Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references.

La perte de masse et de force musculaires liée à l'âge, ou sarcopénie, pourrait être partiellementexpliquée par un défaut de récupération de masse musculaire après des épisodes générateursd'atrophie musculaire. Ainsi, les périodes d'immobilisation qui augmentent avec l'âge (alitement,convalescence, fracture) pourraient être suivies d'une absence de récupération musculaire etcontribuer à la fonte musculaire au cours du vieillissement. Les causes de ce défaut de récupérationimpliquent notamment un déséquilibre du taux de renouvellement protéique et du taux derenouvellement cellulaire. L'objectif de cette thèse a donc été de mettre en évidence les mécanismesresponsables de l'atrophie musculaire chez le rat âgé au cours de l'immobilisation et ceux quiseraient défaillants afin de déceler les mécanismes à cibler pour favoriser la récupérationmusculaire.Des rats âgés ont été immobilisés pendant 8 jours par plâtrage unilatéral de la patte arrière, puislaissés en récupération pendant 40 jours après le déplâtrage. Nous avons montré que chez cesanimaux nourris avec un régime contenant 13% de caséine, l'immobilisation entraîne une atrophiedes muscles immobilisés mais, contrairement au rat adulte, le rat âgé ne récupère jamais la massemusculaire perdue. L'atrophie des muscles immobilisés peut être expliquée par 1/ une augmentationde l'apoptose et de la protéolyse ubiquitine-protéasome-dépendante musculaires, 2/ une diminutionde la régénération des cellules musculaires et 3/ une diminution de la protéosynthèse musculaire àl'état nourri. Tous ces phénomènes pourraient résulter de la présence d'un fort stress oxydant etd'une importante inflammation intramusculaire. Tous ces paramètres sont normalisés dès 10 jours derécupération, ce qui permet de stopper l'atrophie mais ne permet pas d'initier la phase derécupération musculaire. Nous avons donc testé l'effet de différentes supplémentationsnutritionnelles au cours de la période de récupération afin de favoriser un gain de masse musculairepost immobilisation. Des supplémentations en leucine (acide aminé bien connu pour stimuler laprotéosynthèse et inhiber la protéolyse) ont ainsi été réalisées. Chez les rats supplémentés, uneamélioration de la synthèse protéique et une normalisation plus précoce des activités protéolytiquesdu protéasome ont été observées. Cependant cette amélioration du métabolisme protéique ne s'estpas traduite par un gain de masse musculaire. Par contre, la modulation qualitative et quantitative desapports en protéines a pu permettre d'obtenir une récupération significative de masse musculaire :ainsi des régimes contenant 13% de lactosérum et des régimes hyper-protéinés ont permis de gagner50% de la masse perdue et ce, dès 20 jours de récupération.Nos résultats montrent que l'immobilisation chez le rat âgé aggrave la sarcopénie. Une fortealtération du métabolisme protéique permet d'expliquer la perte de muscle et la seule normalisationde la protéolyse et de la protéosynthèse permet d'expliquer l'absence de récupération musculaire.Nous avons montré que la modulation des apports en protéines au cours de la phase de récupérationpouvait permettre un gain de protéines.

Transcription factors are proteins that regulate transcription of genes by binding to specific DNA sequences proximal to the gene. The specificity and affinity of protein-DNA recognition is critical for proper gene regulation. This thesis explores the mechanisms of binding to the sequence 5’CACGTG, a common recognition sequence both in plants where it is known as the G-box and in mammalian cells where it is termed the E-box. This sequence is of clinical interest because it is the target of the transcription factor Myc, an oncogene linked to many cancers. A number of alpha-helical proteins with different dimerization elements, from the basic region-leucine zipper (bZIP), basic region helix-loop-helix leucine zipper (bHLHZ) and basic region helix-loop-helix-PAS (bHLH-PAS) protein families, are capable of binding to this sequence. The basic regions of all these protein families contain residues that contact DNA and determine DNA sequence specificity while the other subdomains are responsible for dimerization specificity. First, the influence of protein-DNA contacts on sequence specificity of the plant bZIP protein EmBP-1 was probed by point mutations in the basic region. Residues that contact the DNA outside the core G-box sequence and residues that contact the phosphate backbone were found to be important for sequence specificity. Second, the impact of the dimerization subdomains of bHLHZ protein Max, the required heterodimerization partner of the Myc protein, and bHLH-PAS protein Arnt was probed by mutation, deletion and inter-family subdomain swapping studies. All studied protein families are intrinsically disordered, forming structure upon dimerization and DNA binding. The dimerization domains were found to indirectly influence DNA binding by affecting folding, dimerization ability or proper orientation of the basic regions relative to DNA. Lastly, a new strategy for selection of G-box binding proteins in the Yeast One-hybrid system is explored. Together, these studies broaden our understanding of the structure-function relationship of the DNA-binding activities of these closely related families of transcription factors. The creation and characterization of mutants with altered specificity, affinity and dimerization specificity may also be useful for biotechnology applications.

