To see the other types of publications on this topic, follow the link: Metalloproteasi.
Dissertations / Theses on the topic 'Metalloproteasi'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Metalloproteasi.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Mazzoni, Annalisa <1979>. "Ruolo delle metalloproteasi nei processi degenerativi dei tessuti duri del cavo orale." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2165/1/mazzoni_annalisa_tesi.pdf.pdf.
Full text
2
Mazzoni, Annalisa <1979>. "Ruolo delle metalloproteasi nei processi degenerativi dei tessuti duri del cavo orale." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2165/.
Full text
3
Orpianesi, E. "STUDIO DELLE CITOCHINE E DELLE METALLOPROTEASI NELLA LINFANGIOLEIOMIOMATOSI E NELLA SCLEROSI TUBEROSA." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/230542.
Full textAbstract:
Tuberous sclerosis complex (TSC), an autosomal dominant disease, is characterized by the formation of hamartomas in various organs such as brain, kidney, skin, retina and heart, and is caused by mutations in TSC1 e TSC2 tumor suppressor genes, encoding hamartin and tuberin, respectively. Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by cystic lung destruction leading to progressive respiratory failure and formation of abdominal tumors. LAM can be sporadic or associated to TSC and LAM lung alterations resulting from proliferation of neoplastic cells bearing mutations in either TSC1 or TSC2 genes. A metastatic process has been proposed in dissemination of LAM and TSC cells to explain the identical TSC2 mutations and loss of heterozigosity (LOH) patterns in LAM cells of lung nodules, angiomyolipomas and lymph nodes of the same sporadic LAM patients, that is consistent with metastatic spread among organs.
The aim of this project is to study the role of cytokines and matrix metalloproteinases in LAM and TSC. LAM/TSC cells, isolated from chylous of a patient affected by LAM associated to TSC, bear a TSC2 germline mutation and do not express tuberin for an epigenetic silencing of the TSC2 second allele that confirms our previously published evidence that an epigenetic alterations of TSC2 can cause the loss of tuberin in TSC cells. Treatment of LAM/TSC cells with 5-azacytidine that inhibits CpG DNA methylation led to the expression of tuberin as consequence of the chromatin remodeling. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death, as we previously showed in cells lacking tuberin, while rapamycin, an mTOR inhibitor, significantly reduced proliferation. LAM/TSC cells have the ability to grow independently from adhesion and survive in adherent and nonadherent condition. For these features these cells are a good model to study the mechanisms of motility in LAM and TSC and the relation to tuberin expression. We studied the effect of anti-EGFR Ab and rapamycin on motility, cytokines and MMPs (Matrix metalloproteinases) expression. LAM/TSC cells spontaneously detach and undergo spontaneous cycles of adhesion and nonadhesion conditions likely for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway. In LAM/TSC cells FAK inhibition caused the reduction of AKT phosphorylation which was followed by inhibition of mTOR phosphorylation and mTOR autophosphorylation and, consequently, by a strong reduction of S6 phosphorilation as it occurs in nonadherent cells. Anti-EGFR antibody and rapamycin reduced the viability of adherent and nonadherent cells while 5-azacytidine slightly increased the percentage of detaching cells. Nonadherent LAM/TSC cells underwent an extremely low proliferation rate consistent with tumour stem cell characteristics. One of the step driving EMT is the repression of E-cadherin, resulting in the loss of cell-cell adhesion. LAM/TSC cells did not express E-cadherin, but they express vimentin, marker of mesenchymal cells.
EMT process controls the migration of cancer cells from primary tumors depending on an inflammatory microenvironment. LAM/TSC cells secreted high amount of IL-6, IL-8 and IL-1α, cytokines crucial for a variety of cancer cells. The levels of IL-6, IL-8 and IL-1α were not affected by rapamycin or anti-EGFR antibody but were regulated by tuberin expression since their levels were reduced by 5-azacytidine incubation. MMPs degrade and modify the extracellular matrix (ECM), facilitating detachment of cells from the tissue. Levels of MMP-2 and MMP-9 in urine are predictive of disease status in a variety of cancers and also in LAM. MMP-2 and MMP-9 levels are high in urine of LAM patients and MMP-2 is substantially up-regulated in their lung tissue. Matrilisin (MMP-7) contributes to tumor progression, invasion and is overexpressed in several types of invasive cancers.
In adherent status LAM/TSC cells expressed higher levels of MMP-2 mRna and lower of MMP-7 than in nonadherent condition. Consistent with ECM degration and invasive features respectively,. MMP-2 and MMP-7 mRna expression appeared to be related to tuberin since they were significantly reduced by 5-azacytidine incubation. Anti-EGFR Ab and rapamycin significantly decreased MMP-2 mRna expression while their effect was the opposite on MMP-7.
Extracellular matrix metalloproteinase inducer (EMMPRIN), is thought to affect tumor progression through its ability to stimulate MMPs expression. EMMPRIN expression, quantified by cytometric analysis, was reduced by 5-azacytidine and was much higher in LAM/TSC cells than in MCF7, breast cancer cells. Anti-EGFR antibody and rapamycin did not change EMMPRIN levels. For the mesenchymal feature of LAM/TSC cells and their ability to survive in adherent and nonadherent conditions, we evaluated LAM/TSC motility in wound healing assay providing an indication of cell migration rate. LAM/TSC cells closed wounds in about 11h, much faster than tumoral cells such as MCF7. Cell motility of LAM/TSC cells is related to tuberin expression since 5-azacytidine treatment, and the consequent induced-tuberin expression, increased the time of wound closure to 19h. However, 5-azacytidine treatment did not have any effect on MMP-2 and MMP-7 mRna expression in wound healing assay. Rapamycin and anti-EGFR Ab decreased the migration rate of LAM/TSC cells leading to the closure of wound in about 25h and causing an induced an increase of MMP-2 and MMP-7 levels. However during wound closure (50%) MMP-2 secretion was increased by anti-EGFR and rapamycin treatments and normalized at complete wound closure.
In conclusion, LAM/TSC cells express invasive and migratory features which appear to be related to tuberin expression . Phosphorylation of FAK and MMP-2 expression was reduced in nonadherent LAM/TSC cells compared to adherent cells suggesting that FAK signaling was required for the activation of MMP-2 expression. MMP-2 and MMP-7 are related to the adherent and nonadherent conditions and are involved in the metastatic process proposed in dissemination of LAM cells. LAM/TSC cells have the ability to migrate and their motility is related to tuberin expression and reduced by anti-EGFR antibody and rapamycin. Rapamycin and anti-EGFR Ab control MMP-2 and MMP-7 expression and secretion during cell migration.
These data suggest a specific role for MMP-2 and MMP-7 in LAM/TSC cell motility and thereby in the pathogenesis of LAM and TSC indicating their involvement in the metastatic process proposed in dissemination of LAM cells.
The understanding of LAM/TSC cell features in motility is important for the assessment of cell invasiveness in LAM and TSC and provide highlights to sustain MMPs pathways as potential target for LAM. TSC and TSC related disorders.
4
Testoni, Blasco di Sciacca Alessandra Lucia Maria. "Metalloproteasi 9, TIMP-1 e Osteopontina quali possibili biomarcatori del carcinoma dell endometrio." Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1217.
Full textAbstract:
Lo scopo del nostro lavoro è stato quello di valutare l esistenza di una diversa espressione tissutale e periferica di metalloproteasi MMP9, TIMP1 (la sua proteina inibitoria) e Osteopontina, quali possibili biomarcatori di progressione tumorale, in donne con patologie benigne dell endometrio, con carcinoma dello stesso, e di un gruppo di donne come controllo.
