Dissertations / Theses on the topic 'Propeptide de la sortiline'
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1
Hivelin, Céline. "Étude des mécanismes de libération du propeptide de la sortiline et de ses effets sur l’homéostasie glucidique." Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4109/document.
Abstract:
En France, l’obésité touche 15% de la population et est en perpétuelle augmentation. Elle est une cause majeure du diabète de type 2. Elle se traduit par un accroissement du nombre de cellules de stockage du gras (adipocytes) et une résistance périphérique à l’insuline. Chez des individus obèses, l’augmentation du nombre d’adipocytes est associée à une diminution de l’expression de la sortiline, protéine transmembranaire dont le clivage entraine la formation du propeptide (PE) et sa libération dans la circulation sanguine. L’analogue synthétique du PE, la spadine, est connu pour moduler l’activité du canal potassique TREK-1. Ce canal étant exprimé dans la cellule bêta pancréatique qui sécrète l’insuline, une hormone participant à la régulation du taux de glucose dans le sang, il est possible que la spadine joue un rôle dans l’homéostasie du glucose. Mes résultats confirment cette hypothèse. En effet, la spadine améliore la tolérance au glucose des souris, en favorisant la libération d’insuline. La spadine est également connue pour interagir avec sortiline, indispensable au trafic du transporteur de glucose Glut4 vers la membrane des adipocytes. Cette interaction spadine-sortiline suggère que la spadine pourrait moduler l’entrée du glucose dans les adipocytes via la sortiline. Mes résultats montrent que la spadine ne modifie pas les capacités de stockage du glucose des adipocytes. En conclusion, la spadine joue un rôle dans la sécrétion de l’insuline et dans la régulation de la glycémie, ce qui peut présenter un intérêt pharmaceutiqueIn France, approximately 15% of the population is obese and this number keeps rising up every year. Obesity is a major cause of diabetes, inducing an increase of the number of fat-filled cells, called the adipocytes, and a peripheral insulin resistance. This increase of the number of adipocytes is associated with a decrease of sortilin expression, a transmembrane protein which is involved in the release of a propeptide (PE) in the blood circulation. Spadin, a synthetic PE analog, is known to modulate the potassium TREK-1 channel activity. Since, this channel is expressed in pancreatic beta cells which secrete insulin, a hormone involved in blood glucose regulation, spadin may play a role in glucose homeostasis. Consistent with this hypothesis, spadin improves glucose tolerance in mice, by stimulating insulin release. Spadin is a natural peptide derived from sortilin, which is known to control the glucose transporter Glut4 trafficking to the plasma membrane of adipocytes. This suggests that spadin may regulate glucose storage in adipocytes by affecting the sortilin function. However, my results show that spadin has no effect on glucose storage. In summary, spadin is involved in insulin secretion and glucose homeostasis and may be an alternative treatment against obesity and diabetes
2
Hivelin, Céline. "Étude des mécanismes de libération du propeptide de la sortiline et de ses effets sur l’homéostasie glucidique." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4109.
Abstract:
En France, l’obésité touche 15% de la population et est en perpétuelle augmentation. Elle est une cause majeure du diabète de type 2. Elle se traduit par un accroissement du nombre de cellules de stockage du gras (adipocytes) et une résistance périphérique à l’insuline. Chez des individus obèses, l’augmentation du nombre d’adipocytes est associée à une diminution de l’expression de la sortiline, protéine transmembranaire dont le clivage entraine la formation du propeptide (PE) et sa libération dans la circulation sanguine. L’analogue synthétique du PE, la spadine, est connu pour moduler l’activité du canal potassique TREK-1. Ce canal étant exprimé dans la cellule bêta pancréatique qui sécrète l’insuline, une hormone participant à la régulation du taux de glucose dans le sang, il est possible que la spadine joue un rôle dans l’homéostasie du glucose. Mes résultats confirment cette hypothèse. En effet, la spadine améliore la tolérance au glucose des souris, en favorisant la libération d’insuline. La spadine est également connue pour interagir avec sortiline, indispensable au trafic du transporteur de glucose Glut4 vers la membrane des adipocytes. Cette interaction spadine-sortiline suggère que la spadine pourrait moduler l’entrée du glucose dans les adipocytes via la sortiline. Mes résultats montrent que la spadine ne modifie pas les capacités de stockage du glucose des adipocytes. En conclusion, la spadine joue un rôle dans la sécrétion de l’insuline et dans la régulation de la glycémie, ce qui peut présenter un intérêt pharmaceutiqueIn France, approximately 15% of the population is obese and this number keeps rising up every year. Obesity is a major cause of diabetes, inducing an increase of the number of fat-filled cells, called the adipocytes, and a peripheral insulin resistance. This increase of the number of adipocytes is associated with a decrease of sortilin expression, a transmembrane protein which is involved in the release of a propeptide (PE) in the blood circulation. Spadin, a synthetic PE analog, is known to modulate the potassium TREK-1 channel activity. Since, this channel is expressed in pancreatic beta cells which secrete insulin, a hormone involved in blood glucose regulation, spadin may play a role in glucose homeostasis. Consistent with this hypothesis, spadin improves glucose tolerance in mice, by stimulating insulin release. Spadin is a natural peptide derived from sortilin, which is known to control the glucose transporter Glut4 trafficking to the plasma membrane of adipocytes. This suggests that spadin may regulate glucose storage in adipocytes by affecting the sortilin function. However, my results show that spadin has no effect on glucose storage. In summary, spadin is involved in insulin secretion and glucose homeostasis and may be an alternative treatment against obesity and diabetes
3
Daziano, Guillaume. "Rôle du propeptide de la sortiline et de ses dérivés dans les mécanismes de survie de la cellule bêta pancréatique." Electronic Thesis or Diss., Université Côte d'Azur, 2021. http://www.theses.fr/2021COAZ6025.
Abstract:
En 2019, la Fédération Internationale du Diabète a révélé que près de 500 millions de personnes étaient atteintes de diabète dans le monde. On estime que cette incidence atteindra les 700 millions de personnes en 2045. Outre, l’aspect financier de la prise en charge, le diabète est un véritable enjeu de santé publique. En effet, l’environnement hyperglycémique délétère associé au diabète est à l’origine de graves complications pouvant altérer le fonctionnement de nombreux organes tels que le cœur, le cerveau ou encore le rein. La résistance à l’insuline associée à la détérioration de la sécrétion d’insuline et à la perte de la masse cellulaire bêta pancréatique constituent les principales caractéristiques du diabète de type 2. Ainsi, afin d’améliorer la prise en charge des patients diabétiques, l’identification d’une approche thérapeutique maîtrisée permettant de protéger la masse cellulaire bêta et de favoriser la sécrétion d’insuline uniquement en réponse au glucose et ce, sans effets secondaires, apparaît idéale.Les précédents travaux du laboratoire ont identifié le PE endogène et ses dérivés synthétiques la Spadine et la Mini-Spadine comme des inhibiteurs sélectifs des canaux potassiques TREK-1 au fort potentiel antidépresseur et impliqués dans la sécrétion de sérotonine, la prolifération et la survie neuronale. Au niveau périphérique, la Spadine a été décrite in vitro et in vivo comme un peptide à l’effet incrétine comparable à celui de l’exendine-4, un antidiabétique couramment utilisé en clinique. Ainsi, à la suite de cette étude et par analogie aux effets protecteurs observés sur le neurone, nous avons émis l’hypothèse que le PE et ses dérivés pouvaient avoir un rôle bénéfique dans les mécanismes de survie de la cellule bêta pancréatique.Dans ce manuscrit, nous démontrons que le PE endogène et ses dérivés protègent les cellules bêta de l’apoptose induite par la présence chronique de l’interleukine pro-inflammatoire et diabétogène IL-1β, ainsi que d’un choc toxique aigu induit par la staurosporine. De plus, l’analyse des mécanismes intracellulaires révèle que ces peptides provoquent une augmentation de la concentration en calcium intracellulaire, activent les voies prolifératives et de survie ERK et Akt, et maintiennent l’activité du facteur transcriptionnel CREB dans un environnement délétère via un mécanisme dépendant des calmodulines kinases.Ainsi, ces travaux de thèse montrent que le PE et ses dérivés synthétiques protègent la cellule bêta pancréatique et initient des processus cellulaires vertueux par une voie de signalisation originale indépendante de la PKA, où le potentiel de membrane et le calcium occupent un rôle crucial. Ces données suggèrent le PE endogène et ses dérivés synthétiques comme une nouvelle classe de peptides protecteurs des cellules bêta pancréatiquesIn 2019, the International Diabetes Federation revealed that nearly 500 million people have diabetes worldwide. It is estimated that this incidence will reach 700 million people by 2045. In addition to the financial aspect of treatment, diabetes is a real public health issue. Indeed, the deleterious hyperglycemic environment associated with diabetes is could induce serious complications, leading to a functional alteration of many organs such as the heart, the brain or the kidney. Insulin resistance associated with the deterioration of insulin secretion and the loss of pancreatic beta cell mass are the main characteristics of type 2 diabetes. Thus, in order to improve the management of diabetic patients, the identification of a controlled therapeutic approach to protect beta cell mass and promote insulin secretion only in response to glucose and without side effects, appears ideal.The laboratory has identified the endogenous PE and its synthetic derivatives Spadin and Mini-Spadin as selective inhibitors of TREK-1 potassium channels. By this mechanism, the peptides showed also a strong antidepressant potential by modulating serotonin secretion, neuronal proliferation and survival. At the peripheral level, Spadin has been described in vitro and in vivo as a peptide with an incretin effect comparable to that of exendin-4, an antidiabetic commonly used in the clinic. Thus, following this study and by analogy to the protective effects observed on the neuron, we hypothesized that PE and its derivatives may have a beneficial role in the survival mechanisms in the pancreatic beta-cell.In this thesis, we demonstrate that endogenous PE and its derivatives protect beta cells from apoptosis induced by the chronic presence of the pro-inflammatory and diabetogenic interleukin IL-1β, as well as from an acute toxic shock induced by staurosporine. Furthermore, analysis of intracellular mechanisms reveals that these peptides cause an increase in intracellular calcium concentration, activate the ERK and Akt proliferative and survival pathways, and maintain CREB transcriptional factor activity in a deleterious environment via a calmodulin kinase-dependent pathway.Thus, this work shows that PE and its synthetic derivatives protect the pancreatic beta-cell and initiate virtuous cellular processes through an original PKA-independent signaling pathway, where membrane potential and calcium play a crucial role. This suggests the sortilin-derived peptides as a new class of pancreatic beta-cell protective molecules
4
Moreno, Sébastien. "Le récepteur 3 de la neurotensine/Sortiline dans la régulation de l’état dépressif." Thesis, Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4136/document.
Abstract:
Le trouble dépressif majeur est une pathologie qui atteint 20% de la population et qui représente le premier facteur de morbidité et d’incapacité au niveau mondial. Récemment, le canal potassique TREK-1 est une cible potentielle avérée dans le traitement de la dépression. La délétion de ce canal ou son blocage par un peptide dérivé résultant de la maturation de la sortiline, le propeptide (PE) ou son analogue synthétique la Spadine, résulte en un phénotype de résistance à la dépression. La sortiline est une protéine capable de s’associer à TREK-1 mais également au facteur neurotrophique BDNF important pour la viabilité neuronale et la régulation de l’état dépressif. La sortiline est donc impliquée dans la régulation de l’adressage intracellulaire de TREK-1 et du BDNF. Mes travaux se sont d’abords focalisés sur les conséquences de la délétion du gène codant pour la sortiline (Sort1-/-) sur l’adressage de TREK-1 et du BDNF, et également sur le système neurotensinergique. Les résultats révèlent au niveau cérébral une diminution de l’expression membranaire de TREK-1 et une augmentation de BDNF. L’ensemble de ces modifications conduisent les souris Sort1-/- à développer un phénotype de résistance à la dépression. De plus, ces souris présentent une augmentation de la concentration en neurotensine cérébrale ainsi que de son récepteur 2, ce qui entraine une résistance à la douleur. Par la suite, nous avons montré une diminution de la concentration sérique du PE chez les personnes dépressives, un niveau restauré après traitement avec des antidépresseurs. En conclusion, la sortiline joue un rôle prépondérant dans la régulation du trouble dépressif et aussi dans la nociceptionMajor depressive disorder is a condition that affects 20% of the population and is the leading cause of morbidity and disability worldwide. Recently, the TREK-1 potassium channel has been shown to be a potential target in the treatment of depression. The deletion of this channel or its blocking by a derived peptide resulting from the maturation of Sortilin, propeptide (PE), or its synthetic analogue Spadin, results in a phenotype of resistance to depression in mice. Sortilin is a protein able to bind with TREK-1 but also with the neurotrophic factor BDNF, an important factor for neuronal viability and depressive state regulation. Sortilin is therefore involved in regulating the intracellular addressing of TREK-1 and BDNF. Initially, my work focused on the consequences of the deletion of the Sortilin gene (sort1-/-) on the TREK-1 and BDNF addressing, and the neurotensinergic system. The results showed a decrease in TREK-1 membrane expression at the cerebral level and an increase in BDNF. All of these changes lead the Sort1-/- mice to develop a phenotype of resistance to depression. In addition, these mice show an increase in brain neurotensin concentration and its receptor 2, leading to increased resistance to pain perception. In a second phase, I was interested in whether PE, a potential antidepressant, showed serum variations in depressed patients and could be an indicator of depressive syndrome. We showed that the serum PE level is significantly reduced in depressed people, a level restored after treatment with antidepressants. In conclusion, Sortilin plays a major key in the regulation of depressive disorder and also in nociception
5
Moreno, Sébastien. "Le récepteur 3 de la neurotensine/Sortiline dans la régulation de l’état dépressif." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4136.