Transcription factors are proteins that regulate transcription of genes by binding to specific DNA sequences proximal to the gene. The specificity and affinity of protein-DNA recognition is critical for proper gene regulation. This thesis explores the mechanisms of binding to the sequence 5’CACGTG, a common recognition sequence both in plants where it is known as the G-box and in mammalian cells where it is termed the E-box. This sequence is of clinical interest because it is the target of the transcription factor Myc, an oncogene linked to many cancers. A number of alpha-helical proteins with different dimerization elements, from the basic region-leucine zipper (bZIP), basic region helix-loop-helix leucine zipper (bHLHZ) and basic region helix-loop-helix-PAS (bHLH-PAS) protein families, are capable of binding to this sequence. The basic regions of all these protein families contain residues that contact DNA and determine DNA sequence specificity while the other subdomains are responsible for dimerization specificity. First, the influence of protein-DNA contacts on sequence specificity of the plant bZIP protein EmBP-1 was probed by point mutations in the basic region. Residues that contact the DNA outside the core G-box sequence and residues that contact the phosphate backbone were found to be important for sequence specificity. Second, the impact of the dimerization subdomains of bHLHZ protein Max, the required heterodimerization partner of the Myc protein, and bHLH-PAS protein Arnt was probed by mutation, deletion and inter-family subdomain swapping studies. All studied protein families are intrinsically disordered, forming structure upon dimerization and DNA binding. The dimerization domains were found to indirectly influence DNA binding by affecting folding, dimerization ability or proper orientation of the basic regions relative to DNA. Lastly, a new strategy for selection of G-box binding proteins in the Yeast One-hybrid system is explored. Together, these studies broaden our understanding of the structure-function relationship of the DNA-binding activities of these closely related families of transcription factors. The creation and characterization of mutants with altered specificity, affinity and dimerization specificity may also be useful for biotechnology applications.

Internalin J (InlJ) gehört zu der Klasse der bakteriellen, cysteinhaltigen (leucine-rich repeat) LRR Proteine. Bei den Internalinen handelt es sich um meist invasions-assoziierte Proteine der Listerien. Die LRR-Domäne von InlJ ist aus 15 regelmäßig wiederkehrenden, stark konservierten Sequenzeinheiten (repeats, 21 Aminosäuren) aufgebaut. Ein interessantes Detail dieses Internalins ist das stark konservierte Cystein innerhalb der repeats. Daraus ergibt sich eine ungewöhnliche Anordnung von 12 Cysteinen in einem Stapel. Die Häufigkeit von Cysteinen in InlJ ist für ein extrazelluläres Protein von L. monocytogenes außergewöhnlich, und die Frage nach ihrer Funktion daher umso brennender. Im Vergleich zum ubiquitären Vorkommen der sogenannten repeat-Proteine in der Natur sind Studien zu ihrer Stabilität und Faltung nicht äquivalent vertreten. Die zentrale Eigenschaft der repeat-Proteine ist ihr modularer Aufbau, der durch einfache Topologie gekennzeichnet ist und auf kurzreichenden Wechselwirkungen basiert. Diese Topologie macht repeat-Proteine zu idealen Modellproteinen, um die stabilitätsrelevanten Wechselwirkungen zu separieren und zuzuordnen. In der vorliegenden Arbeit wurde die Faltung und Entfaltung von InlJ umfassend charakterisiert und die Relevanz der Cysteine näher beleuchtet. Die spektroskopische Charakterisierung von InlJ zeigte, dass dessen Faltungszustand durch zwei Tryptophane im N- und C-Terminus fluoreszenzspektroskopisch gut zugänglich ist. Die thermodynamische Stabilität wurde mittels fluoreszenz-detektierten, Guanidiniumchlorid-induzierten Gleichgewichtsexperimenten bestimmt. Um die kinetischen Eigenschaften von InlJ zu erfassen, wurden die Faltungs- sowie die Entfaltungsreaktion spektroskopisch untersucht. Die Identifizierung der produktiven Faltungsreaktion war lediglich durch die Anwendung des reversen Doppelsprungexperiments möglich. Die Auswertung erfolgte nach dem Zweizustandsmodell, wonach die Faltung dem „Alles-oder-Nichts“ Prinzip folgt. Die Gültigkeit dieser Annahme wurde durch die kinetische Charakterisierung bestätigt. Es wurde sowohl in den Gleichgewichtsexperimenten als auch in den kinetisch erhaltenen Daten eine hohe freie Stabilisierungsenthalpie festgestellt. Die hohe Stabilität von InlJ geht mit hoher Kooperativität einher. Die kinetischen Daten zeigen zudem, dass die hohe Kooperativität hauptsächlich der Faltungsreaktion entstammt. Der Tanford-Wert von 0.93 impliziert, dass die Oberflächenänderung während der Faltung bereits zum größten Teil erfolgt ist, bevor der Übergangszustand ausgebildet wurde. Direkte strukturelle Informationen über den Übergangszustand wurden mit Hilfe von Mutationsstudien erhalten. Zu diesem Zweck wurden 12 der 14 Cysteine gegen ein Alanin ausgetauscht. Die repeats 1 bis 11 von InlJ beinhalten jeweils ein Cystein, deren Anordnung eine Leiter ergibt. Deren Substitutionen haben einen vergleichbar destabilisierenden Effekt auf InlJ von durchschnittlich 4.8 kJ/mol. Die Verlangsamung der Faltung deutet daraufhin, dass die Interaktionen der repeats 5 bis 11 im Übergangszustand bereits voll ausgebildet sind. Demnach liegt bei InlJ ein zentraler Faltungsnukleus vor. Im Rahmen dieser Promotionsarbeit wurde eine hohe Stabilität und ein stark-kooperatives Verhalten für das extrazelluläre Protein InlJ beobachtet. Diese Erkenntnisse könnten wichtige Beiträge zur Entwicklung artifizieller repeat-Proteine leisten, deren Verwendung sich stetig ausweitet. / Internalin J (InlJ) is a member of the family of bacterial cysteine-containing leucine-rich repeat (LRR) proteins. Internalins are invasion-associated surface proteins of Listeria monocytogenes. The LRR domain of InlJ consists of 15 repeating units, which are arranged in tandem. The consensus sequence consists of 21 residues. Interestingly, a leucine residue which is highly conserved among the Internalins is replaced by cysteine. This results in a continuous cysteine ladder of 12 repeats. This frequency of cysteines is remarkable for an extracellular protein of L. monocytogenes. Stability and folding of repeat proteins are not equivalently studied considering their ubiquitous distribution in nature. Their modular structure results in simple topology and is dominated by short-range interactions. These characteristic features of repeat proteins facilitate the separation and identification of stabilizing interactions, making repeat proteins to ideal model systems for folding studies. In this work the folding and unfolding of InlJ has been extensively characterized, shedding light on the relevance of the cysteines. Two tryptophans located in the N- and C-terminus allowed monitoring the folding state of the entire protein via fluorescence. Thermodynamic stability was therefore derived by guanidinium chloride induced equilibrium experiments. Furthermore, the chemically induced unfolding and folding reactions were characterized with respect to their kinetics. Interrupted refolding experiments were essential for tracking the productive folding reaction of InlJ. Analysis of the kinetic and equilibrium data leads to the conclusion that the results are compatible with a two-state model. The study presented here reveals high stability of the protein InlJ in conjunction with high cooperativity. Kinetic data disclosed the origin of high cooperativity in the folding reaction; with a Tanford value of about 0.93. This high value implicates that the major change of the accessible surface area occurs before the transition state is formed. Mutational studies provided more detailed structural information about the transition state. 12 of 14 cysteine residues were mutated to alanine for this purpose. The cysteines in repeats 1 to 11 stack over each other and form a ladder of reduced cysteines. The substitution of one of these cysteines has an average destabilizing effect of 4.8 kJ/mol. The deceleration of the folding reaction by the substitution shows that repeats 5 to 11 are already fully structured in the transition state, pointing to a central nucleus in the folding of the LRR-protein InlJ. The extracellular protein InlJ reveals extreme stability and high cooperativity. The insights into the folding of this LRR motif could facilitate the design of further artificial repeat proteins.

