Dissertations / Theses on the topic 'Calpain'
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Czerwinski, Eric Paul. "Two Proteins Containing Tandem DIII Domains, Calpain 10 and Dictyostelium Cpl, are Involved in Cytoskeletal Regulation." Connect to Online Resource-OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1193689816.
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Dissertation (Ph.D.)--University of Toledo, 2007."In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 117-147.
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Janardhanan, Anitha C. "Gene expression of components of the calpain system m-calpain, [mu]-calpain and calpastatin in male and female broiler skeletal muscle /." Morgantown, W. Va. : [West Virginia University Libraries], 1999. http://etd.wvu.edu/templates/showETD.cfm?recnum=895.
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Thesis (M.S.)--West Virginia University, 1999.Title from document title page. Document formatted into pages; contains vii, 93 p. : ill. (some col.) Includes abstract. Includes bibliographical references (p. 72-80).
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Fernández-Montalván, Amaury Ernesto. "Structural requirements for activation and membrane binding of human m-calpain [mu-calpain]." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973390123.
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Schroeder, Ewald. "Structural studies of #mu#-calpain, a novel calpain substrate, and a papain-leupeptin complex." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386677.
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Huang, Xinhua. "DIII Domain of Calpain 10 and Cpl Towards an Understanding of Calpain 10 Function." University of Toledo Health Science Campus / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=mco1096641027.
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Huang, Xinhua. "DIII domain of calpain 10 and Cpl towards an understanding of calpain 10 function /." Connect to full-text via OhioLINK ETD Center, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1096641027.
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Thesis (Ph. D.)--Medical College of Ohio, 2003."In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Ronald Mellgren. Includes abstract. Document formatted into pages: iv, 126 p. Title from title page of PDF document. Includes bibliographical references (p. 92-124).
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Rose, Robert Edward. "Calpain and lipopolysaccharide mediated hepatitis." Thesis, [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1806.
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Plammootil, Salma Martha. "Mutationen im Calpain-3-Gen." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973536306.
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Douillard, Aymeric. "Implication des calpaïnes lors d'un remodelage musculaire induit par un traitement chronique au clenbutérol." Thesis, Montpellier 1, 2011. http://www.theses.fr/2011MON14001/document.
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Afin de lutter efficacement contre les malversations du dopage, il apparaît essentiel de comprendre les mécanismes conduisant au remodelage musculaire. Dans ce but nous avons analysé les effets d’un β2-agoniste, le clenbutérol, sur le remodelage musculaire et les différentes voies de signalisation qui y sont associées. Nous nous sommes particulièrement intéressés au système des calpaïnes qui a souvent été associé à des phénomènes de remodelage musculaire, principalement dans des modèles d’atrophie. Nous avons montré une sollicitation précoce du système des calpaïnes lors d’un traitement chronique au clenbutérol chez le rat associé à une conversion phénotypique dans les muscles EDL et Soléaire et à une hypertrophie dans le muscle EDL uniquement. Puis, nous avons inhibé l’activité des calpaïnes en parallèle d’un traitement au clenbutérol. Les muscles ayant une activité des calpaïnes diminuée et soumis à un traitement au clenbutérol n’ont pas développé de remodelage musculaire. Ces premiers résultats renforcent l’idée d’une implication des calpaïnes dans le remodelage musculaire induit par un traitement chronique au clenbutérolTo fight doping in an effective manner, it is essential to understand the mechanisms leading to muscle remodeling. For this purpose we analyzed the effects of clenbuterol, on muscle remodeling and various associated signaling pathways. We were particularly interested with the calpain system which has often been associated with muscle remodeling phenomena, mainly in models of atrophy. We have shown that an early calpain system solicitation during chronic treatment with clenbuterol in rats was associated with a phenotypic conversion in the Soleus and EDL muscles and hypertrophy in the EDL muscle. We then inhibited the activity of calpains with a parallel clenbuterol treatment. The muscles with a reduced activity of calpain and treated with clenbuterol did not develop muscle remodeling. These initial results reinforce the idea of an involvement of calpain in the muscle remodeling induced by chronic treatment with clenbuterol
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Arthur, John Simon Campbell. "Regulation of m-calpain by phospholipids." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240569.
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Joshi, Aashish. "SUBSTRATE AND REGULATION OF MITOCHONDRIAL μ-CALPAIN." UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_diss/80.
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μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive.
We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same
submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.
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Chen, Hongyuan. "Design, synthesis and testing of calpain inhibitors for the treatment of cataract." Thesis, University of Canterbury. Chemistry, 2007. http://hdl.handle.net/10092/1405.
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This thesis reports the development of potent and selective inhibitors of m-calpain for the treatment of cataract. SJA6017 has been proven to prevent lens opacity in rat and has been our lead compound. A series of Val-Leu peptidyl aldehyde inhibitors (33a-e, 33g, 33i and 35) have been designed, synthesized, and tested for therapeutic potential as cataract inhibitors. Chapter 1 is an introduction to calpain and the diseases associated with it's over activation. A review of the literature on calpain inhibition is given. Structure activity relationship (SAR) theory is presented. The techniques that have been applied in our research group to drug design include molecular modeling, synthesis, assay and animal studies which are all briefly discussed. The importance of a -strand conformation for an inhibitor to bind to calpain is discussed. Chapter 2 describes the synthesis of m-calpain inhibitors. This comprises the preparation of the Val-Leu dipeptide core 29, Val-Leu dipeptidyl alcohols 31a-g and 31i, and the synthesis of dipeptidyl aldehydes 33a-e, 33g, 33i and 35. The choice of coupling regents and conditions in the coupling reactions is investigated. Sulfur trioxide pyridine oxidation for the conversion of Val-Leu dipeptidyl alcohols to aldehydes is discussed. The molecular modeling and biological assay results are presented.
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Singh, Ranjana. "CALPAIN 5: A NON-CLASSICAL CALPAIN HIGHLY EXPRESSED IN THE CNS AND LOCALIZED TO MITOCHONDRIA AND NUCLEAR PML BODIES." UKnowledge, 2014. http://uknowledge.uky.edu/neurobio_etds/9.
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Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF-hand motif characteristic of the classical calpains, calpain 1 and 2, but retains catalytic and Ca2+ binding non EF domains. Tra-3, an ortholog of CAPN5, is involved in necrotic cell death in C.elegans; although specific role of CAPN5 has not been investigated in the mammalian CNS. I compared relative mRNA levels of calpains in rat CNS, which revealed that CAPN5 is the second most highly expressed calpain. We examined relative levels of CAPN5 from late embryonic day 18 to postnatal day 90 and found lower mRNA but higher protein levels during CNS development. Using X –gal staining in Capn5 +/- mice, immunostaining of rat brain sections and SH-SY5Y cells, and subcellular fractionation of rat brain cortex, we found that CAPN5 is a non-cytoplasmic calpain localized in the nucleus and enriched in synaptic mitochondria. Proteinase K treatment of mitochondria and mitoplasts from B35 rat neuroblastoma cells and rat synaptic mitochondria revealed CAPN5 was localized on the inner mitochondrial membrane and released from mitochondria on membrane permeabilization with alamethicin. We used immunolabelling, confocal imaging, nuclear subfractionation and transient transfections to evaluate the subnuclear localization of CAPN5. CAPN5 was detected in punctate domains and associated with promyelocytic leukemia (PML) protein, a tumor suppressor protein. We further demonstrated that CAPN5 carries a nonconventional bipartite nuclear localization signal. Together, these findings demonstrate that CAPN5 is a non-cytosolic calpain, abundant in the CNS and localized to the mitochondria inner membrane and nuclear PML bodies.
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Fraser-Smith, Emma Louise. "Characterizing the Catalytic Action of μ-Calpain on Myofibrillar Protein Structure." The University of Waikato, 2006. http://hdl.handle.net/10289/2253.
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Solving the problem of inconsistent meat tenderness is a top priority of the meat industry. This requires a greater understanding of the processes that affect meat tenderness and the adoption of such information by the meat industry. It is essential that we understand the mechanism of meat tenderisation of which, the calpain protease system is believed to play a central role. This thesis focuses on three aspects; characterisation of calpain activity, the effect of porcine μ-calpain on myofibril degradation and the effect of μ-calpain on specific proteins desmin and troponin-T. To study the effect of calpain activity, fluorogenic assays were used to determine: μ-calpain concentration for optimal peptide cleavage; calcium requirements and the effect of chelating substances on the activity of μ-calpain. In addition, the affinity of μ-calpain for substrates CalS-I and CalS-III were assessed. The effect of μ-calpain on myofibril degradation was evaluated through the use of myofibrillar fragmentation index and density marker beads. Myofibrils were digested at three different temperatures for varying time periods. Conflicting results were displayed and it was concluded that these methods are not accurate, thus further research should be conducted to ensure inconsistencies are eliminated. Specific proteins desmin and troponin-T have previously been shown to exhibit degradation in the presence of calcium and μ-calpain. SDS-polyacrylamide electrophoresis, western blotting and densitometry measurements were utilized to investigate this effect. It was concluded that μ-calpain plays a significant role in the post mortem proteolysis of myofibrillar protein. This thesis provides information and strives to give a better understanding of the proteolytic changes that occur within muscle. Understanding how these mechanisms affect meat on a cellular level, can help to control the influence they inflict on meat quality.
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Sazili, Awis Qurni. "Calpastatin and meat tenderness in sheep and cattle." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273257.
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Ishak, Reezal. "Calpain-1 : investigating its role in murine neutrophils." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/37448/.