Abbiamo reclutato per il nostro studio 31 pazienti che si erano sottoposte ad un esame isteroscopico o per sanguinamenti anomali, o per un ispessimento endometriale evidenziato durante un ecografia ginecologica. Lo stesso giorno in cui alle pazienti veniva eseguita l isteroscopia della cavità uterina, sono stati prelevati circa 10 ml di sangue venoso periferico, suddivisi in due provette: 2-3 ml per la raccolta del siero e 6-7 ml per la raccolta del plasma, con aggiunta di Na++ eparina. Le provette sono state quindi inviate, entro 30 minuti, per la loro centrifugazione ed i campioni di plasma e siero aliquotati, sono stati conservati a -80°C, fino al momento del saggio. Successivamente sono stati eseguiti i dosaggi in ELISA su siero o plasma delle tre proteine da noi considerate, con Kits disponibili commercialmente secondo la metodica indicata dal produttore (R e D System). In corso di isteroscopia, sono state praticate delle biopsie della cavità uterina o dei polipi endometriali. Un piccolo frammento tissutale veniva raccolto in Rna Later, e conservato ad una temperatura di -80°C fino alle indagini di biologia molecolare (estrazione dell RNA ed Immunoistochimica - Elisa); il restante materiale è stato inviato per effettuare l esame istologico.
In accordo ai dati della letteratura, i valori medi sierici dei tre parametri da noi considerati, nonchè quelli ricavati dalle biopsie della cavità uterina di donne con carcinoma, sono significativamente più elevati rispetto a quelli di donne con patologie benigne dell endometrio ed ancora di più rispetto ai controlli. La MMP-9 e il suo inibitore TIMP-1, hanno mostrato lo stesso andamento di OPN anche se i valori di TIMP-1 circolante, in accordo con i dati riferiti in letteratura hanno mostrato una maggiore correlazione rispetto alla MMP-9.
5
LEONE, LUCIA. "Ruolo del complesso distrofina-distroglicano e delle metalloproteasi nella plasticità neuronale e sinaptica del ganglio cervicale superiore di roditori." Doctoral thesis, La Sapienza, 2005. http://hdl.handle.net/11573/916857.
Full text
6
Paren, Helen. "Inhibitors of metalloproteases." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260150.
Full text
7
Schneider, Richard. "Zielgerichtete Evolution Matrix-Metalloprotease-aktivierbarer Retroviren." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972141375.
Full text
8
Xiang, Yaozu. "Characterisation of the ADAMTS13 metalloprotease domain." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/9835.
Full textAbstract:
Von Willebrand Factor (VWF) is a large multi-domain plasma glycoprotein that is critical for normal platelet tethering during haemostasis. ADAMTS13 is a plasma metalloprotease that regulates VWF multimeric size/function by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. Deficiency of ADAMTS13 causes microvascular thrombosis. The only reported cleavage of VWF by ADAMTS13 is that of the Tyr1605-Met1606 bond. How this high substrate specificity is conferred remains unclear. To date, all the interactions between these molecules that have been described involve VWF residues C-terminal to the scissile bond and the non-catalytic domains of ADAMTS13. I hypothesised that VWF also contains an interaction site for ADAMTS13 N-terminal of the cleavage site to aid both in positioning of the scissile bond in the active site and substrate specificity. Previous studies have suggested that the VWF sequence between Asp1596 and Val1604, N-terminal to the cleavage site, is essential for cleavage by ADAMTS13. My aim was to identify the residues in this region that are important determinants for ADAMTS13 proteolysis. A panel of mutations were introduced into the substrate VWF 115 (VWF residues 1554- 1668). The mutants were expressed purified and their proteolysis by ADAMTS13 analysed. It was found that the proteolysis of VWF 115 variants (L1603A, L1603S, L1603N or L1603K) were all substantially impaired (up to >400 fold reduction). The importance of VWF Leu1603 was confirmed using a synthetic peptide 1596DREQAPNLVY1605, which competitively inhibited proteolysis of VWF 115 by ADAMTS13. A mutant peptide containing the L1603A mutation, 1596DREQAPNAVY1605, had minimal effect. When the VWF L1603A substitution was introduced into the full-length recombinant VWF, proteolysis by ADAMTS13 was again substantially reduced. These findings implied the presence of a subsite (S3) in the ADAMTS13 metalloprotease (MP) domain that interacts with VWF Leu1603. Using molecular modelling, the distance between VWF Leu1603 and the scissile bond was estimated as ~10Å. Structural homology modelling of the MP domain, mutagenesis of 11 candidate residues and functional characterisation of these variants identified two clusters, Leu198/Leu232/Leu274 and Val195/Leu151, as possible subsites interacting with VWF. It is suggested that VWF Leu1603 interacts with Leu198/Leu232/Leu274, while Val195/Leu151 may interact with VWF Tyr1605. I propose a mechanism for VWF cleavage involving remote C-terminal domain interactions that assist initial orientation of the VWF scissile bond within the active site of ADAMTS13, but in which N-terminal hydrophobic interactions between VWF Leu1603 and the S3 subsite in the MP domain of ADAMTS13 are critical for the positioning required for cleavage of the Tyr1605-Met1606 scissile bond.
9
Solomon, Emilia A. "Regulation and proteolytic activity of ADAM12 metalloprotease." Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/2197.
Full text
10
Denkin, Steven Michael. "Regulations of empA metalloprotease expression in Vibrio anguillarum /." View online ; access limited to URI, 2003. http://0-wwwlib.umi.com.helin.uri.edu/dissertations/dlnow/3115626.
Full text
11
Abhinav, Kanishk. "Investigating Invadolysin's activity : discerning mechanism and cellular roles." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25804.
Full textAbstract:
Invadolysin is a novel metalloprotease, which is conserved amongst metazoans and was first identified in the Heck laboratory. Proteases play a variety of roles in normal physiology. Invadolysin is essential for life in Drosophila. Invadolysin has been shown to be essential for cell division and cell migration. Invadolysin is the only metalloprotease that we know of which localizes to lipid droplets, the lipid storage cell organelle. Previous studies have also shown that invadolysin mutants have a lower triglyceride to protein ratio and reduced fat body thickness and cross sectional area. Fat body in Drosophila is the functionally homolog of adipose tissue in higher organisms. Further suggesting a role of invadolysin in metabolism. In the Heck laboratory, invadolysin is studied using model organisms such as Drosophila melanogaster, Danio rerio (zebrafish) and cultured cell lines. During my PhD, my aim was to study the biosynthesis, activity and function of invadolysin and investigate its role in metabolism and adipogenesis. Invadolysin has a conserved metalloprotease motif ‘HEXXH’ and a potential lipase motif ‘GXSXS’. One of the aims of my PhD was to generate mutant versions of the conserved motifs to study their role on the activity of the proteins. I have generated transgenic flies that express wild type or E258A (protease dead) or S266A (lipase dead) versions of invadolysin. These transgenic flies would help in the study of the importance of the metalloprotease ‘HEILH’ and the lipase ‘GFSVS’ motifs in invadolysin’s activity. Transgenic flies overexpressing wild type and lipase dead form of invadolysin accumulate significantly higher levels of triglycerides as compared to control flies and transgenic flies overexpressing protease dead form of invadolysin. Suggesting a role of the protease motif in lipid accumulation. The other aim of my PhD was to study the role of invadolysin in metabolism. I followed up on previous observations in the laboratory that the insulin-signalling pathway is impaired in invadolysin mutant animals – with the hypothesis that invadolysin plays a role in metabolism and adipogenesis. I used Drosophila to study the effect on downstream targets of the insulin-signalling pathway such as triglyceride synthesis, glycogen synthesis and autophagy in invadolysin mutants. Results suggest that the insulin-signalling pathway and the ability to accumulate lipids are impaired in invadolysin mutants. Insulin also regulates adipogenesis by regulating the expression of PPARγ. I used SGBS cells, a human preadipocyte cell line to study the role of invadolysin in adipogenesis. Increase in protein levels of invadolysin during adipogenesis indicates a potential role of invadolysin in adipogenesis. Invadolysin has a predicted N-terminal signal sequence and also a predicted Cterminus GPI anchor site that suggests invadolysin can either be secreted or anchored to a membrane. Also, leishmanolysin, the closest homolog of invadolysin exists in a secreted and membrane bound form apart from a cytosolic form. This encouraged me to investigate the presence of a secreted form of invadolysin. Analysis of vertebrate and invertebrate plasma fractions of blood and hemolymph led to the identification of a novel secreted form of invadolysin. This novel discovery places invadolysin alongside a small group of metalloproteases, which are secreted into the extracellular environment and which play multiple roles in normal physiology and disease states.
