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Immunociblage de la cathepsine D dans le microenvironnement des cancers du sein triple-négatifs / Antibody-targeting of the protease cathepsin D in the tumor microenvironment inhibits breast cancer growthAshraf, Yahya 30 June 2015 (has links)
Le cancer du sein triple-négatif est un sous-type représentant environ 15 % des cancers du sein, qui est caractérisé par l'absence de récepteurs hormonaux aux œstrogènes et à la progestérone et l'absence d'expression du facteur de croissance HER-2. Le cancer du sein triple-négatif est également associé à un profil clinique défavorable avec un haut risque de rechute métastatique précoce. Actuellement, de nombreuses molécules sont testées comme cible thérapeutique dans ce type de cancer. La cathepsine D, une aspartyl protéase lysosome, est un marqueur de mauvais pronostic du cancer du sein associé au risque métastatique. Cette protéase est surexprimée par les cellules cancéreuses mammaires et hypersécrétée dans le microenvironnement tumoral. Notre équipe a apporté des contributions importantes pour élucider le rôle de la cath-D dans le cancer du sein. La cath-D stimule la prolifération des cellules cancéreuses, la croissance et l'angiogenèse tumorales ainsi que la formation des métastases. Au niveau moléculaire, le proenzyme sécrété par la cellule tumorale stimule la croissance invasive du fibroblaste mammaire via le récepteur LRP1 (low-density lipoprotein receptor-related protein-1). De plus, au pH extracellulaire acide des tumeurs, la pro-cathepsine D secrétée dégrade un des plus puissants inhibiteurs des cystéines cathepsines, la cystatine C, stimulant ainsi leurs activités protéolytiques.Mon travail a consisté à développer des anticorps anti-cath-D, présentant un effet thérapeutique dans le cancer du sein triple-négatif. Nous avons utilisé deux approches l'une par la technologie des hybridomes qui a permis d'isoler 50 anticorps, qui se sont avérés être des IgM. L'autre, par phage display, où 8 anticorps fragments scFv anti-cath-D ont été sélectionnés par ELISA. Ces 8 scFv ont été criblés pour leur capacité à bloquer l'activité biologique de la cathepsine D : inhibition de l'interaction avec LRP1 et inhibition du clivage d'un substrat fluorescent. Les anticorps E2 et F1 reformatés en IgG1 inhibent in vitro le phénomène de cicatrisation, la clonogénéicité, la croissance tridimensionnelle dans le matrigel et in vivo la croissance tumorale des cellules de cancer du sein triple-négatif MDA-MB-231. Cette étude est une première étape vers le développement de thérapie anti-cath-D dans le cancer du sein. / Cancer is the leading cause of death in the developed world. This project focuses t on the high incidence triple-negative breast cancers (ER- and PR-negative, HER2-non amplified) who present unsatisfactory treatments. Tumor progression is known to be due to cross-talk between cancer cells and their surrounding supporting tissue, the tumor stroma. The lysosomal aspartic protease cathepsin D is over-produced and abnormally hypersecreted by epithelial breast cancer cells. This protease is an independent marker of poor prognosis in breast cancer correlated with the incidence of clinical metastasis. Cathepsin D stimulates tumor progression by both proteolytic and non-proteolytic molecular mechanisms. Our team has shown that secreted pro-cathepsin D can alter the tumor microenvironment by interacting with partners such as the LRP1 (low-density lipoprotein receptor-related protein-1) receptor, and/or by cleaving endogenous substrates like cystatin C. My pHD project aimed to target extracellular procathepsin D released in breast cancer by developing specific therapeutic antibodies to block its catalytic and binding capacities. We have isolated 50 hybridomas, that secreted IgM, a pentameric antibody format with high avidity but low affinity, and 8 anti-cath-D scFv by phage display. The anti-human cath-D scFv were selected by functional screenings for their ability to inhibit both the proteolytic activity of Cath-D and its binding to the fibroblastic LRP1 receptor. Two scFv cloned under IgG1 format (F1 and E2) inhibited wound healing, colony formation and three-dimensional outgrowth in Matrigel of MDA-MB-231 triple-negative breast cancer cells, and their tumor growth in athymic nude mice. Our findings strongly suggest that antibody-based targeting of Cath-D within the breast tumor microenvironment may have therapeutic efficacy for breast cancer treatment.