Abstract:
Le trouble dépressif majeur est une pathologie qui atteint 20% de la population et qui représente le premier facteur de morbidité et d’incapacité au niveau mondial. Récemment, le canal potassique TREK-1 est une cible potentielle avérée dans le traitement de la dépression. La délétion de ce canal ou son blocage par un peptide dérivé résultant de la maturation de la sortiline, le propeptide (PE) ou son analogue synthétique la Spadine, résulte en un phénotype de résistance à la dépression. La sortiline est une protéine capable de s’associer à TREK-1 mais également au facteur neurotrophique BDNF important pour la viabilité neuronale et la régulation de l’état dépressif. La sortiline est donc impliquée dans la régulation de l’adressage intracellulaire de TREK-1 et du BDNF. Mes travaux se sont d’abords focalisés sur les conséquences de la délétion du gène codant pour la sortiline (Sort1-/-) sur l’adressage de TREK-1 et du BDNF, et également sur le système neurotensinergique. Les résultats révèlent au niveau cérébral une diminution de l’expression membranaire de TREK-1 et une augmentation de BDNF. L’ensemble de ces modifications conduisent les souris Sort1-/- à développer un phénotype de résistance à la dépression. De plus, ces souris présentent une augmentation de la concentration en neurotensine cérébrale ainsi que de son récepteur 2, ce qui entraine une résistance à la douleur. Par la suite, nous avons montré une diminution de la concentration sérique du PE chez les personnes dépressives, un niveau restauré après traitement avec des antidépresseurs. En conclusion, la sortiline joue un rôle prépondérant dans la régulation du trouble dépressif et aussi dans la nociceptionMajor depressive disorder is a condition that affects 20% of the population and is the leading cause of morbidity and disability worldwide. Recently, the TREK-1 potassium channel has been shown to be a potential target in the treatment of depression. The deletion of this channel or its blocking by a derived peptide resulting from the maturation of Sortilin, propeptide (PE), or its synthetic analogue Spadin, results in a phenotype of resistance to depression in mice. Sortilin is a protein able to bind with TREK-1 but also with the neurotrophic factor BDNF, an important factor for neuronal viability and depressive state regulation. Sortilin is therefore involved in regulating the intracellular addressing of TREK-1 and BDNF. Initially, my work focused on the consequences of the deletion of the Sortilin gene (sort1-/-) on the TREK-1 and BDNF addressing, and the neurotensinergic system. The results showed a decrease in TREK-1 membrane expression at the cerebral level and an increase in BDNF. All of these changes lead the Sort1-/- mice to develop a phenotype of resistance to depression. In addition, these mice show an increase in brain neurotensin concentration and its receptor 2, leading to increased resistance to pain perception. In a second phase, I was interested in whether PE, a potential antidepressant, showed serum variations in depressed patients and could be an indicator of depressive syndrome. We showed that the serum PE level is significantly reduced in depressed people, a level restored after treatment with antidepressants. In conclusion, Sortilin plays a major key in the regulation of depressive disorder and also in nociception
6
Massa, Fabienne. "Rôle du système neurotensinergique dans la prolifération et l'adhésion des cellules cancéreuses de colon." Nice, 2011. http://www.theses.fr/2011NICE4055.
Abstract:
La neurotensine (NT) est un peptide qui peut agir autant en périphérie qu’au niveau du système nerveux central. Deux de ses récepteurs appartiennent à la famille des récepteurs à sept domaines transmembranaires couplés aux protéines G (NTSR1 et NTSR2) tandis que le troisième est un récepteur à un seul segment transmembranaire appartenant à la famille des récepteurs de type I (NTSR3 ou sortiline). La NT et le NTSR1 sont très impliqués dans la progression tumorale et sont surexprimés dans un très grand nombre de cancer. Les effets prolifératifs de la NT résultent de l’activation du NTSR1 qui peut transactiver le récepteur de l’Epidermal Growth Factor (EGF). Nous avons démontré que, dans les cellules HT29, des adénocarcinomes de colon humain, la NT ne transactive pas l’EGFR pour induire la prolifération cellulaire. Quant au NTSR3, il est impliqué dans l’adressage des protéines, la prolifération, la différentiation. . . De plus, une fois à la membrane plasmique, le NTSR3 peut être hydrolysé et libéré sous forme soluble dans le milieu extracellulaire (sNTSR3). Lors de ma thèse, j’ai déterminé que le sNTSR3 était une molécule biologiquement active puisqu’elle induit une libération rapide du calcium intracellulaire. Le sNTSR3 active des voies de signalisation intracellulaires comme le complexe FAK-Src, la PKCα et la voie Pi3K aboutissant à une augmentation de l’adhésion des cellules cancéreuses. Par ailleurs, le sNTSR3 augmente l’expression de la E-Cadhérine, l’espacement entre les cellules et potentialise les effets le l’EGF sur la prolifération cellulaire. Ces données nous indiquent que le sNTSR3 serait une molécule impliquée dans la progression tumoraleNeurotensin (NT) can act in periphery and in the central nervous system. Two of this receptors are G protein coupled receptors (NTSR1 and NTSR2) and the third, is a type I receptor with one transmembrane domain (NTSR3 or sortilin). NT and NTSR1 are both implicated in tumoral progression and there are overexpressed in a lot of cancer. NT induces proliferation of cancerous cells which are mediated by NTSR1 which may transactivate the Epidermal Growth Factor Receptor (EGFR). We demonstrated that NT doesn’t transactivate the EGFR in HT29 cell line, a human adenocarcinoma of colon. NTSR3 is a multifunctional protein, is implicated in sorting of proteins, proliferation, differenciation… Moreover, once at the plasma membrane, NTSR3 can be hydrolysed and freed in a soluble form, in the extracellular medium (sNTSR3). During my PhD, I demonstrated that the sNTSR3 is an active molecule with a biological activity, as it induces the release of intracellular calcium. The sNTSR3 activates intracellular signaling like the complex FAK-Src, PKCα and Pi3K pathway, leading to an increase in cancerous cell adhesion. Furthermore, sNTSR3 increases E-Cadherin expression, space between cells and enhances the proliferation induced by EGF. Taken together, these results indicate that the soluble form of NTSR3 can be implicated in tumoral progression
7
Hassan, Noor. "Characterization and engineering of carbohydrate-active enzymes for biotechnological applications." Doctoral thesis, KTH, Industriell bioteknologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-165613.
Abstract:
Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry. In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I). To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS. In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile. Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.QC 20150429
8
Niemelä, Onni. "Aminoterminal propeptide of type III procollagen and basement related antigens in alcoholic liver disease." Oulu : University of Oulu, 1985. http://catalog.hathitrust.org/api/volumes/oclc/14472875.html.
9
Cacciatore, Angela [Verfasser], and Hans-Ruprecht [Akademischer Betreuer] Neuberger. "Prokollagen Propeptide : Marker für atriale Fibrose und Vorhofflimmern? / Angela Cacciatore. Betreuer: Hans-Ruprecht Neuberger." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2013. http://d-nb.info/1052907148/34.
10
Iyappan, Saravanakumar. "The function of the beta6/Pre7 propeptide for 20S proteasome biogenesis in baker's yeast." [S.l. : s.n.], 2004. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11612026.
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Larkin, Heidi. "Rôle de Calnuc dans le triage endosomial des récepteurs lysosomiaux et implication potentielle dans les maladies du lysosome." Thèse, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8204.
Abstract:
Résumé : Calnuc est une protéine ubiquitaire qui lie le calcium et qui est présente au réseau trans-golgien (TGN) ainsi qu'aux endosomes. Notre groupe a précédemment mis en évidence le rôle de Calnuc dans le transport de Low density lipoprotein receptor-related protein 9 (LRP9), un récepteur aux lipoprotéines de faible densité qui cycle entre le TGN et les endosomes. Les récepteurs lysosomiaux au mannose-6-phosphate (MPR) et Sortiline sont bien caractérisés et empruntent également cette voie. À l'image de LRP9, nous avons montré que Calnuc prévient leur dégradation aux lysosomes en participant à leur recyclage à partir des endosomes vers le TGN. En fait, Calnuc est importante pour l'activation et l'association membranaire de Rab7, une petite protéine G qui recrute ensuite le complexe Rétromère responsable du transport rétrograde des récepteurs. La glycoprotéine lysosomiale Ceroid lipofuscinosis neuronal 5 (CLN5) est également impliquée dans ce processus. La structure et la fonction de cette dernière n'étant pas clairement définies, nous avons établi qu'elle est synthétisée sous forme d’une glycoprotéine transmembranaire de type II, mais son domaine N-terminal cytoplasmique et son segment transmembranaire sont rapidement éliminés suivant le clivage du peptide signal de manière à former une protéine CLN5 mature fortement associée à la membrane par une hélice amphipathique (AH). La compréhension des propriétés de base de CLN5 est particulièrement pertinente puisque la protéine est impliquée dans certaines variantes de céroïdes-lipofuscinoses neuronales (NCL), une maladie neurodégénérative rare causée par une surcharge des lysosomes. D'ailleurs, nos données indiquent que les mutants pathologiques de CLN5 dépourvus de cette AH perdent leur association membranaire, sont retenus au réticulum endoplasmique et sont rapidement dégradés. En raison de la similitude des fonctions de Calnuc et de CLN5 au niveau du triage endosomial, nous avons exploré le lien entre les deux protéines. Calnuc cytosolique et CLN5 luminale semblent former un complexe, par l'intermédiaire de la protéine transmembranaire CLN3, de façon à influencer l'activité de Rab7. CLN3 étant aussi associée aux NCL, nous avons finalement exploré la potentielle implication de Calnuc dans la maladie. L'absence de Calnuc entraîne des phénotypes cellulaires typiques des NCL comme un engorgement des lysosomes, une accumulation de matériel autofluorescent et une augmentation de l'autophagie. Les niveaux protéiques de Calnuc sont diminués dans toutes les lignées de fibroblastes de patients atteints de NCL disponibles ce qui indique que Calnuc pourrait être impliquée dans certains types de NCL. La présente thèse couvre donc la découverte de la fonction de Calnuc dans le transport intracellulaire, jusqu'à son implication potentielle dans les NCL, de même qu'une étude topologique de CLN5.Abstract : Calnuc is a ubiquitous Ca2+-binding protein present on the trans-Golgi network (TGN) and endosomes. We previously highlighted the role of Calnuc in the transport of Low density lipoprotein receptor-related protein 9 (LRP9), a low density lipoprotein (LDL) receptor that cycles between the TGN and endosomes. Lysosomal receptors mannose-6-phosphate receptor (MPR) and Sortilin are well-characterized and also use the TGN-to-endosome trafficking pathway. Similarly to LPR9, we showed that Calnuc prevent their degradation in lysosomes by acting in their recycling from endosomes to the TGN. In fact, Calnuc is a important for the activation and the membrane association of Rab7, a small G protein which then recruit the Retromer complex known to be responsible for the retrograde transport of receptors. Lysosomal glycoprotein Ceroid lipofuscinosis neuronal 5 (CLN5) is also involved in this process. Because its structure and function have not yet been clearly defined, we established that it is synthesized as a type II transmembrane (TM) glycoprotein, but its cytoplasmic N-terminus and TM segment are rapidly removed following signal-peptide cleavage to generate mature CLN5 which is tightly associated to membrane through an amphipathic helix (AH). The understanding of the basic properties of CLN5 is particularly important given that CLN5 is involved in some variants of neuronal ceroid lipofuscinosis (NCL), a rare neurodegenerative disease caused by lysosomal overload. Moreover, our data indicate that CLN5 pathological mutants deprived of AH lose their membrane association, are retained in the endoplasmic reticulum, and are rapidly degraded. Based on the similarity featured by Calnuc and CLN5 in endosomal sorting, we explored the link between these two proteins. Cytosolic Calnuc and luminal CLN5 seem to form a complex, through the transmembrane protein CLN3, in order to influence the activity of Rab7. As CLN3 is also associated with NCL, we finally explored the potential involvement of Calnuc in this disease. Canuc depletion leads to typical NCL phenotypes such as lysosome enlargement, accumulation of autofluorescent material and of an increased of autophagy induction. Canuc's levels are decreased in all fibroblasts cell lines of NCL patients available indicating that Calnuc could be involved in some types of NCL. This thesis thus covers the discovery of the function of Calnuc in intracellular transport up to its potential involvement in the NCL, as well as a topological study CLN5.