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Neutrophils are phagocytic white blood cells which act as the first line of defence against entry of foreign microorganisms. Neutrophils are recruited to their target site through the process of spreading, extravasation and phagocytosis involving complex signal transduction within the cells, which might include the activation of the cytosolic Ca2+ activated protease, calpain-1. The work described here investigates the role of calpain-1 in regulating neutrophil functions such as spreading, trans-endothelial migration, chemotaxis, phagocytosis and Ca2+ signalling. Through the work done at European Mutant Mouse Archive (EMMA), Oxford, and by using intracellular sperm injection (ICSI) of calpain-1 deleted gene from mice generated in the USA, and with a selective genotype breeding programme, a colony of homozygous calpain-1 KO mouse has been generated in Cardiff. Homozygous calpain-1 KO neutrophils appeared to have a smaller surface spreading area and their recruitment into the peritoneal cavity of the mouse in vivo was disrupted. In vitro experiments showed significant defects in their ability to cross the ICAM-1 expressing endothelial cells in trans-endothelial migration assay. Disruption in this transmigration was only evident with ICAM-1 upregulated (TNF-treated) endothelial cells, suggesting a specific defect in the β2 integrin-ICAM-1 signalling process. Calpain-1 absence did not affect signal transduction as neutrophils were able to signal cytosolic Ca2+ in response to β2 integrin engagement (C3bi-opsonised zymosan) and also to release intracellular Ca2+ store upon IP3 uncaging. This showed that the IP3 pathway in the cells was not affected by knocking-out calpain-1 and continued to be functional. The key signalling mechanisms from β2 integrin also remained intact and this is consistent with calpain-1 activation by Ca2+ being an important event in trans-endothelial migration. In conclusion, calpain-1 absence has significantly affected the ability of neutrophils to undergo trans-endothelial migration and this effect is directed towards the event which happens downstream to the increase in cytosolic free Ca2+ concentration.
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Olego-Fernandez, S. "A calpain-like multigene family in Trypanosoma brucei." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:5256ea6f-4da0-4d42-b77c-a0d2da6f3af2.
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Trypanosomatid parasites are unicellular eukaryotes characterised by the presence of a subpellicular array of microtubules, a single flagellum, and a kinetoplast (containing the condensed mitochondrial DNA). The majority of trypanosomatid species undergo complex life-cycles, alternating between a mammalian host and an insect vector. Progression through this life-cycle requires the differentiation of trypanosomatids into distinct, niche adapted developmental forms. Differentiation into each life-cycle stage involves important biochemical and morphological changes, including the remodelling of the subpellicular cytoskeleton that defines cell shape. In higher eukaryotes, proteins from the calpain superfamily are involved in developmentally- and environmentally-regulated remodelling of the cytoskeleton and the dynamic organisation of signal transduction cascades. Interestingly, trypanosomatids contain unusually large families of calpain-related proteins, but there is little knowledge about the functional roles of these molecules during the life-cycle of trypanosomatid parasites. In this thesis, I present the results of the bioinformatic analysis of calpain-like proteins in three trypanosomatid parasites, Trypanosoma brucei, Leishmania major and Leishmania infantum. From this analysis, I selected several calpain-related proteins tor RNAi functional analysis, on the bases of their domain composition and conservation across different species. The detailed analysis of the resulting RNAi phenotypes revealed the essential function of some calpain-like proteins for the correct morphogenesis of specific developmental forms of T. brucei, shedding some light on the mechanisms that regulate this parasite differentiation and cytoskeletal remodelling, and providing new putative therapeutic targets for African sleeping sickness.
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Zhang, Siwei. "Calpain in ovarian cancer progression and chemotherapeutic response." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/51279/.
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The calpain system is associated with cancer chemotherapeutic response in both in vivo and in vitro studies. Previous immunohistochemistry (IHC) data conducted in our group indicated that high calpain-2 expression was associated with both the resistance to platinum-based adjuvant chemotherapy and worse patient outcome; moreover calpain-2 appeared as an independent prognostic factor in multivariate analysis. To test the hypothesis that conventional calpain subunits, especially calpain-2, are associated with the chemo-resistance of ovarian cancer cells to platinum-based chemotherapy (cisplatin and/or carboplatin), five ovarian cancer cell lines, with varying platinum-based chemotherapy sensitivities, were chosen as in vitro models: the platinum-sensitive A2780 cells and its resistant counterpart A2780-cis cells; the platinum-resistant SKOV3 cells; and the platinum-sensitive PEO1 cells and its platinum-resistant counterpart PEO4 cells. Western blotting was used to assess the expression of the conventional calpain subunits (i.e. calpain-1, -2 and -4) and calpastatin in this panel of cell lines. Calpain activity was regulated by inhibitor calpeptin and calpain-2 short hairpin RNA (shRNA) was used in attempt to specifically downregulate calpain-2 expression. Calpain activity was assessed by an activity assay using fluorogenic peptidase substrate t-BOC. The role of calpain in proliferation and resistance to platinum-based chemotherapy were examined in vitro using growth curves and colony formation. Moreover the study of calpain-4 expression was added into the current project, in addition to verifying the association between the expression of calpain-1, -2 and calpastatin and clinicopathologic variables (e.g. chemo-resistance and patient outcome) by standard immunohistochemistry (IHC) with a larger patient cohort (n=575). To test the hypothesis that conventional calpain subunits and calpastatin are associated with ovarian tumour metastasis, the effect of calpain inhibition (by calpain inhibitors) and activation (by calcium ionophore) on ovarian cancer cell migration was examined using haptotaxis (scratch wound) migration assay. Based on information from 2016 FASEB calpain conference, microtubule-associated protein 4 (MAP4) and spleen tyrosine kinase (Syk) appeared as potential calpain-related proteins associated with angiogenesis and epithelial-mesenchymal transition. Using IHC, their protein expression (i.e. MAP4 and Syk) was assessed and their associations with clinicopathological variables in ovarian cancer patient samples were studied; besides, their correlations with conventional calpain subunits and calpastatin, together with EMT (epithelial-to-mesenchymal transition)-associated proteins and angiogenesis-associated proteins (n=87, data provided by Dr S. Deen) were analysed. Significant variations of calpain system protein expression levels were observed between the different cell lines. Among the 5 cell lines, A2780 and A2780-cis cells (likely to be endometrioid carcinoma cell lines) expressed very low levels of the conventional calpain subunits and calpastatin; whilst PEO1 and PEO4 cells (high-grade serous carcinoma cell lines) expressed comparatively higher level of these proteins. Thus, different expression of the calpain system seemed to be associated with ovarian cancer histological subtypes, which was supported by the IHC study. No significant difference of the calpain system expression was detected and calpeptin caused a similar inhibition of cell proliferation between chemo-sensitive ovarian cancer cells and their resistant counterparts. Because SKOV3 and PEO4 cells expressed the highest levels of calpain-2, shRNA was used for specific knockdown of calpain 2 in these two cell lines, unfortunately numerous attempts proved unsuccessful. Although with the optimised concentration and treatment duration, calpeptin could inhibit approximately 30% of calpain activity, down-regulation of calpain activity via calpeptin could not sensitise ovarian cancer SKOV3, PEO1 and PEO4 cells to cisplatin and carboplatin. Hence, to revisit the question as to whether conventional calpains and calpastatin are associated with chemoresponse and patient survival, a larger cohort was used to validate the previous study. In the current study, the expression of the conventional calpain subunits and calpastatin were positively associated with each other. Calpain-2, -4 and calpastatin expression were associated with overall survival (OS) but none of them was an independent marker of OS in multivariate analysis. Neither the conventional calpain subunits nor calpastatin expression was found associated with resistance to platinum-based adjuvant chemotherapy. Since low calpain-1 expression was associated with low tumour stage, cellular processes that involved in cancer spread were studied. Again calpeptin was used for the inhibition of calpain activity, but no significant inhibition was observed on ovarian cancer cell migration. In contrast, upregulating calpain activity by A23187 using optimised concentration was found able to significantly inhibit the migration of SKOV3 cells. The recently found calpain-related proteins MAP4 and Syk were then included into the current study. Like calpain-1, neither MAP4 nor Syk expression was associated with patient outcome. Next, cases were grouped by clinicopathological variables for the examination of the association between the protein expression and survival; in such a case only high nuclear Syk expression was significantly associated with better patient outcome in certain subgroups. Low calpain-1 expression was associated with low tumour stage, so were MAP4 and Syk expression. MAP4, Syk and calpain-1 expression were significantly associated with tumour histological subtypes and their expression were significantly correlated with each other. Integrin α2β3 was moderately correlated with calpain-1, MAP4 and cytoplasmic DARC expression. In conclusion, although in both the previous and the current study, calpain-2 expression was adversely associated with OS of ovarian cancer patients, the current results did not support the initial hypothesis that calpain can sensitise ovarian cancer cells to cisplatin/carboplatin. The roles that calpain system played in cancer cell haptotactic migration appeared to vary with cell context. Calpain-1, MAP4 and Syk expression were significantly correlated with each other and were closely related to ovarian cancer spread.
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Wan, Feng. "[Rôle du sytème calpaïne /calpastatine dans le remodelage cardiovasculaire]." Thesis, Paris Est, 2013. http://www.theses.fr/2013PEST0067.
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Rôle du Système de calpaïne/calpastatine dans l'hypertension pulmonaire induite par l'hypoxie chez la souris. Les souris calpaïnes knockout ont montré des effets protecteurs dans l'hypertension pulmonaire (HP) induite par l'hypoxie. Cependant, le modèle animal avec une surexpression de calpastatine (cast) n'a jamais été étudié. Notre objectif est d'utiliser des souris transgéniques CMV-cast qui surexpriment constitutivement la calpastatine intracellulaire sous le contrôle du promoteur CMV dans tous les types cellulaires pour étudier les effects de la calpaïne intracellulaire. Nous utilisons aussi des souris transgéniques CRP-cast qui surexpriment la calpastatine extracellulaire sous le contrôle du promoteur CRP (protéine C-réactive) pour étudier les effets de la calpaïne extracellulaire. Finalement, nous examinons les effets d'un traitement avec le PD150606, inhibiteur de calpaïne, chez des souris C57BL/6j (WT) hypoxiques et SM22-5HTT+ qui dévélopent l'HP spontanément. Nous avons constaté que les protéines calpaïne et calpastatine sont augmentées immédiatement dans un état hypoxique. Les calpaïnes ont ensuite culminé le jour 8 et sont restées élevées jusqu'au jour 18 chez les souris WT hypoxiques, alors que la calpastatine a augmenté du jour 1 au jour 3, retournant au niveau basal jusqu'au jour 18. Les activités des calpaïnes intra- et extra-cellulaires ont augmenté progressivement pour atteindre un sommet au jour 8, restant aux niveaux élevés jusqu'au jour 18 chez les souris WT hypoxiques. En utilisant l'immunofluorescence, nous avons constaté que l'augmentation des calpaïnes sont principalement colocalisés avec les CML vasculaires pulmonaires (α-SMA+). Cependant, chez la souris CMV-cast, la surexpression de la calpastatine a atténué le développement d'HP. Chez les souris CMV-cast hypoxiques les niveaux de calpastatine sont restés plus élevés que ceux des souris WT à tous les moments de l'hypoxie. Les niveaux de calpastatine plus élevés chez les souris CMV-cast ont empêché de manière significative une augmentation des niveaux de protéines calpaïnes et des activités intra- et extra-cellulaires de la calpaïne au cours de l'hypoxie. Les résultats d'immunofluorescence également ont confirmé que moins de calpaïnes colocalisent avec les CML vasculaires pulmonaires (α-SMA+) chez la souris CMV-cast. Après 18 jours hypoxie, CRP-cast mice ont attenué le développement d'HP. En outre, cette surexpression a montré des effets similaires par rapport à celle intracellulaire. Cependant, le PD150606 a eu que des effets supplémentaires chez les souris WT hypoxiques par rapport aux souris CMV-cast et CRP-cast hypoxiques. Chez les souris SM22-5HTT+, les niveaux de calpastatine, de calpaïnes ainsi que des activités intra- et extra-cellulaires de la calpaïne ont été significativement augmentés dans les poumons. Le PD150606 n'a pas modifié les niveaux de calpastatine, mais il a diminué de manière significative des calpaïnes ainsi que des activités intra- et extra-cellulaire de calpaïne. Les niveaux de calpastatine et des calpaïnes ont également paru augmentées dans les vessaux pulmonaires remodelés chez les patients atteints de maladie pulmonaire chronique par rapport à ceux nonremodelés. En résumé, nos résultats indiquent un nouveau rôle des calpaïnes extracellulaires dans la prolifération des CML-AP dans l'HP. Les stratégies d'inhibition des calpaïnes extracellulaires sembleraient être une stratégie thérapeutique dans le traitement de la progression de l'HPTargeting the Calpain/Calpastatin System to Protect against Hypoxia-induced Pulmonary Hypertension in Mice. Calpain knockout mice exhibited protective effects against hypoxia-induced PH. Our aim was to study the role of the calpain/calpastatin system on PH development in mice. To this end, we used mice ubiquitously overexpressing intracellular calpastatin (cast) under the control of a CMV promoter (CMV-cast) to explore the effects of intracellular calpains. We also used mice overexpressing extracellular calpastatin under the control of a CRP (C-reactive protein) promoter (CRP-cast) to explore the effect of extracellular calpains. Finally, we examined the effects of treatment with PD150606, an inhibitor of calpain, in WT mice exposed to hypoxia and in SM22-5HTT+ mice with spontaneous PH. During time-course of hypoxia, we found that calpain and calpastatin protein levels increased immediately after hypoxic exposure. Calpain protein levels then peaked on day 8 and remained elevated until day 18 in hypoxic WT mice; however, calpastatin protein levels increased from day 1 to day 3, and returned to basal level until day 18. Both intra- and extra-cellular calpain activities were upregulated gradually and peaked on days 8, and still markedly remained in high levels until day 18 in hypoxic WT mice. By using immunofluorescence, we found that increased calpains were predominantly colocalized with α-SMA positive pulmonary vascular SMCs. In CMV-cast mice, intracellular calpastatin overexpression successfully attenuated PH development. In CMV-cast mice, calpastatin protein levels remained higher than those in WT mice at all time points of hypoxia. The higher calpastatin protein levels in CMV-cast mice did significantly prevent an increase in calpain protein levels and calpain intra- and extra-cellular activities during hypoxia. After 18 days hypoxia, CRP-cast mice exhibited less PH severity. Moreover, extracellular calpastatin overexpression showed similar effects as intracellular calpastatin overexpression. Treatment with PD150606 induced an additional protective effect in hypoxic WT mice but not CMV-cast and CRP-cast mice. In SM22-5HTT+ mice, lung calpastatin and calpain proteins as well as calpain intra- and extra-cellular activities were significantly increased. PD150606 did not alter lung tissue calpastatin. However, it significantly decreased calpain protein levels as well as calpain intra- and extra-cellular activities. In summary, our present results demonstrate that calpain inhibition prevents PH development. Either increasing extracellular calpastatin or increasing both extra- and intra-cellular calpastatin is efficient to attenuate PH. Treatment with PD150606 which inhibits both extra- and intra-cellular calpain activities may be useful in teh setting of PH
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Hewitt, Kimberley E. "The role of calpain in excitotoxic neuronal cell death." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0017/NQ46523.pdf.
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Adams, Sarah Elizabeth. "The synthesis and evaluation of novel calpain-I inhibitors." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/53271/.
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The calcium activated cysteine protease calpain-I has a pivotal role in a variety of physiological processes within the human body. In particular calpain-I enables the cell spreading and subsequent chemotaxis behaviour of neutrophils in response to tissue damage. Neutrophils are linked to the pathological condition rheumatoid arthritis and so calpain-I is considered be a valuable therapeutic target. Many inhibitors of calpain-I are highly non-selective with the exception of two small molecule synthetic inhibitors. A phenyl and an indole-based α-mercaptoacrylic acid have shown a slight selectivity towards calpain-I over other cysteine proteases. In this work 24 novel monohalogenated α-mercaptoacrylic acid inhibitors were prepared based on these lead structures using Vilsmeier-Haack chemistry followed by Knoevenagel condensation of the resulting aromatic aldehydes as key steps. The thiols within the α-mercaptoacrylic acid moiety demonstrated a tendency to form disulfide bridges in solution. Analysis of this disulfide formation through 1H NMR spectroscopy, UV-Vis spectrophotometry and HPLC showed that the monomeric form was active under the reducing conditions used in subsequent assays. The analogues were tested as inhibitors of calpain-I revealing that bromoindole based inhibitors were the most potent. Selected compounds showed ~10 fold selectivity towards calpain-I versus calpain-II. In live neutrophils they were capable of slowing the cell spreading process by up to 70%. When live neutrophils containing the inhibitor were irradiated with 410 nm light, the cells completely lost the ability to spread. To show that these compounds were allosteric inhibitors the calcium binding domain PEF(S) was expressed in E. coli and purified using anion exchange chromatography and size exclusion chromatography. Solution of X-ray co-crystal structures of the calpain PEF(S) domain with two different inhibitors revealed that they bind to the protein in a similar fashion as an α-helical domain of calpastatin, the endogenous inhibitor of calpain.
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Chen, Hongyuan. "Development of macrocyclic β-strand calpain cysteine protease inhibitors." Thesis, University of Canterbury. Chemistry, 2011. http://hdl.handle.net/10092/5582.
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The work in this thesis reports studies directed to developing a calpain cysteine protease
inhibitor that could be of value in slowing cataract development in humans. The work
focuses on the development of macrocyclic compounds which can have advantages over
acyclic compounds due to their resistance to proteolytic hydrolysis, improved selectivity,
bioavailability and membrane permeability. A review of X-ray crystal structures of
natural and synthetic calpain inhibitors complexed with the cysteine protease calpain
show the inhibitors generally bind in the enzyme active site in an extended β-strand
conformation.
The calpain inhibitor SJA-6017 has been identified as a suitable lead compound. The
importance of the para-fluoro group in SJA-6017 has been investigated. Modifications
have been made to constrain this basic structure within a macrocycle and restrict the
peptide chain as a β-strand conformation. Macrocycle CAT811 is a potent calpain 1 and
2 inhibitor and shows promise in slowing the progression of cortical cataract in trials with
sheep having a hereditary propensity towards the development of cataract.
In this thesis I report studies directed to improve the yield of the key RCM
macrocyclisation step in the synthesis of aldehyde CAT811 and of three ester analogues
(2.1, 2.3 and 2.4).
I also report the development of a more commercial route to CAT811 not involving
RCM but using intramolecular nucleophilic cyclisation.
This intramolecular nucleophilic cyclisation strategy was attempted for the preparation of
a histidine containing macrocyclic ester (4.1a) but was unsuccessful. An alternate
strategy involving intramolecular lactamization proved successful for the synthesis of
histidine-based macrocyclic esters (4.1a-4.3a). Reduction to the corresponding alcohols
(4.1b-4.3b) was successful and oxidation of (4.1b and 4.3b) afforded the corresponding
aldehydes (4.1c and 4.3c) for biological assay against ovine calpain 2.
Aldehyde 4.3c has an IC50 of 1 μM and the corresponding alcohol 4.3b shows no activity
(IC50 > 50 μM) consistent with the modelling which indicated that these two compounds
did not adopt a β-strand conformation in the docking studies. Aldehyde 4.1c, on the other
hand, shows significant inhibitory activity with an IC50 of 238 nM but as expected the
corresponding alcohol 4.1b shows little activity (IC50 = 29 μM). Modelling studies
showed that both the aldehyde 4.1c and the alcohol 4.1b on docking can form a β-strand
with appropriate H-bonding interactions. The aldehyde is more active than the alcohol
due to the reactivity of the aldehyde warhead allowing for the reversible formation of a
hemiacetal. A similar difference in reactivity is observed for CAT811 (30 nM) and its
alcohol analogue (700 nM).
These results demonstrate the value of molecular modelling as a screening mechanism
before unproductive synthetic work is considered.
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Norton, Luke. "Calpain-10 and insulin resistance in human skeletal muscle." Thesis, University of Nottingham, 2007. http://eprints.nottingham.ac.uk/11536/.
Full textAbstract:
Variation in the calpain-10 gene has been linked to a three-fold increased risk for type 2 diabetes in Pima Indian and some European populations. Furthermore, reduced skeletal muscle expression of calpain-10 is associated with reduced insulin mediated glucose disposal and carbohydrate oxidation. The skeletal muscle specific calpain-3 plays a key role in skeletal muscle integrity and has also been linked to insulin resistance in humans and rodents. The major aims of this thesis were to 1) investigate the hypothesis that alterations in insulin sensitivity in healthy humans would lead to significant changes in the mRNA and protein expression of calpain-10 and -3, 2) investigate the effect of hyperinsulinaemia and lipid availability on calpain-10 and -3 expression, 3) further address the role of genetic variation in the calpain-10 gene on glucose utilisation in humans and finally 4) investigate the expression of calpain-10 in skeletal muscle of type 2 diabetic patients. The studies in this thesis show for the first time that insulin resistance as a result of short term fasting or high fat availability is not associated with changes in calpain-10 and -3 mRNA and protein expression, providing evidence against an adaptive role for these genes in the development of fasting- and lipid-induced insulin resistance.
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Parr, Timothy. "Calpain proteinase mRNA and beta-agonist induced muscle growth." Thesis, University of Nottingham, 1991. http://eprints.nottingham.ac.uk/11445/.
Full textAbstract:
The mechanism by which β-agonists induce skeletal muscle hypertrophy is believed largely to be through a reduction in protein degradation. These growth promoters are also known to effect the activity of the calcium dependent proteinases (calpains) and their specific endogenous inhibitor calpastatin. The changes in activity appear to be toward a decrease in the calpain system's proteolytic potential. In this study attempts were made to determine whether the altered activity of the enzymes and inhibitor were brought about by induced changes in gene expression as reflected by altered levels of specific mRNAs. Various strategies were employed to generate oligonucleotide and cDNA probes to calpain I and II and calpastatin which would detect their respective mRNAs in L. dorsi total RNA samples originating from a bovine growth trial using the ß-agonist cimaterol. Semi-quantiative measurements of specific mRNAs using Northern blot analysis were related to enzyme and inhibitor activities. In addition ß-agonist-mediated effects on muscle RNA and expression of actin and myosin light chain 2 mRNAs were determined. Using a human calpain cDNA specific hybridization was detected for bovine calpain II mRNA which increased by 34% in the L. dorsi of cimaterol treated animals, similar to the increase in the enzyme activity, 28%. A novel bovine-specific calpastatin cDNA was generated by the polymerase chain reaction and sequence analysis allowed comparison to those already published for other species. Using this PCR cDNA as a probe multiple calpastatin mRNAs were detected in cattle L.dorsi, as had been observed in rabbit. The predominant mRNA increased by 160% in cimaterol treated steers compared to a 76% change in inhibitor activity. There changes were in contrast to the essentially unchanged response of muscle total RNA and actin and myosin light chain 2 specific mRNAs in treated animals. The implications for the calpain system in ß-agonist induced hypertrophy are discussed.
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Liu, Wen. "The characterisation of calpain-like proteins in Trypanosoma brucei." Thesis, University of Hull, 2010. http://hydra.hull.ac.uk/resources/hull:6899.
Full textAbstract:
Calpains are a ubiquitous family of calcium-dependent cysteine proteases involved in a wide range of cell regulatory and differentiation processes. In many protozoan organisms, atypical calpains have been discovered that lack the characteristic calcium-binding penta-EF-hand motif of typical vertebrate calpains and most of these novel calpain-like proteins are non-enzymatic homologues of typical calpains. The gene family is particularly expanded in ciliates and kinetoplastids, comprising 25 members in the parasite Trypanosoma brucei. Unique to kinetoplastids, some calpain-like proteins contain N-terminal dual myristoylation/palmitoylation signals, a protein modification involved in protein-membrane associations. We analysed the expression of calpain-like proteins in the insect (procyclic) and bloodstream-stage of T. brucei using quantitative real time PCR and identified the differential expression of some of the calpain genes. We also present a comprehensive analysis of the subcellular localisation of selected members of this protein family in trypanosomes. Here, of particular interest is the role of protein acylation for targeting to the flagellum. We show that, although acylation is important for flagellar targeting, additional signals are required to specify the precise subcellular localisation.
26
Joyce, Peter. "Characterisation of the atypical calpain family of C elegans." Thesis, University of Bristol, 2008. http://hdl.handle.net/1983/fd3f92bf-ab35-4564-84d8-f36227fc0640.
Full textAbstract:
Regulated proteolysis plays an important role in modulating the activities of receptors, cytoskeletal proteins and transcription factors during cell growth and development. A family of calcium-regulated thiol proteases, known as calpains, has been shown to promote this process in mammals, and other eukaryotes. The importance of calpains is highlighted through their involvement in a number of pathologies in humans.
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Drouet, Saltos Domenica Elizabeth. "Calpain-Calpastatin System in Peripheral Nerve Myelination and Demyelination." Wright State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wright1559220437439116.
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Redpath, Gregory. "Calpain cleavage and subcellular characterisation of the ferlin family." Thesis, The University of Sydney, 2015. http://hdl.handle.net/2123/14278.
Full textAbstract:
The ferlins are a family of C2-domain containing proteins. C2 domains regulate vesicle fusion in synaptotagmins, and animal models of ferlin deficiency display pathologies related to Ca2+-dependent vesicle fusion. Dysferlin mutations cause limb-girdle muscular dystrophy due to defective membrane repair. Our group has previously shown that Ca2+-dependent proteases, calpains, cleave dysferlin following membrane injury, releasing mini-dysferlinC72, that we hypothesise mediates membrane repair. Otoferlin mutations cause non-syndromic deafness, while no pathology causing mutations have been identified in other ferlins. My project establishes that dysferlin and myoferlin, type-I ferlins, are present at the plasma membrane and endo-lysosomal pathway while otoferlin and Fer1L6, type-II ferlins, are present at the plasma membrane and recycling trans-Golgi compartments. I also show that dysferlin is cleaved to mini-dysferlinC72 following injury in all cell types by the ubiquitous calpains-1 and -2 in the alternatively spliced exon 40a, indicating dysferlin cleavage is a fundamental response to membrane injury. Exon 40a-containing dysferlin recruits to sites of membrane injury in myotubes, indicating mini-dysferlinC72 may function directly at sites of injury. Finally, I have shown that calpains also cleave otoferlin and myoferlin. Cleavage of other ferlins indicates ferlin cleavage is an evolutionarily conserved event, predating the split between type-I and type-II ferlins.
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Robilotto, Anthony T. "Calpain activation following cryopreservation an initial investigation into their roles in cell death and cell adhesion /." Diss., Online access via UMI:, 2005. http://wwwlib.umi.com/dissertations/fullcit/1425587.
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Klanchantra, Mutita. "Design and synthesis of beta-strand conformationally constrained calpain inhibitors for cataract treatment via metathesis ring closure." Thesis, University of Canterbury. Chemistry, 2006. http://hdl.handle.net/10092/1609.
Full textAbstract:
This thesis summarises the progress made in the design and synthesis of conformationally constrained β-strand peptidomimetic compounds using ring closing metathesis methodology under microwave irridation conditions. The best macrocycle were elaborated into an inhibitor for a specific protease target. Calpain was used as an example of protease targeting cataract disease. Chapter One introduces proteases in general centring on the general context of protease inhibitor design. The significant of the β-strand 'bioactive' conformation is discussed in details in particular the exploitation of conformationally constrained to potential lock the 'bioactive' conformation. Chapter Two illustrates in silico methods used to design a series of β-strand macrocycle 2.1-2.7. The analysis of these is performed using molecular modelling software Schrodinger suite (2005). A brief discussion of ring closing metathesis methodology is also included. Chapter Three describes the synthesis of the precursor required for RCM reactions (tripeptides dienes). Various types of allylated amino acid side chains were synthesised. The tripeptides were obtained using standard peptide coupling methodology utilising reagents such as HATU, EDC and HOAT. Chapter Four describes the application of ring closing metathesis for the synthesis of β-strand macrocycles. The development of a new reaction conditions to optimise the ring closing metathesis reaction is discussed. In particular the effect of the use of a Lewis acid (chlorodicyclohexylborane) additive in RCM reactions is investigated. Chapter Five discusses the mechanism of cataract formation, cataract treatment and the potential development of calpain inhibitors. One of the macrocycles synthesised in chapter 4 is elaborated into a calpain inhibitor. The in-vitro assay result of this is presented and this compound is currently undergoing in vivo evaluation.
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Kim, Hyun Woo. "Characterization of genes involved in molting and limb regeneration in land crab, Gecarcinus lateralis." Access citation, abstract and download form; downloadable file 6.77 Mb, 2004. http://wwwlib.umi.com/dissertations/fullcit/3131680.
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Hanouna, Guillaume. "Rôle des calpaïnes dans le vieillissement et la réponse anti-tumorale." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066385/document.
Full textAbstract:
Les calpaïnes 1 et 2 sont des protéases à cystéine ubiquitaires et la calpastatine est leur inhibiteur naturel, également ubiquitaire. Les calpaïnes sont impliquées dans le développement de la réponse inflammatoire via l’activation par protéolyse partielle de plusieurs substrats (activation de NF-κB par le clivage de I-κBα, remodelage du cytosquelette des cellules inflammatoires, clivage de la protéine chaperonne HSP 90…).Il a été précédemment démontré que les calpaïnes favorisent le vieillissement neuronal. Nous avons pu montrer dans un modèle murin que l’inhibition in vivo des calpaïnes par la surexpression de calpastatine limite le vieillissement notamment rénal et vasculaire. L’inflammation liée au vieillissement ou « inflammaging » est considérablement réduite par l’inhibition spécifique des calpaïnes. Ceci est dû, au moins en partie, à l’effet des calpaïnes sur la production de cytokines pro-inflammatoires et sur la maturation de l’interleukine-1.Si les calpaïnes intracellulaires exercent un rôle pro-inflammatoire, les calpaïnes externalisées ont un effet anti-inflammatoire via le clivage de TLR2. Les calpaïnes peuvent en effet être excrétées hors des cellules via les transporteurs ABCA1. Dans le cadre d’un modèle murin de mélanome, nous avons pu montrer que l’inhibition des seules calpaïnes extracellulaires par la surexpression de calpastatine extracellulaire préserve TLR2 et limite ainsi la progression de la tumeur.Les calpaïnes intra- et extracellulaires sont des médiateurs majeurs de la réponse inflammatoire et modulent « l’inflammaging » ainsi que la réponse immune anti-tumoraleCalpain 1 and 2 are cysteine proteases and calpastatin is their natural inhibitor. Calpains and calpastatin are ubiquitous. Calpains are involved in inflammatory response development via activation by partial proteolysis of several substrates (NF-kappaB activation by I-kappaBalpha cleavage, remodeling of inflammatory cells cytoskeleton, cleavage of chaperone protein HSP90 ... ). It has been previously shown that calpains promote neuronal aging. We have shown in a mouse model that inhibition of calpain by calpastatin overexpression limits renal and vascular aging. The inflammation associated with aging or "inflammaging" is considerably reduced by specific inhibition of calpain. This is due, at least in part, to calpain effect on production of pro-inflammatory cytokines and in maturation of interleukin-1 alpha. If intracellular calpains are pro-inflammatory, secreted calpains have an anti-inflammatory effect via cleavage of TLR2. Calpains can indeed be excreted out of the cells via the transporter ABCA1. In the context of a mouse model of melanoma, we have shown that inhibition of extracellular calpain by only extracellular calpastatin overexpression preserves TLR2 and thus limit the progression of the tumor.Calpains intra- and extracellular are major mediators of inflammatory response and modulate the "inflammaging" and the anti-tumor immune response
33
Vanhooser, Lisa M. "Engineering Calpastatin to Develop a Sensor to Detect Active Calpain." Fogler Library, University of Maine, 2006. http://www.library.umaine.edu/theses/pdf/VanhooserLM2006.pdf.
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Nakagawa, Yasuaki. "Calcium-Dependent Neutral Proteinase(Calpain)in Fracture Healing of Rats." Kyoto University, 1994. http://hdl.handle.net/2433/168857.
Full textAbstract:
本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものであるKyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第5555号
医博第1525号
新制||医||577(附属図書館)
UT51-94-C13
京都大学大学院医学研究科外科系専攻
(主査)教授 畑中 正一, 教授 岡 正典, 教授 山室 隆夫
学位規則第4条第1項該当
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Davis, Benjamin. "Genetic and Functional Analysis of Calpain-14 in Eosinophilic Esophagitis." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1447688593.
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Pasalic, Dario. "No Calpain, No Gain: Newly Developed Procedures for the Separation and Characterization of The Calpain Family of Proteins in Human Dystrophic and Non-dystophic Muscle." Thesis, The University of Arizona, 2010. http://hdl.handle.net/10150/146022.
Full textAbstract:
Muscular dystrophy is a disease which gradually deteriorates skeletal muscle cells, leading to the eventual death of such cells and the surrounding tissue. Calpains are Ca2+- dependent proteases and together with the Ca2+-dependent specific inhibitor of calpains, calpastatin, are widely distributed in eukaryotic cells. It has been suggested that part of the enhanced deterioration in the dystrophic state is due to enhanced calpain activity; therefore analysis of normal and dystrophic muscle was essential. Conventional techniques for the isolation and characterization of calpain and calpastatin utilize relatively large muscle samples (>100g), whereas biopsy or post-mortem samples are considerably less than this. Thus, the initial and main objective of this project was to develop methods suitable for purification and analysis of the calpain family from limited muscle samples. With these restrictions in mind, techniques were developed for samples ranging from 0.2-1g, a realistic biopsy extraction. The hypothesis to be further evaluated is that some dystrophies are characterized by increased calpain activity, caused either by an increased expression of m- or μ- calpain or decreased inhibition by calpastatin, or both. The procedures are now in place to test this hypothesis further and extensive analyses are required using defined dystrophic types and increased sampling numbers.
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Mazon, Madeline Rezende. "Efeitos da Imunocastração e de agonistas beta-adrenérgicos sobre a qualidade da carne de bovinos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/74/74131/tde-18052016-095207/.
Full textAbstract:
Os agonistas beta-adrenérgicos (βAA) são conhecidos por aumentar a hipertrofia muscular e lipólise, neste caso uma maneira de se reduzir o efeito da lipólise seria a imunocastração. Dessa forma, o objetivo deste projeto foi avaliar o efeito dos βAA e da imunocastração sobre a qualidade da carne de bovinos Nelore. Foram utilizados noventa e seis bovinos Nelore, sendo que metade dos animais (n=48) receberam uma dose da vacina de imunocastração, e após 30 dias receberam a segunda dose. A outra metade dos animais (n=48) não recebeu nenhuma dose da vacina. Durante 70 dias os animais foram alimentados com uma dieta padrão composta de 24% volumoso e 76% de concentrado. Após 70 dias de confinamento os animais foram divididos em três grupos, dentro de bloco (peso inicial) e condição sexual e foram alimentados por 30 dias, com umas das seguintes dietas: CON - dieta padrão utilizada na fase anterior, sem a adição de βAA; ZIL - dieta padrão acrescida de 80 mg/dia Cloridrato de Zilpaterol; RAC - dieta padrão acrescida de 300 mg/dia Cloridrato de Ractopamina. Ao final desse período os animais foram abatidos e colhidas amostras do músculo Longissimus dorsi para as avaliações de qualidade de carne, lipídeos totais, perfil de ácidos graxos, análise sensorial do consumidor, perfil morfométrico muscular, expressão dos genes calpaína e calpastatina, comprimento de sarcômero. Para a maioria das características avaliadas não foram observadas interações entre os tratamentos. Ao avaliar o efeito da condição sexual, os animais imunocastrados apresentaram maiores intensidades de cor L, a e b, lipídios totais, ácidos oleico, palmítico e total de monoinsaturados e maior frequência para as fibras oxidativas (FO) e glicolíticas (FG) em relação aos não-castrados. Contudo, os animais não-castrados tiveram uma tendência a apresentarem uma carne mais macia na análise sensorial e obtiveram maior frequência das fibras oxidativasglicolíticas (FOG) em relação aos imunocastrados. Quanto ao efeito dos βAA, o grupo ZIL apresentaram uma carne menos macia na força de cisalhamento, maiores concentrações de ácidos heptadecanoico, linoleico, araquidonico ácido C20:3 N6C8C11C14, ômega 6, maior frequência para as FO e menor para FG em comparação ao grupo RAC e CON. No entanto, os animais do grupo CON e ZIL apresentaram maior área para as FO em comparação ao grupo RAC, enquanto que para as FOG, os animais do grupo CON tiveram maior área do que os animais do grupo RAC e ZIL. Na análise sensorial, os grupos RAC e ZIL receberam menores notas para os atributos textura e qualidade global em relação ao CON. Não foi observado efeito da condição sexual e dos βAA sobre a expressão dos genes e comprimento de sarcômero. Conclui-se que a condição sexual e a suplementação com os βAA podem alterar a qualidade da carne, perfil de ácidos graxos e morfométrico muscular, sem, contudo, alterar a expressão dos genes e do comprimento de sarcômero.The Beta adrenergic agonist (βAA) are knowed for increase muscle hypertrophy and lipolysis, in this case on way for decrease the lipolysis effect is use the immunocastration. The objective of this research was evaluated the effect of βAA and immunocastration on meat quality of Nellore . Ninety-six Nellore were fed in this trial; half of the animals (n = 48) received one dose of immunocastration vaccine on d 0, and received another dose at d 30. The other half of animals (n = 48) received no vaccine. Animals were fed with a standard diet consisting of 24% forage and 76% concentrate for 70 d. After 70 d of the standard diet, animals were divided into three groups, and were fed 30 d with one of the following diets: CON - standard diet used in the previous phase, without the addition of βAA; ZIL - standard diet plus 80 mg/d Zilpaterol hydrochloride; RAC - standard diet plus 300 mg/d Ractopamine hydrochloride. After this period, animals were harvested and the Longissimus dorsi sample were colleted to evaluate meat quality, total lipid content, fatty acid profile, consumer sensory analysis, muscle morfometric profile, genes expression of calpain and calpastatin and sarcomere length. For almost of characteristics evaluated, were not observed interactions between treatments. The effect of sexual condition, imunocastrated animals showed higher intensity of color L, a and b, total lipidics, oleic, palmitic and total monounsaturated acids and more frequency for oxidative fibers (FO) and glycolytic fibers (FG) in relation at noncastrated. However, non-castrated animals had a tendency to show a meat tender in sensory analysis and more frequency of oxidative-glicolytics fibres (FOG) in relation to imunocastrated. The βAA effect, ZIL group showed a meat less tender, higher concentrations of heptadecanoic, linoleic, araquidic acids, C20:3 N6C8C11C14, ômega 6, higher frequency for FO and less for FG than RAC and CON group. Animals of CON and ZIL group showed more FO area than RAC group, while for the FOG, animals from COM group showed more area than animals from RAC and ZIL group. In the sensory analysis, RAC and ZIL group received lower grades for tenderness and global quality in relation to COM group. Was no observed effect of sexual condition and βAA for genes expressions and sarcomere length. As conclusion, sexual condition and βAA affected the meat quality, fatty acid profile, muscle fibers, but not affect genes expression and sarcomere lenght.
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Millar, Tarek Lawson. "Synthesis and evaluation of CA clan cysteine inhibitors." Thesis, University of Canterbury. Chemistry, 2008. http://hdl.handle.net/10092/1911.
