Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Trz1 chez levure est responsable du clivage endonucléolytique à l'extrémité 3 'au cours du processus de maturation des ARNt. Trz1 appartient à la famille des RNases de type b-lactamase, caractérisé par la présence d'un motif de séquence HxHxDH qui est impliqué dans la fixation des ions de zinc catalytique. La famille des RNaseZ est partagée en deux sous-familles de longueur de séquence différente: les formes courtes (300-400 acides aminés) et les formes longues (700-900 acides aminés). Les structures cristallines des enzymes RNaseZ de forme courte ont montré qu'ils sont actifs comme des homodimères. Une sous-unité englobe les ARNt substrat en utilisant un bras en saillie et l'autre fournit le site catalytique. Nous présentons ici la structure cristalline de Trz1, la première pour une RNase Z de forme longue. Trz1 est organisé en deux domaines reliés par un long peptide charnière. Chaque domaine est composé d'un repliement de type β-lactamase. Le domaine N-terminal a perdu ses résidus catalytiques au cours de l’évolution, mais il contient le bras long qui est important pour la liaison de l'ARNt; tandis que c’est l'inverse pour le domaine C-terminal. À partir des études protéomiques, on sait que Trz1 forme un complexe ternaire avec NUC1, une nucléase mitochondriale impliquée dans l'apoptose, et avec une mutarotase (codée par YMR099C). Nous avons purifié le complexe ternaire Trz1/NUC1/mutarotase caractérisé ses propriétés biochimiques. Trz1/NUC1/mutarotase forme in vitro un heterohexamère très stable en solution. A partir de nos données SAXS et MALLS nous proposons que l'homodimère NUC1 est au centre du complexe et que chaque sous-unité interagit avec une copie de Trz1 et une copie de mutarotase. / Yeast Trz1 is responsible for the endonucleolytic cleavage at the 3’-end during the maturation process of tRNAs. Trz1 belongs to the family of β-lactamase type RNases characterized by the presence of a HxHxDH sequence motif that is involved in the ligand formation of the catalytical required Zn-ions. The family consists of two subfamilies: the short forms with sequence lengths between and the long forms. A few crystal structures of short form RNase Z enzymes showed that they are active as homodimers. One subunit embraces the substrate tRNA using a protruding arm and the other provides the catalytic site. We here present the crystal structure of Trz1, the first of a long form RNase Z. Trz1 is organized in two domains connected by a large linker. Each domain is composed of a beta-lactamase type fold. The N-terminal domain has lost its catalytic residues, but contains the long arm that is important for tRNA binding; while it is the other way around of the C-terminal domain. From proteomics studies it is known that Trz1 forms a ternary complex with NUC1, a mitochondrial nuclease involved in apoptosis, and with a mutarotase (encoded by YMR099C). We purified the ternary Trz1/Nuc1/mutarotase complex and characterized its biochemical properties. Trz1/Nuc1/mutarotase forms in vitro a very stable heterohexamer in solution. From our SAXS and MALLS data we propose that the Nuc1 homodimer is at the centre of the complex and that each subunit interacts with one copy of Trz1 and mutarotase.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xi, 183 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Chlamydiae are important human bacterial pathogens with an intracellular life cycle that consists of two distinct bacterial forms, an infectious form (EB) that infects the eukaryotic host cell, and a non-infectious form (RB) that allows intracellular proliferation. To be successful, chlamydiae need to alternate between EB and RB to generate infectious EB’s which are competent to infect new host cells. Chemical genetics is an attractive approach to study bacterial pathogenesis; in principal this approach relies on an inhibitory compound that specifically inhibits a protein of interest. An obstacle in using this approach is target identification, however whole genome sequencing (WGS) of spontaneous mutants resistant to novel inhibitory compounds has significantly extended the utility of chemical genetic approaches by allowing the identification of their target proteins and/or biological pathways. In this thesis, a chemical genetics approach is used, I have found that heme and glucose metabolism of C. trachomatis is specifically important for the transition from the RB form to the infectious EB form. Heme and glucose metabolism are both coupled to energy metabolism, which suggests a common link between the RB-to-EB transitions. In connection with the above findings I have developed strategies that enable the isolation of isogenic C. trachomatis mutant strains. These strategies are based on WGS of spontaneous mutant populations and subsequent genotyping of clonal strains isolated from these mutant populations. Experiments with the mutant strains suggest that the uptake of glucose-6-phosphate (G-6-P) regulates the RB-to-EB transition, representing one of the first examples where genetics has been used to study C. trachomatis pathogenesis. Additional experiments with the mutant strains indicate that G-6-P promotes bacterial growth during metabolic stress. In concert with other findings presented in this thesis, I have fine-tuned methods that could be employed to reveal how novel inhibitory chemical compounds affect chlamydiae. In a broader context, I suggest that C. trachomatis could be used as a model organism to understand how new inhibitory drugs affect other bacterial pathogens. In addition, I observed that C. pneumoniae infections resulted in generalized bone loss in mice and that these mice display a cytokine profile similar to infected bone cells in vitro. Thus, this study indicates that C. pneumoniae potentially can infect bone cells in vivo, resulting in bone loss, alternatively, the inflammatory responses seen in vivo could be the causative factor of the bone loss observed.