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Role of Cathepsin G in AtherosclerosisRafatian, Naimeh January 2013 (has links)
Angiotensin II (Ang II) is an important modulator for development of atherosclerosis from early stage foam cell formation to advanced stage plaque rupture. Recently, the importance of locally generated Ang II, especially in macrophages, has become more evident. Generation of Ang II by several enzymes other than ACE and renin has been shown mainly in vitro. Cathepsin G is one these enzymes which is expressed in neutrophils and macrophages. Macrophages are one of the primary and crucial cells in atherosclerotic lesions which become lipid-laden foam cells through lipoprotein uptake. We hypothesized that activation of nuclear factors in foam cells increases Ang II by modulation of the renin angiotensin system (RAS) genes and cathepsin G. We also hypothesized that cathepsin G, through its Ang II generating activity and its other catalytic functions, promotes atherosclerosis.
The present study assessed the Ang I and II levels and expression of the RAS genes in THP-1 cells, a human acute monocytic leukemia cell line, and in peritoneal and bone marrow-derived macrophages after exposure to acetylated LDL (ac-LDL). I also evaluated how RAS blockade would affect foam cell formation in THP-1 cells. In parallel, I assessed the role of cathepsin G in Ang II generation and in the progression of atherosclerosis in cathepsin G heterozygous knockout mice on an Apoe-/- background (Ctsg+/-Apoe-/- mice).
Ac-LDL treatment increased Ang I and Ang II levels in cell lysates and media from THP-1 cells but not in peritoneal or bone marrow-derived macrophages from wild type C57BL/6 mice. In ac-LDL-treated THP-1 cells, ACE and cathepsin G mRNA levels and activities were elevated. Angiotensinogen mRNA is increased but not the angiotensinogen protein concentration. Renin mRNA level and activity were not altered by ac-LDL treatment. Blocking RAS by an AT1 receptor blocker, ACE inhibitors or a renin inhibitor decreased cholesteryl ester content of THP-1 cells after exposure to ac-LDL. To confirm that the Ang II effect on foam cell formation was not unique to ac-LDL, we treated the THP-1 macrophages with a renin inhibitor or an AT1 receptor inhibitor after exposure to oxidized LDL (ox-LDL). RAS blockade in ox-LDL-treated cells also abolished cholesteryl ester formation. To see how Ang II plays a role in foam cell formation we assessed the effect of RAS inhibitors on SR-A, the principal receptor for mediating ac-LDL entry into the cells and on acyl-CoA:cholesterol acyl transferase (ACAT-1), the enzyme responsible for intracellular cholesterol esterification. RAS blockade in both ac-LDL- and ox-LDL-treated cells decreased SR-A and ACAT-1 protein levels.
Cathepsin G partial deficiency on an Apoe-/- background did not change Ang II levels in peritoneal or bone marrow-derived macrophage cell lysates or media. This deficiency also did not affect immunoreactive angiotensin peptide levels in atherosclerotic lesions. After 8 weeks on a high fat diet Ctsg+/-Apoe-/- mice were similar to Ctsg+/+Apoe-/- mice in terms of lesion size and serum cholesterol levels but the Ctsg+/+Apoe-/- mice had more advanced lesions with more collagen and smooth muscle cells and fewer macrophages. Moreover, Ctsg+/+Apoe-/- mice had more apoptotic cells than their Ctsg+/-Apoe-/- littermates.
Overall, our findings indicate that Ang II is increased in foam cells and this endogenous Ang II is involved in cholesteryl ester formation, possibly by regulating the levels of ACAT-1 and SR-A. We did not find any role for cathepsin G in generation of Ang II in mice but cathepsin G does, nevertheless, promote the progression of atherosclerotic lesions to a more advanced stage.
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Cancer du sein et micro-environnement tumoral : rôle de la protéase cathepsine D adipocytaire et de son récepteur LRP1 / Breast cancer and tumoral micro-environment : role of the cathepsin-D protease and its LRP1 receptor in adipocytesMasson, Olivier 15 January 2010 (has links)
L’aspartyl protéase cathepsine D, surexprimée et hyper-sécrétée par les cellules épithéliales cancéreuses mammaires est un facteur de mauvais pronostic des cancers du sein et stimule la prolifération des cellules cancéreuses et la formation des métastases. Elle affecte également le micro-environnement tumoral en induisant la croissance invasive des fibroblastes. Les travaux de l’équipe ont montré que le LDL-receptor related protein 1, LRP1, est le récepteur fibroblastique de la cathepsine D. LRP1 est fortement exprimé par les adipocytes. Les études cliniques indiquent que l’obésité est un facteur de risque dans de nombreux cancers, dont le cancer du sein chez la femme ménopausée.Dans cette thèse, nous avons étudié le rôle de la cathepsine D et du LRP1 dans les adipocytes, type cellulaire prédominant du micro-environnement tumoral mammaire. Nos résultats indiquent une surexpression de la cathepsine D et du LRP1 dans le tissu adipeux humain et murin obèse. De plus, l’expression de la cathepsine D est augmentée pendant la différenciation adipocytaire. Finalement, l’extinction de l’expression de la cathepsine D et du LRP1 inhibe l’adipogenèse indiquant leurs rôles clefs dans ce processus.L’ensemble de ces résultats suggère que la cathepsine D et son récepteur LRP1 pourraient être des cibles thérapeutiques potentielles dans le traitement de l’obésité. / The aspartyl protease cathepsin D, overexpressed and hyper-secreted by epithelial breast cancer cells is a poor prognosis factor in breast cancers and stimulates cancer cell growth and metastasis formation. It also affects the tumor microenvironment, inducing the fibroblasts invasive outgrowth. Our works have shown that the LDL receptor-related protein1, LRP1, is the fibroblastic receptor for cathepsin D. LRP1 is highly expressed in adipocytes. Clinical studies indicate that obesity is a risk factor in many cancers, including breast cancer in postmenopausal women.During this thesis, we studied the role of cathepsin D and LRP1 in adipocytes, which are the prominent cell type in the tumor microenvironment of breast cancers. Our results indicate that cathepsin D and LRP1 are overexpressed in human and mouse obese adipose tissue. Furthermore, the expression of cathepsin D is increased during adipocyte differentiation. Finally, the inhibition of the cathepsin D and LRP1 expression inhibits adipogenesis indicating their key role in this process.All these results suggest that cathepsin D and its receptor LRP1 could be potential therapeutic targets in the treatment of obesity.
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Regulation of Cathepsin L expression and activity by cell confluence and the circadian clockGaikwad, Prashant 15 May 2023 (has links)
No description available.
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Increased stability of class II MHC-peptide complexes in macrophages infected with <i>Mycobacterium avium</i> and the examination of a novel role for Cathepsin L in the innate immune response to <i>Francisella Novicida</i> infectionFlorence, William C. 08 March 2007 (has links)
No description available.