12
Kowatz, Thomas. "Mechanisms of silicate polymerisation, carbohydrate epimerisation and metalloprotease inhibition." Thesis, St Andrews, 2009. http://hdl.handle.net/10023/771.
Full text
13
Yu, Angel Chia-yu. "Structural analysis of an enterohemorrhagic Escherichia coli metalloprotease effector." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42821.
Full textAbstract:
Mucins are proteins that contain dense clusters of α-O-GalNAc-linked carbohydrate chains
and are the major component of the mucosal barrier that lines the mammalian
gastrointestinal tract from mouth to gut. A critical biological function of mucins is to protect
the underlying epithelial cells from infection. Enterohemorrhagic Escherichia. coli O157:H7
(EHEC), a bacterial pathogen that causes severe food and water borne disease, is capable of
breaching this barrier and adhering to intestinal epithelial cells during infection. StcE
(secreted protease of C1-esterase inhibitor) is a ~100 kDa zinc metalloprotease virulence
factor secreted by EHEC and plays a pivotal role in remodelling the mucosal lining during
EHEC pathogenesis. StcE also dampens the host immune response by targeting the mucinlike
region of C1-INH, a key complement regulator of innate immunity. To obtain further
mechanistic insight into StcE function, I have determined the crystal structure of the fulllength
protease to 2.5Å resolution. This structure shows that StcE adopts a dynamic, multidomain
architecture featuring an unusually large substrate binding cleft. Electrostatic surface
analysis reveals a prominent polarized charge distribution highly suggestive of an
electrostatic role in substrate targeting. The observation of key conserved motifs in the active
site allows us to propose the structural basis for the specific recognition of α-O-glycan
containing substrates, which have been confirmed by glycan array screening to be Oglycosylation
of the mucin-type. Complementary biochemical analysis employing domain
variants of StcE further extends our understanding of the substrate binding stoichiometry and
distinct substrate specificity of this important virulence-associated metalloprotease.
14
Ishimwe, Egide. "Functional characterization of the Cydia pomonella granulovirus matrix metalloprotease." Thesis, Kansas State University, 2014. http://hdl.handle.net/2097/18229.
Full textAbstract:
Master of ScienceDepartment of Biology
A. Lorena Passarelli
Cydia pomonella granulovirus (CpGV) is a member of the Baculoviridae family of viruses. The CpGV open reading frame 46 (CpGV-ORF46) predicts a 545 amino acid protein that shares homology with matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. In silico analyses revealed the presence of putative mmp genes in all species from the Betabaculovirus genus, while no mmps were identified in members of the Alphabaculovirus, Gammabaculovirus or Deltabaculovirus genera. Unlike most cellular MMPs, baculovirus MMPs do not have a propeptide domain, a domain involved in regulating MMP activation, or a hemopexin-like domain, which is necessary for substrate binding and specificity in many MMPs. However, Betabaculovirus MMPs do contain a predicted conserved zinc-binding motif (HEXGHXXGXXHS/T) within their catalytic domain. The function of CpGV-MMP and its effects on baculovirus replication in cultured cells and insect larvae were investigated. CpGV-MMP was expressed in and purified from Escherichia coli, and activity was measured using a generic MMP substrate in vitro. CpGV-MMP had in vitro activity and its activity was specifically inhibited by MMP inhibitors. To study the effects of CpGV-MMP on virus replication and dissemination, CpGV-MMP was expressed from Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of a strong and constitutive promoter, the Drosophila heat shock 70 protein promoter. Expression of CpGV-MMP did not affect virus replication in cultured cells. The effects of expressing CpGV-MMP from AcMNPV during larval infection were evaluated in the presence or absence of the AcMNPV chitinase and cathepsin genes. Insect bioassays showed that the absence of cathepsin resulted in a significant delay in larval time of death; however, this delay was compensated by expression of CpGV-MMP. In addition, larval time of death was accelerated when cathepsin, chitinase, and CpGV-MMP were all expressed. Finally, we determined the effects of CpGV-MMP on larvae melanization and liquefaction. CpGV-MMP was able to promote larvae melanization in the absence of cathepsin. CpGV-MMP, in the absence of cathepsin, was not able to promote larvae liquefaction. When chitinase was engineered to be secreted from cells, CpGV-MMP rescued liquefaction in the absence of cathepsin. In conclusion, CpGV-MMP is a functional MMP which can enhance larvae mortality with the presence of cathepsin. In addition, CpGV-MMP can promote larvae melanization; however, it can only promote liquefaction when chitinase is engineered to be secreted from cells.
15
Sahli, Stefan. "Strukturbasiertes Design und Synthese von nichtpeptidischen Inhibitoren der Metalloprotease Neprilysin /." [S.l.] : [s.n.], 2004. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=15727.
Full text
16
Yu, Bin. "Characterisation of Invadolysin, a novel essential metalloprotease in Drosophila melanogaster." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/25353.
Full textAbstract:
The IX-14 mutant was first found as a mutation affecting neuroblasts and imaginal discs and later characterised as a mutant affecting chromosome condensation. Base on its sequence, IX-14 may be classified as a metalloprotease that is a homologue of leishmanolysin. IX-14 have a wide range of functions in cell cycle and cell migration, probably through its alternative spliced forms or its diverse substrates. In this study, I have showed that IX-14 mutants have a chromosome structure defect besides a length-wise chromosome hyper-condensation phenotype. The turnover of three nuclear envelope proteins (lamin Dm0, otefin and lamin A/C) was affected in IX-14 mutants. One of them, lamin Dm0, has been shown to be cleaved by IX-14 in vitro, which suggests it is a substrate of IX-14. I have also shown Drosophila IX-14 mutant embryos have a germ cell migration defect, germ cells failing to form gonads at stage 13 after passing through the midgut. This was consistent with the suggestion that IX-14 might play a migration role based on the observation that IX-14 localized to the leading edge of macrophages and as invadopodia-like structures in some HeLa cells. Due to the presence of a signal sequence at the N-terminus and a GPI anchor sequence at the C-terminus of DmIX-14, I could not purify DmIX-14 since it keeps losing its tags on both the N-terminus and the C-terminus in the baculovirus system. Methods to express large scale active IX-14 still need to be found.
17
Wilson, David P. M. "Angiotensin II and matrix metalloprotease involvement in proliferative vascular disease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ45013.pdf.
Full text
18
Chang, Ching-Wen. "Studies on invadolysin : a novel metalloprotease localizing to lipid droplets." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4214.
Full textAbstract:
Invadolysin (INV) is a member of the M8 family of metzincin metalloproteases. The gene was discovered in the Heck laboratory. Based on studies in Drosophila, INV is important for mitotic progression, nuclear envelope protein dynamics, and germ cell migration. INV-like immunoreactivity has shown its association with lipid droplets (LDs), which are intracellular organelles for lipid and protein storage. INV is the first metalloprotease found on LDs. Thus, INV’s role and LD-associated pathways are the puzzles we would like to investigate. The formation of LDs is dependent on the nutritional status of cells and starvation can disrupt the generation of LDs. Based on this concept, I established a starvation / re-feeding system. When nutrition is sufficient, LDs were surrounded by INV, whereas no INV or LDs were found in the majority of starved cells. With a supply of oleic acid (OA), LDs re-appeared and so did INV localized to LDs. In this system, inhibition of protein kinase C (PKC) disrupts INV’s re-localization to LDs. As I found INV to be phosphorylated by PKC in vitro (residues within the N-terminus might be phosphorylated by PKC), I conclude that PKC might regulate INV’s re-localization in the starvation / re-feeding system. 3T3-L1 mouse fibroblasts can differentiate into adipocytes in vitro; this is termed adipogenesis. Since INV is a LD associated protein, the role of INV in adipogenesis is of interest. INV localized on LDs in the early stage of differentiation but disassociated from LDs in mature adipocytes. The levels of INV mRNA and protein were significantly increased upon differentiation to adipocytes. On the other hand, INV decreased when adipocyte differentiation was inhibited by PKC and PI3K inhibitors, suggesting that the increase of INV is required for adipocyte differentiation. I was interested to examine the possible role of INV in InR/PI3K/Akt signalling, and therefore compared wild type with mutant INV (Drosophila INV4Y7). Decreased levels of phospho-Akt and phospho-S6K, and increased mRNA levels of d4E-BP were observed in INV4Y7 mutant larvae, suggesting that INV may be required for InR/PI3K/Akt signalling. In addition, a decreased level of Lsd2 (LD binding protein) was found in INV4Y7 mutants. These correlations between INV and molecules important for signaling suggest that INV might be a mediator of nutritional metabolism. In light of these data, I speculate that INV plays a homeostatic role, possibly by affecting the InR/PI3K/Akt signaling pathway. In conclusion, the localization of INV to LDs is dependent on the activity of PKC. An increase in invadolysin accompanies adipogenesis, in which PKC and PI3K may be mediators. Examining mutant Drosophila, I found INV to be involved in InR/PI3K/Akt signalling. Collectively, I conclude that INV may serve as a regulator in adipogenesis and the InR/PI3K/Akt signaling pathway.
