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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
211

Development of Overhauser-enhanced magnetic resonance imaging in vivo : application to molecular imaging of proteolysis. / Développement de l'imagerie par résonance magnétique rehaussée par l'effet Overhauser in vivo : application à l'imagerie moléculaire de la protéolyse.

Koonjoo, Neha 08 October 2015 (has links)
Ce travail fait l’objet d’une avancée scientifique dans le développement de la technique d’IRM rehaussée par l’effet Overhauser dans la souris à 0,2 T. Cette dernière repose sur le transfert de polarisation des spins électroniques saturés d’un radical libre vers les spins des protons (généralement de l’eau) voisins pour rehausser le signal RMN du proton. Notre équipe a développé cette technique pour détecter une activité protéolytique au travers de deux stratégies. La première partie de la thèse a été de détecter pour la première fois une activité protéolytique in situ dans des souris saines et in vitro sur cellules vivantes. L’efficacité du rehaussement par effet Overhauser repose sur le temps de corrélation des spins des électrons non-appariés. Un radical nitroxyde greffé à l’élastine a été utilisé comme substrat. La protéolyse de ce dernier par des élastases pancréatiques a conduit l’observation en 3D d’un rehaussement du signal RMN de plus de 10 fois dans le tube digestif de souris vivantes. De plus, des développements méthodologiques, tels que l’implémentation de la séquence TrueFISP, le sous-échantillonnage par la méthode “Keyhole”, et la reconstruction des données en 3D ont été faits. La deuxième stratégie repose sur des molécules de nitroxyde ayant l’unique propriété de pouvoir décaler leurs pics de résonance après hydrolyse. Un nitroxyde phosphorylé en position Béta pouvant être détecté à deux fréquences spécifiques différentes avant et après hydrolyse d’un groupement chimique a été synthétisé par des chimistes à Marseille. L’hydrolyse de cette macromolécule a été observée in vivo dans l’estomac de souris saines avec des rehaussements de plus de 400% et imagée en 3D avec une bonne résolution spatio-temporelle. Ainsi, une prochaine étape serait de poursuivre ce travail sur un modèle pathologique et développer cette technique à un champ magnétique plus bas. / This work relates the continuity and advances in the implementation of the Overhauser-enhanced Magnetic Resonance Imaging technique on a 0.2 T scanner. Briefly, OMRI technique is based on polarization transfer of saturated electronic spins from free nitroxide radicals to proton spins of surrounding water molecules in the aim to drastically enhance proton NMR signal. To this technique, our research team has merged specific strategies for proteolytic activity detection. The first strategy relies on a 3D visualization of proteolytic activity happening in intact living cells or in vivo in healthy mice. With an Overhauser switch based upon changes in molecular tumbling time, high Overhauser enhancements of 10-fold were observed in the intestinal tract of mice after that elastolytic activity of our probe: the nitroxide-labeled elastin macromolecule took place. In addition, MRI developments - TrueFISP sequence implementation, undersampling Keyhole method and data reconstruction were carried out for imaging these rapid biological processes. A second exquisite strategy is also described using nitroxides with shifting resonant peaks. Here, a Beta-phosphorylated nitroxide molecule was specifically detected at two distinct frequencies: one for its substrate and the other for its product once hydrolysis took place. This hydrolysis was imaged in 3D in the stomach of living mice with Overhauser enhancements of more than 400% and with a good spatiotemporal resolution. The perspectives of this work lie on a future detection of a pathological proteolytic activity in vivo and eventually and development of very low magnetic field OMRI.
212

Efeito de autólise de culturas lácticas na proteólise do queijo Prato / Autolysis effect of lactic cultures proteolysis in cheese dish