12
Blondy, Sabrina. "Implication de la sortiline dans la résistance des cellules cancéreuses au 5-Fluorouracile. Cas du cancer colorectal." Thesis, Limoges, 2019. http://www.theses.fr/2019LIMO0020.
Abstract:
Le cancer colorectal (CCR) représente la seconde cause de mortalité par cancer dans le monde. Les tumeurs sont classées selon les grades histologiques (état de différenciation cellulaire), en bas et haut grades. Les grades les plus avancés sont associés à de faibles taux de survie globale après chimiothérapie à base de 5-Fluorouracile (5-FU). Les taux de réponse aux traitements basés sur l’utilisation du 5-FU en première ligne de traitement demeurent relativement faibles (20–30%) et associés à de mauvais pronostics et des échappements thérapeutiques, dus à la résistance des cellules cancéreuses. Ces données soulignent donc la nécessité d’identifier de nouveaux biomarqueurs de résistance des cellules de CCR au 5-FU. Les travaux réalisés au cours de cette thèse ont porté sur l’étude de la sortiline, une protéine « multi-tâches » appartenant à la famille des récepteurs à domaine VPS10P avec sorLA et sorCS1-3, qui agit comme un (co)récepteur et un régulateur du traffic et de la sécrétion de protéines. La sortiline (et non sorLA), constitue un biomarqueur d’agressivité dans le CCR et est de mauvais pronostique. In vivo et in vitro, au sein des tumeurs et des cellules de CCR (lignées cellulaires et cultures primaires) résistantes au 5-FU, seule la sortiline apparait surexprimée au niveau transcriptomique et protéique. Ces données indiquent que la sortiline constitue également un biomarqueur de résistance au 5-FU, quels que soient les grades, stades et statuts mutationnels. Cette surexpression de la sortiline est majoritairement localisée au sein de l’appareil de Golgi des cellules résistantes et semble être régulée transcriptionnellement via une down-régulation d’ATF-3. L’inhibition génétique (shRNA) ou pharmacologique (CADA) de la sortiline potentialise les effets cytotoxiques du 5-FU, résultant en une forte augmentation de la mort cellulaire. La sortiline semble donc être également impliquée dans la résistance des cellules de CCR au 5-FU ; l’expression de sorLA ne variant pas après son inhbition. Ces résultats suggèrent que la sortiline pourrait constituer un biomarqueur d’agressivité et de résistance des cellules de CCR au 5-FU, mais aussi une nouvelle cible thérapeutique potentielleWorldwide, colorectal cancer (CRC) is the second leading cause of cancer-related deaths. Colorectal adenocarcinomas are divided into low and high grades. More advanced tumor grade is associated with poorer global survival following 5-fluorouracil (5-FU)-based systemic chemotherapy. Even when 5-FU is used as the first-line molecule, responsiveness is only 20–30%, and drug resistance contributes to both poor patient prognosis and relapses, emphasizing the need to identify biomarkers involved in this process. In this study, we focused on sortilin, a multifunctional protein among VPS10P domain-related receptors with sorLA and sorCS1-3, that acts as a receptor and co-receptor as well as a regulator of intra- and extracellular protein sorting and trafficking. We obtained evidence that sortilin, but not sorLA, is a biomarker of CRC aggressiveness associated with poor clinical outcomes. In vivo and in vitro, in both 5-FU–resistant CRC cell lines and primary cultures, only sortilin was overexpressed at both protein and mRNA levels, indicating that it could serve as a biomarker of 5-FU resistance regardless of tumor grade and mutational status. Sortilin overexpression is especially localized in Golgi apparatus of 5-FU resistant cells and seems to be transcriptionally regulated through ATF-3 downregulation. Genetic (shRNA) and pharmacological (CADA) inhibition of sortilin potentiated 5-FU–induced cytotoxicity, resulting in a significant increase in cancer cell eradication, implying that sortilin is also actively involved in 5-FU resistance. Moreover, sorLA expression is unchanged upon sortilin inhibition. Our findings indicate that sortilin is a promising biomarker of 5-FU resistance in CRC, as well as a potential therapeutic target for overcoming 5-FU resistance
13
Fabre, Emmanuelle. "Roles du propeptide dans la secretion de la protease alcaline exocellulaire de la levure yarrowia lipolytica." Paris 6, 1992. http://www.theses.fr/1992PA066140.
Abstract:
Notre travail a permis la caracterisation des roles que le propeptide de la protease alcaline exocellulaire (aep) de la levure yarrowia lipolytica assure au cours de la secretion et de l'activation de l'aep. La protease est synthetisee sous forme de prepro-proteine. Les maturations successives du precurseur par un signal peptidase, une dipeptidyl aminopeptidase et une endoprotease de type kex2p entrainent la secretion de la protease mature et active dans le milieu exocellulaire. Nous montrons que le propeptide assume une fonction essentielle lors du transit de la protease a travers les compartiments precoces de la voie de secretion. Des mutations creees dans la sequence propeptidique deletions ou destruction du site unique de n-glycosylation conduisent a l'accumulation intracellulaire de formes precurseurs inactives. Celles-ci franchissent la premiere etape du parcours de secretion car le clivage de leur peptide signal s'accompagne d'une translocation complete dans le lumen du re. Elles sont en revanche incapables d'atteindre l'appareil de golgi ou devrait s'effectuer leur maturation par la dpap et l'endoprotease de type kex2p. Elles ne parviennent pas en outre a acquerir la conformation finale de la protease, resistante a la proteinase k. Nous definissons la contribution du propeptide au cours du transport de l'aep grace a une experience de complementation en trans. La co-expression d'une construction codant pour le seul peptide pro, et de celle codant pour un precurseur pro-delete, provoque le retablissement du transit intracellulaire du precurseur mute. Dans une souche trans-complementee comme dans une souche sauvage, la secretion de la forme mature s'accompagne, en quantite equimolaire, de celle du propeptide. L'analyse des cinetiques de secretion etablit une etape limitante lors du sauvetage des precurseurs mutes: avant la proteolyse par kex2p. Ces resultats suggerent que le propeptide est necessaire a une etape precoce du parcours de secretion afin de procurer aux precurseurs une conformation compatible avec leur transport. Lorsque leur transit est retabli, les precurseurs pro-deletes acquierent une activite enzymatique partielle. L'activite recuperee s'accompagne d'un changement de conformation car les formes secretees sont devenues resistantes a la proteinase k. Ceci suggere que le propeptide assume une seconde fonction aupres des precurseurs proteasiques, au cours de l'acquisition d'une configuration active
14
Kawar, Susannah Louise. "Inhibition of myostatin (GDF-8) via myostatin propeptide minigenes : a potential gene therapy for Duchenne muscular dystrophy." Thesis, Royal Holloway, University of London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497826.
Abstract:
The objective of this project was to evaluate the alleviation of muscle wasting phenotypes by hepatic delivery and expression of a recombinant mutated myostatin propeptide mini gene in wild type mice. Initial work focused on using naked plasmid DNA as a vector for delivery of the therapeutic gene. Various β-galactosidase reporter plasmids were tested including pCMVβ, pCAGGSβ, pA2β and a liver specific pSV40 Albuminβ for systemic gene transfer to liver tissue. Following hydrodynamic intravenous administrations, pCAGGSβ proved to be the optimum plasmid and a cDNA encoding a mutated form of the myostatin propeptide (ProFcD/A) was subsequently cloned into a CAGG based plasmid (pAAVCAGGProFcD/A). The expression and bioactivity of the propeptide were assessed using in vitro cell proliferation experiments which showed the ProFcD/A was capable of increasing both proliferation and differentiation in a range of cell types. An initial in vivo study involved intravenous hydrodynamic injections of pAAVCAGGProFcD/A into MF-1 male mice and monitoring growth rates over time. A significant increase was observed in growth rate of the treated mice compared to controls with an accompanying increase in tibialis anterior (TA) muscle mass. Fibre analysis revealed pAAVCAGGProFcD/A treated mice to contain significantly larger fibres than controls owing to muscle hypertrophy. Transgene product levels were detected strongly 7 days post injection in the serum, which declined steadily over the following 21 days. By day 28, levels of transgene product were barely detectable. There was no indication response to either the vector or transgene product. The pAAVCAGGProFcD/A was then used to produce an AAV serotype 8 viral vector containing the ProFcD/A gene (AAV8ProFc) and assessed in initial intravenous tail vein injections of AAV8ProFc into MF-1 male mice. Growth rates were measured over four weeks with a significant increase in the AAV8ProFc treated mice compared to the pCAGG empty plasmid controls. In addition, significant weight increases in both TA and gastrocnemius muscles were seen in AAV8ProFc treated mice over the pCAGG control mice at week 4. Serum showed high levels of transgene expression after 7 days which was sustained up to 28 days post injection with no indication of immune response. Levels of the myogenic protein MyoD also remained higher in AAV8ProFc than pCAGG treated mice. Future studies may involve intravenous injection of both pAAVCAGGProFcD/A and AAV8ProFc into mdx mice, with subsequent monitoring of growth rates, creatine kinase levels, tissue necrosis and muscle strength over time.
15
Al-Akhrass, Hussein. "Un rôle inédit de la sortiline dans le contrôle du transport rétrograde de l'EGFR pour limiter la croissance tumorale." Thesis, Limoges, 2017. http://www.theses.fr/2017LIMO0035/document.