19
Atherton, Ruth Elizabeth. "Catalytic activity and maturation of the metalloprotease-disintegrin protein, MDC9 /." Access full-text from WCMC, 1999. http://proquest.umi.com/pqdweb?did=733095101&sid=8&Fmt=2&clientId=8424&RQT=309&VName=PQD.
Full text
20
Liu, Zhiwen. "Matrix metalloproteases and cell motility in malignant mesothelioma /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-061-3/.
Full text
21
Kaminska, Monika. "New activity-based probes to detect matrix metalloproteases." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS538/document.
Full textAbstract:
Les Métallo Protéases Matricielles (MMP) en tant qu'endopeptidases à zinc ont une large gamme de fonctions biologiques allant du remodelage tissulaire à la modulation de la réponse cellulaire. Une modification de leur activité protéolytique est souvent associée à de nombreux désordres biologiques. In vivo, ces protéases sont soumises à de nombreuses modifications post-traductionnelles. Elles sont sécrétées sous formes latentes à l'extérieur des cellules pour être ensuite transformées en forme fonctionnelles. Ces dernières sont ensuite inhibées par des inhibiteurs endogènes. En raison de leur sécrétion dans l’espace extra cellulaire, les MMP sous formes actives ont longtemps été considérées comme de simples ciseaux moléculaires capable de dégrader uniquement la matrice extracellulaire. Cependant, le remodelage tissulaire ne constitue pas la fonction unique et encore moins la fonction principale de ces enzymes. Elles peuvent en effet cliver une grande variété de substrats non matriciels et à ce titre sont impliquées dans la progression tumorale, l'immunité et l'inflammation. Pour ajouter une complexité supplémentaire à la biologie des MMP, il a été récemment montré que certaines MMP ont une localisation intracellulaire associée à des fonctions non protéolytiques. Ces observations, mais aussi celles montrant que ces protease participent à la progression de la maladie alors que d'autres ont une fonction protectrice, soulignent la nécessité de mieux documenter leur activation spatiale et temporelle dans divers contextes biologiques.Le profilage protéique basé sur l'activité vise à analyser l'état fonctionnel des protéines dans des échantillons biologiques complexes. À cette fin, des sondes basées sur l'activité (ABP), qui réagissent avec les enzymes en s’appuyant sur leur mécanisme catalytique, ont été développées pour la détection d’enzymes sous formes actives, notamment dans le cas des protéases à sérine et à cystéine. Une sonde basée sur l’activité (ABP) est classiquement composée : i) d’un groupement réactif conduisant à la modification covalente de résidus au sein du site actif de l’enzyme, ii) d’un motif de liaison imposant la sélectivité au groupement réactif et iii) d’un groupement rapporteur permettant la détection des enzymes ciblées. Cette approche ne s’applique toutefois pas aux MMP, pour lesquelles il n’existe pas de résidus nucléophiles conservés au sein du site actif. À cet égard, tous les ABP ciblant les MMP comportent un groupement photo activable qui, sous irradiation UV, favorise la formation du complexe covalent. De telles sondes photo sensibles ont permis de détecter les MMP sous leurs formes actives dans des tissus et des fluides, mais pas chez les animaux vivants au sein desquels l’étape de photo-activation ne peut être réalisé.Dans ce contexte, en nous appuyant sur un contexte structural favorable et en exploitant la chimie de l'acyl imidazole (LDAI) dirigée par un ligand, nous avons identifié une nouvelle série de sondes capables de modifier de manière covalente les MMP sans recourir à la photo-activation. Nous avons ainsi validé la capacité de ces sondes à marquer de manière sélective et efficace la MMP12 humaine in vitro et dans des protéomes complexes. Dans ce dernier cas, jusqu’à 50ng de hMMP12 correspondant à 0,05% du protéome total peuvent être détectés. Nous avons également déterminé l'identité de l’unique résidu modifié de façon covalente au sein du site actif de la hMMP-12 et vérifié que cette modification avait peu d'impact sur l’activité protéolytique de cette dernière. Nous avons démontré que cette approche permettait de détecter des MMP endogènes. Enfin, nous avons étendu cette stratégie de marquage à un panel plus large de MMP.En développant la première stratégie de marquage des formes actives de MMP «sans photo-activation», il semble maintenant possible d’envisager la détection de ces enzymes à la fois dans les protéomes complexes et in vivoMatrix MetalloProteases (MMPs) as zinc endopeptidases have a wide range of biological functions, and changes in their proteolytic activity underlie many biological disorders. Since their proteolytic activity has to be tightly controlled to prevent tissue destruction, theses proteases are subjected to numerous posttranslational modifications in vivo. They are secreted under latent forms outside of the cells, and are subsequently processed into their functional form that can be further inhibited by endogenous inhibitors. Due to their delineated area of activation, MMP active forms have long been considered for their unique ability to degrade extracellular substrates. However, turnover and breakdown of the extracellular matrix are neither the sole nor the main function of MMPs. These enzymes can indeed process a wide variety of non-matrix substrates and are involved in the regulation of multiple aspects of tumor progression, immunity and inflammation. To add further complexity to MMPs biology, some members within the family were recently reported to have intracellular localization associated to non-proteolytic functions. These observations but also those evidencing that some MMPs participate in disease progression while others have a protective function, stress the need to better document their spatial and temporal activation in various biological contexts.Activity-based protein profiling (ABPP) aims to analyze the functional state of proteins within complex biological samples. To this purpose, activity-based probes (ABPs) that react with enzymes in a mechanism-based manner have been successfully developed for the profiling of several enzymes including serine and cysteine proteases. A typical Activity-Based probe (ABP) is composed of i) a reactive warhead, which reacts in a covalent manner with enzyme active site residues, ii) a targeting moiety that imposes selectivity upon the reactive group and iii) a detectable group for subsequent analyses. This approach is not applicable to MMPs, which lack a targetable nucleophile involved in the catalysis. In this respect, all ABPs directed to MMPs are affinity-based probes (AfBPs) containing within their structure a photo cross-linking group that promotes the formation of a covalent complex upon UV-irradiation. Such photoactivatable probes have been successfully developed for the detection of MMPs under their active forms in fluids and tissue extracts, but not in living animals where the photo-activation step is not feasible.By relying on a favorable structural context and by exploiting the ligand-directed acyl imidazole (LDAI) chemistry, we have identified a novel series of AfBPs capable of covalently modifying matrix metalloproteases without making use of photo-activation. These active-site-directed probes whose structure was derived from that of a MMP12 selective inhibitor harbored a reactive acyl imidazole in their P3' position. They demonstrated their labelling specificity in vitro by covalently modifying a single Lysine residue within the MMP-12 S3' region. We also showed that these probes only targeted functional states of hMMP-12 and spared forms whose active site was occluded either by a synthetic or a natural inhibitor. We have validated the ability of these chemical probes to efficiently label human MMP12 in complex proteomes. In this case, down to 50 ng of hMMP12 corresponding to 0.05% of the whole proteome can be labelled and detected by in-gel fluorescence analysis. We demonstrated that this approach also allowed detecting endogenous MMPs secreted by stimulated-macrophages. In addition, by modifying the nature of the targeting moiety, we have extended this affinity-labeling approach to six other MMPs.By developing the first “photo activation-free” strategy to covalently modify active forms of MMPs, the unresolved proteomic profiling of native MMPs should be now accessible both in complex proteomes and in preclinical model in which MMPs are potential relevant targets
22
Kaw, Semiko. "Endothelin converting and degrading enzymes." Thesis, Oxford Brookes University, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261235.
