Indiana University-Purdue University Indianapolis (IUPUI) / The goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.

No description available.

Thesis (Ph. D.)--University of New South Wales, 2000. / Also available online.

Thesis (Ph. D.)--University of Minnesota, 1923. / Cover title. Vita.

Metabolic fuel use in rainbow trout (𝘖𝘯𝘤𝘰𝘳𝘩𝘺𝘯𝘤𝘩𝘶𝘴 𝘮𝘺𝘬𝘪𝘴𝘴 W.) was investigated using closed system respirometry and proximate body analysis. During short term starvation (15 days, routine activity) the utilization of protein as a substrate, as determined by respirometry, increased from 14 to 24% of total fuel supply. However, even by the end of the experiment, the contribution of protein (24%) did not approach the classically reported values for fish of between 40 and 90%. Indeed, from respirometry data, during the first quarter of the experiment lipid contributed the majority of the fuel ( >60%) while carbohydrate contributed about 20%. Thereafter, lipid and carbohydrate became essentially equivalent in importance (about 37% each). However, from proximate body analysis, a more traditional fuel mixture was found (protein, 58%; lipid, 40%; carbohydrate, 2%) suggesting the possibility that the two procedures were measuring fundamentally different parameters. Instantaneous fuel use during sustainable swimming at different speeds was investigated by respirometry using three day test periods. While protein catabolism remained constant over time, and uniform between groups, its relative contribution tended to increase with time as total M₀₂ declined with sustained swimming. Protein catabolism was highest in nonswimming fish (30-45%) and lowest in the high speed swimmers (20%); lipid was the most abundant (41-55%) fuel used in all groups at all times. In the nonswimmers and lowspeed swimmers, lipid use tended to increase slightly over time whereas in the high speed swimmers, lipid use dropped from 54 to 44%. Carbohydrate use (up to 38%) was higher than predicted by earlier literature, but decreased greatly in both the nonswimmers and low speed swimmers over the three days, whereas in the high speed swimmers the contribution increased with time. The low speed swimmers from the last set of experiments were used as controls for the final set of experiments in which another group of fish were trained for two weeks at 1.0 L·s⁻¹ prior to testing using an otherwise similar regime. Even though there was no difference in gas exchange, the make-up of the fuel mixture was different for the two groups. Protein use was significantly lower, while lipid use was higher in the trained fish. In addition, relative protein use in the trained fish was constant over the three day period, a feature found only in the the high speed swimmers of the previous experiment. A critical evaluation of the respiratory quotient is given since its use by fish physiologists has been without complete conversion from that used by the mammal physiologists. In addition, the often quoted term "fuel use" is differentiated into 𝘪𝘯𝘴𝘵𝘢𝘯𝘵𝘢𝘯𝘦𝘰𝘶𝘴 𝘧𝘶𝘦𝘭 𝘶𝘴𝘦 and 𝘤𝘰𝘮𝘱𝘰𝘴𝘪𝘵𝘪𝘰𝘯𝘢𝘭 𝘧𝘶𝘦𝘭 𝘶𝘴𝘦 since the two describe fundamentally different principles, though this has not always been recognized in the literature. / Thesis / Master of Science (MS)