Abstract:
Le cancer du poumon est le troisième cancer le plus fréquent chez les femmes et le deuxième chez les hommes, il est la cause principale de décès par cancer dans le monde, avec une mortalité annuelle supérieure à 1 million. Malgré des progrès remarquables dans la thérapie ciblée, la majorité des patients atteints de cancer du poumon sont diagnostiqués dans un stade avancé où ils ne connaissent pas d'amélioration significative de leur survie globale. Les récepteurs à domaine tyrosine kinase, tels que le récepteur du facteur de croissance épidermique (EGFR), traduisent les informations du microenvironnement dans la cellule en activant des voies de signalisation homéostatiques. L'internalisation et la dégradation de l'EGFR suite à la liaison du ligand limitent l'intensité de la signalisation proliférative, ce qui contribue à maintenir l'intégrité cellulaire. Dans les cellules cancéreuses, la dérégulation du trafic de l’EGFR aboutit à des effets divers sur la progression tumorale. Dans cette étude, nous avons identifié la sortiline comme un régulateur clé de l'internalisation de l'EGFR à partir de la membrane plasmique. La perte de l’expression de la sortiline dans les cellules tumorales augmente la prolifération cellulaire en soutenant la signalisation de l’EGFR à la surface de la cellule, ce qui accélère la croissance tumorale. Chez les patients atteints de cancer du poumon, l'expression de la sortiline diminue avec l’augmentation du grade pathologique et l’expression de son gène, SORT1, est fortement corrélée à une meilleure survie globale, en particulier chez les patients présentant une forte expression de l'EGFR. Ainsi, la sortiline représente un nouveau régulateur du trafic intracellulaire de l’EGFR, elle agit en contrôlant l'internalisation de ce récepteur et en limitant la croissance tumoraleLung cancer is the third most common cancer in women and the second in men, it is the leading cause of cancer-related death worldwide, with an annual mortality of more than 1 million. Despite remarkable advances in targeted therapy, the majority of patients with lung cancer are diagnosed at an advanced stage where they do not experience a significant improvement in overall survival. Tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) transduce information from the microenvironment into the cell and activate homeostatic signalling pathways. Internalisation and degradation of EGFR after ligand binding limits the intensity of its proliferative signalling, thereby helping to maintain cell integrity. In cancer cells, deregulation of EGFR trafficking has a variety of effects on tumour progression. Here, we report that sortilin is a key regulator of EGFR internalisation. Loss of sortilin in tumour cells promotes cell proliferation by sustaining EGFR signalling at the cell surface, ultimately accelerating tumour growth. In lung cancer patients, sortilin expression decreases with increased pathologic grade, and the expression of SORT1 (the gene encoding sortilin) is strongly correlated with a better survival, notably in patients with high EGFR expression. Thus, sortilin is a novel regulator of EGFR intracellular trafficking acting by controlling receptor internalisation and limiting tumour growth
16
Koclar, Gulden. "Pcr Cloning And Heterologous Expression Of Scytalidium Thermophilum Laccase Gene In Aspergillus Sojae." Master's thesis, METU, 2005. http://etd.lib.metu.edu.tr/upload/3/12607079/index.pdf.
Abstract:
In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding intervening sequences is very GC-rich (60.8% G+C) and encodes a preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 amino acid predicted propeptide. lccS gene was amplified using specific primers to exclude the signal and pro-peptide coding regions and ligated to expression vector pAN52-4. The recombinant plasmid was used to transform Aspergillus sojae ATCC11906 (pyrG-). Heterologuos expression was observed in glucose-containing media, under the control of the glyceraldehydes 3-phosphate dehydnogenese promoter and the secretion signal of glucoamylase gene. Laccase gene is an important step towards the high level expression of this enzyme in a GRAS eucaryotic host and for further biotransformation and enzyme engineering studies. In this study also bioinformatic analysis of N-terminal and C-terminal propeptide cleavage sites of fungal proteins including laccases were studied.
17
Gäddnäs, F. (Fiia). "Insights into healing response in severe sepsis from a connective tissue perspective:a longitudinal case-control study on wound healing, collagen synthesis and degradation, and matrix metalloproteinases in patients with severe sepsis." Doctoral thesis, University of Oulu, 2010. http://urn.fi/urn:isbn:9789514262548.
Abstract:
Abstract
Sepsis is a major challenge for healing responses maintaining homeostasis. Coagulation and inflammation are activated at the whole-body level, even in undamaged tissues. Despite constantly growing knowledge and advances in care, high mortality in severe sepsis remains. It was hypothesised that tissue regeneration processes may also be altered in severe sepsis.
The study population consisted of 44 patients with severe sepsis and 15 healthy controls. Serum samples were obtained during ten days of severe sepsis and twice again, three months and six months later. Experimental suction blisters were performed twice during severe sepsis and at 3 and 6 months. Serum samples were obtained and suction blisters were induced once in controls. Biochemical analyses were performed to assess the level of procollagen I and III aminoterminal propeptides (PINP, PIIINP), reflecting the synthesis of corresponding collagens; in serum and suction blister fluid. In addition collagen I degradationproduct in serum was measured. Physiological measurements of transepidermal water loss and blood flow were done in order to evaluate the re-epithelisation rate and blood flow in an experimental wound. Levels of matrix metalloproteinases (MMPs) 2, 8 and 9 were measured from serum and suction blister fluid.
Decrease in water evaporation from an experimental blister wound was slower in sepsis than in controls. On the fourth day the sepsis patients had higher blood flow in the blister wound than the controls (both in the healing wound and in the newly induced wound). The procollagen III aminoterminal propeptide (PIIINP) levels were increased in serum in severe sepsis, whereas procollagen I aminoterminal propeptide (PINP) levels were not, making up a pronounced PIIINP/PINP ratio. PIIINP and PINP levels were associated with disease severity and outcome. In addition, collagen I degradation measured with ICTP assay was increased in severe sepsis and PINP/ICTP ratio was lower. Furthermore, the overall protein concentration and PINP and PIIINP levels were low in suction blister fluid, which implies that the balance of the extracellular matrix consistence is disturbed in uninjured skin in severe sepsis. Then again in survivors the levels of PINP and PIIINP were up-regulated at three months but returned to normal by six months. MMP-9 levels in serum and skin blister fluid were lower in severe sepsis than in controls during the ten days studied. The MMP-2 levels were found to be increased both in serum and in skin blister fluid in severe sepsis in comparison to the controls and MMP-2 was associated with disease severity and outcome. MMP-8 was increased in serum and in skin blister fluid.
In conclusion, the balance of collagen turnover is altered in severe sepsis in serum and skin and epidermal re-epithelisation is delayed. The levels of MMP-2 and MMP-8 are increased whereas levels of MMP-9 are depressed.
18
Cioffi, U. "Effects of chronic inflammatory bowel diseases on left ventricular structure and function : dottorato di ricerca in fisiopatologia chirurgica." Doctoral thesis, Università degli Studi di Milano, 2003. http://hdl.handle.net/2434/69556.
Abstract:
Background: Experimental evidences suggest an increased collagen deposition in inflammatory bowel diseases (IBD). In the hypothesis that chronic inflammation and increased collagen metabolism are reflected also in the systemic circulation, we aimed this study to evaluate the effects on left ventricular wall structure by assessing splanchnic and systemic collagen metabolism (procollagen III), its deposition (ultrasonic tissue characterization), and cardiac function (echocardiography) in patients with different longstanding history of IBD, before and after surgery.Methods: Twenty-seven patients affected by active IBD, 17 with Crohn and 10 with ulcerative colitis, submitted to surgery were enrolled in the study in a double blind fashion. They were studied before the surgical operation and will be controlled at 6 and 12 months after surgery. A control group of 14 healthy age and gender-matched subjects were also studied. Blood was drawn from a peripheral vein prior to surgery. Immediately before intestinal resection, additional samples were drawn for the mesenteric vein draining the affected intestinal segment . Before surgery a transthoracic echocardiogram was recorded for the subsequent determination of cardiac function and collagen deposition. Results: Before surgery, serum PIIIP level in IBD patients was 5.0 ± 1.9 vs 2.7 ± 0.7 mcg/l of healthy controls (p=0.0001). No significant differences were found when comparing IBD subgroups, respectively Crohn = 5.0 ± 1.6 vs ulcerative colitis = 4.9 ± 2.4 mcg/l, (p=ns). During surgery, serum PIIIP level in IBD patients was 5.5 ± 2.6 mcg/l. No significative differences are found when coumparing the two subgroups, respectively Chron 5.4 +2.3 is ulcerative colits 5.7+3.1 mcg/l, (p=ns). Conclusion : the present study provides evidence that the altered intestinal collagen metabolism in Chrons diseases and ulcerative colits is reflected also in the local systemic circulation.
19
Demont, Yohann. "Le ProNGF dans le cancer du sein." Thesis, Lille 1, 2009. http://www.theses.fr/2009LIL10181/document.
Abstract:
Le NGF (nerve growth factor), membre prototypique des neurotrophines, a été largement décrit pour son rôle dans le système nerveux où il active la survie et la différenciation neuronale via son récepteur tyrosine kinase TrkA et le récepteur commun à toutes les neurotrophines p75NTR. Plus récemment, il a été décrit que le précurseur du NGF, le proNGF, peut être sécrété et induire la mort neuronale en se liant à p75NTR et à la sortiline. Plusieurs études ont montré l’implication des neurotrophines dans la pathologie tumorale et les travaux pionniers de notre laboratoire ont démontré plus particulièrement l’effet mitogénique et l’action anti-apoptotique du NGF dans le cancer du sein. Néanmoins, aucune donnée n’a été rapportée concernant le rôle du proNGF dans ce cancer, c’est pourquoi nous avons décidé de mener cette étude. Nous avons démontré que le proNGF est produit et sécrété par les cellules cancéreuses mammaires. Ainsi, des western-blots ont permis de mettre en évidence une bande de 25 kDa qui diminue après transfection de siRNA contre le proNGF. De plus, par immunocytochimie, nous avons observé que le proNGF est localisé dans des vésicules intracellulaires qui sont libérées suite à un traitement avec un inducteur de sécrétion comme la ionomycine. En outre, le proNGF est détecté par spectrométrie de masse dans le milieu conditionné des cellules cancéreuses. Par ailleurs, en analysant 1423 biopsies humaines normales et cancéreuses par immunohistochimie, nous avons observé, en comparaison avec les tissus bénins et normaux, une surproduction du proNGF dans les cellules épithéliales mammaires cancéreuses associée avec l’envahissement lymphatique. De façon intéressante, en utilisant des chambres de Boyden, nous avons validé, in vitro, nos observations concernant la métastase lymphatique, en montrant que le proNGF permet de stimuler la migration et l’invasion des cellules cancéreuses de façon autocrine. Enfin, l’invalidation de ses récepteurs a révélé le rôle essentiel de la sortiline pour conduire l’effet pro-invasif du proNGF alors que nos données ont montré que p75NTR n’intervenait pas. En conclusion, notre travail démontre que le proNGF est produit dans les tumeurs mammaires où il stimule l’invasion des cellules cancéreuses mammaires participant ainsi au processus métastatique. De plus amples investigations précliniques et cliniques devront être menées afin de déterminer la valeur pratique du proNGF comme un marqueur et/ou cibleNerve growth factor (NGF), the prototypical neurotrophin, has extensively been studied for its role in the nervous system where it activates neuron survival and differentiation through the tyrosine kinase receptor TrkA and the neurotrophin receptor p75NTR. More recently, it was described that the precursor of NGF (proNGF) can be secreted and elicits neuronal apoptosis by engaging with p75NTR and sortilin receptors. Outside of the nervous system, several studies have indicated the implication of neurotrophins in tumors and in particular, our laboratory was pioneer in demonstrating the pro-survival and mitogenic effects of NGF for breast cancer cells. Nevertheless, nothing was reported about the role of the proNGF in breast cancer and this is what we have studied here. We demonstrated that proNGF was produced and secreted by breast cancer cells. Western blots analysis revealed a 25 kDa band that was decreased after transfection with siRNA against proNGF. Immunocytochemical observation showed proNGF localization in intracellular vesicles which were released upon treatment with the inducer of secretion ionomycin and proNGF was detected by mass spectrometry in conditioned medium of breast cancer cell. In addition, immunohistochemical analysis of 1423 normal vs human tumor biopsies showed proNGF overproduction in the epithelial compartment of malignant tumors when compared with benign and normal tissues, with a relationship to lymph node metastasis. Interestingly, in vitro, using Boyden chambers and RNA interference we showed that proNGF was a potent autocrine stimulator of breast cancer cell migration and invasion. Besides, impairment of its receptors revealed the essential role of sortilin in conducting proNGF pro-invasive effect whereas p75NTR was found not involved. In conclusion, our work demonstrates that proNGF is produced in breast tumors where it can stimulate cancer cell invasion, hence participating to metastasis. Further preclinical and clinical investigations will now have to be performed to determine the practical value of proNGF as a marker and/or therapeutic target
20
Boice, Emily. "The Role of the Propeptide and its Residues in Activation and Secretion of Elastase, an M4 Metalloprotease Secreted by Pseudomonas aeruginosa." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/217.