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PREPARATION AND EVALUATION OF PEPTIDYL ACYLOXYMETHYL KETONES FOR CATHEPSIN B IMAGINGEdem, Patricia 10 1900 (has links)
<p>This thesis describes the initial steps towards the use of dipeptidyl acyloxymethyl ketones as a platform to develop molecular imaging (MI) probes for cancer. Initially the synthesis of an AOMK was performed following a literature procedure which resulted in an epimerized product. This issue was addressed by optimizing an alternative method yielding all intermediates in yields similar or better to those reported in the literature (final product yield of 67%). An AOMK derivative that can be used to evaluate target expression levels was synthesized by linking a fluorescent dye to the ε-amine group of lysine in accordance to a literature procedure describing the synthesis of an optical imaging probe in 24% yield. A second generation derivative AOMK was prepared by linking 4-fluoro-benzoic acid to the same amino group yielding a model of a PET MI probe.</p> <p>An endpoint colorimetric assay was developed and optimized to test cathepsin B inhibitors. Due to the fact that the AOMKs exhibit time-dependent inhibition these assay conditions did not prove to be adequate for the assessment of the cathepsin B binding. Steps toward developing a continuous assay that would be better suited for these compounds were achieved. Factors such as the relationship between the formation of the assay product vs enzyme concentration and determination of the Michelis-Menten constant (K<sub>m</sub> = 390 ± 30 nM) were established. These parameters can be used to determine the optimal enzyme and substrate concentration that should be used to test the AOMK based probes.</p> / Master of Science (MSc)
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Class II MHC function in macrophages and mice infected with mycobacteriumNepal, Rajeev Mani 15 March 2006 (has links)
No description available.
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Einfluss des Aktin-bindenden Proteins Synaptopodin-1 auf die Prognose des Pankreaskarzinoms / Impact of the actin-binding protein Synaptopodin-1 on pancreatic cancer's prognosisRommel, Anna Friederike 08 January 2019 (has links)
No description available.
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Avaliação da imunogenicidade da proteína BYCr (Boophilus York pro-Cathepsin) expressada por vetores eucariotos.Medeiros, Maria Lúcia Schiaffino January 2008 (has links)
O carrapato Rhipicephalus (Boophilus) microplus é o principal ectoparasita bovino e causa importantes perdas econômicas nas criações de bovino. O controle imunológico é estudado como um método alternativo para seu controle, no entanto, uma vacina eficaz ainda necessita ser desenvolvida. A proteína BYC (Boophilus Yolk Pro-Cathepsin) é uma aspártico proteinase presente no ovo do carrapato e envolvida na embriogênese, já tendo sido testada como imunógeno vacinal. O propósito deste estudo foi avaliar se a inoculação de plasmídeos para expressão em células eucarióticas (BYCr-PC e BYCr-PME) contendo a região codificante para a proteína BYC poderiam gerar uma resposta imune específica. A região codificante da proteína BYC foi amplificada por PCR e clonada em dois vetores de expressão eucariotos (pcDNA3 e pME18Neo). Os clones, BYCr-PC e BYCr-PME foram utilizados para testes de inoculação de camundongos BALB/C por via intramuscular. Os camundongos receberam duas inoculações de 100 μg das construções (BYCr-PC ou BYCr-PME) e os controles negativos receberam somente PBS, pcDNA3 ou pME18Neo. A produção de anticorpos, após a inoculação, foi avaliada por Western Blotting e ELISA, sendo detectados anticorpos contra a proteína BYC nos camundongos inoculados com a construção BYCr-PC. A imunolocalização da proteína BYC nas amostras de músculo no local da inoculação foram realizadas com o monoclonal BrBm5 (anti-BYC). Estes resultados mostraram que a inoculação com o plasmídeo BYCr-PC induz a produção de anticorpos específicos e possibilita testar o uso de uma vacina de DNA como um método alternativo para o controle de carrapatos. / The Rhipicephalus (Boophilus) microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. However, an effective vaccine remains to be developed. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of Rhipicephalus (Boophilus) microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the immunization containing rBYC (rBYC-PC and rBYC-PME) could elicit a specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18Neo). The clones, rBYC-PC and rBYC-PME, were produced in large scale for immunoassays. To evaluate the immunogenicity of BYC, BALB/c mice were immunized with DNA vaccine by intramuscular injection. The mice received two intramuscular inoculations of 100μg plasmids DNA (rBYCPC or rBYC-PME) and the negative controls received only PBS, pcDNA3 or pME18Neo. The production of antibody after the immunizations was evaluated by Western Blotting and ELISA. Antibodies against BYC in mice inoculated with rBYC-PC were detected. Immunolocalization of the rBYC protein in muscle samples from the injection site with rBYCPC was detected with monoclonal BrBm5 anti-BYC. These results show that DNA immunization produced specific anti-BYC antibodies and suggest that a DNA vaccine could prove useful to develop an alternative method for tick control.