Full textAbstract:
This investigation involved the synthesis of potential CA clan cysteine inhibitors of m-calpain and cathepsin B. Inhibitors 2.1.3a-j were based on the SJA-6017 construct containing the N-(4-fluorobenzenesulfonyl) moiety at the P₃ address region. The inhibitor 2.1.3k was based on CAT-0059 a novel dipeptide dialdehyde inhibitor containing the 5-formyl pyrrole moiety at the P₃ address region. Chapter 1 introduces proteases in particular m-calpain and cathepsin B implicated in human pathologies cataract and tumour metastasis respectably. Structure, disease processes and known inhibitors for m-calpain and cathepsin B are presented and described. The chapter also describes drug design and rational including the requirement of the β-strand conformation for enzyme substrate binding. Chapter 2 details the synthesis of m-calpain and cathepsin B inhibitors, N-(4-fluorobenzenesulfonyl) peptide aldehyde 2.1.3a-j and the dipeptide dialdehyde 2.1.3k. The synthesis involved the preparation of the N-(4-fluorobenzenesulfonyl) α-amino acids 2.1.8a-f, the N-(4-fluorobenzenesulfonyl) peptide esters of 2.1.10a-g, the peptide alcohols 2.1.11a-k and the peptide aldehydes 2.1.3a-k. Specific coupling reagents for amide bond formation are also discussed. The oxidation of the alcohols 2.1.11a-k with sulfur trioxide and pyridine complex are also addressed. The results from molecular modelling and enzymatic assays of the inhibitors 2.1.3a-k with m-calpain and cathepsin B are presented and discussed.
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Stuart, Blair Gibb. "Molecular Modelling for Enzyme Inhibition: A Search for a New Treatment for Cataract and New Antimicrobials and Herbicides." Thesis, University of Canterbury. Chemistry, 2010. http://hdl.handle.net/10092/4551.
Full textAbstract:
There have been several reports that cataract development results from unregulated Ca2+ mediated degradation of lens crystallins. The calpain isoform m-calpain, a cysteine protease, is known to be a major player in cataract formation in rodent lenses and recent evidence indicates that over-activation by Ca2+ causes cataractogenesis in other mammals. Molecular modelling studies of seventeen analogues of compound SJA6017 (our lead compound) in a calpain model are compared to measured IC50 values against ovine calpain. The studies validated the potential of the ‘model’, method and defined activity criteria that could be used as a tool to select molecules to synthesize as potential calpain inhibitors. Using this screening methodology and two virtual libraries of potential inhibitory molecules led to the synthesis of several inhibitors including macrocyclic 811. In vitro sheep eye lens culture experiments showed that macrocycle 811 possessed the characteristics to slow cataractogenesis.
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Draper, Kati Elizabeth. "Increased structure-bound proteolytic activity in maturing dystrophic skeletal muscle." Thesis, Virginia Tech, 2004. http://hdl.handle.net/10919/31735.
Full textAbstract:
Duchenne Muscular Dystrophy (DMD) is a severe X-linked progressive muscle wasting disease resulting from the absence of the membrane-associated protein dystrophin and the secondary components of the dystrophin-glycoprotein complex. Although the genetic basis of the disease has been known for over 15 years, the onset mechanism of the disease is not yet known and no treatment is yet available to significantly increase the lifespan of DMD patients.
Increased levels of intracellular calcium have been noted in dystrophic muscle (Turner et al., 1991) and increased intracellular levels of calcium in skeletal muscle lead to increased levels of calcium-dependent proteolysis (Zeman et al., 1985). Increased levels of calpain, a calcium-dependent protease have been reported as early as age 4 weeks in mdx (dystrophin-deficient) mice (Spencer et al., 1995). Increased calpain activity has been demonstrated in mdx myotubes (Alderton et al., 2000a). There is also evidence of a role for calpain in DMD, but the contribution of calpain activity to the onset of DMD has not yet been determined.
The purpose of this study was to test the hypothesis that increased calpain activity contributes to the onset of DMD in maturing (birth to weaning) dystrophic skeletal muscles and to determine if increased calpain activity was due to the relative distribution of calpain and calpastatin, calpainâ s endogenous inhibitor. Calpain activity was assessed in quadriceps and diaphragm muscle homogenate supernatant and pellet fractions from C57BL/6 control and mdx mice at ages 7, 14, and 21 days. Total calpain and calpastatin content were determined by Western analysis.
In both the quadriceps and diaphragm samples, calpain activity in the supernatant increased with age. There was a significant increase (47.7%; p<0.05) in calciumdependent calpain activity in mdx quadriceps pellet compared to control at age 7 days. In the quadriceps at age 7 days, calpain activity in the pellet in the presence of calcium was significantly greater than at age 14 (61.2%) and 21 days (52.6%; p<0.05). In the diaphragm, there were no significant differences in pellet activity in either the presence or absence of calcium at any age between control and mdx samples. In both control and mdx diaphragms, pellet calpain activity in the absence compared with the presence of calcium was significantly greater at both age 7 (control, 46.4%; mdx, 45.4%) and 14 days (control, 42.4%; mdx, 43.6%; p<0.05). At age 21 days, both control and mdx calpain activities in the diaphragm supernatants in the presence of calcium were significantly greater than those at ages 7 (control, 66.7%; mdx, 72.1%) and 14 days (control, 39.9%; mdx 49.5%; p<0.05). In general, there were no differences in total calpain and calpastatin content that would account for the differences in calpain activity. There were similar patterns of calpain activity and total calpain and calpastatin content in both control and mdx samples, suggesting a similar pattern of development in control and mdx muscle from ages 7-21 days. The increase in calcium-dependent calpain activity in mdx quadriceps pellet compared to control at age 7 days may be due to differences in regulation and/or distribution of the calpain system early in mdx maturation compared to control. From the present study, the role of calpain in the onset of DMD appears to be minor if global calcium-dependent activity is evaluated.Master of Science
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Wang, Qiong. "The activity and content of calpains in maturing dystrophic muscle membranes." Thesis, Virginia Tech, 2005. http://hdl.handle.net/10919/42729.
Full textAbstract:
Increased calcium-activated calpain proteolysis in the sarcolemma membrane is thought to be a primary mechanism in the pathophysiology of Duchenne Muscular Dystrophy (DMD). However, few studies have tested this possibility prior to the overt signs of the dystrophy. The purpose of this study was to test the hypothesis that there is greater calpain content and total relative calpain activity in membranes obtained from dystrophic (mdx; mdx:utrophin-deficient (mdx:utrn-/-)) compared to wildtype (wt) mouse skeletal muscles during maturation at ages 7- and 21-d,and at a post-maturation age of 35-d. Calpain activity was determined as the calcium-dependent cleavage of the flurogenic substrate SLY-AMC, and content was determined by Western analysis with an anti-calpain antibody. There were several intriguing findings:
1. There was an inverse relationship between calpain content and relative activity in the whole muscle in both wt and mdx mice from age 7- to 35-d: calpain content decreased, and relative calpain activity increased as the mice aged. This suggests a similar role for calpain in both genotypes, which might relate to specific maturation processes, possibly up to age 21-d. Although the inverse relation was evident at 35-d, the targets for calpain in mdx compared to wt likely differed.
2. The increased relative calpain activity in the membrane fraction of mdx mice at age 35-d (26.73 Arbitrary Units, (AU)) compared to that of age 7- (4.9AU; p<0.05) and 21-d (8.74AU; p<0.05) is temporally related to degeneration and regeneration processes, and may also indicate activation of apoptosis, in mdx muscles at this age.
3. At age 7-d, there were no significant differences in either calpain content or relative calpain activity in all subcellular fractions for wt and mdx mice. This result might suggest similar calpain distribution and activities that are related to the regulation of muscle maturation and differentiation in both genotypes. (Note:data were not obtained for the mdx:utrn-/- mice at age 7-d because of insufficient animals).
4. At age 21-d, there was greater relative calpain activity in the myofibrillar supernatant fraction in mdx (15.13AU) than wt mice (1.18AU; p<0.05). This could indicate calpainâ s role in the initiation of myofibrillar protein turnover and the proteolysis of submembranous networks in the mdx muscles.
5. At age 21-d, greater calpain content in the mdx (1.40ìg) compared to wt (0.23 ìg; p<0.05) membrane fraction might suggest a broader distribution of calpain along membranes that contributes to the onset of dystrophy in the mdx muscles.
6. At age 35-d, there was greater calpain content in the mdx:utrn-/- compared to the wt membrane (0.48ìg vs 0.13 ìg), cytosolic (0.88ìg vs 0.30ìg), and myofibrillar supernatant (0.49ìg vs 0.17ìg; p<0.05 ) fractions This increased content and broad distribution across several subcellular fractions may reflect degeneration and regeneration processes, and potentially activation of apoptosis, in the mdx:utrn-/- muscles.
These data suggest that calpain activity contributes to dystrophic pathophysiology mainly in the membrane fraction of mdx skeletal muscles at age ~21-d, but appears to contribute later at 35-d and in more subcellular fractions in mdx:utrn-/- skeletal muscles.Master of Science
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Sun, ChunHui. "Régulation des réponses calciques et de la cytotoxicité par l’effecteur de type III IpgD durant l’invasion des cellules épithéliales par Shigella." Thesis, Université Paris-Saclay (ComUE), 2015. http://www.theses.fr/2015SACLS179.
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Shigella, l’agent de la dysenterie bacillaire, envahit la muqueuse colique, causant une inflammation intense et destruction tissulaire. Afin de promouvoir l’infection, Shigella injecte des facteurs de virulence par l’intermédiaire d’un appareil de sécrétion de type III (T3SS) dans les cellules hôte, qui permettent la réorganisation du cytosquelette d’actine, régulent l’inflammation et l’intégrité du tissu. Parmi ces facteurs injectés par la bactérie, la protéine IpgD agit comme une phosphatidylinositol (PI) (4,5) bisphosphate phosphatase déphosphorylant le PIP2 en PI(5)P. L’hydrolyse du PI(4,5)P2 favorise la polymérisation de l’actine durant l’invasion des cellules épithéliales et régule négativement la migration des lymphocytes T. Il a également été démontré que le PI(5)P produit par IpgD active la voie de survie cellulaire dépendant de la PI(3) kinase et de la kinase AKT. Mon projet de thèse a pour but de caractériser le rôle d’IpgD dans les contrôles des réponses calciques durant l’invasion des cellules épithéliales par Shigella. Nous avons montré qu’IpgD limite le recrutement de récepteurs à l’inositol-triphosphate (InsP3) au site d’invasion. Des expériences d’imagerie calcique indiquent que durant les étapes précoces de l’invasion, IpgD favorise l’émission de réponses locales aux sites d’invasion de Shigella et limite celle de réponses globales. Des expériences de recouvrement de fluorescence après photo-blanchiment (FRAP) indiquent que les foyers d’invasion induits par le mutant ipgD montrent une diffusion moins restreinte que celle observée dans les foyers induits par la souche sauvage. Des études de modélisation supportent la notion qu’en limitant la production locale d’InsP3 et sa diffusion aux sites d’invasion, IpgD participe au confinement des réponses calciques locales en empêchant l’émission de réponses globales. Nous avons montré que lors de cinétiques d’infection prolongée, IpgD inhibe les réponses calciques globales dépendant de l’InsP3 induites par Shigella ou par des agonistes. L’inhibition de ces réponses conduit à un retard de l’activation de la calpaine, de la dégradation de la taline, une protéine des adhérences focales, ainsi que de la mort des cellules durant la réplication bactérienne intracellulaire. Nos résultats indiquent qu’IpgD est un effecteur critique de Shigella permettant de réguler la transition des réponses calciques locales vers des réponses globales et préservant les cellules infectées de la mort liée à une perte d’adhérenceShigella, the agent of bacillary dysentery, invades colonic epithelial cells where it disseminates causing an intense inflammation and tissue destruction. To efficiently promote infection, Shigella injects virulence effectors via a type III secretion system (T3SS) into host cell to divert processes involved in cytoskeletal rearrangements and processes regulating inflammatory signals or tissue integrity. Among the Shigella T3SS effectors, IpgD acts as a phosphatidylinositol (PI) (4, 5) bisphosphate phosphatase, which dephosphorylates PI (4,5) P2 to generate PI(5)P. IpgD-mediated hydrolysis of PI (4, 5) P2 favors actin polymerization during cell invasion and negatively regulates the migration of T cells. PI(5)P produced by IpgD was also shown to up-regulate the PI3K/AKT cell survival pathway and recycling of the Epidermal Growth Factor receptor. My project thesis focuses on the role of IpgD in the control of calcium responses during Shigella infection. We show that that IpgD is responsible for a decrease in the recruitment of inositol-triphosphate (InsP3) receptors at invasion sites. Ca2+-imaging experiments indicate that during the early stages of bacterial invasion, IpgD favors the elicitation of local Ca2+responses at Shigella invasion sites, and limits the induction of global Ca2+ responses. Fliuorescence Recovery After Photobleaching (FRAP) experiments indicate that actin foci induced by the ipgD mutant show faster diffusion kinetics than those in foci induced by WT. Modeling studies support the notion that the local decrease in InsP3 levels and of its diffusion kinetics triggered by IpgD at entry sites accounted for the modulation of local to global Ca2+ responses during Shigella invasion. We show that over prolonged infection kinetics, IpgD inhibits InsP3-dependent global Ca2+ responses induced by Shigella or agonists. IpgD-mediated inhibition of Ca2+ signals delays the activation of calpain, the degradation of the focal adhesion protein talin, and cell death during bacterial intracellular replication. Our results provide evidence that IpgD is a critical T3SS effector of Shigella regulating the transition from local to global Ca2+ signals, and preserving cells from death linked to loss of adhesion
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Allcock, Stephen M. J. "Interactions between calpain proteases #beta#-adrenergic receptor and skeletal muscle growth." Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358286.
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Liu, Zhao. "PALMITATE INDUCED ENDOTHELIAL DYSFUNCTION: THE ROLE OF CALPAIN, AMPK AND ENOS." Master's thesis, Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/288932.
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PhysiologyM.S.
Obesity is a serious health problem worldwide. Consumption of fat rich food is a common cause of obesity. Some of the food components (i.e. saturated free fatty acids (SFAs)) have been identified as inflammatory inducers (Egger G at al., 2010). After a meal, absorbed free fatty acids (FFAs) will be stored in the liver and adipose tissue. On the luminal surfaces of endothelium in adipose tissue microcirculation, lipoprotein lipase hydrolyses absorbed triglycerides into FFAs. Then, in order to be available for adipocyte storage, FFAs have to cross the capillary endothelium barrier, which connected by tight junctions (Stremmel W et al., 2001). Increased leukocyte infiltration is a featured sign of adipose tissue inflammation found in obesity. Endothelial adhesion molecules up-regulation contributes to leukocyte infiltration during inflammation. Some clinical data suggested an increase of leukocyte-endothelium interaction in healthy volunteers after ingestion of high-fat meals (Shimabukuro M et al., 2007). Other lab results also showed that neutrophil infiltration occurred at a very early stage with high-fat feeding in mice (Talukdar S et al., 2012). However, the detailed mechanism of the above phenomena is still unknown. This thesis provides exciting preliminary data which will guide the further study in this area. First of all, we successfully established a stable protocol that CD31 antibody conjugated microbeads were used to isolate primary microvascular endothelial cells from fresh mice lung tissue. After second sorting, CD31+ cells reach 83.3% by FACS analysis. Previous literatures showed that FFAs activate recruitment of inflammatory cells through up-regulation of endothelial adhesion molecules via reduced eNOS derived eNO production (Rizzo NO et al., 2010; Davenpeck KL et al., 1994; Ahluwalia A et al., 2004). In this thesis, it was found that SFAs palmitate exposure dose dependently reduced endothelial AMPK thr172 and eNOS ser1177 phosphorylation by western blot. Moreover, our study demonstrated that endothelial calpain, a calcium dependent protease associated with endothelial dysfunction, was activated by palmitate, specifically its μ-calpain isoform. Altogether, these data suggested that a new role of calpain as a key mediator of palmitate induced endothelial dysfunction and indicated both AMPK and eNOS1177 phosphorylation contribute to this pathological process. Further investigations are still needed to explore connections among those molecules. This thesis may also lead to a novel way of clinical treatment for the obese related vascular diseases.
Temple University--Theses
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Cataldo, Francesca. "Role of calpain in USP1 stability regulation and genome integrity maintenance." Doctoral thesis, Università degli studi di Trieste, 2012. http://hdl.handle.net/10077/7860.
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2010/2011The calpains are a family of intracellular cysteine proteases, among which the best studied isoforms, micro- (CAPN1) and milli-calpain (CAPN2), are heterodimers consisting of a catalytic subunit and a common regulatory subunit, CAPNS1, required for function. Calpain is involved in many processes important for cancer biology, such as autophagy, indeed in calpain-depleted cells autophagy is impaired, with a subsequent increase in apoptosis sensitivity. Calpain is also important in all the stages of the stress response. A proteomic approach was employed for the identification of novel CAPNS1 interacting proteins. Proteins immunoprecipitating with endogenous CAPNS1 in HT1080 cell lysates were analyzed by Mass Spectrometry. We identified novel partners among which the deubiquitinating enzyme USP1, a key regulator of the DNA damage response and genome integrity maintenance via its specific action on FANCD2, involved in DNA repair and protection from chromosome instability, and PCNA, involved in the regulation of translesion DNA synthesis (TLS), that bypasses DNA lesions with low stringency basepairing requirements. We performed co-IP assays in lysates of 293T cells and confirmed that the interaction was specific. Furhermore, we observed that calpain is able to bind a USP1 C-terminal deleted mutant, suggesting that USP1 first 523 aminoacids were sufficient for the binding. To understand what is the effect exerted by calpain upon USP1, we depleted calpain activity in a series of cell lines, and followed the fate of endogenous USP1. We transfected CAPNS1 specific siRNAs, or treated cells with a specific inhibitor of calpain, and we observed a strong decrease in USP1 protein levels. This effect should be at a post-transcriptional level, since any significant change in USP1 mRNA levels is detected. We also obtained the same result by transfecting a siRNA specific for CAPN1, the gene encoding for the catalytic subunit micro-calpain. Moreover, we studied the role of calpain in the PCNA-mediated switch between high fidelity replication and TLS upon UV irradiation. In mouse embryonic fibroblasts knockout for CAPNS1, USP1 downregulation is coupled to an increase in PCNA monoubiquitination. Moreover, CAPNS1-depleted U2OS cells showed an increase in the percentage of nuclei containing PCNA-induced foci upon UV irradiation. Since we demonstrated that calpain can modulate an important regulator of DNA damage response such as USP1, we investigated if calpain could have a role in genome integrity maintenance. CAPNS1 depleted cells showed a reduced rescue in DNA repair compared to control cells, suggesting that increased levels in PCNA monoubiquitination could lead to an increased amount of errore-prone TLS. Calpain plays an important role in autophagy, so we asked if USP1 degradation in absence of calpain activity could involve autophagic pathways. We first blocked macroautophagy by silencing ATG5, and we observed that USP1 was downregulated, suggesting that the depletion of ATG5 could lead to an increased activity of other degradation pathways. To impaire chaperone-mediated autophagy (CMA), we silenced a protein important for autophagosome formation, LAMP-2A. Also in this case we observed a decrease in USP1 protein levels, thus suggesting that USP1 is alternatively degraded by different pathways. However, we observed that USP1 is stabilized upon inhibition of lysosomal enzymes, suggesting that USP1 may be degraded in the lysosome. To better understand the mechanism by which calpain affect USP1 stability we search for an effect of calpain upon USP1 co-factor and activator UAF1/WDR48. CAPNS1-depleted cells showed WDR48 downregulation, but WDR48 overexpression only partially rescue USP1 protein levels in this cells. Furthermore, we provided evidences that calpain regulation of p35/p25 activator of Cdk5 can affect Cdh1 phosphorylation and thus APC/Cdh1 activity, leading to a regulation of USP1 stabilization. In conclusion, we identified USP1 as a novel interactor of calpain, and we found that calpain is important for USP1 stability, since in its absence USP1 is downregulated. The importance of this novel regulation is strengthened by the recent findings that unveiled a role of USP1 in maintenance of a mesenchymal stem cell program in osteosarcoma, and thus placing calpain in a crucial regulatory position for cancer development.