Abstract:
Pseudomonas aeruginosa secretes several proteases associated with pathogenesis, but the most abundant and active is elastase (M4 metalloendopeptidase). Elastase (lasB), is first synthesized as a preproenzyme, with a signal peptide, an 18-kDa N-terminal propeptide, and a 33-kDa mature domain. The propeptide functions as an intramolecular chaperone that is required for the folding and secretion of elastase, but ultimately is proteolytically removed and degraded. Previous research has identified the conserved residues in the propeptide of elastase as compared to other M4 protease precursors and showed some among them to be important for the production of active elastase. In this project, the ability of the propeptide alone to fold into a defined secondary structure was explored and a molecular model was created. Furthermore, the effects of substitutions on conserved residues in the propeptide of plasmid-encoded lasB pro alleles were assessed by expressing them in a lasB propeptide mutant. The kinetics of elastase activity in culture supernatants was quantitated using a fluorescent substrate, Abz-AGLA-p-Nitro-Benzyl-Amide, to provide an accurate assessment of the effects of mutant propeptides. In vitro refolding studies were also performed to determine the effects of specific substitutions on foldase activity of the propeptide. When wild-type propeptide and mature elastase were denatured as separate proteins in guanidine-HCl buffer and renatured together, restoration of activity of the refolded elastase was measured, which was propeptide-dependent. Several mutant propeptides have now been shown to have defects using this in vitro foldase assay. Additional mutants were near wild-type activity level suggesting their role in recognition by the secretion apparatus. Residue locations were determined on a molecular model of the complex and confirmed the role of the secretion mutants as residues on the exterior. Residues that had diminished ability to refold in the in vitro assay were found to be in the interior parts of the complex, confirming their ability to be critical residues at the interface of the proteins or important in the stability of the propeptide’s intrinsic structure. The goal was to perform a series of comprehensive analyses of the propeptide and its conserved residues in order to determine its role as an intramolecular chaperone.
21
Sloves, Pierre-Julien. "La sortiline et les voies endosomales apparentées sont les éléments clefs pour la biogenèse des organites apicaux et la virulence chez Toxoplasma gondii." Thesis, Lille 2, 2012. http://www.theses.fr/2012LIL2S020/document.
Abstract:
Toxoplasma gondii, l'agent de la toxoplasmose, représente un modèle appartenant aux parasites Apicomplexa, phylum dans lequel on retrouve Plasmodium falciparum, l'agent causal du paludisme, et de nombreux agents opportunistes dangereux. Les parasites de ce phylum possèdent la même organisation avec des organites situés à l'apex, spécialisés dans l'interaction hôte-pathogène et qui sont cruciaux pour permettre l'infection de l'hôte: les rhoptries et micronèmes. Les protéines contenues dans ces organites sécrétoires sont acheminées via le système de type endolysosomal. Toutefois, les récepteurs qui jouent un rôle clé dans le tri des protéines et la biogenèse des organites apicaux restaient à identifier. La sortiline est un récepteur transmembranaire de type I, plus connu chez l'Homme dans le tri, le transport des protéines et ses rôles dans la maladie de Parkinson, d'Alzheimer ou du diabète ont largement été décrits. Nous avons déterminé que l'homologue de la sortiline chez Toxoplasma TgSORTLR pour «Toxoplasma gondii Sortiline Like Receptor» est localisé au niveau de l'appareil de Golgi et du réseau post-Golgien. TgSORTLR se lie spécifiquement aux protéines de rhoptries et de micronèmes à l’aide du domaine N-terminus, probablement au niveau de l'appareil de Golgi. Nous avons montré que le domaine C-terminus est déterminant pour la localisation de TgSORTLR et que l'expression d'une version tronquée de la protéine dépourvue du domaine C-terminus induit la complète délocalisation des protéines de rhoptries et de micronèmes. Nous avons également identifié les protéines cytosoliques qui interagissent avec le domaine C-terminus de TgSORTLR et démontré que ces protéines cytoplasmiques sont pour la plupart connues pour leurs rôles importants dans le transport antérograde ou rétrograde des vésicules du trafic intracellulaire. L’absence de TgSORTLR obtenue grâce à une stratégie de «knock-out » conditionnel entraîne non seulement une totale délocalisation des protéines de rhoptries et de micronèmes mais aussi à une disparition des organites apicaux, ce qui conduit à la forte atténuation de la virulence du parasite chez la souris et surtout à l'absence complète de symptômes toxoplasmiques. Des expériences de complémentations réalisées dans la souche dépourvue de TgSORTLR ont montré que l’extrémité N-terminus (acides aminés 39 à 202) de TgSORTLR est également nécessaire pour sa localisation correcte et pour ses fonctions de récepteur intracellulaire chez T. gondii. Enfin, nous avons caractérisé, par des techniques de « knock in » ou remplacement du gène endogène par une copie étiqueté avec l’épitope HA, puis montré que des protéines homologues de μ1-adaptine, Sec23, Vps9, Vps26 et Vps35 de T. gondii co-localisent et se fixent spécifiquement à TgSORTLR. Ces résultats récents confirment que l'extrémité C- terminus et cytoplasmique du récepteur TgSORTLR se comporte d'une manière similaire aux sortilines des cellules de mammifère. L’étude de ces partenaires de TgSORTLR nous permet de mieux comprendre le trafic de TgSORTLR, de reconstituer en partie le système endolysosomal et son rôle dans le transport et la biogenèse des organites vitaux de T. gondiiToxoplasma gondii, the causative agent of toxoplasmosis belongs to the phylum named Apicomplexa that also contains Plasmodium falciparum, the causative agent of malaria and others parasites such as Cryptosporidium or Eimeria. Apicomplexan parasites are uniquely characterized by specific organelles, rhoptries and micronemes, which are located at the apical end of the parasite. These organelles are involved in the control of host-pathogen interactions. The proteins in these secretory organelles are trafficked through the endolysosomal system. However, the receptors that play key roles in protein sorting and biogenesis of these apical organelles remain to be identified. Sortilin is a type I transmembrane receptor known in humans for protein sorting and trafficking. Here, we report that the homologue of sortilin in T. gondii designated TgSORTLR for “Toxoplasma gondii SORTilin Like Receptor” is localized to Golgi and post-Golgi compartments and transports proteins into rhoptries and micronemes via their specific interactions with its luminal domain. We demonstrate that the C-terminus of TgSORTLR is also important for its subcellular localization through binding to cytosolic components of vesicular trafficking proteins known to be involved in anterograde and retrograde transports. The depletion of TgSORTLR using conditional knock-out strategy causes a complete rmis-localization of proteins of both rhoptries and micronemes, leading to the loss of these apical organelles. These mutants display a strong attenuation of the parasite virulence in mice with the absence of acute toxoplasmosis symptoms. Complementation of the strain lacking TgSORTLR showed that N-terminal region between 39-202 amino acids indicated that the N-terminus, similarly to the C-terminus is essential for its correct localization. We conclude that the full-length TgSORTLR protein is required for its biological functions as intracellular sorting receptor in T. gondii
22
Corbet, Cyril. "Etude de la signalisation du proNGF dans les cellules de cancer du sein." Thesis, Lille 1, 2012. http://www.theses.fr/2012LIL10120/document.
Abstract:
Le NGF (Nerve Growth Factor) induit la croissance des cellules cancéreuses de sein, alors qu’il est sans effet sur les cellules normales. Il participe également au développement tumoral in vivo et est une cible thérapeutique potentielle dans le cancer du sein. Le NGF agit via les récepteurs TrkA et p75NTR. De même, le précurseur du NGF, le proNGF, peut être sécrété et induire la mort neuronale en se liant à p75NTR et à la sortiline. Néanmoins, aucune donnée n’a été rapportée sur l’expression et les effets potentiels du proNGF dans le cancer du sein. Au cours de ma thèse, nous avons démontré que le proNGF est produit et sécrété par les cellules cancéreuses de sein. Nous avons observé une surproduction du proNGF dans les biopsies cancéreuses malignes par rapport aux biopsies normales et aux pathologies bénignes. Cette surexpression est associée à l’envahissement des nœuds lymphatiques.J’ai confirmé, in vitro, que le proNGF, comme le NGF, favorise l’invasion des cellules cancéreuses de sein. Néanmoins, ces effets sont induits via des voies de signalisation distinctes. Ainsi, j’ai montré le rôle essentiel de la sortiline dans l’effet pro-invasif du proNGF. De même, le proNGF, comme le NGF, est capable d’activer la phosphorylation de TrkA mais ceci conduit à des voies de transduction différentes. Une analyse de l’interactome de TrkA a permis d’identifier des protéines différentiellement recrutées en fonction du ligand. Ainsi, l’ensemble des résultats obtenus a permis de mettre en évidence l’intervention du proNGF dans le cancer du sein. La discrimination des voies induites par le proNGF et par le NGF offre la possibilité de nouvelles modulations thérapeutiques dans ce cancerNerve Growth Factor (NGF) induces the growth of breast cancer cells, whereas it has no effect on normal breast epithelial cells. NGF acts also on the tumor development in vivo and is considered as a potential therapeutic target in breast cancer. To exert its effects, NGF binds the receptors TrkA and p75NTR. More recently, proNGF, the NGF precursor, has been found to be secreted and to induce neuronal cell death by binding to a sortilin/p75NTR complex. Nevertheless, so far no data has been reported on the expression and the putative effects of proNGF in breast cancer cells. During my thesis, we have demonstrated that proNGF is produced and secreted by breast cancer cells. Moreover, we revealed an overproduction of proNGF in malignant breast tumors, in comparison to benign tumors and normal biopsies. Interestingly, a statistically significant association was obtained between the presence of proNGF and lymph node invasion by breast cancer cells. I confirmed that proNGF, but also NGF, induces in vitro breast cancer cell invasion. However, both proNGF and NGF induce their effects through distinct signaling pathways. I found that sortilin is essential for the proNGF pro-invasive effect while p75NTR is not necessary. Interestingly, proNGF, like NGF, is able to activate TrkA phosphorylation but this leads to different transduction cascades. TrkA interactome analysis allowed the identification of proteins differentially recruited on the receptor, depending on the ligand. Thus, our results demonstrate the first implication of proNGF in breast cancer. Deciphering of the pathways induced by proNGF and NGF would give the opportunity for new therapeutic modulations in breast cancer
23
López, Pelegrín Maria del Mar. "Mechanisms of proteolytic activity regulation exerted via a unique propeptide in matrix metalloproteinases and intra/intermolecular interactions in a novel family of minimal gluzincins." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/347212.
Abstract:
Metallopeptidases are major players in the physiology and pathology of all living organisms. Their exquisite regulation is therefore essential for proper function and to prevent misdirected temporal and/or spatial proteolytic activity, which may lead to disease. This regulation is achieved through a wide variety of mechanisms, including their biosynthesis as inactive precursors, also known as zymogens. In the present thesis, various mechanisms of metallopeptidase regulation were studied by using a combination of biochemical, biophysical, and structural techniques. In the first project, the crystal structure of a zymogen fragment of Tannerella forsythia karilysin revealed the shortest propeptide reported to date for a metallopeptidase. Additional biochemical and biophysical assays allowed ascertaining the importance of the propeptide in protein expression, folding and stability. In the second project, a novel family of minimal prokaryotic metallopeptidases termed “minigluzincins” was discovered, providing a minimal soluble scaffold for gluzincin metallopeptidases and integral-membrane metallopeptidases. Two members of this family, called proabylysin and projannalysin, showed two unique zymogenic forms of latency maintenance exerted, respectively, via intramolecular and intermolecular interactions through their C-terminal segments. In the third project, a further member of this novel family, called selecase, evinced selective and specific proteolytic activity against casein. A set of biophysical and structural studies showed that selecase reversibly commutes between several conformations of defined three- dimensional structure, which are associated with loss of enzymatic activity due to autoinhibition (i.e. active monomers vs. inactive dimers, tetramers and octamers). Overall, the present thesis contributes substantially to the field broadening previous knowledge at the molecular level on metallopeptidases and their regulatory mechanisms, which paves the way for the design of specific inhibitors to modulate their activity as part of therapeutic approaches.Les metal·lopeptidases participen de manera decisiva en la fisiologia i patologia de tots els organismes vius. La seva estricta regulació és, per tant, essencial pel correcte funcionament d’aquestes i per a prevenir una activitat proteolítica inadequada en el temps i/o espai que podria causar malalties. Aquesta regulació s’aconsegueix mitjançant un ampli ventall de mecanismes, incloent la seva biosíntesis com a precursors inactius, també coneguts com a zimògens. En la present tesi s’han estudiat diversos mecanismes de regulació de metal·lopeptidases, tot combinant tècniques bioquímiques, biofísiques i estructurals. En el primer projecte, l’estructura cristal·lina d’un fragment zimogènic de la proteïna “karilysin” de Tannerella forsythia ha revelat el propèptid més curt descrit fins al moment per una metal·lopeptidasa. Assajos bioquímics i biofísics addicionals han permès determinar la importància del propèptid en l’expressió, plegament i estabilitat de la proteïna. En el segon projecte, s’ha descobert una nova família de metal·lopeptidases mínimes d’origen procariota, que s’han anomenat “minigluzincins”, les quals proporcionen un model estructural per a metal·lopeptidases pertanyents al clan de les gluzincines i per a d’altres integrals de membrana. Dues membres d’aquesta família, anomenades “proabylysin” i “projannalysin”, han exhibit formes úniques de manteniment de latència exercides, respectivament, de forma intra- i intermolecular mitjançant llurs segments C-terminals. En el tercer projecte, una altra membre d’aquesta nova família, anomenada “selecase”, ha presentat una activitat proteolítica selectiva i específica sobre caseïna. La conjunció d’estudis duts a terme, tant biofísics com estructurals, ha evidenciat que “selecase” transita de manera reversible entre diverses conformacions d’estructura tridimensional definida, associades amb una disminució d’activitat enzimàtica deguda a autoinhibició (és a dir, monòmers actius i dímers, tetràmers i octàmers inactius). En definitiva, aquesta tesi contribueix de manera substancial a ampliar el coneixement previ sobre metal·lopeptidases a nivell molecular i a entendre els mecanismes que en regulen l’activitat, facilitant així el disseny d’inhibidors específics com a part d’aproximacions terapèutiques.