Full text
23
Manoury, Boris. "Analyse cellulaire et moléculaire des mécanismes physiopathologiques conduisant au remodelage tissulaire et au développement de la fibrose pulmonaire chez la souris : étude de l'implication des métalloprotéinases et des radicaux libres oxygènes." Rennes 1, 2005. http://www.theses.fr/2005REN1B066.
Full textAbstract:
De nombreuses pathologies graves du poumon impliquent le développement d'une fibrose. Celle-ci est caractérisée par un remodelage progressif du parenchyme alvéolaire associée à l'accumulation excessive de matrice extracellulaire (MEC). La dégradation de la matrice extracellulaire est assurée par un grand nombre d'enzymes qui agissent de manière complémentaire. Les métalloprotéinases de la matrice (MMPs) sont une famille d'enzymes impliquées dans le remodelage de la MEC lors de processus biologiques, notamment dans certaines pathologies respiratoires telles que la fibrose pulmonaire. Une famille de protéines inhibitrices spécifiques des métalloprotéinases, les inhibiteurs tissulaires des métalloprotéinases (TIMPs), régulent ces enzymes. Nos travaux (modèle expérimental de fibrose pulmonaire chez la souris) nous ont permis de mettre en évidence le rôle essentiel de la production des espèces réactives de l'oxygène dans le développement de la fibrose pulmonaire, via le complexe enzymatique NADPH-oxxydase. En outre une augmentation de la quantité de TIPM-1 pourrait fortement contribuer à l'accumulation de matrice extracellulaire observée au cours du processus. Ces résultats confortés par les données de la littérature, pourraient ouvrir la voie à de nouvelles perspectives en terme de réversion du développement de la fibrose pulmonaire chez l'homme.
24
McCarthy, Conor Neil, and n/a. "Regulatory Elements Controlling Lipase and Metalloprotease Production in Pseudomonas fluorescens B52." Griffith University. School of Health Science, 2003. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20031015.124744.
Full textAbstract:
Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d2, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.
25
Cakebread, Julie Ann. "Genetic and molecular characterisation of A Disintegrin and Metalloprotease (ADAM) 33." Thesis, University of Southampton, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436931.
Full text
26
Effenberger, Timo [Verfasser]. "Die Rolle der Metalloprotease ADAM17 während der zellulären Seneszenz / Timo Effenberger." Kiel : Universitätsbibliothek Kiel, 2014. http://d-nb.info/1053326246/34.
Full text
27
Li, Shanghao. "Theoretical Insight into Mechanisms of Natural and Artificial Metalloproteases." Scholarly Repository, 2011. http://scholarlyrepository.miami.edu/oa_theses/265.
Full textAbstract:
In this study, theoretical and computational approaches have been utilized to investigate the mechanisms of natural and artificial metalloproteases. The active sites of most natural metalloproteases contain a tetrahedral zinc center, coordinated by three amino acid residues combinated from His(N), Cys(S), Glu(O), and Asp(O) with a water molecule as the fourth ligand. However, the roles played by the ligands environment in the catalytic functions of enzyme are not clear. In this study, the effects of different ligand combinations (NS2, N2S, N2O, N3, S3, NO2 and NSO) in the mechanism were investigated energy barriers were compared. The machanism and energetics of the substrate bound artificial metalloproteases Ni(II)cyclen (cyclen: 1,4,7,10-tetraazacyclododecane) and Cd(II)cyclen have been investigated. In addition, the mechanism of hydrolysis of Phe-Phe peptide bond catalyzed by another artificial metalloprotease [Pd(H2O)4]2+ has also been studied.
28
Cornet, Joséphine. "Conception, synthese et etude pharmacologique d'inhibiteurs non peptidiques des metalloproteases matricielles (doctorat : pharmacochimie)." Lille 2, 1990. http://www.theses.fr/1999LIL2P271.
Full text
29
Gandham, Lyngrace. "Characterization of the secretion and anchoring domains of Caulobacter crescentus SapA metalloprotease." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/24820.
Full textAbstract:
The Caulobacter crescentus type I secretion system can be used to display foreign peptides at high density on the bacterium’s surface as part of the S-layer. Certain recombinant proteins, however, are subject to proteolytic cleavage by SapA, a unique S-layer associated metalloprotease. SapA (71 kDa) is an unstable protein that breaks down to a 45-kDa product when over-expressed. It needs to be secreted before it can become an active enzyme that anchors to the cell. A point mutation adjacent to the protease’s active site reduced SapA processing, indicating that SapA is self-processing. The last 10 and 50 amino acids were removed and prevented secretion, indicating SapA was a type I secreted protein. Further, SapA secretion was blocked in an S-layer type I secretion deficient strain. Lack of secretion prevents this protease from becoming an active enzyme evidenced by the type I defective clones, which are not processed at all. The last 100 amino acids of the protease are sufficient for anchoring, as determined by immunofluorescence. Interestingly, SapA could be detected on the cell surface by immunofluorescence only in an S-layer negative, O-antigen deficient strain. This suggests that SapA is localized on the cell membrane, beneath the S-layer and is hidden by smooth LPS. A fusion protein, containing a 242 amino acid protein G peptide attached to the last 238 amino acids of SapA secreted and anchored to the cell surface of C. crescentus. This fusion was detectable an anti-IgG antibody. SapA is the first identified self-processing protease that uses its C-terminus for both type I secretion and anchoring.
30
Edwin, Aaron. "Structural and functional studies of the secreted metalloprotease PrtV from Vibrio cholerae." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-84553.
Full textAbstract:
Cholera, an acute diarrheal diseases caused by the intestinal infection of the pathogenic bacterium Vibrio cholerae, continues to be a global killer in the world today. PrtV, a secreted zinc metalloprotease, is a potent cytotoxic virulence factor of V. cholerae. The 102 kDa full length multi-domain PrtV protein undergoes several N and C terminal modifications before being secreted as a 81 kDa pro-protein. The activation of the pro-protein is calcium dependent. The removal of calcium triggers auto-proteolysis to give a stable active protease with the catalytic zinc binding domain. The aim of the thesis was to study the structure and function of the PrtV protein. The results from paper I, identified the end product of the maturation of PrtV as the stable 37 kDa M6 active domain, and not a 55 kDa complex as reported earlier. Results also showed the this 37 kDa active M6 domain alone was sufficient for catalytic activity. A revised model for the maturation of PrtV was proposed. Individual domains were isolated from the PrtV protein by domain phasing methods. This included the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 589-839). The isolated domains were recombinantly over expressed as fusion proteins to increase expression and solubility. The PKD1 domain was purified to homogeneity and crystallized. The structure of the PKD1 domain reported in paper II, was solved by X-ray crystallography at an atomic resolution of 1.1 Å. From the structure, a previously unknown calcium binding site was identified at the N-terminal of the PKD1 domain. The structure also revealed two conformations for the PKD1 domain depending on free or bound calcium. From the structure, a function of the PKD1 domain as a protector of the cleavage site in the linker region between the M6 domain and the PKD1 domain in the presence of calcium was elucidated. A new model for the activation of PrtV was given. In paper III, the structure of the N-terminal domain solved by NMR spectroscopy was reported. The structure revealed two well defined helices but a third predicted helix was found to be unstructured.
31
Billson, Jeremy Paul. "The design, synthesis, and evaluation of some conformationally constrained matrix metalloprotease inhibitors." Thesis, University of Exeter, 1997. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.535965.
Full text
32
Arai, Hiroyuki. "Metalloprotease-dependent attenuation of BMP signaling restricts cardiac neural crest cell fate." Doctoral thesis, Kyoto University, 2020. http://hdl.handle.net/2433/245816.
