of bacteria in vitro for 30 days, although transient, possibly favored the expression of nitrate transporters ScNRT1.1 and ScNRT2.1, and the ammonium transporter ScAMT1.1, resulting in accumulation of 15N-ammonium nitrate in \'SP70-1143\'. A bacterial community associated with in vitro plants of \'SP70-1143\' and \'Chunee\' was detected with different composition between genotypes, and which changed with artificial inoculation. For plants grown in the field, the bacterial community was affected by N fertilization but not by inoculation with diazotrophic. These results indicate that the inoculation with diazotrophic bacteria appears to induce the expression of the major ammonium and nitrate transporters genes in \'SP70-1143\' plants resulting in higher uptake of inorganic N sources.

Dans le but de maintenir leur métabolisme et leur prolifération exacerbée, les tumeurs sont dépendantes d’un apport accru en acides aminés. Afin d'optimiser cet apport, les tumeurs surexpriment certains transporteurs clés tels que l'hétérodimère multifonctionnel CD98/LAT1. CD98 (SLC3A2) agit comme co-récepteur des intégrines β et amplifie leur signalisation régulant ainsi la migration et l'adhésion cellulaire. La protéine LAT1 (SLC7A5) est quant à elle responsable du transport des acides aminés (AA) essentiels. Des études antérieures ont suggéré que la fonction CD98/intégrines du complexe est essentielle à la croissance tumorale, alors que LAT1 aurait un rôle mineur dans ce contexte. Cependant, les besoins nutritifs accrus des cellules tumorales nous ont conduit à émettre l’hypothèse contraire selon laquelle l’avantage prolifératif donné par ce complexe serait en réalité supporté par l’activité du transporteur LAT1 et non pas par l’interaction CD98/intégrine. Dans ce contexte, j’ai montré que l’invalidation génétique ou pharmacologique de LAT1 dans différentes lignées tumorales entraine une suppression totale du transport de la leucine, sodium-indépendant. Ceci entrainant une perte d’homéostasie des AA avec l’activation de la voie de stress GCN2, l’inhibition de mTORC1 et la suppression de la croissance tumorale. De plus, l’invalidation génétique de CD98 ne s’est traduite par aucun phénotype visible. Cependant, la suppression de l'activité résiduelle de LAT1 de ces cellules est suffisante pour abolir leur potentiel tumoral. Ainsi, mes résultats démontrent le rôle clé de LAT1 dans la croissance tumorale en faisant ainsi une cible thérapeutique prometteuse. / Tumours rely on external amino acids (AA) uptake to maintain their exacerbated metabolism and proliferation. To optimize AA uptake, tumors overexpress key carriers such as the multifunctional CD98/LAT 1 heterodimer. CD98 (SLC3A2) acts as a co-receptor of β integrins and enhances signaling that promotes cellular migration and invasion. LAT1 (SLC7A5) is responsible for the transport of essential AA. Previous studies have suggested that the CD98/integrin axis of the complex is essential for tumour growth, while LAT1 activity is dispensible. However, the increased nutritional requirements of tumor cells led us to hypothesize that the proliferative advantage given by this complex is in fact supported by the AA transporter activity of LAT1 and not by the CD98/integrin activity. In this context, I have shown that genetic or pharmacological invalidation of LAT1 in various tumor cell lines leads to a complete removal of the sodium-independent leucine transport. This leads to a loss of AA homeostasis with activation of the GCN2 stress pathway, inhibition of mTORC1 and supression of tumour growth. In addition, genetic invalidation of CD98 did not result in any detectable phenotype. However, inhibition of the residual activity of LAT1 in CD98 knockout cells is sufficient to abolish their tumorigenicity. Thus, my results clearly demonstrate the fundamental role of LAT1 in tumour growth and advocate the pharmacology development of LAT1 transporter inhibitors as very promising anticancer agents.