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Avaliação da imunogenicidade da proteína BYCr (Boophilus York pro-Cathepsin) expressada por vetores eucariotos.Medeiros, Maria Lúcia Schiaffino January 2008 (has links)
O carrapato Rhipicephalus (Boophilus) microplus é o principal ectoparasita bovino e causa importantes perdas econômicas nas criações de bovino. O controle imunológico é estudado como um método alternativo para seu controle, no entanto, uma vacina eficaz ainda necessita ser desenvolvida. A proteína BYC (Boophilus Yolk Pro-Cathepsin) é uma aspártico proteinase presente no ovo do carrapato e envolvida na embriogênese, já tendo sido testada como imunógeno vacinal. O propósito deste estudo foi avaliar se a inoculação de plasmídeos para expressão em células eucarióticas (BYCr-PC e BYCr-PME) contendo a região codificante para a proteína BYC poderiam gerar uma resposta imune específica. A região codificante da proteína BYC foi amplificada por PCR e clonada em dois vetores de expressão eucariotos (pcDNA3 e pME18Neo). Os clones, BYCr-PC e BYCr-PME foram utilizados para testes de inoculação de camundongos BALB/C por via intramuscular. Os camundongos receberam duas inoculações de 100 μg das construções (BYCr-PC ou BYCr-PME) e os controles negativos receberam somente PBS, pcDNA3 ou pME18Neo. A produção de anticorpos, após a inoculação, foi avaliada por Western Blotting e ELISA, sendo detectados anticorpos contra a proteína BYC nos camundongos inoculados com a construção BYCr-PC. A imunolocalização da proteína BYC nas amostras de músculo no local da inoculação foram realizadas com o monoclonal BrBm5 (anti-BYC). Estes resultados mostraram que a inoculação com o plasmídeo BYCr-PC induz a produção de anticorpos específicos e possibilita testar o uso de uma vacina de DNA como um método alternativo para o controle de carrapatos. / The Rhipicephalus (Boophilus) microplus tick is the major bovine ectoparasite and causes important economical losses on cattle breeding. The immunologic control has been studied as an alternative method for the tick control. However, an effective vaccine remains to be developed. BYC (Boophilus Yolk Pro-Cathepsin) is an aspartic proteinase found in eggs that is involved in the embryogenesis of Rhipicephalus (Boophilus) microplus, and it has been proposed as a probable antigen in vaccine development. The purpose of this study was to evaluate whether the immunization containing rBYC (rBYC-PC and rBYC-PME) could elicit a specific anti-BYC immune response in vivo. The cDNA of BYC was amplified by PCR and it was cloned into two eukaryotic expression vectors (pcDNA3 and pME18Neo). The clones, rBYC-PC and rBYC-PME, were produced in large scale for immunoassays. To evaluate the immunogenicity of BYC, BALB/c mice were immunized with DNA vaccine by intramuscular injection. The mice received two intramuscular inoculations of 100μg plasmids DNA (rBYCPC or rBYC-PME) and the negative controls received only PBS, pcDNA3 or pME18Neo. The production of antibody after the immunizations was evaluated by Western Blotting and ELISA. Antibodies against BYC in mice inoculated with rBYC-PC were detected. Immunolocalization of the rBYC protein in muscle samples from the injection site with rBYCPC was detected with monoclonal BrBm5 anti-BYC. These results show that DNA immunization produced specific anti-BYC antibodies and suggest that a DNA vaccine could prove useful to develop an alternative method for tick control.
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