Full textAbstract:
京都大学0048
新制・論文博士
博士(医学)
乙第13301号
論医博第2190号
新制||医||1039(附属図書館)
京都大学大学院医学研究科医学専攻
(主査)教授 妻木 範行, 教授 木村 剛, 教授 影山 龍一郎
学位規則第4条第2項該当
Doctor of Medical Science
Kyoto University
DFAM
33
Hall, Ronelle Jean. "ADAM 10 : expression and functional analysis of a metalloprotease during embryonic morphogenesis /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2003. http://uclibs.org/PID/11984.
Full text
34
Hong, Yue. "Exploring the roles of zinc metalloproteases in prostate cancer progression." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511149.
Full text
35
Müller, Miryam [Verfasser]. "The role of a disintegrin and metalloprotease 10 in the liver / Miryam Müller." Kiel : Universitätsbibliothek Kiel, 2018. http://d-nb.info/1169132553/34.
Full text
36
Maretzky, Thorsten. "Funktionelle Analyse der proteolytischen Spaltung von Adhäsionsmolekülen durch Disintegrin-ähnliche Metalloproteasen." [S.l.] : [s.n.], 2005. http://e-diss.uni-kiel.de/diss_1613/d1613.pdf.
Full text
37
Ohler, Anke [Verfasser]. "Aktivität der humanen Meprin-Metalloproteasen unter Berücksichtigung potentieller Aktivatoren / Anke Ohler." Mainz : Universitätsbibliothek Mainz, 2011. http://d-nb.info/1025055578/34.
Full text
38
Wardle, Fiona Claire. "Regulation of the BMP signalling pathway by BMP-1 related metalloproteases." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287477.
Full text
39
Pini, Rodolfo <1985>. "Terapie farmacologiche nell'attivita degenerativa della metalloproteinasi-9 negli aneurismi dell'aorta addominale." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amsdottorato.unibo.it/8864/1/TESI%20DOTTORATO%20FINALE%20pini.pdf.
Full textAbstract:
Introduzione.Lo sviluppo degli aneurismi dell’aorta addominale (AAA) è da associarsi prevalentemente ad un’eccessiva proteolisi della matrice extracellulare (ECM), mediata da metallo-proteinasi (MMPs). Differenti farmaci hanno mostrato un’attività modulatoria rispetto alla produzione delle MMP-9, principalmente coinvolta nello sviluppo degli AAA, che potenzialmente possono influenzare la crescita dell’aneurisma stesso. Lo scopo dello studio è stato valutare influenza di differenti attività farmacologiche sulla produzione di MMP nei tessuti aortici di aneurismatici.
Metodi. Sono stati prelevati segmenti di parete aortica aneurismatica da pazienti portatori di AAA e sottoposti a trattamento chirurgico open. Da tali tessuti sono state ricavate le cellule staminali mesenchimali (CSM – identificate come maggiormente associate alla produzione di MMP-9) e saggiate per l’espressione genica di MMP-9 a livelli basali. Sono stati effettuate valutazioni di vitalità e espressione genica di MMP-9, mediante Real Time PCR in associazione a tre differenti farmaci: Pioglitazone, Sinvastatina e Doxiciclina, a differenti concentrazioni.
Risultati. Lo studio è stato approvato dal comitato etico locale e sono stati utilizzati 12 prelievi da pazienti portatori di AAA. Da tutti i segmenti sono state ricavate CSM e saggiate mediante Real Time PCR. Nel campione basale in assenza di farmaco si è riscontrata un’iperespressione di MMP-9, 400 volte superiore rispetto ai controlli sani (P=.0001). Tutti i farmaci testati si sono associati al mantenimento della vitalità cellulare testata e hanno dimostrato una significativa riduzione dell’espressione di MM-9 (P<0,001) con valori risultanti dall’analisi della Real Time mediante il metodo comparativo del 2—ΔCt, di 0,46 per Pioglitazone (10 μM), 0,1 per Doxiciclina (25 μM) e 0,58 per Sinvastatina (10 μM)
Conclusioni.
L’attività farmacologica testata si è associata a una significativa riduzione dell’espressione di MMP-9 da parte delle CSM mantenendo la vitalità cellulare, questa valutazione pone le basi per un possibile studio in vivo delle differenti attività farmacologiche nel rallentamento dello sviluppo degli AAA.Introduction. The development of abdominal aortic aneurysms (AAA) is associated with excessive proteolysis activity of the extracellular matrix (ECM), mediated by metal-proteinase (MMPs). Different drugs have shown modulatory effect on the production of MMP-9, mainly involved in the development of AAA, which can potentially influence the growth of the aneurysm itself. The aim of the study was to evaluate the influence of different pharmacological activities on the production of MMP in aortic aneurysmal tissues. Methods. Aneurysmal aortic wall segments were taken from AAA patients undergoing open surgical treatment. Mesenchymal stem cells (CSMs - associated with the production of MMP-9) derived from aortic wall were assayed for MMP-9 gene expression at baseline levels. Assessments of viability and gene expression of MMP-9 were than performed, using Real Time PCR in association with three different drugs: Pioglitazone, Sinvastatin and Doxycycline, at different concentrations. Results. The study was approved by the local ethics committee and 12 specimens of aortic wall from different patients were analysed. From all the segments, CSMs were obtained and tested using Real Time PCR. At the baseline sample without medication, an over-expression of MMP-9 was found, 400 times higher than in healthy controls (P = .0001). All the drugs tested were associated with the maintenance of the cellular vitality and showed a significant reduction in the expression of MM-9 (P <0.001) with values resulting from the analysis of Real Time by the comparative method of 2-ΔCt, of 0.46 for Pioglitazone (10 μM), 0.1 for Doxycycline (25 μM) and 0.58 for Sinvastatin (10 μM). Conclusions. The pharmacological activity tested was associated with a significant reduction of MMP-9 expression by the CSMs while maintaining the cell viability, this assessment let speculate a possible in vivo study of the different pharmacological activities in the slowing down of the AAA development.
40
Vieira, Helena Soraia Fernandes. "Caracterização de enzimas proteoliticas produzidas por bactérias de origem marinha." Master's thesis, ISA, 2013. http://hdl.handle.net/10400.5/6476.
Full textAbstract:
Mestrado em Engenharia Alimentar - Instituto Superior de AgronomiaProteases from marine microorganisms have received increased attention due to their advantages compared to other sources. The objetive of this work was the characterization of an extracellular protease produced by a marine strain. The bacterial growth was performed in a liquid culture media, containing bacteriological peptone (1, 2 and 4%), yeast extract (0.5%) and sugar (0.5% saccharose or glucose). Bacterial growth and enzymatic activity were followed for 50 h. Best results were obtained with saccharose, yeast extract and 2% of peptone. The optimum temperature and pH were 60ºC and 6.5, respectively. However, another proteolytic activity maximum was recorded at pH 7.6. The protease was stable at 40ºC for approximately 15h, as well as when pre-incubated for 1h at temperatures between 6 and 50 ºC. However, this protease was quickly denaturated at 60ºC. The incubation of the protease at different pH showed that it was stable at pH 6.5-10.6. This protease was inhibited by EDTA and phenantroline indicating that this protease is a metalloprotease. The addition of ions like Ca2+ and Mg2+ led to an significantly increase of enzyme activity. The characteristics of this protease indicate that this enzyme may be further exploited for various industrial applications.
41
deGroot, Rens. "The role of the Metalloprotease and Disintegin-like domains in ADAMTS13 activity and specificity." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508488.
Full text
42
Howard, Linda. "Molecular cloning and characterisation of a bovine metalloprotease with homology to snake venom proteins." Thesis, Open University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240359.
Full text
43
SANTAVICCA, MARIA. "Etude fonctionnelle de la stromelysine-3, une metalloprotease matricielle impliquee dans la progression cancereuse." Université Louis Pasteur (Strasbourg) (1971-2008), 1996. http://www.theses.fr/1996STR13036.
Full textAbstract:
La stromelysine-3 (st3) est une metalloprotease matricielle (mmp) exprimee dans le stroma de nombreux carcinomes. Elle se distingue des autres mmps par des proprietes fonctionnelles inhabituelles. En immunisant des souris avec de la st3 produite dans un systeme procaryotique, nous avons obtenu une serie d'anticorps monoclonaux. L'un d'eux (5st-4a9) est utilise en immunohistochimie semi-quantitative afin de determiner le taux d'expression de la st3 sur des coupes minces de carcinomes mammaires et ainsi evaluer la valeur pronostique de l'expression de la st3. Des etudes anterieures ayant montre que la st3 ne degradait aucun des constituants majeurs de la matrice extracellulaire, nous avons entrepris d'identifier les determinants proteiques susceptibles d'etre responsables de ce comportement insolite. En ayant recours a des experiences de mutagenese dirigee, nous avons demontre que la st3 de souris acquiert une activite proteolytique a condition d'etre deletee d'au moins 175 acides amines a son extremite c-terminale. Pour observer des activites proteolytiques comparables avec la st3 humaine, il est necessaire de proceder a une mutagenese supplementaire dans laquelle le residu alanine en position 235, caracteristique de la st3 humaine et localise au niveau du met-turn, est remplace par une proline. La st3 differe egalement des autres mmps en ce qui concerne l'activation de sa proforme. Nous avons montre que la st3 etait secretee sous une forme potentiellement active, ayant perdu son prodomaine n-terminal. Ce comportement particulier est associe a un mecanisme moleculaire inedit pour une mmp. Nous avons localise dans le prodomaine de la st3, une sequence de dix acides amines comportant un site de reconnaissance et de clivage par la furine, une proproteine convertase presente dans le reseau trans-golgien. A l'aide de transfectants cellulaires stables et transitoires, nous avons teste et confirme la capacite de la furine a activer la st3
44
Menon, Vasudev Ramdas. "Analysis of the role and regulation of disintegrin metalloproteases in renal fibrosis." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3872/.
Full textAbstract:
Chronic Kidney Disease (CKD) affects about 10-20% of the adult population of the developed world. The underlying molecular mechanisms contributing to its varied pathophysiology CKD are still unclear. Without early diagnosis and therapeutic intervention, patients with CKD risk progressing to end stage renal failure (ESRF) requiring renal replacement therapy such as dialysis and eventually a renal transplant. Tubulointerstitial fibrosis is the common end point in most progressive renal diseases and has consistently been shown to be the best histological predictor of progression towards end stage renal failure. This 'point of no return' involves atrophy of the tubulointerstitium, leukocyte infiltration, persistent fibroblast proliferation and activation and dysregulation of extracellular matrix (ECM) resulting ultimately in scarring and loss of function. TGFB initiates and is involved in the entire course of fibrosis pathogenesis. Critical mediators of TGFB regulation are the intracellular signal transducers - the Smads which regulate gene transcription, and the recently discovered small neucleotide RNAs - microRNAs (miRs). miRs are a post transcriptional gene regulatory system and demonstrate distinct spatio-temporal expression and their expression is associated with crucial developmental and pathophysiological processes. Disintegrin metalloproteases (ADAMs) are members of the calss of zinc-ion proteases that can regulate key cellular and acellular processes including chemotaxis, adhesion and fusion, and modulate auto and paracrine signalling pathways by regulating ligand/receptor availability. Therefore, ADAMs can potentially regulate both inflammatory and fibrotic changes associated with renal disease. However, evidence towards the role of ADAMs in renal disease remains largely descriptive. While TGFB regulation of MMPs and TIMPs in renal disease has been well studies, the regulation or mechanisms of action of ADAMs in renal disease remains unknown. This thesis aims to demonstrate the involvement of proteolytically active ADAMs in renal fibrosis and provide mechanistic evidence towards transcriptional and post-transcriptional regulation of these ADAMs by canonical TGFB signalling.
45
Gururaja, Rao Shubha. "Characterization of Invadolysin in flies and human cells : new insights into a novel, essential metalloprotease." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/24659.
Full textAbstract:
I have analysed the functions of a novel, conserved metalloprotease named Invadolysin, previously reported by the Heck laboratory to be involved in mitosis and cell migration. I carried out a second site non-complementation screen in Drosophila melanogaster to identify the interactors of Invadolysin utilizing the availability of a collection of genome-wide deficiency stocks. As a result of this screen, I identified non-stop, a ubiquitin protease, as a genetic interactor of invadolysin. I characterized similar mutant phenotypes of invadolysin and non-stop and showed that the abnormal chromosomal architecture observed in both mutants might be a result of histone ubiquitination. I also investigated the involvement of Invadolysin in Notch pathway. The Notch extracellular domain levels were normal in the invadolysin mutants, but the intracellular domain levels were greatly reduced suggesting that the Notch pathway was inactive, or compromised. The levels of the Notch ligand Delta were abnormally high in invadolysin mutants, suggesting a block of Notch activity through cis-inactivation due to a possible failure in endocytosis of the Delta ligand. In human cells, I showed that Invadolysin co-localized with Rab11 and Itch proteins, which have been shown to influence the Notch pathway. Considering the possible involvement of Invadolysin in Delta endocytosis, I examined the co-localisation of Invadolysin with endocytosis machinery in human cells. I found that Invadolysin rings partially localised with Caveolin-1, and the caveolar endocytosis marker cholera toxin B was found inside the Invadolysin ring-like structures, suggesting that the inner region of Invadolysin rings might be a lipid based entity. When the lipid droplet marker BODIPY was utilized with Invadolysin, I established that Invadolysin surrounds lipid droplets.
46
Mitchell, Sally A. "Immunolocalisation within neural tissue of MADM : a novel metalloprotease with a potential integrin-binding domain." Thesis, University of Leicester, 1996. http://hdl.handle.net/2381/34168.
Full textAbstract:
A metalloprotease previously isolated from bovine brain was shown to be a member of a family which includes both mammalian proteins and snake venom metalloproteases containing a potential integrin-binding (disintegrin) domain. The bovine enzyme was named MADM for Mammalian-Disintegrin Metalloprotease (Howard et al., 1996; Biochemical Journal 371; 45-50). The present studies were undertaken to generate antibodies reactive with the rat MADM homologue and to use these for immunolocalisation of the protein in rat central nervous system (CNS). A truncated rat MADM homologue was initially isolated from a commercial brain cDNA library, and the sequence encoding the mature protein was completed using the technique of 5'-RACE (rapid amplification of cDNA ends). It was found that the rat MADM sequence had a very high degree of identity at both the nucleic acid and the amino acid level with bovine MADM, possibly implying a critical function for this protein. An antigenicity plot of the mature rat protein was used to design peptides for anti-MADM antibody production. Although the level of immunogenicity was generally low, two specific polyclonal antisera were produced in rabbits, and used in Western blotting experiments to demonstrate the presence of MADM protein in a range of rat tissues and cultured cell lines. Within adult rat CNS, the MADM protein was localised by immunohistochemistry to microglial cells of the white matter. The patterns of expression of 1, 2, 3, and 4-integrin subunits within the CNS were also examined, with the intention of identifying potential candidates for MADM's cognate integrin; only 2 integrins have been shown to co-localise with MADM to microglia. Ramified microglia of normal adult rat brain were shown to express low levels of MADM, and the protease was not demonstrated to be upregulated by reactive microglia either during early postnatal development or in the vicinity of a cerebral stab wound. Fluorescent confocal microscopy used to examine MADM expression by isolated microglia demonstrated that in vitro, MADM was found intracellularly, and could not be induced to move to a cell surface location even on microglia activated by cytokines; this intracellular localisation is in direct contradiction of the structural evidence pointing to a transmembrane position for MADM. However, MADM was found to be expressed on the surface of cultured mouse NSO myeloma cells, a result which may reflect a facet of the immortalisation process, or may imply that MADM is dependent on specific integrin expression for its surface localisation.
47
Lu, Xiaohong. "Molecular cloning and expression study of MADM : a metalloprotease with a potential integrin-binding domain." Thesis, University of Leicester, 1996. http://hdl.handle.net/2381/34142.
Full textAbstract:
Mammalian disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. From cDNA libraries of human U937 lymphoma cells and human (adult) brain temporal cortex, a novel integral membrane metalloprotease named MADM (Mammalian Disintegrin-Metalloprotease) was cloned by probing with the bovine cDNA homologue. MADM contains several distinct protein domains: preproprotein, metalloprotease, disintegrin (potential integrin-binding domain), cysteine- rich, transmembrane and cytoplasmic tail. The deduced bovine and human mature protein sequences are 97 % identical while a unique insertion domain (52 residues) was found in the prodomain of human MADM. Northern blotting analysis revealed that two different sizes (4.5 kb and 3.2 kb) of MADM mRNA transcripts were expressed at low levels in a variety of transformed human cell lines. Western blotting of various mammalian cells with an antiserum to a peptide in the disintegrin domain detected a glycoprotein of 62 kDa. Immunolocalisation of MADM showed that it is expressed mainly in an intracellular compartment but was also expressed on the surface of a small percentage of some human cell lines. Three approaches were tried to express recombinant MADM in eukaryotic cells in order to study putative interactions of MADM with cognate integrins and pro tease substrates. Recombinant bovine MADM was expressed transiently in monkey COS cells but most of the expressed proteins were the precursor of MADM and the expression levels of recombinant mature protein were not sufficiently increased over endogenous level to allow functional studies. In a second approach, bovine MADM was expressed stably in murine erythroleukaemia (MEL) cells. Nine individual stably transfected MEL cell clones appeared to express recombinant bovine MADM but the lack of reactivity of this protein with the monoclonal antibody CG4 suggested that some of its disulphide bonds had misformed. The third approach was to achieve high expression of MADM in a baculovirus/insect cell system. A recombinant baculovirus containing a soluble disintegrin-cysteine rich domain polypeptide of human MADM (hDC) was successfully generated and conditions established to isolate high levels of recombinant hDC protein either from cell lysates or from the conditioned medium. Recombinant hDC protein would provide means to investigate putative interactions of MADM with integrins or other proteins and produce antibody reactive with native human MADM.
48
Schneider, Matthias [Verfasser], and Georg [Akademischer Betreuer] Krohne. "Characterisation of Metalloprotease-mediated EGFR Signal Transactivation after GPCR Stimulation / Matthias Schneider. Betreuer: Georg Krohne." Würzburg : Universitätsbibliothek der Universität Würzburg, 2011. http://d-nb.info/1014892147/34.
Full text
49
Mesclet-Cladière, Laurence. "Identification et analyse de facteurs de virulence d'une bactérie entomopathogène, Photorhabdus temperata." Paris 11, 2003. http://www.theses.fr/2003PA112028.
Full textAbstract:
Photorhabdustemperata est une bactérie à Gram négatif, de la famille des Entérobactéries et pathogène pour les insectes. Elle vit en symbiose dans l'intestin de nématodes de la famille des Hétérohabditidae qui sont capables de pénétrer à l'intérieur de la larve d'insectes. Une fois à l'intérieur, les nématodes relâchent les bactéries qui tuent l'insecte et se multiplient rapidement, puis hydrolysent les organes, permettant aux nématodes de se reproduire et de se développer dans des conditions idéales. Finalement, les nématodes et les bactéries entrent à nouveau dans une interaction symbiotique, et sortent du corps de l'insecte, à la recherche d'un nouvel hôte. P. Temperata possède deux phases phénotypiques en laboratoire: la phase 1 et la phase II. Pour essayer d'élucider les facteurs et les mécanismes impliqués dans la variation de phase, une approche, le RAP (RNA fingerprint by arbitrarely primed PCR), a été employée dans le but de comparer les gènes exprimés par la phase 1 et ceux par la phase II de P. Temperata en milieu LB. Plusieurs gènes ont été découverts lors de cette étude, dont deux gènes homologues aux gènes MR/P de Proteus mirabilis. Les fimbriae jouent un rôle important dans la virulence et par conséquent, nous avons voulu poursuivre l'étude de ces fimbriae chez P. Temperata K122. Parallèlement à ce travail, nous avons étudié une métalloprotéase à zinc, PrtA, fortement sécrétée dans le milieu de culture de P. Tempe rata. Cette dernière fait partie de la famille des RTX protéines (Repeats in Toxin). Enfin, nous avons réalisé une soustraction génomique entre deux souches de Photorhabdus très proches, P. Temperata K122 et P. Luminescens W14. Malgré des similitudes dans la pathologie des insectes, elles montrent de nombreux traits phénotypiques différents, laissant supposer que des facteurs de virulence peuvent être spécifiques d'une seule souche, bien qu'elles puissent employer des facteurs communsPhotorhabdus temperata is a Gram negative entomopathogenié bacterium of the Enterobactericae. P. Temperata lives in symbiosis in the intestine of nematodes of the family, Heterorhabditae, which, in turn, are capable of penetrating into the interior of insect larvae. Within the insect, nematodes release the bacteria which rapidly multiply, killing the larva then hydrolysing the internai structure, providing ideal conditions for nematode reproduction and development. Finally, nematodes and bacteria re-enter a symbiotic interaction and they leave 1the cadaver in search of a new hosto P. Temperata exhibits two distinct phenotypic phase variants in the laboratory, termed phase 1 and II. " ln an attempt to elucidate the factors and mechanisms implicated in phase variation we initially performed RAP PCR (RNA fingerprint by Arbitrarily Primed PCR) in order to identify genes specifically expressed in the two phase variant forms of P. Temperata in laboratory LB medium. Several genes were identified during this study, including two clones with homology to a major subunit and one similar to a minor subunit of mannose resistant fimbriae encoded by MR/P genes of Proteus mirabilis. Fimbriae typically play an important Tale in virulence and thus we continued with a more detailed study of these structures in P. Temperata. Concomitant with the above investigations, we also studied a zinc metalloprotease, : PrtA, secreted to the culture medium by P. Tempe rata. PrtA is a member of the RTX (Repeats. 1 in Toxin) family. Finally, a study was undertaken using the technique of genomic DNA subtractive C hybridisation between two species of Photorhabdus, P. Temperata K122 and the closely related P. Luminescens W14. Despite having a similar pathology in insect infections, they ~ exhibit many different phenotypic traits, leading to the hypothesis that, although they may ~ employ many similar virulence factors, some may be species specific
50
Berthelot, Thomas. "Conception et évaluation de nano-systèmes de détection d'acitivités métalloprotéasiques." Bordeaux 1, 2005. http://www.theses.fr/2005BOR13120.
Full textAbstract:
La détection précoce du cancer permettrait un meilleur diagnostic et mise en place de traitements plus sélectifs. Actuellement, l'imagerie du cancer se tourne vers une approche moléculaire. De nombreux travaux présentent les métalloprotéases de la matrice extracellulaire (principalement les MMP-2 et -9) comme des cibles biologiques centrales pour la détection des processus tumoraux. Nous avons conçu des nano-systèmes fluorescents pour évaluer et imager l'activité métalloprotéasique. Nous avons synthétisé des acides aminés originaux fluorescents incorporables directement en synthèse peptidique en phase solide (SPPS). Leur utilisation en SPPS, au sein d'une séquence reconnue et clivée par des MMP, permet d'avoir accès facilement et rapidement à des substrats originaux. L'activité protéolytique a pu être visualisée grâce à un phénomène de transfert de fluorescence par énergie de résonance (FRET). Ce système a été utilsé pour l'élaboration de substrats originaux et hautement sélectifs pour la MMP-1 (modèle) et la MMP-9. Afin d'augmenter la stabilité et la sélectivité, nous avons synthétisé de nouveaux substrats cycliques incorporant deux fois la séquence cible de la MMP. Pour leur conception, nous avons élaboré une méthode originale, efficace et rapide pour leur synthèse en SPPS. Nous avons également décrit la synthèse de puces organosiliciées fonctionnalisées par nos substrats. Nous avons obtenu la première biopuce permettant de suivre l'activité protéolytique de la MMP-9. Pour caractériser l'état de surface de nos puces et suivre la protéolyse, nous avons utilisé une approche originale, fondée sur l'imagerie IR-TF et l'illipsométrie.