24
Thurairaja, Ramesh. "The use of serum aminoterminal propeptide of type 1 procollagen (P1NP) and magnetic resonance imaging (MRI) in the management of patients with prostate cancer." Thesis, Queen Mary, University of London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435933.
25
Jemel, Ikram. "La phospholipase A2 sécrétée de groupe X : maturation protéolytique et rôles fonctionnels." Nice, 2009. http://www.theses.fr/2009NICE4096.
Abstract:
Les phospholipases A2 constituent une superfamille de protéines comprenant au moins onze phospholipases A2 sécrétées (sPLA2) et douze phospholipases A2 intracellulaires. Ces protéines catalysent l'hydrolyse des phospholipides en position sn-2, libérant un acide gras et un lysophospholipide. Elles contrôlent ainsi la production d'une variété de médiateurs lipidiques qui sont importants pour de multiples fonctions cellulaires dans différents contextes physiologiques ou physiopathologiques (maladies inflammatoires et cancer). La sPLA2 de groupe X a été clonée au laboratoire en 1997 et possède des propriétés moléculaires uniques. Son ARN messager est présent dans différents tissus, mais semble peu régulé par des stimuli proinflammatoires. L'enzyme est unique dans sa capacité à libérer des médiateurs lipidiques à partir des phopholipides cellulaires ou des lipoprotéines et possède aussi des propriétés antimicrobiennes variées. Un élément clé de la régulation fonctionnelle de la sPLA2 de groupe X est vraisemblablement lié à la présence d'un propeptide dans sa partie N-terminale. Le lieu de la maturation protéolytique de sPLA2 de groupe X (dans la cellule avant sécrétion ou à l'extérieur après sécrétion du proenzyme), les protéases impliquées et la régulation de cette maturation dans des conditions physiologiques ou physiopathologiques sont inconnus. Le travail de cette thèse a permis de mieux comprendre comment peut s'effectuer la maturation de la sPLA2 de groupe X et quels sont ses rôles physiologiques et physiopathologiques. Concernant la maturation, nos études in vitro sur protéines purifiées et en cellules transfectées (HEK293) ont permis de montrer qu'une protéase de type furine contribue de façon majeure à l'activation de l'enzyme, vraisemblablement au cours de sa sécrétion. Nos travaux suggèrent aussi qu'une maturation est possible par d'autres protéases et dans le milieu extracellulaire comme par exemple dans les cellules LOVO. Nous avons aussi tenté de mettre en évidence cette maturation dans des tissus murins dans certaines conditions physiologiques et physiopathologiques. Nous avons notamment trouvé que la sPLA2-X était la sPLA2 majeure présente dans l'acrosome des spermatozoïdes. Enfin nous avons observé un polymorphisme présent dans le propeptide de la sPLA2 humaine qui conduit à la formation d'une protéine inactive et rapidement dégradée. Dans la deuxième partie de cette thèse, nous avons montré que la forme active de la sPLA2 de groupe X, mais pas son proenzyme était capable i) de stimuler la prolifération cellulaire dans un contexte de cancer colorectal, ii) d'exercer une action toxique contre le parasite de la malaria P. Falciparum lors de l'infection de globules rouges humains, et iii) de contrôler la réaction acrosomique des spermatozoïdes de souris, avec un impact important sur le taux de fécondité dans des tests de fécondation in vitroThe superfamily of phospholipase A2 currently consists of at least eleven secreted phospholipases A2 (sPLA2s) and twelve intracellular phospholipases A2. These proteins catalyze the hydrolysis of phospholipids at the sn-2 position, releasing free fatty acids and lysophospholipids. Furthermore, phospholipase A2 controls the production of a variety of lipid mediators which are important for multiple cellular functions in various physiological or physiopathological contexts such as inflammatory diseases and cancer. Group X sPLA2 was cloned in 1997 and have peculiar molecular properties. SPLA2-X has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. SPLA2-X is produced as a proenzyme and has a propeptide of 11 amino acids ending with a dibasic motif. The cellular location and the protease (s) involved in proenzyme conversion have remained unclear. The work of this thesis has allowed to better defining the way by which the maturation of group X sPLA2 can be made and how this sPLA2 functions at both physiological and physiopathological levels. Our in vitro studies with recombinant group X proenzymes and transfected cells (HEK293) have shown that furin-like protease plays a major role in the activation of the enzyme during its secretion. Our work also suggests that the removal of the propertied can also occur via other proteases and extracellularly as found when using the human colon cancer cell line LOVO as a cellular model. We also studied the maturation of sPLA2-X in mouse tissues in various physiological and physiopathological conditions. Of note, we found that sPLA2-X is stored in large amount and likely in an active form in the acrosome of mouse sperm cells. Finally, we discovered a human polymorphism in the propeptide sequence mutating Arg-38 into cysteine and leading to the secretion of an inactive protein that is rapidly degraded after synthesis. In the second part of this thesis, we have shown that the active form of sPLA2-X, but not its proenzyme is able i) to stimulate the proliferation of various mouse colon cell lines, including Colon-26 cancer cells, ii) to protect human red blood cells from infection by P. Falciparulmn the parasite of malaria, and iii) to control the acrosomal reaction of mouse sperm during capacitation with an important impact on the outcome of in vitro fertilization
26
Lebel, Carl. "Étude des effets d'un propeptide muté de la myostatine sur la greffe de myoblastes : dans le cadre du développement d'un traitement pour la dystrophie musculaire de Duchenne." Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/26882/26882.pdf.
27
Otite, Ugo. "Evaluation of serum and urinary human chorionic gonadotrophin beta, urinary beta core fragment and amino terminal propeptide of type 1 procollagen in the clinical management of prostate cancer." Thesis, Queen Mary, University of London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406511.
28
Vulin, Adeline. "Restauration de la dystrophine par saut d'exons chez le modèle canin GRMD ; Augmentation de la masse musculaire par inhibition de la myostatine : rationnel thérapeutique pour DMD ?" Paris 12, 2005. https://athena.u-pec.fr/primo-explore/search?query=any,exact,990003942550204611&vid=upec.
Abstract:
La dystrophie musculaire de Duchenne (DMD) est une maladie progressive et sévère qui demeure incurable malgré le développement de différentes stratégies thérapeutiques. Nous avons décidé de travailler sur deux approches innovantes. Notre premier objectif était de mettre au point des outils moléculaires pour une correction post-transcriptionnelle du gène de la dystrophine. La technique du saut d'exon permet de restaurer le cadre de lecture dans le cas de nombreuses mutations et d'obtenir une protéine plus courte mais fonctionnelle. Nous avons obtenu un saut d'exon(s) efficace et stable après une seule administration du vecteur AAV exprimant les séquences anti-sens liées à un petit ARN nucléaire U7. Nos résultats montrent une restauration de la dystrophine fonctionnelle chez le modèle canin GRMD et localement la correction de la dystrophie musculaire. Notre second objectif était d'améliorer le phénotype DMD en augmentant la masse musculaire grâce à la sous-expression de la myostatine et de vérifier une possible amélioration de la régénération musculaire. Nous avons démontré que le propeptide de la myostatine était un agent efficace pour l'accroissement de la masse musculaire et le bénéfice fonctionnel reste à établir dans les modèles dystrophiques murin et caninDuchenne Muscular Dystrophy (DMD) is a progressive devastating disorder that remains incurable in spite of development of different therapeutic strategies. We decided to work on two original approaches. Our first aim was to setup molecular tools for post-transcriptional correction by targeting exon skipping of frequent out-of-frame deletions in the dystrophin gene and obtain a shorter but functional protein. We have achieved persistent exon skipping by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA. Our results show the sustained production of functional dystrophin at physiological levels in injected muscles of GRMD dog model and the correction of the muscular dystrophy. Our second aim was to improve the DMD phenotype by increasing the skeletal muscle mass thanks to the down-regulation of myostatin and verify a possible improvement of the muscular regeneration. We have demonstrated that the propeptide of myostatin is an effective agent for increasing muscle mass and the functional benefit continue to be established in dystrophic mouse and dog models
29
Ikram, Jemel. "La phospholipase A2 sécrétée de groupe X : Maturation protéolytique et rôles fonctionnels." Phd thesis, Université de Nice Sophia-Antipolis, 2009. http://tel.archives-ouvertes.fr/tel-00496969.
Abstract:
Les phospholipases A2 constituent une superfamille de protéines comprenant au moins onze phospholipases A2 sécrétées (sPLA2) et douze phospholipases A2 intracellulaires. Ces protéines catalysent l'hydrolyse des phospholipides en position sn-2, libérant un acide gras et un lysophospholipide. Elles contrôlent ainsi la production d'une variété de médiateurs lipidiques qui sont importants pour de multiples fonctions cellulaires dans différents contextes physiologiques ou physiopathologiques (maladies inflammatoires et cancer). La sPLA2 de groupe X a été clonée au laboratoire en 1997 et possède des propriétés moléculaires uniques. Son ARN messager est présent dans différents tissus, mais semble peu régulé par des stimuli proinflammatoires. L'enzyme est unique dans sa capacité à libérer des médiateurs lipidiques à partir des phospholipides cellulaires ou des lipoprotéines et possède aussi des propriétés antimicrobiennes variées. Un élément clé de la régulation fonctionnelle de la sPLA2 de groupe X est vraisemblablement lié à la présence d'un propeptide dans sa partie N-terminale. Le lieu de la maturation protéolytique de la sPLA2 de groupe X (dans la cellule avant sécrétion ou à l'extérieur après sécrétion du proenzyme), les protéases impliquées et la régulation de cette maturation dans des conditions physiologiques ou physiopathologiques sont inconnus. Le travail de cette thèse a permis de mieux comprendre comment peut s'effectuer la maturation de la sPLA2 de groupe X et quels sont ses rôles physiologiques et physiopathologiques. Concernant la maturation, nos études in vitro sur protéines purifiées et en cellules transfectées (HEK293) ont permis de montrer qu'une protéase de type furine contribue de façon majeure à l'activation de l'enzyme, vraisemblablement au cours de sa sécrétion. Nos travaux suggèrent aussi qu'une maturation est possible par d'autres protéases et dans le milieu extracellulaire comme par exemple dans les cellules LOVO. Nous avons aussi tenté de mettre en évidence cette maturation dans des tissus murins dans certaines conditions physiologiques et physiopathologiques. Nous avons notamment trouvé que la sPLA2-X était la sPLA2 majeure présente dans l'acrosome des spermatozoïdes. Enfin nous avons observé un polymorphisme présent dans le propeptide de la sPLA2 humaine qui conduit à la formation d'une protéine inactive et rapidement dégradée. Dans la deuxième partie de cette thèse, nous avons montré que la forme active de la sPLA2 de groupe X, mais pas son proenzyme était capable i) de stimuler la prolifération cellulaire dans un contexte de cancer colorectal, ii) d'exercer une action toxique contre le parasite de la malaria P.falciparum lors de l'infection de globules rouges humains, et iii) de contrôler la réaction acrosomique des spermatozoïdes de souris, avec un impact important sur le taux de fécondité dans des tests de fécondation in vitro.
30
Milne, Trudy Jane. "Purification and characterisation of Tex31, a conotoxin precursor processing protease, isolated from the venom duct of Conus textile." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/16960/1/Trudy_J_Milne_Thesis.pdf.
Abstract:
The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides or “conotoxins”. Conotoxins are bioactive peptides found in the venom duct of Conus spp. Like other neuropeptides, conotoxins are expressed as propeptides that undergo posttranslational proteolytic processing. Peptides derived from propeptides are typically cleaved at a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys or Arg-Lys) by proteases found in secretory vesicles. However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes. As many of the substrate-specific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible. In order to understand more about the substrate specificity of conotoxin substrate-specific proteases a characterisation study of one such endoprotease isolated from the venom duct of Conus textile was undertaken. The C. textile mollusc was chosen as a good source from which to isolate the endoprotease for two reasons; firstly, these cone shells are found in great abundance on the Great Barrier Reef (Queensland, Australia) and are readily obtainable and secondly, a number of conotoxin precursors and their cleavage products have been previously identified in the venom duct. In order to purify the endoprotease an activity-guided fractionation protocol that included a para-nitroanilide (p-NA) substrate assay was developed. The p-NA substrate mimicked the cleavage site of the conotoxin TxVIA, a member of the C. textile O-superfamily of toxins. The protocol included a number of chromatographic techniques including ion exchange, size-exclusion and reverse-phased HPLC and resulted in isolation of an active protease, termed Tex31, to >95% purity. The purification of microgram quantities of Tex31 made it possible to characterise the proteolytic nature of Tex31 and to further characterise the O-superfamily conopeptide propeptide cleavage site specificity. Specificity experiments showed Tex31 requires a minimum of four residues including a leucine in the P4 position (LNKR↓) for efficient substrate processing. The complete sequence of Tex31 was determined from cDNA. A BLAST search revealed Tex31 to have high amino acid sequence similarity to the CAP (abbreviated from CRISP (Cysteine-rich secretory protein), Antigen 5 and PR-1 (pathogenesis-related protein)) superfamily and most closely related to the CRISP family of mammalian and venom proteins that, like Tex31, have a cysteine-rich C-terminal domain. The CAP superfamily is widely distributed in the animal, plant and fungal kingdoms, and is implicated in processes as diverse as human brain tumour growth and plant pathogenesis. This is the first report of a biological role for the N-terminal domain of CAP proteins. A homology model of Tex31 constructed from two PR-1 proteins, Antigen 5 and P14a, revealed the highly conserved and likely catalytic residues, His78, Ser99 and Glu115. These three amino acids fall within a structurally conserved N-terminal domain found in all CAP proteins. It is possible that other CAP proteins are also substrate-specific proteases. With no homology to any known proteases, Tex31 may belong to a new class of protease. The sequence alignment of five Tex31-like proteins cloned from C. marmoreus, C. litteratus, C. arentus, C. planboris, and C. omaria show very high sequence similarity to Tex31 (~80%), but only one weakly conserved serine residue was identified when the conserved residues of the new Tex31-like protein sequences were aligned with members of the CAP superfamily. Future work to identify members of catalytic diad or triad, e.g. by site-directed mutagenesis, will rely on the expression of active recombinant Tex31. In this study neither Escherichia coli nor Pichia pastoris expression systems yielded active recombinant Tex31 protein, possibly due to the number of cysteine residues hindering the expression of correctly folded active Tex31. This study has shown Tex31 to be highly sequence specific in its cleavage site and it is likely that this high substrate specificity has confounded previous attempts to identify the proteolytic nature of other CAP proteins. With the proteolytic nature of one member of the CAP protein family confirmed, it is hoped this important discovery may lead the way to discovering the role of other CAP family members.
31
Milne, Trudy Jane. "Purification and characterisation of Tex31, a conotoxin precursor processing protease, isolated from the venom duct of Conus textile." Queensland University of Technology, 2008. http://eprints.qut.edu.au/16960/.
Abstract:
The venom of cone snails (predatory marine molluscs of the genus Conus) has yielded a rich source of novel neuroactive peptides or “conotoxins”. Conotoxins are bioactive peptides found in the venom duct of Conus spp. Like other neuropeptides, conotoxins are expressed as propeptides that undergo posttranslational proteolytic processing. Peptides derived from propeptides are typically cleaved at a pair of dibasic residues (Lys-Arg, Arg-Arg, Lys-Lys or Arg-Lys) by proteases found in secretory vesicles. However, many precursor peptides contain multiple sets of basic residues, suggesting that highly substrate specific or differentially expressed proteases can determine processing outcomes. As many of the substrate-specific proteases remain unidentified, predicting new bioactive peptides from cDNA sequences is presently difficult, if not impossible. In order to understand more about the substrate specificity of conotoxin substrate-specific proteases a characterisation study of one such endoprotease isolated from the venom duct of Conus textile was undertaken. The C. textile mollusc was chosen as a good source from which to isolate the endoprotease for two reasons; firstly, these cone shells are found in great abundance on the Great Barrier Reef (Queensland, Australia) and are readily obtainable and secondly, a number of conotoxin precursors and their cleavage products have been previously identified in the venom duct. In order to purify the endoprotease an activity-guided fractionation protocol that included a para-nitroanilide (p-NA) substrate assay was developed. The p-NA substrate mimicked the cleavage site of the conotoxin TxVIA, a member of the C. textile O-superfamily of toxins. The protocol included a number of chromatographic techniques including ion exchange, size-exclusion and reverse-phased HPLC and resulted in isolation of an active protease, termed Tex31, to >95% purity. The purification of microgram quantities of Tex31 made it possible to characterise the proteolytic nature of Tex31 and to further characterise the O-superfamily conopeptide propeptide cleavage site specificity. Specificity experiments showed Tex31 requires a minimum of four residues including a leucine in the P4 position (LNKR↓) for efficient substrate processing. The complete sequence of Tex31 was determined from cDNA. A BLAST search revealed Tex31 to have high amino acid sequence similarity to the CAP (abbreviated from CRISP (Cysteine-rich secretory protein), Antigen 5 and PR-1 (pathogenesis-related protein)) superfamily and most closely related to the CRISP family of mammalian and venom proteins that, like Tex31, have a cysteine-rich C-terminal domain. The CAP superfamily is widely distributed in the animal, plant and fungal kingdoms, and is implicated in processes as diverse as human brain tumour growth and plant pathogenesis. This is the first report of a biological role for the N-terminal domain of CAP proteins. A homology model of Tex31 constructed from two PR-1 proteins, Antigen 5 and P14a, revealed the highly conserved and likely catalytic residues, His78, Ser99 and Glu115. These three amino acids fall within a structurally conserved N-terminal domain found in all CAP proteins. It is possible that other CAP proteins are also substrate-specific proteases. With no homology to any known proteases, Tex31 may belong to a new class of protease. The sequence alignment of five Tex31-like proteins cloned from C. marmoreus, C. litteratus, C. arentus, C. planboris, and C. omaria show very high sequence similarity to Tex31 (~80%), but only one weakly conserved serine residue was identified when the conserved residues of the new Tex31-like protein sequences were aligned with members of the CAP superfamily. Future work to identify members of catalytic diad or triad, e.g. by site-directed mutagenesis, will rely on the expression of active recombinant Tex31. In this study neither Escherichia coli nor Pichia pastoris expression systems yielded active recombinant Tex31 protein, possibly due to the number of cysteine residues hindering the expression of correctly folded active Tex31. This study has shown Tex31 to be highly sequence specific in its cleavage site and it is likely that this high substrate specificity has confounded previous attempts to identify the proteolytic nature of other CAP proteins. With the proteolytic nature of one member of the CAP protein family confirmed, it is hoped this important discovery may lead the way to discovering the role of other CAP family members.
32
Lindahl, Katarina. "Osteogenesis Imperfecta : Genetic and Therapeutic Studies." Doctoral thesis, Uppsala universitet, Metabola bensjukdomar, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-208942.
Abstract:
Osteogenesis imperfecta (OI) is a heterogeneous disease of connective tissue, the cardinal symptom being fractures and severity ranging from mild to lethal. Dominant mutations in collagen I, encoded by COL1A1 and COL1A2, cause >90% of cases. To delineate genotype-phenotype correlations and pharmaco-genetic response, collagen I was sequenced in 150 unrelated Swedish families and clinical data were collected in Paper I. Mutation type, gene affected, and N- to C-terminal location correlated with phenotype and severity. Bisphosphonate response assessed by calculated yearly change in lumbar spine bone mineral density (BMD) was inversely related to age and BMD at treatment initiation. Mutations associated with a more severe phenotype exhibited an increased response after 2 years; however, all types of OI responded well. To investigate the effect of naturally occurring variations in collagen I, the only common coding single nucleotide polymorphism (rs42524 in COL1A2) was genotyped in 2004 healthy men in Paper II. Heterozygous genotype was associated with decreased BMD and an increased risk of stroke. An adolescent with repeated fractures despite a markedly high BMD harbored a unique C-terminal procollagen cleavage-site mutation in COL1A1, which motivated extensive investigations in concert with a similar COL1A2 case in Paper III. The probands were found to have impaired procollagen processing, incorporation of collagen with retained C-propeptide in matrix and increased mineral to matrix ratio, which demonstrates that C-propeptide cleavage is crucial to normal bone mineralization and structure. Bisphosphonate therapy has insufficient effect in OI, and as classical OI is a dominant disorder severe cases would benefit from silencing of the mutated allele. In Paper IV and V small interfering RNAs (siRNAs) were used to allele-specifically target primary human bone cells heterozygous for I) a coding polymorphism in COL1A2 and II) insertion/deletions in the 3’UTR of COL1A1 and COL1A2. Results were promising with altered allele ratios and decreased mRNA levels in the predicted fashion. To summarize, this thesis found that collagen I is crucial to bone and connective tissue and that collagen I mutations create markedly diverse phenotypes. Age, BMD and pharmaco-genetic effects influence the response to bisphosphonate therapy in individuals with OI; however, novel approaches are needed. Utilizing allele-specific siRNAs may be a way forward in the treatment of severe OI.
33
Hsu, Yi-Lin, and 許裔林. "Screening and Characterization of Streptomyces Transglutaminase Propeptide Mutants that Affect Mature Enzyme Activity." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/54324008049819498920.
Abstract:
碩士國立中興大學
分子生物學研究所
104
Transglutaminase is an acyl-transferase enzyme (amine γ-glutaminyltransferas, EC2.3.2.13; TGase) that catalyzes intra- or intermolecular crosslinking between glutamine and primary amines in proteins. TGase enzymes from microbial source have been widely used in food processing, textile, biomedical and pharmaceutical industries. The Streptomyces TGases are secreted as zymogen and stepwise processed into mature enzyme after cleavage of the pro-peptide by endogenous proteases. The pro-peptide of TGase has been demonstrated to play dual roles as having both molecular chaperone and inhibitor activities. In this study, random and site-directed mutagenesis in the propeptide regions of Streptomyces mobaraense (Sm) and Streptomyces netropsis (Sn) TGases were performed to identify mutants that maintain chaperone function and weaken their interactions with mature enzyme. The strategy of error-prone PCR and LIC (ligation-independent cloning) were applied to generate random mutations in the pro-peptide regions of Sm and Sn TGases. By coexpression of the plasmids containing mutated proTGase and TVMV protease genes in E. coli, mature TGases could be purified by one-step Ni2+-NTA affinity column. The purified Sm and Sn mature TGases that enhanced their enzymatic activities or have higher yields were identified. Preliminary screening revealed that 7 Sm and 50 Sn mature TGases have higher specific activities than that of the wild type after Ni2+-NTA affinity purification. DNA sequence analyses revealed that most of the mutants have one to two amino acids changes in the conserved domain of the TGase propeptide. Two of the Sn TGase propeptide mutants identified in the conserved domain, D22 and A13, were chosen for further site-directed mutagenesis. Results showed that replacement of D22 with G, A, L, K and Y facilitate high yield and produced mature enzymes with the same specific activities as wild type. Replacement of A13 with H, W, Y, F, E and T revealed that the Ni2+-NTA affinity purified mature TGases have higher specific activities but the yields were lower than that of the wild type.