La contribution des cellules souches au développement tumoral est une percée conceptuelle récente dans notre compréhension des mécanismes moléculaires et cellulaires impliqués dans la carcinogenèse. En ce sens, il est reconnu depuis quelques années qu'une sous-population de cellules souches mésenchymateuses (MSC) mobilisables en réponse à des facteurs de croissance tumoraux pourrait contribuer au développement tumoral. Les recherches rapportées dans ce mémoire nous ont permis d'étudier certains partenaires clé dans la régulation de la migration et de la survie cellulaire des MSC. L'observation préalable d'une modulation conjointe de l'expression d'une métalloprotéase matricielle de type membranaire (MT1-MMP) et du transporteur microsomal de glucose-6-phosphate (G6PT) nous a permis d'évaluer la contribution respective de ces joueurs dans la signalisation affectant la chimiotaxie des cellules souches ainsi que des cellules tumorales cérébrales. De plus, nous avons évalué l'impact de certains « mannosides » synthétisés en vue de cibler spécifiquement les fonctions de surfaces de MT1-MMP et qui pourraient être à l'origine de nouvelles approches thérapeutiques anticancéreuses affectant le recrutement des cellules souches au foyer tumoral. Finalement, l'importance de l'axe de signalisation MT1-MMP/G6PT dans la mobilisation du calcium intracellulaire en réponse à la sphingosine-1-phosphate, un lipide bioactif synthétisé par des niveaux d'expression élevés de sphingosine-kinase retrouvé au niveau tumoral, permet également de concevoir le ciblage effectif de l'un ou l'autre de ces partenaires dans la progression tumorale. L'ensemble de nos résultats permettra de mieux comprendre les phénomènes régulant la survie et le recrutement des MSC aux sites de foyers tumoraux, en plus de fournir de précieux renseignements sur un nouvel axe original de signalisation liant les fonctions de MT1-MMP à celles, inattendues, de G6PT. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Cellules souches, Cancer, MT1-MMP, G6PT, Sphingosine-1-phosphate.

L'une des caractéristiques des cellules souches mésenchymateuses (MSC) est leur capacité à survivre en conditions hypoxiques et à contribuer au développement tumoral. Le transporteur du glucose-6-phosphate (G6PT) exerce, par ailleurs, un contrôle métabolique contribuant à la mobilisation et à la survie des MSC. Les effets d'un environnement faible en oxygène (1,2% O₂) sur l'expression de G6PT sont inconnus et pourraient expliquer en partie la contribution et le recrutement des MSC au centre d'un foyer tumoral hypoxique. Nous avons découvert que l'expression génique de G6PT ainsi que de la sous-unité catalytique G6PC-3 (β) du système glucose-6-phosphatase sont significativement exprimés, tandis que l'expression des isoformes G6PC-I (α) et G6PC-2 (IGRP) était indétectable. L'environnement hypoxique ainsi que l'hypoxie artificielle induite suite à un traitement au chlorure de cobalt (CoCl₂), induisent à la fois l'expression de G6PT, du facteur de croissance de l'endothélium vasculaire (VEGF), et du facteur inductible hypoxique-l α (HIF-1 α), mais pas de G6PC-3 (β). L'analyse de la séquence promotrice du gène de G6PT révèle la présence de sites potentiels de liaison pour HIF-1 α. Nous démontrons également, à l'aide d'ARN interférant (siRNA) dirigé contre le gène HIF-1 α, que l'induction de G6PT ainsi que de VEGF dans les MSC en conditions de cultures hypoxiques est antagonisée. Finalement, nous avons généré un modèle de MSC exprimant constitutivement HIF-1 α, et avons observé une augmentation significative des niveaux endogènes de l'expression de G6PT ainsi que de la mobilité cellulaire. De plus, nous avons démontré que l'inhibition de la fonction de G6PT par un dérivé de la mumbaïstatine (AD4-015) permettrait ainsi de cibler préférentiellement la mort des cellules possédant un niveau de G6PT élevé. Nos résultats démontrent un axe de régulation métabolique de G6PT dépendant de HIF-1 α. Cet axe contribuerait à la flexibilité métabolique caractérisant les MSC et leur permettrait de survivre dans des conditions telles l'ischémie ou le développement tumoral, caractérisées par l'hypoxie et la privation en nutriments. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Cellules souches mésenchymateuses, Facteur inductible hypoxique-1 α, Transporteur du glucose-6-phosphate, Migration des cellules souches, Tumeur cérébrale.

Abstract Plant cells contain plastids, organelles dedicated to performing specific biochemical processes including photosynthesis, starch and oil biosynthesis. Fatty acid biosynthesis in oil seeds occurs in one type of plastid termed the leucoplast. Anabolic metabolism in leucoplasts includes the production of fatty acids and amino acids that depend on the availability of reductants such as NADPH. NADPH can be generated in plastid by glucose 6-phosphate dehydrogenase (G6PDH) which is the chief control enzyme and first step in the Oxidative Pentose Phosphate Pathway (OPPP). G6PDH catalyses the reaction of NADP+ and glucose 6-phosphate to NADPH and 6-phosphogluconate. At least two compartment-specific isoforms of G6PDH exist in plants, a cytosolic and a plastidic form. In this study, castor oil seed (COS) (Ricinus communis L.) was used as a model enzyme system for the ongoing study of oil biosynthesis in plants. This is the first ever report of the full-length clone of the plastidic isoform of G6PDH being isolated from a castor cDNA library using polyclonal potato plastidic G6PDH antiserum. The full-length cDNA was sequenced and compared to other G6PDH genes from higher plants, the castor sequence reveals conserved regions and conserved cysteine residues similar to other higher plant G6PDH. Over expression of the recombinant cleaved fusion protein in an E. coli expression system from the isolation of the cDNA clone shows it is enzymatically active, stable and unlike other plastid G6PDH’s dithiothreitol insensitive. In fact this G6PDH shows increased activation in the presence of dithiothreitol. Initial kinetic characteristics shows that it behaves in a similar fashion enzymatically when compared to other higher plant chloroplast G6PDH. The gene sequence and initial kinetic findings for castor G6PDH concur with other higher plant, non-photosynthetic, plastidic isoforms. / Thesis (Master, Biology) -- Queen's University, 2007-09-19 13:41:54.584

Many of the physiological responses concomitant with exercise are understood. Similarly, many of the changes characterizing the aging process have been established. However, the combination of the two (ie. effects of aging on exercise or vice versa) presents a myriad of questions, of which many remain unanswered.The objective of this study was to establish the differences between previously untrained young and adult male Fischer 344 rats following an exhaustive swim for the following parameters: 1) muscle glycogen, an essential fuel substrate; 2) Glucose-6-phosphate dehydrogenase (G6PDH), a marker of inflammation and tissue damage; 3) citrate synthase (CS), an integral enzyme of the Kreb's cycle and a respiratory chain marker; 4) muscle protein; and 5) percent muscle dry weight.The rats were divided into two groups by age. Young (3 mo., n=16) and adult (12 mo., n=17) rats were randomly divided into sedentary (young sed (YSD) n=7 and adult sed (ASD) n=9) or exercised groups (young swimmers (YSW) n=8 and adult swimmers (ASW) n=8). Rats in the swimming groups were given a brief exposure to the water one week prior to their exhaustive swim to minimize the stress and confusion during the actual exercise bout. On the study days one randomly selected swimmer from each age group was swum to exhaustion and sacrificed via pneumothorax. One animal from each of the respective sedentary age groups was also randomly selected and sacrificed as above. The plantaris, rectus femoris, red vastus, soleus, triceps, and liver were surgically excised from each animal and frozen in liquid nitrogen for later analysis.While the younger animals had lower glycogen stores initially, following the exhaustive swim their reduction in muscle glycogen was approximately 150% that of the adult animals for any given muscle. Muscle glycogen levels in ASD and YSD rats were significantly higher than those of the YSW animals for all muscles with the exception of the YSD's soleus. However, the percent decrease in liver glycogen following the swim for the two age groups was almost identical (a reduction of 55.05% and 58.59% for the adult and young age groups, respectively).Although the adult animals were significantly heavier than the younger rats, this did not appear to cause a significant difference in their swim time to exhaustion. No significant differences were observed between the groups for muscle protein or G6PDH. Levels of CS were significantly higher in the YSD plantaris when compared to the ASW. Similarly, the ASD rectus femoris CS levels were significantly greater than those of the ASW. Although significant differences between groups in percent muscle dry weight existed for the plantaris, rectus femoris, and triceps such differences seemed to have little bearing on the two age group's swim to exhaustion times.On the basis of this study it was concluded that although starting with greater glycogen stores prior to exercise, adult animals use less of this substrate prior to exhaustion than do younger animals. While the mechanism for such a phenomenon was not discovered it is believed to be enzymatic in nature. Furthermore, the adult animals do not appear to exhibit significantly more tissue damage following an exhaustive swim than that seen in younger animals. / School of Physical Education

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains x, 125 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 123 p. : ill. (some col.). Includes abstract. Includes bibliographical references.