XXIV Ciclo
1983
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MARCASSA, ELENA. "Dissection of the role of calpain in the regulation of autophagy." Doctoral thesis, Università degli Studi di Trieste, 2016. http://hdl.handle.net/11368/2908036.
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Lo scopo principale del progetto di dottorato è stato quello di studiare il ruolo delle calpaine ubiquitarie nella regolazione del processo autofagico. Differenti tecniche biochimiche, di microscopia confocale e elettronica, ci hanno portato all'identificazione di una proteina target della micro-calpaina: Bif-1. L'azione proteasica su Bif-1 sembra sia indispensabile per la fissione e il trasporto di porzioni del Golgi verso il sito di formazione dell'autofagosome.
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Nettersheim, Jo-Ann. "Interplay between the translesion DNA polymerase η and the calpain system." Electronic Thesis or Diss., Strasbourg, 2020. http://www.theses.fr/2020STRAJ078.
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L’ADN est constamment altéré du fait du métabolisme oxydatif de la cellule ou de l’exposition à des agents génotoxiques. Malgré l’existence de systèmes de réparation efficaces, certaines lésions présentes lors de la phase S bloquent la progression des fourches de réplication. La synthèse translésionnelle (TLS) permet la reprise de la réplication. Pol η est une ADN polymérase TLS capable de franchir et sans erreur les lésions induites par les UV mais pol η est mutagène lorsqu’elle réplique l'ADN non endommagé. Par conséquent, son activité doit être strictement régulée. La thèse présente l’étude de l’interaction entre pol η et CAPNS1, la sous-unité des calpaïnes 1 et 2. Ces protéases ubiquitaires dépendantes du calcium régulent de nombreux processus cellulaires. Nous montrons que CAPNS1 est colocalisée avec pol η dans le noyau. La calpaïne clive pol η in vitro et in vivo après l’acide aminé 465, laissant le domaine catalytique intact et le motif PIP1. De manière surprenante, l'inhibition de la calpaïne entraîne une diminution de la formation de foyers pol η et de la survie cellulaire. Ces résultats suggèrent que la calpaïne est impliquée dans la TLS dépendante de pol ηCells are constantly exposed to DNA damaging agents causing lesions, which are repaired by a range of DNA repair pathways. If DNA damages prevail during replication, they can cause replication fork breakdowns and mutations. One mechanism to prevent this is the translesion synthesis (TLS). Pol η is a translesion DNA polymerase which is capable to circumvent UV-induced lesions, to be repaired at a later time. However, pol η is error prone on non-damaged DNA and, therefore, needs to be tightly regulated. This thesis present an interaction between pol η and CAPNS1 and our investigation of its role in regulating pol η. CAPNS1 is the small subunit of the calcium dependent calpain 1 and 2. We demonstrate that CAPNS1 is colocalized with pol η in the nucleus and calpain 1/2 can cleave pol η in vitro and in vivo. The proteolysis of pol η is found to occur at position 465 leading to a truncated protein encompassing the catalytic domain and the PIP1 motif. Interestingly, inhibition of calpain leads to a perturbed pol η foci formation and decreased cell survival. Taken together, these results suggest an important positive role for calpain in pol η dependent TLS
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Andrique, Caroline. "La calpaine- 6 identifie et maintient la population de cellules souches des necones osseux en contrôlant les processus d'autophagie et de sénescence." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC306/document.
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Les cellules souche cancéreuses contribuent au développement des sarcomes, mais le manque de marqueurs spécifiques empêche leur caractérisation et la possibilité de cibler ce type de cellules. Nous avons utilisé la séquence régulatrice de la calpaïne-6 dans des systèmes rapporteurs pour identifier les cellules exprimant la calpaïne-6. Ces cellules étaient des cellules initiatrices de tumeurs et se comportaient comme des cellules souche, au sommet de la hiérarchie cellulaire. L'expression de la calpaïne-6 dépend d’un programme génique de cellules souche qui implique Oct4, Nanog et Sox2 et est activée par l'hypoxie. L’inhibition de la calpaïne-6 a bloqué le développement tumoral et a induit la diminution du nombre de cellules souche cancéreuses dans les sarcomes osseux. L’expression de la calpaïne-6 était inversement corrélée à l'expression de marqueurs de sénescence mais était associé à un flux autophagique dynamique. L’inhibition de la calpaïne-6 a induit l'entrée des cellules en sénescence et a supprimé le flux autophagique. Nos résultats révèlent que le calpaïne-6 identifie les cellules souche des sarcomes et joue un rôle important dans le maintien des cellules souche cancéreuses en contrôlant les processus d’autophagie et de sénescence. La calpaïne-6 semble être une cible thérapeutique prometteuse pour éradiquer les cellules souche dans les sarcomesCancer stem cells contribute to sarcoma development, but lack of specific markers prevents their characterization and the possibility of targeting. We used the regulatory sequence of calpain-6 in reporter constructions to identify calpain-6–expressing cells. These cells were tumor-initiating cells and behaved like stem cells at the apex of the cellular hierarchy. Calpain-6 expression depended on the stem-cell transcription network that involves Oct4, Nanog, and Sox2 and was activated by hypoxia. Calpain-6 knockdown blocked tumor development and induced depletion of sarcoma stem cells. Calpain-6 was inversely associated with expression of senescence markers but was associated with a dynamic autophagy flux. Calpain-6 knockdown induced cell entry into senescence and suppressed autophagy flux. Our results reveal that calpain-6 identifies sarcoma stem-cell and plays an important role as a regulator of cancer cell fate driving a switch between autophagy and senescence. Calpain-6 may be a promising therapeutic target to eradicate sarcoma stem cells
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Carvalho, Minos Esperândio. "Caracterização da freqüência de polimorfismos em genes ligados à maciez da carne em bovinos da raça Nelore." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/74/74131/tde-23062008-082501/.
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O objetivo deste trabalho foi avaliar o potencial de utilização de marcadores moleculares em genes candidatos da calpaína (CAPN) e calpastatina (CAST) como ferramenta auxiliar para programas de melhoramento de características relacionadas ao crescimento e maciez da carne. Foram avaliados 605 bovinos da raça Nelore, pertencentes à Agropecuária CFM Ltda, com idade média ao abate de 24 meses. Após a extração do DNA de amostras de sangue, por desproteinização em presença de NaCl, a identificação e determinação do polimorfismo para os marcadores moleculares CAPN316, CAPN530, CAPN4751, CAPN4753 e UOGACAST1, foi realizada pelo sistema de detecção TaqManTM utilizando-se PCR em Tempo Real. A análise de maciez da carne, aos 7, 14 e 21 dias de maturação, foi realizada com amostras de carne do Longissimus dorsi, retiradas entre a 12ª e 13ª costela e cisalhadas utilizando-se um Warner Bratzler Shear Force. Nenhum efeito significativo dos marcadores avaliados foi observado para as características de crescimento. Foi verificado efeito significativo, em relação à maciez da carne, para os seguintes polimorfismos: aos 7, 14 e 21 dias de maturação para o marcador CAPN4751; aos 21 dias para o marcador CAPN4753 e aos 14 e 21 dias para o marcador UOGCAST1. Em relação aos efeitos das combinações genotípicas para os marcadores dois a dois, os resultados foram significativos para a combinação CAPN4751/UOGCAST1 nos três tempos de maturação. Para a combinação de marcadores CAPN4753/UOGCAST1 também foram verificados resultados significativos para carnes maturadas aos 14 e 21 dias. Os resultados observados neste trabalho sugerem a possibilidade da utilização de seleção assistida por marcadores (MAS), visando o aumento da qualidade da carne em bovinos da raça Nelore.The objective of this study was to evaluate the potential utilization of molecular markers on candidate calpain and calpastati n genes as an auxiliary tool for breeding programs on traits related to growth and meat tenderness. A total of 605 Nellore animals, raised by CFM Agro-pecuária Ltda, were used in this study and slaughtered with 24 months in average. After DNA blood samples extraction, by desproteinization in presence of NaCl, the polymorphism (CAPN316, CAPN530, CAPN4751, CAPN4753 and UOGACAST1) identification and determination was realized by TaqManTM detection system using real time PCR. The meat tenderness analysis, at the 7, 14 and 21 days of maturation was realized with Longissimus dorsi meat samples, taken at the 12th and 13th rib interval and Warner Bratzler Peak Shear Force measurements were used. There were no significant effects of molecular markers in growth traits. There were significant effects, regarding to meat tenderness, for following polymorphisms: at 7, 14 and 21 days of maturation, for CAPN4751 marker; at 21 days of maturation, for CAPN4753, and finally, at 14 and 21 days of maturation, for UOGCAST1 marker. In respect to genotypic combination effects analysis for pairwise marker, the results were significant for CAPN4751/UOGCAST1 in three days of maturation. In combination effects for CAPN4753/UOGCAST1 markers, significant effects were also observed for meat tenderness at 14 and 21 days. Theses results suggest that marked selection assisted (MAS) can be used to improve meat quality in Nellore beef cattle.
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Xu, Jin. "Synthesis of novel sulfonamide-based calpain inhibitors and their potential as anti-tumor agents." View the abstract Download the full-text PDF version, 2007. http://etd.utmem.edu/WORLD-ACCESS/Xu/2007-028-Xu.pdf.
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Thesis (M.S. )--University of Tennessee Health Science Center, 2007.Title from title page screen (viewed on July 28, 2008). Research advisor: Dr. Isaac O. Donkor, Ph.D. Document formatted into pages (xii, 80 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 65-80).