Izildinha Moreno 10 April 2003 (has links)
Nesta pesquisa, estudaram-se as variações ocorridas na relação entre autólise de culturas lácticas e o desenvolvimento da proteólise de queijo Prato produzido em quatro regiões brasileiras: Santa Catarina (Queijo A), Goiás (Queijo B), São Paulo (Queijo C) e Minas Gerais (Queijo D). A análise quantitativa da população de bactérias lácticas durante a maturação mostrou perfis microbiológicos similares para todas as amostras de queijos examinadas. Após 5 dias de maturação, lactococos e estreptococos estavam presentes em números mais elevados do que lactobacilos mesofílicos e termofílicos, leuconostoc e fermentadores de lactato. Contudo, essas populações aumentaram consideravelmente no final do processo de maturação. Enterococos e fermentadores de citrato permaneceram em números relativamente reduzidos ao longo da maturação. A análise qualitativa mostrou a predominância de \"non starter lactic acid bactéria\" (NSLAB) nos queijos das quatro origens, principalmente de Lactobacillus sp. Outros gêneros foram identificados em menor proporção: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. e Streptococcus sp. O queijo C diferiu dos demais por não apresentar Pediococcus sp. e Streptococcus sp. As culturas lácticas adicionadas Lactococcus lacfis sp. e Leuconostoc sp. estavam presentes em populações menores. A autólise foi estudada pela determinação da atividade de aminopeptidases e detecção de autolisinas por zimogramas e de enzimas intracelulares por \"imunoblotting\". Uma maior liberação de aminopeptidases ocorreu no queijo D, seguido dos queijos C, B e A. Não foram detectadas bandas de atividade lítica nos zimogramas dos queijos A e B em todas as condições avaliadas. Nos zimogramas, detectou-se uma banda de 30 KDa nos queijos C e D a pH 7,4 e 44°C, e uma outra de 40 KDa, exclusiva no queijo D, ambas de fraca intensidade. A pH 6,8 e 42°C, detectou-se bandas de 40KDa de fraca intensidade no queijo C e forte no D, além de mais duas de fraca intensidade de 90KDa e 110KDa no queijo D. Na análise em \"imunnoblotting\" com o antisoro anti-Lc, foi observado apenas sinais fracos de reação positiva e em números inferiores àquelas reveladas com o citoplasma bruto de Lac. Lacfis subsp. lacfis (controle positivo), indicando que a autólise foi praticamente inexistente. Com o antisoro anti-D-LDH, também não se detectou sinais de reação positiva nos queijos A e B, enquanto nos queijos C e D, verificou-se sinais positivos de 37KDa, de forte intensidade e correspondentes à proteína D-LDH, indicando a lise de Lab. helveticus. A evolução da proteólise foi determinada quantitativamente durante a maturação e avaliada com base nos índices: NS-pH4,6/NT% e NNP/NT%, teor de tirosina, eletroforese (Uréia-PAGE) e quantificação de aminoácidos individuais livres. Não foram detectadas diferenças significativas entre os queijos A. B, C e D no início da maturação. Contudo, com a fragmentação das proteínas, ocorreu um aumento gradual desses índices, tendo-se observado valores mais elevados no queijo D, seguido dos queijos C, B e A. Os perfis eletroforéticos de proteínas foram similares para os queijos das quatro origens e mostraram claramente que o coagulante e a plasmina foram os responsáveis pela degradação inicial das caseínas. A taxa de degradação da αs1- e β-caseína apresentou a seguinte ordem: D > C ≥ B > A. O acúmulo de aminoácidos livres também foi mais rápido no queijo D, seguido dos queijos C, B e A. Portanto, a autólise de Lab. helveticus no queijo D acelerou a proteólise, diminuindo o período de maturação em 45% e não afetando negativamente o desenvolvimento de \"flavour\" e nem a textura. No final da maturação (45 dias), os compostos voláteis foram determinados por meio de cromatografia gasosa com espectrometria de massa (CG-MS). Com raras exceções, os queijos das quatro origens continham os mesmos compostos voláteis, embora em quantidades distintas. Os álcoois e ésters foram os compostos majoritários nos queijos A e B e benzaldeído, 3-metil-butanal-2 e hexanal nos C e D. O perfil de textura instrumental (TPA) e a análise sensorial descritiva e quantitativa foram realizados. Os queijos B, C e D apresentaram características mais típicas de queijo Prato, independentemente do fato de que o aroma de manteiga e o sabor doce serem mais acentuados no queijo D. O queijo A foi classificado como tendo as menores características de queijo Prato e apresentou maior nível de defeitos de \"flavour\", principalmente residual e amargor. Os queijos avaliados não apresentaram diferenças significativas quanto à elasticidade e coesividade. Pequenas alterações na composição físico-química dos queijos, principalmente os teores de umidade e de caseína, influenciaram nos parâmetros como a firmeza e a adesividade. O presente estudo demonstrou pela primeira vez a ausência de autólise de Lc. Lacfis sp. em queijo Prato de quatro origens, bem como a ocorrência de autólise de Lab. helveticus nos queijos de duas origens, C e D. A pronunciada autólise dessa espécie teve um impacto positivo na proteólise e foi a responsável pelo aumento da concentração de aminoácidos livres nesses queijos. As diferenças na evolução da proteólise observada entre os queijos C e D, com taxas mais baixas no queijo C, independentemente da autólise pronunciada de Lab. helveticus, foram atribuídas à falta de uniformidade na composição físico-química dos queijos, principalmente pH e os teores de sal na umidade (S/U). / This paper reports a study aimed at evaluating the variations that occur in the interrelationship between autolysis of lactic starter bacteria and the development of proteolysis in Prato cheese produced in four different regions of Brazil: Santa Catarina (Cheese A), Goiás (Cheese B), São Paulo (Cheese C) and Minas Gerais (Cheese D). Quantitative analysis of microbial population yielded similar microbiological profiles for all the cheese samples investigated. After 5 days ripening, lactococci and streptococci were present in higher numbers than mesophilic and thermophilic lactobacilli, leuconostoc and lactate fermenting bacteria. However, the populations of the latter species had considerably increased by the time the ripening process completed 45 days. Enterococci and citrate fermenting bacteria remained present in relatively low numbers throughout ripening. The findings from qualitative analysis confirmed the predominance of non-starter lactic acid bacteria (NSLAB) in the cheeses from four different origins, especially Lactobacillus sp. Other genera of non-starter lactic acid bacteria (NSLAB) were identified in smaller proportions: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. and Streptococcus sp. Cheese C differed from the cheeses in that it no evidence was found of the presence of Pediococcus sp. and Streptococcus sp. The bacteria of the lactic starter culture Lactococcus lactis sp. and Leuconostoc sp. were also found to be present, although in lower numbers. Autolysis was studied by: (1) determination of aminopeptidase activity; (2) detection of autolysins by zymograms and (3) detection of intracellular enzymes by immunoblotting. The release of aminopeptidase was highest in cheese D, followed by C, B and A. No bands of lytic activity were appeared in the zymograms of Cheeses A and B in all conditions evaluated. At pH 7,4 and 44°C, a low-intensity band of 30 KDa was found in cheeses C e D, whereas another low-intensity band was observed only in cheese D. At pH 6,8 and 42°C, bands of 40KDa were observed in cheese C (low intensity) and cheese D (high intensity), in addition to two more low-intensity bands of 90KDa and 110KDa in cheese D. Immunoblotting with antiserum anti-Lc produced only minor signs of positive reaction, evidenced by the formation of low-intensity bands of 100 Kda in cheeses A and B and two high-intensity bands of 75 Kda and 100 Kda in cheeses C and D. Since these were present in smaller numbers to those revealed with crude cytoplasm of Lac. lactis subsp. lactis, it was concluded that autolysis did practically non occur. Immunoblotting with antiserum anti-D-LDH also detected sings of positive reaction in cheeses A and B, whereas in cheeses C e D positive high-intensity signs of 37Kda were found relative to D-LDH protein, indicating lysis of Lab. helveticus. The evolution of proteolysis was determined quantitatively during the ripening process and evaluated on the basis of the following parameters: NS-pH4,6/NT% and NNP/NT% indexes, tyrosine content, electrophoresis (Urea-PAGE) and quantification of free amino acids. No significant differences were found between cheeses A, B, C and D in the ear1y stages of ripening. However, with the on-going fragmentation of proteins during ripening, a gradual increase of the ripening indexes occurred, with the highest values being observed in cheese D, followed by C, B e A. The electrophoretic profiles were similar for the four cheeses investigated and clear1y showed that the clotting agent or milk coagulant and plasmin were responsible for the initial breakdown of the caseins. The degradation rate of Q.sl- and p-casein followed the following order: D > C ≥ B > A. The buildup of free amino acids was also faster in cheese D, followed by cheeses C, B e A. At the end of the ripening process studied (45 days), the volatile compounds were identified using gas chromatography and mass spectrometry (GC-MS), whereas the instrumental texture profile was measured and evaluated by Texture Profile Analysis (TPA). Cheese samples were evaluated by descriptive and quantitative sensory analysis. With rare exceptions, the cheeses of four different origins contained the same volatile compounds, although in different quantities. Alcohols and esters were the predominant volatile compounds in cheeses A and B and benzaldehyde, 3-methyl-butanal-2 and hexanal in cheeses C and D. Autolysis of Lb. helveticus accelerated proteolysis in cheese D, thereby reducing ripening time by 45% without any negative effect on either flavor or texture development. Cheeses B, C and D exhibited the most typical Prato cheese characteristics, in spite of the fact that the buttery aroma and sweet taste were more pronounced in cheese D. Cheese A was rated as the cheese with the less typical overall Prato cheese profile and was also the one that exhibited the highest degree and number of flavor defects, notably aftertaste and bitterness. The cheeses investigated did not present any significant differences as to elasticity and cohesiveness. Minor changes in the physical-chemical composition of the cheeses - mainly related to the moisture and casein levels - influenced parameters such as firmness and adhesiveness. The present study demonstrates for the very first time the absence of autolysis of Lc. Lactis sp. in Prato cheese from four different origins, as well as the occurrence of autolysis of Lb. helveticus in two of the cheeses analyzed (cheeses C and D). The pronounced autolysis of this species had a positive impact on proteolysis and was responsible for the release of increased quantities of free amino acids in these cheeses. The differences in the evolution of proteolysis observed between cheeses C and D - lower rate of proteolysis in cheese C, in spite of pronounced autolysis of Lb. helveticus - were attributed to poor uniformity of the physical-chemical composition of this cheese, particularly as related to pH and the salt and moisture levels (S/M).
213

The multifaceted proprotein convertases PC7 and furin : identification of new substrates and physiological relevance

Duval, Stéphanie 04 1900 (has links)
Les proprotéines convertases (PCs) sont responsables de la maturation de plusieurs protéines précurseurs et sont impliquées dans divers processus biologiques importants. Durant les 30 dernières années, plusieurs études sur les PCs se sont traduites en succès cliniques, toutefois les fonctions spécifiques de PC7 demeurent obscures. Afin de comprendre PC7 et d’identifier de nouveaux substrats, nous avons généré une analyse protéomique des protéines sécrétées dans les cellules HuH7. Cette analyse nous a permis d’identifier deux protéines transmembranaires de fonctions inconnues: CASC4 et GPP130/GOLIM4. Au cours de cette thèse, nous nous sommes aussi intéressé au rôle de PC7 dans les troubles comportementaux, grâce à un substrat connu, BDNF. Dans le chapitre premier, je présenterai une revue de la littérature portant entre autres sur les PCs. Dans le chapitre II, l’étude de CASC4 nous a permis de démontrer que cette protéine est clivée au site KR66↓NS par PC7 et Furin dans des compartiments cellulaires acides. Comme CASC4 a été rapporté dans des études de cancer du sein, nous avons généré des cellules MDA-MB-231 exprimant CASC4 de type sauvage et avons démontré une diminution significative de la migration et de l’invasion cellulaire. Ce phénotype est causé notamment par une augmentation du nombre de complexes d’adhésion focale et peut être contrecarré par la surexpression d’une protéine CASC4 mutante ayant un site de clivage optimale par PC7/Furin ou encore en exprimant une protéine contenant uniquement le domaine clivé N-terminal. Finalement, des résultats provenant de base de données de patients atteint de cancer du sein ont démontrés que l’expression élevé des gènes CASC4 et PCSK7 corrélaient à un mauvais prognostique, tandis qu’une expression élevée de CASC4 mais faible de PCSK7 était associée un meilleur prognostique. Dans le chapitre III, nous avons démontré que GPP130 est aussi clivé par PC7 et Furin mais au niveau des motifs H67RSRLEK73↓SL et K274PTR277↓EV dans les endosomes/TGN ou à la membrane plasmique. Récemment, GPP130 a été rapporté comme étant impliqué dans la prolifération cellulaire de cancers de la tête et du cou. Nos analyses provenant de la banque de données cBioPortal ont montré que le gène GPP130/GOLIM4 était surexprimé dans 35% des cas de cancer du poumon. Nous avons aussi montré qu’une réduction de GPP130 dans les cellules de cancer de poumon A549 augmente légèrement la prolifération cellulaire. Nous étudions actuellement l’hypothèse que GPP130 transporterait des cargos qui pourraient influencer la prolifération cellulaire. Finalement, durant le chapitre IV de cette thèse, nous avons poursuivi les études comportementales chez les souris PC7 KO afin d’investiguer si ces souris seraient protégées d’un effet anxiogène causé par l’obésité induite par l’alimentation. Nous avons montré que les souris PC7 KO ont une tendance à être moins affectées par la diète riche en gras saturé. Nous avons aussi montré que les souris PC7 ont une réponse déficiente face au stress. En conclusion, nos travaux de recherche ont permis d’identifier de nouveaux substrats de PC7 afin de mieux comprendre son rôle biologique, / The proprotein convertases (PCs) are responsible for the maturation of precursor proteins and are involved in multiple biological processes. Over the past 30 years, the PCs have had great translational achievements, but the physiological roles of PC7, the seventh member of the family, are still obscure. Searching for new PC7 substrates, a quantitative proteomics screen for selective enrichment of N-glycosylated polypeptides secreted from hepatic HuH7 cells identified two type-II transmembrane-proteins of unknown function(s): Cancer Susceptibility Candidate 4 (CASC4) and Golgi Phosphoprotein of 130 kDa (GPP130/GOLIM4). The chapters II and III of this thesis will focus on the investigation of CASC4 and GPP130 shedding by PC7 and Furin, and their corresponding physiological functions. In chapter IV we pursued the PC7 KO mice behavior phenotyping. Concentrating on CASC4 in chapter II, its mutagenesis characterized the PC7/Furin-shedding site to occur at KR66↓NS, in HEK293 cells. We further defined PC7 and Furin activity and demonstrated that CASC4 shedding occurs in acidic endosomes and/or trans-Golgi Network. Since CASC4 has been reported in breast cancer studies, we generated MDA-MB-231 cells stably expressing CASC4 WT and we showed a significant reduction of migration and invasion, caused by an increased number of paxillin-positive focal adhesions. This phenotype was reversed in cells overexpressing an optimally PC7/Furin-cleaved CASC4 mutant, or upon overexpression of CASC4 N-terminal domain. In accord, breast cancer patients’ datasets show that high CASC4 and PCSK7 expression levels predict a significantly worse prognosis compared to high CASC4 but low PCSK7 levels. In chapter III, we demonstrated that GPP130 is also cleaved by PC7 and Furin at similar and distinct motifs (H67RSRLEK73↓SL and K274PTR277↓EV) within acidic endosomes or at the TGN. GPP130 is predicted to be trafficking cargos and is responsible for the binding and retrograde trafficking of the Shiga toxin. In addition, GPP130 was recently reported to be implicated in cell proliferation in head and neck cancer cells. Our analysis from cBioPortal for Cancer Genomics has shown that the GPP130/GOLIM4 gene is amplified in up to 35% of the patients with lung cancer. During this chapter we also showed that GPP130 knockdown in A549 cells slightly increases cell proliferation. We are currently investigating that GPP130 transports important cargos that would influence cell proliferation. Finally, during the chapter IV of this thesis we have pursued the characterization of the PC7 KO mice anxiolytic phenotype that was previously described, and we investigated a possible protection from diet-induced obesity anxiety-like behavior. Interestingly, we have shown that the PC7 KO mice have a tendency to be less affected by the saturated high-fat anxiogenic diet. Also, we showed that the PC7 KO mice have an impaired stress-coping response that will need further investigations. In conclusion, we identified new PC7 substrates to better understand its biology but we also investigated more deeply known substrates, such as BDNF in the PC7 KO mice to fully grasp the physiological functions of this enigmatic proprotein convertase.
214

The implication of GPP130 shedding by PC7 and Furin in lung cancer progression

Prabhala, Priyanka 08 1900 (has links)
Le cancer du poumon est la principale cause de mortalité par cancer au Canada et entraîne un taux de mortalité important chez les patients. En effet, l’Organisation Mondiale de la Santé (OMS) indique que le cancer du poumon est la principale cause de décès liés au cancer dans le monde, avec 2.21 millions de diagnostics par année qui conduisent en moyenne à 1.8 millions de décès par an. Initialement asymptomatique, ce cancer évolue rapidement, devenant très invasif et métastatique, et est alors responsable de plus de morts par an que ces quatre cancers meurtriers combinés : côlon, sein, prostate et pancréas. Les Proprotéines Convertases (PCs) sont une famille de 9 sérines protéases qui jouent un rôle dans la maturation des précurseurs de protéines. Les PCs activent/désactivent ces précurseurs en les clivant à un unique ou une paire de résidus d’acides aminés et sont ainsi essentiels pour divers processus biologiques, tels que l’activation de facteurs de croissance qui jouent un rôle vital dans la transformation cellulaire et les risques de formation de tumeurs. Parmi les neufs membres des sérines protéases identifiées, les rôles physiologiques du septième membre de la famille, PC7, restent encore largement méconnus à ce jour. Afin d'identifier davantage de substrats de PC7, un criblage protéomique quantitatif a été réalisé pour l'enrichissement sélectif de polypeptides Nglycosylés, sécrétés par les cellules hépatiques HuH7. Deux protéines transmembranaires de type II clivées par PC7/Furine, et sécrétées sous forme soluble, ont alors été identifiées : CAncer Susceptibility Candidate 4 (CASC4) and Golgi PhosphoProtein de 130 kDa (GPP130). Des études ultérieures menées sur CASC4 par iii notre laboratoire ont mis en évidence son rôle protecteur contre la migration et l'invasion du Cancer du Sein Triple Négatif. GPP130 est une protéine transmembranaire de type II avec un domaine luminal contenant des déterminants endosomaux et de récupération du Golgi, lui offrant une voie de trafic cellulaire unique. Jusqu'à présent, le rôle de GPP130 a principalement été étudié dans la liaison et le trafic rétrograde des Shiga-toxines. Un récent rapport a cependant aussi montré son implication dans la progression du cycle cellulaire et dans la prolifération des cellules du cancer de la tête et du cou. Ainsi, notre analyse du cBioPortal pour Cancer Genomics a révélé que GPP130 est amplifié jusqu'à 35% chez les patients atteints de cancer du poumon. Le travail présenté ici montre les implications du clivage de GPP130 par PC7 et Furine dans la progression du cancer du poumon, en identifiant la région de GPP130 responsable de la croissance cellulaire. Ce projet dévoile ainsi des stratégies thérapeutiques potentielles ciblant la prolifération cellulaire induite par GPP130. / Lung Cancer is the leading cause of cancer death in Canada and causes significant morbidity in patients. Globally, World Health Organization (WHO) reports lung cancer as the leading cause of cancer-related deaths, with 2.21 million diagnoses/year, resulting in approximately 1.8 million deaths/year. Initially asymptomatic, it progresses to a highly invasive and quickly metastasizing cancer. It is responsible for more deaths per year than the combined death of the four deadly cancers: colon, breast, prostate, and pancreas. Proprotein Convertases (PCs) are a family of nine serine proteases that play a role in the maturation of secretory precursor proteins. Basic amino acid-specific PCs activate/inactivate precursor proteins by cleaving them at single or paired basic aminoacid residues. They are crucial for various biological processes, including the activation of growth factors that play a vital role in cellular transformation and the likelihood of tumor formation. Of the nine serine proteases identified, the physiological functions of the seventh member of the family, PC7, currently remain mostly unidentified. To further identify novel PC7 substrates, a quantitative proteomics screen for selective enrichment of N-glycosylated polypeptides, secreted from hepatic HuH7 cells, was performed. This identified two type-II transmembrane proteins, which were shed into soluble secreted forms by PC7/Furin: CAncer Susceptibility Candidate 4 (CASC4) and Golgi PhosphoProtein of 130 kDa (GPP130). Subsequent studies on CASC4 by our laboratory reported the protective role that CASC4 plays against migration and invasion in Triple Negative Breast Cancer. v GPP130 is a type-II transmembrane protein with a luminal domain containing endosomal and Golgi-retrieval determinants enabling a unique subcellular trafficking route. So far, the role of GPP130 has only been extensively studied in the binding and retrograde trafficking of Shiga toxin. However, recent reports have shown its implication in cell-cycle progression and cellular proliferation of head and neck cancer cells. Our analysis from cBioPortal for Cancer Genomics revealed that GPP130 is amplified in up to 35% of patients with lung cancer. The work presented here shows the implications of shedding GPP130 by PC7 and Furin in lung cancer progression by identifying the region of GPP130 responsible for cellular growth and unravelling potential therapeutic strategies for GPP130-induced cellular proliferation.
215

Struktur-Funtionsbeziehung in den spleißosomalen Proteinen des U4/U6•U5 tri-snRNP / Structure-functionrelationship of the Proteins within the U4/U6•U5 tri-snRNP

Müllers, Nina 02 November 2006 (has links)
No description available.
216

Rôle des facteurs physico-chimiques du micro-environnement intestinal et des boucles inter-hélicales du Domaine I dans l’activité de la toxine insecticide Cry9Ca du bacille de Thuringe

Brunet, Jean-Frédéric 11 1900 (has links)
Une fois ingérées par un insecte sensible, les toxines insecticides du bacille de Thuringe doivent être activées par les protéases intestinales de cet insecte. Leur premier domaine, un ensemble de sept hélices-α amphipathiques, est responsable de leur insertion dans la membrane luminale de certaines cellules de l’intestin médian, ce qui crée des pores peu sélectifs. La toxicité et la capacité à former des pores d’une telle toxine, la Cry9Ca, de ses mutants simples R164A et R164K et d’un fragment de 55 kDa résultant d’un clivage protéolytique au niveau de son résidu 164 ont été étudiées à l’aide d’une combinaison de modélisation par homologie, de bioessais, d’expériences de gonflement osmotique avec des vésicules de membrane en bordure en brosse de larves de sphinx du tabac et de mesures électrophysiologiques sur des intestins isolés. Ni les mutations simples ni le clivage protéolytique n’ont altéré la toxicité de la Cry9Ca. Dans une solution à faible force ionique, toutefois, la formation des pores dépend fortement du pH : une augmentation de celui-ci de 6,5 à 10,5 a entraîné une baisse irrégulière et par étapes successives de la perméabilité membranaire. Les quatre préparations de toxine ont néanmoins dépolarisé la membrane apicale d’intestins médians fraîchement isolés baignant dans une solution contenant 122 mM de KCl à pH 10,5. L’activité de la Cry9Ca, et des mutants R164A et R164K, a été grandement stimulée lorsque les expériences ont été effectuées en présence de suc intestinal, de lipides extraits d’un volume équivalent de suc intestinal ou d’un cocktail d’inhibiteurs de protéases solubles dans l’eau. De plus, le rôle des boucles inter-hélicales du Domaine I lors de l’insertion dans la membrane a été étudié avec des mutants doubles de la Cry9Ca dont les mutations introduisaient, neutralisaient ou renversaient une charge électrique. À l’exception de trois d’entres eux, tous ces mutants ont conservé une toxicité et une capacité à former des pores comparables à celles de la toxine parentale. L’ensemble de ces résultats suggère que le micro-environnement de l’intestin médian contribue à minimiser l’influence des charges de surface portées par les résidus des boucles inter-hélicales du Domaine I sur la capacité des toxines du bacille de Thuringe à former des pores. Il indique aussi que, d’une part, selon le site de clivage et les conditions expérimentales utilisées, des protéolyses supplémentaires de la toxine Cry9Ca activée peuvent soit stimuler, soit nuire à son activité et que, d’autre part, le suc intestinal du sphinx du tabac contient probablement un inhibiteur de protéases qui pourrait jouer un rôle important dans l’activité des toxines du bacille de Thuringe. / Once ingested by susceptible insects, Bacillus thuringiensis insecticidal toxins must be activated by the insect’s intestinal proteases. Their first domain, a bundle of seven amphipathic -helices, is responsible for their insertion into the luminal membrane of midgut cells, thereby creating poorly selective pores. The toxicity and pore-forming ability of one such toxin, Cry9Ca, its single-site mutants, R164A and R164K, and of the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using a combination of homology modeling, bioassays, osmotic swelling experiments with Manduca sexta larval midgut brush border membrane vesicles and electrophysiological measurements on isolated midguts. Neither the single mutations nor the proteolytic cleavage altered Cry9Ca toxicity. In low ionic strength solutions however, pore formation was highly dependent on pH: increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization. All four toxin preparations nevertheless depolarized the apical membrane of freshly isolated midguts bathing in a solution containing 122 mM KCl at pH 10.5. The activity of Cry9Ca, R164A and R164K was greatly enhanced when the experiments were conducted in the presence of midgut juice, the lipids extracted from an equivalent volume of midgut juice or a cocktail of water-soluble protease inhibitors. Additionally, the role of the interhelical loops of Domain I in membrane insertion was investigated with Cry9Ca double mutants with mutations that either introduced, neutralized or reversed an electrical charge. All but three mutants retained a toxicity and a pore-forming ability that were comparable to those of their parental toxin. Overall, the results suggest that the midgut microenvironment contributes to minimizing the influence of surface charges carried by Domain I interhelical loop residues on B. thuringiensis toxins pore-forming ability. They also indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.
217

Effekten av 10 veckors styrketräning på markörer för hypertrofi, translation och proteolys

Väisänen, Daniel January 2016 (has links)
Det har forskats mycket på olika signalvägar i det mänskliga genomet, trotts detta finns det många frågetecken som kvarstår. Denna uppsats undersöker några av dem. Syfte: Undersöka förändringar i genuttryck och mRNA-nivåer för hypertrofi- (MRF4) translations- (5.8S & 18S) och proteolysreglerande gener (MuRF1 & GDF-8) efter en 10 veckor lång styrketräningsperiod hos kvinnor och män. Frågeställningar: (1) Finns det en förändring i total mängd RNA före och efter en 10 veckors styrketräningsintervention. (2) Finns det en förändring i uttryck av MRF4, 5.8S, 18S, MuRF1 samt GDF-8 efter en 10 veckors styrketräningsintervention. (3) Finns det en könsskillnad i förändringen av total mängd RNA samt aktivering av MRF4, 5.8S, 18S, MuRF1 och GDF-8 efter en 10 veckors styrketräningsintervention. Metod: Urvalet för analysen bestod av 16 otränade försökspersoner varav 8 var män och 8 var kvinnor. Försökspersonerna utförde unilateral styrketräning av nedre extremiteten under 10 veckor, under 2 av dessa veckor utfördes ocklusionsträning.  Träningsperiodiseringen var vågformig (70-90% av 1RM, 5-12 rep, 3 ggr/vecka). Muskelbiopsier togs i det arbetande benet före träningsperiodens start samt 3-7 dagar efter träningsperiodens avslut. Genuttryck analyserades med qPCR. Resultat: Det fanns ingen signifikant skillnad i förändring mellan män och kvinnors totala RNA eller genuttryck. Total RNA ökade signifikant (p<0,01) med 19,2 %. Kvinnorna hade en signifikant ökning (P<0,05) av RNA på 27,6 % medan männen hade en signifikant ökning (p<0,05) på 14 %. MRF4 hade en signifikant (P>0,05) procentuell ökning i genuttryck med 55,7 % och kvinnor för sig hade en signifikant (P>0,05) ökning på 64 %. GDF-8 ökade signifikant (P>0,05) med 55,5 % medan GAPDH ökade signifikant (P>0,05) för båda könen tillsammans med 70,6 % och för män med 87,8 %. MuRF1 och 5.8S hade inga signifikanta förändringar i genuttryck. Slutsats: Det verkar som att både män och kvinnor får en liknande procentuell förändring av total RNA och mRNA genuttryck 3-7 dagar efter en 10 veckors hypertrofistyrd styrketräningsperiod. För att mäta genuttryck av translationsgenen MRF4 verkar 3-7 dagar efter en 10 veckors styrketräningsperiod vara en tidpunkt då det fortfarande pågår hypertrofi av skelettmuskulaturen.  Av de proteolysreglerande generna GDF-8 och MuRF1 sågs en uppreglering av GDF-8 vilket skulle kunna vara ett tecken på att hypertrofin börjar hämmas. Ett oväntat fynd var att GAPDH visade sig vara olämplig som kontrollgen vid en styrketräningsintervention på 10 veckor och att 18S var väldigt stabil. Detta kan betyda att GAPDH inte skall användas vid längre styrketräningsinterventioner. / There have been much research on signaling pathways in the human genome, but there still remain many questions. This paper examines some of them. Aim: Investigate changes in gene expression and mRNA levels of hypertrophy (MRF4), translation (5.8S & 18S) and proteolysis regulating genes (GDF-8) after a 10-week strength training period in men and women. Research questions: (1) Is there a change in the total amount of RNA before and after a 10-week strength training intervention. (2) Is there a change in the expression of MRF4, 5.8S, 18S, Murf1 and GDF-8 after 10 weeks of strength training. (3) Is there a gender difference in the change of total RNA and the expression of MRF4, 5.8S, Murf1 and GDF-8 after a 10-week long strength training intervention. Method: The sample for analysis consisted of 16 untrained subjects, of whom 8 were men and 8 were women. The subjects performed unilateral resistance training of lower extremities for 10 weeks, during two of these weeks blood flow restriction training were performed. The training was undulating (70-90% of 1RM, 5-12 cord, 3 times / week). Muscle biopsies were taken from the working leg before the start and 3-7 days after the training period. Gene expression was analyzed by qPCR. Results: There was no significant gender difference in total RNA or gene expression. Total RNA was significantly increased (p <0.01) with 19.2 %. The women had a significant increase (P <0.05) of RNA at 27.6 %, while the men had a significant increase (p <0.05) at 14 %. MRF4 had a significant (P> 0.05) percentage increase in gene expression by 55.7 %, and women had a significant (P> 0.05) increase of 64 %. GDF-8 increased significantly (P> 0.05) with 55.5 %, while GAPDH increased significantly (P> 0.05) for both sexes with 70.6 % and for men with 87.8 %. Murf1 and 5.8S had no significant changes in gene expression. Conclusions: It seems that both men and women experience a similar percentage difference of total RNA and mRNA gene expression 3-7 days after a 10 weeks long strength training period. To measure the gene expression of MRF4 3-7 days after a 10-week weight-training period seems to be a time when there still is a anabolic responses in the skeletal muscle. Of the proteolysis regulating genes GDF-8 and Murf1 there was an upregulation of GDF-8, which could be a sign that the inhibition of hypertrophy started. An unexpected finding is that GAPDH was found to be unsuitable as a control gene at a strength training intervention at 10 weeks and rRNA 18S was very stable, which could mean that GAPDH should not be used as control gene in longer strength training studies.
218

Purification and crystallization of spliceosomal snRNPs / Reinigung und Kristallisation von spleißosomalen snRNPs

Weber, Gert 01 July 2008 (has links)
No description available.
219

Rôle des facteurs physico-chimiques du micro-environnement intestinal et des boucles inter-hélicales du Domaine I dans l’activité de la toxine insecticide Cry9Ca du bacille de Thuringe

Brunet, Jean-Frédéric 11 1900 (has links)
Une fois ingérées par un insecte sensible, les toxines insecticides du bacille de Thuringe doivent être activées par les protéases intestinales de cet insecte. Leur premier domaine, un ensemble de sept hélices-α amphipathiques, est responsable de leur insertion dans la membrane luminale de certaines cellules de l’intestin médian, ce qui crée des pores peu sélectifs. La toxicité et la capacité à former des pores d’une telle toxine, la Cry9Ca, de ses mutants simples R164A et R164K et d’un fragment de 55 kDa résultant d’un clivage protéolytique au niveau de son résidu 164 ont été étudiées à l’aide d’une combinaison de modélisation par homologie, de bioessais, d’expériences de gonflement osmotique avec des vésicules de membrane en bordure en brosse de larves de sphinx du tabac et de mesures électrophysiologiques sur des intestins isolés. Ni les mutations simples ni le clivage protéolytique n’ont altéré la toxicité de la Cry9Ca. Dans une solution à faible force ionique, toutefois, la formation des pores dépend fortement du pH : une augmentation de celui-ci de 6,5 à 10,5 a entraîné une baisse irrégulière et par étapes successives de la perméabilité membranaire. Les quatre préparations de toxine ont néanmoins dépolarisé la membrane apicale d’intestins médians fraîchement isolés baignant dans une solution contenant 122 mM de KCl à pH 10,5. L’activité de la Cry9Ca, et des mutants R164A et R164K, a été grandement stimulée lorsque les expériences ont été effectuées en présence de suc intestinal, de lipides extraits d’un volume équivalent de suc intestinal ou d’un cocktail d’inhibiteurs de protéases solubles dans l’eau. De plus, le rôle des boucles inter-hélicales du Domaine I lors de l’insertion dans la membrane a été étudié avec des mutants doubles de la Cry9Ca dont les mutations introduisaient, neutralisaient ou renversaient une charge électrique. À l’exception de trois d’entres eux, tous ces mutants ont conservé une toxicité et une capacité à former des pores comparables à celles de la toxine parentale. L’ensemble de ces résultats suggère que le micro-environnement de l’intestin médian contribue à minimiser l’influence des charges de surface portées par les résidus des boucles inter-hélicales du Domaine I sur la capacité des toxines du bacille de Thuringe à former des pores. Il indique aussi que, d’une part, selon le site de clivage et les conditions expérimentales utilisées, des protéolyses supplémentaires de la toxine Cry9Ca activée peuvent soit stimuler, soit nuire à son activité et que, d’autre part, le suc intestinal du sphinx du tabac contient probablement un inhibiteur de protéases qui pourrait jouer un rôle important dans l’activité des toxines du bacille de Thuringe. / Once ingested by susceptible insects, Bacillus thuringiensis insecticidal toxins must be activated by the insect’s intestinal proteases. Their first domain, a bundle of seven amphipathic -helices, is responsible for their insertion into the luminal membrane of midgut cells, thereby creating poorly selective pores. The toxicity and pore-forming ability of one such toxin, Cry9Ca, its single-site mutants, R164A and R164K, and of the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using a combination of homology modeling, bioassays, osmotic swelling experiments with Manduca sexta larval midgut brush border membrane vesicles and electrophysiological measurements on isolated midguts. Neither the single mutations nor the proteolytic cleavage altered Cry9Ca toxicity. In low ionic strength solutions however, pore formation was highly dependent on pH: increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization. All four toxin preparations nevertheless depolarized the apical membrane of freshly isolated midguts bathing in a solution containing 122 mM KCl at pH 10.5. The activity of Cry9Ca, R164A and R164K was greatly enhanced when the experiments were conducted in the presence of midgut juice, the lipids extracted from an equivalent volume of midgut juice or a cocktail of water-soluble protease inhibitors. Additionally, the role of the interhelical loops of Domain I in membrane insertion was investigated with Cry9Ca double mutants with mutations that either introduced, neutralized or reversed an electrical charge. All but three mutants retained a toxicity and a pore-forming ability that were comparable to those of their parental toxin. Overall, the results suggest that the midgut microenvironment contributes to minimizing the influence of surface charges carried by Domain I interhelical loop residues on B. thuringiensis toxins pore-forming ability. They also indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.
220

Human β<sub>1</sub>-adrenergic receptor:biosynthesis, processing and the carboxyl-terminal polymorphism

Hakalahti, A. (Anna) 20 September 2011 (has links)
Abstract The β1-adrenergic receptor (β1AR) belongs to the large family of G protein-coupled receptors. It is activated by epinephrine and norepinephrine and thus has a central role in mediating the effects of the sympathetic nervous system. β1AR is the predominant adrenergic receptor in the heart, where it mediates positive inotropy and chronotropy. Thus, it is the most important target receptor for β-adrenergic antagonists, which are widely used in the treatment of cardiovascular diseases. Furthermore, β1AR is also expressed in the brain, where it has a crucial role in regulating memory formation and synaptic plasticity. Human β1AR (hβ1AR) has two polymorphisms, one at each terminus. The carboxyl-terminal (C-terminal) Arg389Gly8.56 polymorphism has previously been shown to have functional significance. Despite the clinical importance of hβ1AR, its biosynthetic profile and post-translational processing have not been well characterized to date. The aims of the present study were to shed light on these events, focusing on the limited proteolysis of hβ1AR and the impact of β-adrenergic ligands on receptor processing. In addition, the C-terminal polymorphism and its associations with certain parameters were investigated in a population consisting of survivors of acute myocardial infarction (AMI). By using a heterologous expression system, hβ1AR biosynthesis was revealed to be efficient and rapid. The N-terminus of the mature receptor was modified with O-glycans and one N-glycan, but despite these modifications it was subject to cleavage at the cell surface that resulted in two C-terminal fragments. The cleavage was mediated by a metalloproteinase, and importantly, it also occurred in vivo. Moreover, receptor activation enhanced the cleavage, which suggests that it represents a novel regulatory mechanism of hβ1AR. Interestingly, those ligands that enhanced the cleavage stabilized intracellular hβ1AR precursors, possibly via a pharmacological chaperone activity. Thus, the present study demonstrates that β-adrenergic ligands can have different regulatory effects on distinct hβ1AR forms. Among the AMI survivors, the Arg3898.56 homozygotes had significantly increased left ventricular mass indexes, when compared to the Gly3898.56 carriers, which suggests an association between Arg3898.56 and left ventricular hypertrophy (LVH). When euglycemic and diabetic patients were analyzed separately, the association existed among the euglycemic patients but was not present in diabetic patients. Diabetes is one of several risk factors that have previously been shown to influence the progression of LVH. Here, diabetes was shown to have a stronger effect on the development of LVH, when compared with the Arg3898.56 variant of hβ1AR. / Tiivistelmä β1-adrenerginen reseptori (β1AR) kuuluu laajaan G-proteiineihin kytkettyjen reseptorien perheeseen. β1AR on tärkeässä asemassa sympaattisen hermoston toiminnassa. Sydämessä β1AR on vallitseva adrenerginen reseptori, ja sydänlihaksen supistusvireys sekä -taajuus voimistuvat β1AR:n aktivaation kautta. Siten se edustaa sydän- ja verisuonisairauksissa käytettävien β-salpaajien tärkeintä kohdereseptoria. β1AR:n luontaisia agonisteja ovat lisämunuaisytimestä ja hermopäätteistä vapautuvat adrenaliini ja noradrenaliini. Sydänlihaksen lisäksi β1AR:a ilmennetään myös aivoissa, jossa reseptorilla on keskeinen asema muistin ja synaptisen muovautuvuuden kannalta. Ihmisen β1AR (hβ1AR) sisältää kaksi polymorfismia, joista toinen (Arg389Gly8.56) sijaitsee reseptorin karboksyyli- (C-) terminaalissa solulimassa. Tällä polymorfismilla on havaittu olevan toiminnallista merkitystä. Vaikka hβ1AR:n kliininen merkitys on huomattava, sen biosynteesistä ja translaationjälkeisestä muokkauksesta ei ole tähän mennessä ollut juurikaan tutkimustietoa. Tämän väitöskirjatyön tavoite oli kuvata näitä tapahtumia ja erityisesti keskittyä hβ1AR:n solunulkoisen amino- (N-) terminaalin rajoitettuun proteolyysiin. Lisäksi haluttiin tutkia, onko β-adrenergisillä ligandeilla vaikutusta reseptorin prosessointiin. Tutkimuksen kliinisessä osiossa kartoitettiin C-terminaalisen polymorfian yhteyttä valikoituihin muuttujiin aineistossa, joka koostui akuutin sydäninfarktin (AMI) sairastaneista potilaista. hβ1AR:n biosynteesin havaittiin olevan tehokas ja nopea heterologisessa systeemissä. Kypsän reseptorin N-terminaalissa havaittiin useita O-kytkennäisiä ja yksi N-kytkennäinen glykaani. Glykosyloinnista huolimatta N-terminaali pilkkoutui solun pinnalla, mikä tuotti kaksi solukalvolla sijaitsevaa, C-terminaalista reseptoripalasta. Pilkkoutumista, joka havaittiin myös in vivo, katalysoi metalloproteinaasi. Reseptorin aktivaatio kiihdytti pilkkoutumista, joka siten todennäköisesti edustaa uudenlaista hβ1AR:n säätelymekanismia. Ligandit, jotka kiihdyttivät pilkkoutumista, toisaalta stabiloivat solunsisäisiä hβ1AR:n epäkypsiä muotoja toimien luultavasti ns. farmakologisina kaperoneina. Näin ollen väitöskirjatyö osoittaa, että β-adrenergisillä ligandeilla voi olla erilaisia säätelyvaikutuksia eri hβ1AR-muotoihin. Kliinisessä tutkimuksessa Arg3898.56-homotsygooteilla potilailla havaittiin merkittävästi suurentunut vasemman kammion massaindeksi Gly3898.56-kantajiin verrattuina, mikä puoltaa Arg3898.56-polymorfismin ja vasemman kammion hypertrofian (LVH) välistä yhteyttä. Kun euglykeemisiä potilaita ja diabeetikkoja tutkittiin erikseen, yhteys ilmeni vain euglykeemisessä ryhmässä. Diabetes on riskitekijä, joka vaikuttaa LVH:n kehittymiseen. Tässä tutkimuksessa diabeteksellä havaittiin olevan voimakkaampi vaikutus LVH:n kehittymiseen Arg3898.56 -polymorfismiin verrattuna.

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