Résumé: L’autophagie est un processus essentiel au maintien de l’homéostasie cellulaire. Elle permet de dégrader et recycler aussi bien des organelles entières que des composants cytoplasmiques non fonctionnels. De plus, l’augmentation d’autophagie en condition de stress constitue une réponse adaptative favorisant la survie cellulaire. Chez les cardiomyocytes, l’autophagie en condition basale est indispensable au renouvellement, entre autres, des mitochondries et des protéines formant les sarcomères. De plus, les stress tels l’ischémie cardiaque ou la carence en nutriments induisent une augmentation de l’autophagie protectrice. Dans certaines conditions extrêmes, il a été suggéré qu’un surcroît d’autophagie puisse toutefois exacerber la pathologie cardiaque en provoquant la mort des cardiomyocytes. Considérant l’importance de ce processus dans la physiopathologie cardiaque, l’identification des mécanismes signalétiques régulant l’autophagie chez les cardiomyocytes a été le sujet de recherches intenses. À cet effet, l’activation des Mitogen-Activated Protein Kinase (MAPK) a été démontrée pour réguler, avec d’autres voies signalétiques, l’autophagie et l’apoptose des cardiomyocytes. Il est donc probable que les Dual-Specificity Phosphatase (DUSP), enzymes clés contrôlant l’activité des MAPK, participent aussi à la régulation de l’autophagie. Afin de vérifier cette hypothèse, nous avons induit l’autophagie chez des cardiomyocytes isolés de rats nouveau-nés en culture. L’analyse de marqueurs d’autophagie par immunobuvardage démontre que l’activation des MAPK ERK1/2 et p38 corrèle avec l’activité autophagique chez les cardiomyocytes. Dans ces conditions, la diminution d’expression de la majorité des ARNm encodant les différentes DUSP retrouvées chez les cardiomyocytes contraste de façon marquée avec l’augmentation d’expression de l’ARNm Dusp5. De plus, nous avons démontré par une étude de gain de fonction que l’activation soutenue de p38 par surexpression d’un mutant MKK6 constitutivement actif stimule l’autophagie chez les cardiomyocytes. De façon surprenante, la perte de fonction de p38 obtenue par surexpression d’un mutant p38 dominant négatif n’altère en rien la réponse autophagique initiatrice dans notre modèle in vitro. Nos résultats suggèrent que les DUSP puissent réguler, via leurs actions sur les MAPK, d’importantes étapes du processus autophagique chez les cardiomyocytes. / Abstract: Autophagy is a process essential to the maintenance of cellular homeostasis. It helps degrade and recycle whole organelles and nonfunctional cytoplasmic components. In addition, the adaptative up regulation of autophagy in stress condition promotes cell survival. In cardiomyocytes basal autophagy is essential to the renewal of, among others, mitochondria and proteins forming sarcomeres. In addition, stresses such as ischemic heart or nutrient deficiency induce an increase in protective autophagy. In extreme conditions, it has been suggested that autophagy may exacerbate cardiac disease causing the death of cardiomyocytes. Considering the importance of this process in cardiac pathophysiology, identify ing safety mechanisms regulating autophagy in cardiomyocytes has been the subject of intense research. To this end, activation of mitogen-activated protein kinase (MAPK) has been demonstrated to regulate, with other signaling pathways, autophagy and cardiomyocyte apoptosis. It is therefore likely that Dual-Specificity Phosphatases (DUSPs), key enzymes that control the activity of MAPKs, also participate in the regulation of autophagy. To test this hypothesis, we have induced autophagy in isolated cardiomyocytes of newborn rats in culture. Analysis of autophagy markers by immunoblotting demonstrated that the activation of MAPKs ERK1/2 and p38 correlates with autophagic activity in cardiomyocytes. Under these conditions, the decrease in expression of the majority of mRNAs encoding different DUSPs found in cardiomyocytes contrast sharply with the increase mRNA expression of Dusp5. Furthermore, we demonstrated by again of function study that sustained activation of p38 by overexpression of a constitutively active MKK6 mutant stimulates autophagy in cardiomyocytes. Surprisingly, the loss of p38 function obtained by overexpression of a dominant negative p38 mutant does not affect the autophagic response in our in vitro model, but increases the lipidation of autophagosomes marker LC3. Our results suggest that DUSPs can regulate, through their actions on MAPKs, important stages of autophagy in cardiomyocytes.

No description available.

No description available.

No description available.

Phosphorus is an essential element for plant growth and development. Due to its low availability, solubility and mobility, phosphate is often the limiting macronutrient for crops and other plants. Plants have evolved several responses to phosphate deficiency. However, very little is known about the molecular basis of these responses. Here, I study the expression of PAP12, its role in the phosphate starvation response and the interaction of its promoter with nuclear factors. Analysis of a PAP12 T-DNA insertion line (pap12-1) revealed PAP12 is responsible for the majority of the acid phosphatase activity detected by the standard in-gel assay. RNA gel blots showed that PAP12 was induced only by Pi deficiency, and not by general nutrient stress. PAP12 expression, at the RNA and protein level, reflected endogenous phosphate levels in two mutants with altered phosphate accumulation. In the pho1 mutant, PAP12 expression and activity were up-regulated with respect to wild-type plants, and in the pho2 mutant, PAP12 expression and activity were reduced. Analysis of the PAP12 promoter using promoter-GUS fusions revealed expression in leaves, roots, flowers, hydathodes, root tips, and pollen grains. This broad pattern of expression suggests that PAP12 functions throughout the plant in response to low phosphate concentrations. The results showed PAP12 does not play a major role in phosphate remobilization, acquisition or in helping plants cope with low phosphate environments. Instead, the major phenotype associated with PAP12 deficiency was a significant delay in flowering in the low-phosphate pho1 background and a slight acceleration of flowering in the high-phosphate pho2 background over-expressing PAP12. These results suggest that PAP12 may have a role in linking phosphate status with the transition to flowering. Finally, I used promoter deletion and DNA-protein interaction assay to understand PAP12 expression upon phosphate starvation. A 35-bp region of the PAP12 promoter was identified as an important phosphate regulatory cis-element required for induction during phosphate starvation. We isolated a 23.5 kDa nuclear factor, which binds to this 35-bp region of the PAP12 promoter in a phosphate-dependent manner. The work presented here will add to our knowledge about the molecular processes that regulate phosphate nutrition.