For all living systems, there is a requirement to recycle and regulate proteins. In eukaryotic organisms this is accomplished by the proteasome. The p97 ATPase is another highly conserved and essential complex present throughout the eukaryotic cell. In Paper I we utilized UFD fluorescent substrates to address the role of p97 and cofactors in soluble proteasome degradation. Results using RNAi and Drosophila p97 mutants propose p97 to function upstream of the proteasome on cytosolic proteasome targets as an important unfoldase together with its Ufd1/Npl4 cofactors. The results implicate p97 to be important for degradation of proteasome substrates lacking natural extended peptide regions. In Paper II we focused on identifying transcription factors essential for production of proteasomal subunits and associated proteins in Drosophila S2 cells. We utilized an RNA library targeting 993 known or candidate transcription factors and monitored RNAi depleted Drosophila S2 cells expressing the UFD reporter UbG76VGFP. We identified a range of potential candidates and focused on the bZIP transcription factor Cnc-C. RNAi and qrt-PCR experiments implicated Cnc-C to be involved in transcription of proteasomal subunits. In Paper III we applied our knowledge gained from Paper I about p97 dependent substrates and set up a high-throughput microscopy screening method to potentially find inhibitors specifically targeting the p97 proteasomal sub-pathway. Utilizing UFD substrates with and without C-terminal peptide tails we determined if compounds inhibited the core proteasomal machinery or the p97 pathway specifically. Through a primary and secondary round of screening we identified several new compounds inhibiting the ubiquitin-proteasome pathway though none from our initial screening had specificity for p97. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 187 pages. Includes vita. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 137-154).

Orientador: Juliany Gomes Quitzan / Resumo: A diminuição da complacência da bexiga está relacionada com modificações no equilíbrio dos componentes da matriz extracelular e na concentração das fibras de colágeno, acarretando em espessamento da parede vesical e fibrose. O objetivo do estudo é analisar a relação entre suplementação oral dos glicosaminogicanos (GAGs), distribuição de pequenos proteoglicanos ricos em leucina (SRLPs) e síntese de colágeno em bexigas saudáveis e fibrosadas por obstrução parcial submetidas a cistotomia. Foram utilizados 56 ratos da linhagem Wistar, fêmeas, divididos em cinco grupos experimentais- G1 (8) – controle, sem procedimento, G2 (12) – animais não suplementados, submetidos ao procedimento de obstrução vesical e posterior cistotomia, G3 (12) – animais suplementados e submetidos ao procedimento de obstrução vesical e posterior cistotomia, G4 (12) – animais não suplementados e submetidos ao procedimento de cistotomia e G5 (12) animais suplementados e submetidos ao procedimento de cistotomia. A suplementação de glicosaminoglicanos foi realizada a cada 24 horas por via oral durante 28 dias pós cistotomia, quando então os animais foram eutanasiados. O colágeno foi avaliado pela Microscopia Cars e biglican, decorina, lumican e fibromodulina pelo método de imunofluorescência. Para análise estatística foi utilizado o teste ANOVA e T-STUDENT, com nível de significância p <0,05. Os animais dos grupos G2 e 3 foram os que apresentaram diferença estatística na seguinte constante: peso bexiga final /p... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre