Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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The purpose of this qualitative study was to explore the experiences of conservative orthodox Christian students attending public, secular, accredited counseling and counseling psychology graduate programs. Conservative orthodox Christian students who were attending public, secular, accredited counseling and counseling psychology graduate programs were recruited by email. A prescreening interview was conducted with each respondent. Seven respondents participated in three rounds of individual interviews. The overall research question was: What are the experiences of conservative orthodox Christian students attending public, secular, accredited counseling and counseling psychology graduate programs? Follow-up questions explored the participants' perceptions and experiences in depth. Data were analyzed through within case and cross case displays using a phenomenological approach. Emergent categories, themes, and descriptors were gathered from each round of interviews. Data were organized into three major categories: reflections on secular programs, experiences attending secular programs and Christian identity, and further organized into underlying themes and descriptors. Implications related to conservative orthodox Christian students attending public, secular, accredited counseling and counseling psychology graduate programs were discussed. Finally, suggestions for future research were provided.

Abstract The peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric protein of β-oxidation possessing 2-enoyl-CoA hydratase-1, Δ3-Δ 2-enoyl-CoA isomerase, and (3S)-hydroxyacyl-CoA dehydrogenase activities. The amino-terminal part of perMFE-1 shows sequence similarity to mitochondrial 2-enoyl-CoA hydratases (ECH-1) and Δ3-Δ 2-enoyl-CoA isomerases, and belongs to the hydratase/isomerase superfamily. Family members with known structures are either homotrimers or homohexamers. The purpose of this work was to elucidate the structure-function relationship of the rat perMFE-1 with special reference to the hydratase/isomerase fold. The structural adaptations required for binding of a long chain fatty acyl-CoA were studied with rat ECH-1 via co-crystallization with octanoyl-CoA. The crystal structure revealed that the long chain fatty acyl-CoA is bound in an extended conformation. This is possible because, a flexible loop moves aside and opens a tunnel, which traverses the subunit from the solvent space to the intertrimer space. Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by ECH-1. In the present work the enzymological properties of Glu144Ala and Glu164Ala variants of ECH-1 were studied. The catalytic activity of hydration was reduced about 2000-fold. It was also demonstrated that rat ECH-1 is capable of catalyzing isomerization. The replacement of Glu164 with alanine reduced the isomerase activity 1000-fold, confirming the role of Glu164 in both the hydratase and isomerase reactions. The structural factors favoring the hydratase over the isomerase reaction were addressed studying the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. These mutants had similar enzymatic properties to wild type, thus the catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on interaction with the Gln162 side chain. The perMFE-1 was divided into five functional domains based on amino acid sequence comparisons with the homologous proteins with known structures. Deletion variants of perMFE-1 showed that the folding of an enzymatically active amino-terminal hydratase/isomerase domain requires stabilizing interactions from the two carboxy-terminal domains of perMFE-1. The last carboxy-terminal domain is also required for the folding of the dehydrogenase part of perMFE-1. The dehydrogenase part of perMFE-1 was crystallized.

Trypanosoma brucei, parasite protozoaire flagellé appartenant à l’ordre des kinétoplastidés, est responsable de la maladie du sommeil, ou trypanosomiase humaine africaine (THA). Son cycle de vie fait intervenir un insecte vecteur hématophage (la mouche tsé-tsé ou glossine) qui lors d’un repas sanguin sur un individu infecté ingère des parasites. Après plusieurs étapes de différentiation, les parasites sont injectés à un hôte lors d’un autre repas sanguin. Nous avons étudié le métabolisme intermédiaire et énergétique de la forme procyclique de T. brucei, forme présente dans l’appareil digestif de l’insecte vecteur. Chez ce parasite, la dégradation du glucose aboutit à la production d’acétate dans l’unique mitochondrie, et de succinate dans la mitochondrie et les glycosomes, organelles spécifiques des trypanosomatidés dans lesquels la glycolyse est compartimentalisée. T. brucei utilise une "navette acétate" permettant de transférer l’acétyl-CoA produit dans la mitochondrie vers le cytosol pour initier la biosynthèse de novo des acides gras et la production d’acétate est essentielle à la croissance du parasite. La navette acétate fait intervenir dans la mitochondrie l’acétate:succinate CoA-transférase (ASCT), qui converti l'acétyl-CoA produit à partir du glucose en acétate. Nous avons identifié et caractérisé une autre enzyme mitochondriale contribuant aussi à la production d’acétate à partir du glucose : l’acétyl-CoA thioesterase (ACH). Le double mutant n’exprimant ni l’ACH ni l’ASCT ne produit plus d’acétate et n’est plus viable, confirmant le rôle essentiel de la production d’acétate. Par ailleurs, nous avons montré que l’ASCT, grâce au cycle formé avec la succinyl-CoA synthétase (SCoAS), contribue à la production d’ATP par phosphorylation au niveau du substrat dans la mitochondrie, mais l’ACH n’est pas impliqué dans la production d’ATP. La thréonine est l’acide aminé le plus rapidement consommé par le parasite et sa dégradation aboutit à la production d’acétate et de glycine. En utilisant des outils de génétique inverse et des analyses métaboliques par RMN du proton et HPTLC, nous avons caractérisé la première étape enzymatique de cette voie, catalysée par la thréonine déshydrogénase (TDH), et nous avons montré que la thréonine est la principale source de carbone pour la production d’acétate, pour la biosynthèse de novo des acides gras et des stérols. L’acétyl-CoA est produit dans la mitochondrie à partir du pyruvate provenant de la dégradation du glucose par le complexe pyruvate déshydrogénase (PDH) et à partir de la thréonine dont la dégradation est initiée par la TDH. L’acétyl-CoA provenant de la dégradation du glucose ou de la thréonine est converti en acétate par les mêmes enzymes, l’ACH et l’ASCT. Nous avons montré que la voie de dégradation de la thréonine est sous régulation métabolique. L’activité et l’expression de la TDH ainsi que la production d’acétate à partir de la thréonine sont diminuées dans le mutant knock out de la phosphoenolpyruvate carboxykinase (PEPCK) dans lequel le flux glycolytique est redirigé vers la production d’acétate. De plus, contrairement au glucose, la dégradation de la thréonine ne participe pas à la production d’ATP dans la mitochondrie du parasite. Nos résultats nous amène à l’hypothèse d’un channeling mitochondrial des voies de dégradation du pyruvate et de la thréonine pour la production d’acétate. Les trypanosomes ont développé une voie de biosynthèse de novo des acides gras faisant appel aux élongases du réticulum endoplasmique et un précurseur inhabituel, le butyryl-CoA dont la voie de biosynthèse n’est à l’heure actuelle pas connue chez les trypanosomatidés. Nous avons reconstitué une voie de biosynthèse hypothétique à partir de l’acétyl-CoA dans la mitochondrie. La dernière enzyme de cette voie, l’isovaléryl-CoA déshydrogénase (IVDH), a été caractérisée, et nos premiers résultats indiquent que cette enzyme est impliquée dans la production du butyryl-CoA. / Trypanosoma brucei, a flagellated protozoan parasite of the kinetoplastidae order, is responsible for human sleeping sickness or human african trypanosomiasis (HAT). Its life cycle is complex and involves a haematophageous insect vector (tse-tse fly or Glossina), which ingests parasites during a blood meal on an infected host. After a series of differentiations, the parasites are injected to another host during another blood meal. We studied the energy and intermediary metabolism of the procyclic form of T. brucei, which is present into the midgut of the tse-tse fly. In this parasite, glucose degradation produces acetate into the mitochondria of the parasite and succinate into both the mitochondria and the glycosomes. Glycosomes are specific organites of trypanosomatids in which the glycolysis is compartimentalized. T. brucei uses an "acetate shuttle" to transfer acetyl-CoA from the mitochondrion to the cytosol to feed de novo fatty acids biosynthesis. This acetate production is essential for cell viability. The "acetate shuttle" involves inside the mitochondrion, the acetate:succinate CoA-transferase (ASCT), which converts glucose-derived acetyl-CoA into acetate. We identified and characterised a new mitochondrial enzyme involved in acetate production from glucose, in addition to ASCT: the acetyl-CoA thioesterase (ACH). Indeed, a double mutant affecting expression of both ACH and ASCT doesn’t produce anymore acetate and is lethal, which confirms the essential role of mitochondrial production of acetate. In addition, we showed that ASCT, via the ASCT/SCoAS (succinyl-CoA synthetase) cycle, contributes to mitochondrial ATP production by substrate phosphorylation, while ACH is not involved in ATP production. We also observed that contribution of the ASCT/SCoAS cycle and oxidative phosphorylation by the mitochondrial F0-F1-ATP synthase to ATP production are similar. Threonine is the most rapidly consumed amino acid by the procyclic trypanosomes and its degradation produces acetate and glycine. Using a combination of reverse genetics, proton NMR metabolic profiling and HPTLC, we characterized the first enzymatic step of the pathway, catalysed by the threonine dehydrogenase (TDH) and showed that threonine is the main carbon source for acetate production, de novo fatty acids and sterol biosynthesis. Acetyl-CoA is produced into the mitochondrion from glucose-derived pyruvate by the pyruvate dehydrogenase complex (PDH) and by the two first steps of the threonine degradation pathway, including TDH. Both glucose-derived and threonine-derived acetyl-CoA is then converted into acetate by the same enzymes, ACH and ASCT. We also found that the threonine degradation pathway is under metabolic control. Indeed, TDH activity, TDH expression and threonine-derived acetate production are reduced in the phosphoenolpyruvate carboxykinase (PEPCK) knock out mutant, in which glycolytic flux is redirected towards acetate production. In addition, we showed that, as opposed to glucose-derived acetyl-CoA, metabolism of threonine-derived acetyl-CoA doesn’t contribute to ATP production into the mitochondrion of the parasite. Our results suggest the existence of mitochondrial metabolic channelings, which disconnect pyruvate and threonine degradation pathways leading to acetate production. Trypanosomes developed a specific de novo fatty acids biosynthesis pathway using elongases located in the endoplasmic reticulum and an unusual primer, butyryl-CoA. The biosynthesic pathway of butyryl-CoA has not been investigated so far in trypanosomatids. Genomic data mining of the T. brucei database, highlights an hypothetical mitochondrial biosynthesis pathway from acetyl-CoA to butyryl-CoA. The last enzyme of this pathway, isovaleryl-CoA dehydrogenase (IVDH), was characterised and our first results suggest that this enzyme is indeed involved into butyryl-CoA production.

Class of 2006 Abstract / Objectives: To evaluate the efficacy of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, in reducing all-cause mortality and death due to rejection when administered to thoracic organ transplant patients. Methods: Using the following Medical Subject Heading (MeSH) terms and text words: hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, heart transplantation, and lung transplantation, the following data bases were searched: Cochrane Central Register of Controlled Trials (First Quarter 2006), Cochrane Database of Systematic Reviews (First Quarter 2006), Database of Abstracts and Reviews of Effects (First Quarter 2006), ACP Journal Club (1991to January/February 2006), International Pharmaceutical Abstracts (1970-February 2006), and Medline (1966 to February 2006) for English language reports. Three prospective randomized controlled trials (RCTs) and 3 retrospective observational studies were identified as using statins to reduce mortality and death due to fatal rejection in thoracic organ transplant patients. Results: Using all 6 studies (n= 1770 patients), statins decreased mortality by 77% (OR=0.23; [95% confidence interval 0.16-0.34] Z test, P<0.001). Sub-analysis using only RCT heart transplant data showed that statins decreased mortality by 69% (OR=0.31; [95% confidence interval 0.09-1.07] Z test, P<0.003). Sub-analysis using retrospective heart transplant data showed that statins decreased mortality by 75% (OR=0.25; [95% confidence interval 0.16-0.39] Z test, P<0.001). Retrospective lung transplant results (1 study) showed statins decreased mortality by 90% (OR=0.10; [95% confidence interval 0.03-0.34] Z test, P<0.001). Statins also significantly reduced death due to rejection (OR=0.22; [95% confidence interval 0.13-0.37]). Using all 6 studies (n= 1770 patients), statins decreased death due to rejection by 78%. Conclusions: In patients undergoing thoracic organ transplantation, statins significantly decrease all-cause mortality and death due to rejection. Therefore, statins should be routinely administered to these patients following transplant surgery.

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Entwicklung eines experimentellen Systems zur Untersuchung der subzellulärenLokalisierung der Alpha-Methylacyl-CoA-Racemase (AMACR) (Methode der retroviralen Transfektion von transformierten, embryonalen Mausfibroblasten) / Development of a experimental system for the investigation of the subcellular localisation of alpha-methylacyl-CoA racemase (amacr) (retroviral transfection of mouse fibroblasts)