34
Knobloch, James Edward. "Activation of vitamin K-dependent carboxylase by the propeptide from clotting factor X." 1987. http://catalog.hathitrust.org/api/volumes/oclc/17376038.html.
Abstract:
Thesis (M.S.)--University of Wisconsin--Madison, 1987.Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 93-97).
35
Benedix, Yvonne. "Untersuchung zur Selektivität der Hemmung von Cathepsin L-ähnlichen Cysteinproteasen durch die dazugehörigen Propeptide /." 2006. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=015669108&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
36
Schlabrakowski, Anne [Verfasser]. "Spezifität der Inhibition von Cathepsin-L-ähnlichen Cysteinproteasen durch ihre Propeptide / von Anne Schlabrakowski." 2003. http://d-nb.info/97008580X/34.
37
Chen, I.-An, and 陳奕安. "The Effects of Recombinant Porcine Myostatin Propeptide on Myogenesis in C2C12 Myoblast and Mice Animal Model." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/92924064777476895019.
Abstract:
碩士國立中興大學
生命科學系所
99
Myostatin (MSTN) is a dominant negative regulator of skeletal muscle development and growth. Myostatin propeptide (MSPP) is a peptide fragment cutting from the N-terminal of myostatin precursor. MSPP is able to bind to MSTN and blocking its function. In previous studies, over-expressed MSPP in transgenic mice would dramatically promote muscle growth through myostatin inhibition. Therefore, we aimed to develop a convenient and effective method to increase the meat production of livestock by feeding or injecting them with MSPP. This procedure may reduce the production costs, provide a clinical potential application of human muscular dystrophies therapy, and moreover, prevent diet-induced metabolic syndrome like obesity. In this study, we used a pGAPZαA expression cassette harboring mspp cDNA and large-scale production of MSPP by yeast Pichia pastoris fermentation. The product was further purified as a 37 kDa recombinant protein by nickel cloumn, and the production rate of fermentation was 0.9 mg/L. In the in vitro functional study, treated C2C12 myoblasts with the purified MSPP (10 μg/ml) in the regular culture medium could dramatically promote cell proliferation by cell proliferation assay analysis. We further investigated the downstream cell proliferation signals of myostatin propeptide by monitoring the mRNA levels of some transcription factors and myogenic regulatory factors (MRFs) in grow medium (GM) and differentiation medium (DM). We found that Cdk2, p21, MyoD and Myf5 mRNA levels were upregulated after treated MSPP (10 μg/ml) in GM and DM. And Cdk2 was significant upregulated (p<0.05) in GM. In in vivo study, we fed mice with normal fat diet (NFD) or high fat diet (HFD), and gave them the crude extract MSPP by oral gavage or intraperitoneal injection (i.p). In the first experiment, mice treated with MSPP significantly increased both of body and muscle weight in HFD group, and the cross-section areas of myotube were increased. In the second experiment, commercialized HFD was highly palatability. Mouse body weight was more than control group after feeding HFD for 4 days and the glucose tolerance dramatically got worse after 21 days. The groups treated with MSPP could prevent HFD-induced glucose tolerance reduction. However, the MSPP concentration were insufficient in crude extract to increase the body and muscle weight by oral gavage and i.p manipulations significantly. In order to facilitate the meat production efficiency for livestock with the MSPP treatment, we will further apply the genetic engineering techniques to modify the gene sequence (D76 mutated into A). With this manipulation, we can further reduce the degradation rate of MSPP in animals and also increase the working half-life to promote the application for MSPP in the economic meat production in the future.
38
Benedix, Yvonne [Verfasser]. "Untersuchung zur Selektivität der Hemmung von Cathepsin-L-ähnlichen Cysteinproteasen durch die dazugehörigen Propeptide / von Yvonne Benedix." 2006. http://d-nb.info/989327485/34.
39
Kaulmann, Guido [Verfasser]. "The structure of human procathepsin S : crystallographic investigations on the functional role of the propeptide / von Guido Kaulmann." 2004. http://d-nb.info/976431319/34.
40
Tao, Wei-Yu, and 陶威宇. "Expression of PCSK and Proteolytic Processing of BMP Propeptide on Dorso-Ventral Patterning of Helobdella Neuroectoderm: a Preliminary Study." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/47271389432792250729.
Abstract:
碩士國立臺灣大學
生命科學系
103
Bone morphogenetic protein (BMP) morphogen is known to play a vital role in body axis patterning during embryogenesis. Unlike BMP4/Dpp functions as a morphogen patterning embryo dorsoventral (DV) axis in vertebrate and fly, BMP5-8 in leech, the key BMP signaling ligand specifies DV patterning, acts in a “cell-cell contact” fashion. Evidences indicate that the ligand BMP proprotein’s differential proteolytic processing by proprotein convertases (PCs or PCSKs) influences its signaling range. Further, different signaling ranges were found between teloblast lineages (N/Q and O/P), above which make the proteolytic processing of BMP5-8 in leech embryo DV patterning an unsolved question. To characterize the role of PCSK-mediated cleavage in leech DV patterning, I carried out the following experiments. First, by reverse transcription-PCR and whole mount in situ hybridization, expressions of all 12 PCSK-related genes in leech have been confirmed and localized at embryogenesis stage 8. Second, a cell surface-linked indicator of proteolysis assay (CLIP) is prepared to test PC activity in vivo. Third, ectopic expression of cleavage site-mutated BMP5-8 in leech has been used. The above results indicate that the proteolytic processing of BMP5-8 is not required for development of normal neuroectodermal, which is also a key event of body axis formation.
41
Iyappan, Saravanakumar [Verfasser]. "The function of the β6-Pre7 [beta-6-Pre7] propeptide for 20S proteasome biogenesis in baker's yeast / vorgelegt von Saravanakumar Iyappan." 2005. http://d-nb.info/973920521/34.
42
Wu, Hsin-Shan, and 吳欣珊. "The Effect of Recombinant Wild-type Porcine Myostatin Propeptide and Its Mutant Form on Growth and Differentiation Gene Expression in C2C12 Myoblast." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/ukhc98.
Abstract:
碩士國立中興大學
生命科學系所
103
Myostatin (MSTN) is an extracellular autocrine produced by cardiac muscle, and circulates in the blood in an inactive form throughout the body to inhibit muscle proliferation and differentiation; it is not only restricted to skeletal muscle but also detected in other tissues (e.g. adipose tissue). Recent study indicated that myostatin can accelerated fatty acid oxidation to prevent diet-induced obesity and stimulated adipogenesis in bone mesenchymal stem cells to improved multipotent cell differentiation. Previous studies mostly using microinjection or transgenic expression of myostatin inhibitor or myostatin propeptide (MSPP) cDNA to animals ameliorate cachexia due to myostatin disequilibrium or high-fat diet induced metabolic syndrome but this technology is too expensive and not easy to do. Therefore, we aimed to develop an alternative low cost and effective method. We used a pPICZαA expression cassette harboring WT-MSPP cDNA and its mutant form designed to be resistant to BMP-1/TLD metalloproteinase (D75A-MSPP) by yeast Pichia pastoris fermentation, which can efficiently secret to the fermentation medium. The products was further purified as 35.4 kD recombinant protein by size exclusion column. In addition, C2C12 myoblasts treated with the crude extract WT-MSPP or D75A-MSPP (10 μg/mL) in the regular culture medium could dramatically promote cell proliferation. We further investigated the downstream cell proliferation signals of MSPP by monitoring the mRNA levels and protein levels of some transcription factors and myogenic regulatory factors (MRFs) in grow medium (GM) and differentiation medium (DM). We found that Cdk2, Pax7, MyoD and Smad3 mRNA levels were upregulated after treated WT-MSPP or D75A-MSPP (10 μg/mL) in GM and DM (p < 0.05). In addition, MyoD and Myogenin protein levels were significantly upregulated (p < 0.05), and Erk, p-Erk, Akt and p-Akt were obviously downregulated (p < 0.05) in GM. Moreover, Myogenin, Akt and p-Akt were significantly increased in DM. The myosin heavy chain immunofluorescence stain result indicated that WT-MSPP and D75A-MSPP can enhance the ability of C2C12 myoblasts differentiation to myotube, especially in D75A-MSPP. In vivo study, we will fed C57BL/6 mice with normal fat diet (NFD) or high fat diet (HFD), and give them the crude extract WT-MSPP or D75A-MSPP by intraperitoneal injection (i.p), measure their blood biochemistry values and sacrifice after 1 month to detect the body weight, muscle weight, organ weight and histochemical stains to determine that control MSTN whether or not a possible way to promote muscle growth, decrease fat accumulation and ameliorate obesity-induced type2 diabetes.
43
Suzuki, Shana T. N. "Effects of enhanced muscle growth by myostatin propeptide trangene and dietary fat content on gene expression of adiponectin, adiponectin receptors, PPAR-α and PPAR-γ." Thesis, 2007. http://hdl.handle.net/10125/20774.
44
Tsai, Sen-Wei, and 蔡森蔚. "Establishment a Muscle Atrophy Rat Animal Model Caused by Botulinum Toxin And Evaluation The Therapeutic Effects of Treadmill Exercise and Myostatin Propeptide in Neuromuscular Junction and Muscle Physiology after Intramuscular Botulinum Toxin Injection." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/54746132896260361343.
Abstract:
博士國立中興大學
生命科學系所
101
Botulinum toxin type A (BoNT-A), a kind of botulinum toxin, is used for spasticity block recently. The mechanism of this drug is to block the secretion of pre-synaptic acetylcholine, thus makes muscle weakness. Botulinum toxin injection is usually performed in cerebral palsy to reduce spasticity. Treadmill training for cerebral palsy is a well-documented program. Although botulinum toxin injection and treadmill training have usually been used in combination, the effects of treadmill training in muscles after intramuscular botulinum toxin injection are not well established. We showed that after Botulinum toxin type A injection, treadmill training improved the sciatic functional index, the maximal contraction force of the gastrocnemius muscle, and the percentage of activated muscle fibers which was demonstrated by differences in amplitude and area of compound muscle action potential. Upregulation of GAP-43, IGF-1, Myo-D, Myf-5, myogenin, AChR subunits α and β were also found following treadmill running which may have contributed to enhanced recovery of gastrocnemius strength. In clinical practice, when considering the therapeutic strategies of combining these two treatments, clinicians should take this potential counteraction effect into consideration. Muscle atrophy is a common symptom associated with reduced skeletal muscle activity following bone fracture, trauma, ligament tear, inflammatory disease and nerve injury. To date, there are no pharmacological strategies for preventing or treating muscle atrophy. Myostatin is a potent inhibitor of skeletal muscle growth. Myostatin propeptide (MPro) is showed to improve muscle growth. However, the underlying mechanism of muscle atrophy attenuation effects of myostatin propeptide in atrophied muscles is not well established. We constructed both wild type MPro (MSPP) and mutant MPro construct (MSPPD75A) with resistance to proteinase. Using the C2C12 in vitro cell model and BoNT-induced muscle atrophy model, we evaluated the effects of myostatin propeptide gene therapy. We also observed changes in gene activation associated with neuromuscular junction, muscle and nerve. We showed that through MSPPD75A gene delivery, gastrocnemius muscle atrophy caused by botulinum toxin was attenuated. Through the delivery of either MSPP or MSPPD75A, the expressions of downstream proteins in ubiquitine-proteosome pathway Smad3 and MuRF1 were decreased, and the expressions of the muscle regulatory factors, IGF-1, GAP43, and acetylcholine receptors were increased. The data suggested that gene therapy may be a promising treatment for muscle atrophy. And the results could be used as basic knowledge for clinical rehabilitation, as a basis for developing treatment strategy of gene therapy.
45
Verlan, Inna. "The Role of Cardiotrophin-Like Cytokine Factor 1 on the Development of Atherosclerosis." Thèse, 2017. http://hdl.handle.net/1866/21386.