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Gewies, Andreas. "Investigation of the ubiquitin-specific protease UBP41 and of the lysosomal cysteine proteases cathepsin-L and cathepsin-B as potential mediators of proapoptotic signalling." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-16836.
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Norbury, Luke James, and s9806495@student rmit edu au. "Structure, Function and Evolutionary Studies of Fasciola Cathepsin L-like Proteases." RMIT University. Applied Science, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081204.160915.
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Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.
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Carrière, Julie. "Characterization of oxyanion hole mutants of the cysteine proteases papain and cathepsin B." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61324.
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It is well accepted that papain and cathepsin B work by stabilizing the transition state of the substrate formed during catalysis. This species carries a formal negative charge which, in the serine proteases family, is stabilized by an oxyanion hole formed by the enzyme. The same structure has been proposed to exist for cysteine proteases. Because the side chain of a Gln residue contributes one stabilizing hydrogen bond to the transition state in the oxyanion hole of papain and cathepsin B, site directed mutagenesis was used in this work to change this residue to Ala and Ser. It was found that this Gln contributes between 2.5 and 3.7 kcal/mol to the transition state stabilization in papain and 3.7 kcal/mol for cathepsin B. The pH profile of cathepsin B is also shifted to higher pH indicating a destabilization of the catalytic ion-pair caused by the mutation. These results demonstrate that the oxyanion hole plays an active role in catalysis by cysteine proteases. (Abstract shortened by UMI.)
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Lockhart, Brent E. "Expression, Purification, and Characterization of the Mast Cell Proteases Chymase and Cathepsin G." Digital Commons @ East Tennessee State University, 2008. https://dc.etsu.edu/etd/1922.
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Human mast cells have been associated with wound healing, allergies, inflammation, and defense against pathogens and have been detected in tissues close to blood vessels especially in the areas between the inside of the body and the external environment, such as the skin, lungs, digestive tract, mouth, and nose. Previous studies have shown that mast cells contain large granules filled with histamine, heparin, cytokines, eicosanoids, and the serine proteases, tryptase, Chymase, and cathepsin G (CatG). These proteases are stored and released from mast-cell granules upon activation by antigen binding to IgE immunoglobulins on the cell surface or by direct injury. In this study, chymase and CatG were expressed as active enzymes in the yeast Pichia pastoris by homologous recombination of the cDNA coding for the mature active proteases into the Pichia genome. Methanol induction resulted in the secretion of active enzyme into the Pichia growth media and increasing levels of enzyme were detected in the media for 5 days. Cells that secreted the highest levels of activity were selected by kinetic assay. Active chymase was purified from the culture media with a 22% yield of activity by a simple two-step procedure that involved hydrophobic-interaction chromatography followed by affinity chromatography on immobilized heparin. The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE. The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media. Active CatG was partially purified from culture media using an ultrafiltration. Mass Spectroscopy (Maldi-Tof) data confirmed that the major protein band was CatG, resulting in the first active human CatG to be produced recombinantly. Additionally, the partially purified enzyme was active against both chymotrypsin and trypsin substrates, and its reaction with inhibitors was consistent with CatG. Although the protein yields were low, these results confirm that CatG was recombinantly expressed.
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Zarella, Bruno Lara. "Papel das proteases na erosão dentinária." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/25/25149/tde-22062017-201442/.
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Na dentina, a matriz orgânica desmineralizada tem um papel protetor contra desafios erosivos subsequentes. Porém, essa camada pode ser degradada por proteases, como as metaloproteinases da matriz (MMPs) e cisteína catepsinas (CCs). Recentemente, o uso de inibidores de proteases da matriz surgiu como uma importante ferramenta preventiva contra a erosão dentinária. Entretanto, o(s) mecanismo(s) exato(s) pelo(s) qual(is) os inibidores de proteases podem prevenir a erosão dentinária, bem como os tipos de proteases mais envolvidas neste processo ainda não são completamente conhecidos. O projeto foi desenvolvido em 2 subprojetos, com os seguintes objetivos: A)Subprojeto 1:Avaliar o papel das proteases na progressão da erosão dentária; B)subprojeto 2: Testar o potencial inibitório do NaF em CCs dentinárias. Para cumprir esses objetivos, foram utilizadas dentina de terceiros molares humanos para a preparação dos espécimes. A)Subprojeto1:Blocos de dentina (4 X 4 x 2 mm) (n=119) foram obtidos de raízes. Os espécimes foram divididos em 7 grupos de acordo com o seu tratamento (E-64, inibidor especifico II de catepsinas B, clorexidina, galardina NaF, placebo) ou sem tratamento, géis foram aplicados uma única vez sobre a superfície e feito o desafio erosivo (90s, 4x por dia por 5 dias) e feita analise perfilométrica. Os espécimes foram incubadas em solução contendo colagenase de Clostridium histolyticum tipo VII por 96hrs e então feita uma segunda analise perfilometrica para se determinar a espessura da MOD. Dois espécimes foram separados para análise de microscopia eletrônica de varredura. B)Subprojeto 2: Palitos de dentina (6 mm X 2 mm X 1 mm) (n=60) foram cortados da porção médio coronária dos dentes e completamente desmineralizados por imersão em EDTA 0,5 M (pH7,4) por 30 dias e lavados em água deionizada sob constante agitação a 4ºC por 72 h. Os espécimes foram divididos em 6 grupos (E-64, NaF e controle negativo, pH 5,5 ou 7,2) e incubados em saliva artificial contendo seus respectivos inibidores por 24 h 7 dias e 21 dias; ao termino de cada período, os espécimes eram pesados para avaliar a perda de massa e analisada a presença de CTX. A)Subprojeto 1: a perda de tecido desmineralizado (m, média± SD) foi: CHX 8,4±1,7b, Gala 8,6±1,9b, IECB 9,6±1,4a, E64 9,9±1,3a, NaF 9,9±1,7a, P 10,9±2,2a, ST 11,0±1,5a. A perda de tecido mineralizado foi: CHX 15,4±2,2b, Gala 16,0±1,8b, IECB 17,6±2,4a, E64 17,6±2,0a, NaF 17,3±2,8a, P 19,1±2,1a, ST 18,9±2,4a. Os inibidores de MMP reduziu significativamente a perda de matriz orgânica e tecido mineralizado em comparação com os outros grupos (p<0,05). Não foi achada diferença significante entre a espessura da matriz orgânica desmineralizada remanescente (p=0,845). B)Subprojeto 2: Na perda de massa houve diferença significante em relação ao inibidor (F=20,047, p<0,0001) e tempo de incubação (F=222,462, p<0,0001) com significante interação entre esses critérios, nos período de menor tempo de incubação, a perda foi similar para todos os grupos testados, no período de maior tempo de incubação, o grupo contendo NaF demostrou os melhores resultados. Na analise de CTX, houve diferença significante em relação aos inibidores (F46,543, p<0,0001), pH (F=14,836, p<0,0004) e tempo de incubação (F=161,438, p<0,0001) com significante interação entre esses critérios, como ocorrido na perda de massa, não houve diferença estatística nos períodos de menor incubação. No período de maior tempo de incubação, mais uma vez o grupo NaF mostrou os melhores resultados. No valor acumulado de CTX, os grupos E64 e controle negativo tiveram os maiores valores de CTX acumulado, o grupo NaF, independente do pH mostrou redução significante em relação aos demais grupos. Após analise dos resultados dos dois subprojetos, podemos indicar que as MMPs são as proteases de maior importância na progressão da erosão dentinária, assim, sua inibição é de maior importância para a redução desta patologia. Mesmo as CCs não exercendo papel direto para a progressão da erosão, elas são efetivas na cascata da ativação de outras proteases, como as próprias MMPs. Com isso, sua inibição também pode ser importante para a redução indireta da progressão da erosão. Neste presente estudo, pudemos comprovar que o NaF tem potencial inibitório sobre as CCs dentinárias, assim, sugerindo um novo inbidor de CCs. Com os resultados deste estudo, podemos afirmar que as MMPs são as principais proteases na progressão da erosão dentinária e que o NaF tem potencial inibitório nas CCs dentinárias.In the dentine, the demineralized organic matrix has a protector part against the following erosive challenges. Nevertheless, this layer can be degraded by proteases, like the matrix metalloproteinases (MMPS) and cystein cathepsins (CCs). Recently, the use of proteases of the matrix´s inhibitors, emerged as an important preventive tool against the dentinária erosion. However, the exact mechanisms from which the inhibitors of the proteases may prevent the dentin erosion, as much as the kinds of proteases more involved in this process are not completely known yet. Therefore, the general objective of this project was to investigate the part of the two main proteases of the matrix (MMPs and CCs) in the dental erosion. The project was developed in 2 subprojects, with the following objectives: A)Subproject 1: Evaluate the part of the proteases in the progression of the dental erosion; B)subproject 2: To test the NaF inhibitory potencial in the dentin CCs. To accomplish these objectives, human third molar dentin were used for the preparation of the specimens, obtained in the surgery and urgency clinics of FOB-USP (subproject 1) or granted by the University of Oulu (subproject 2). A) Subproject 1: Dentine blocks 4 X 4 X 2 mm) (n=119) were obtained from the roots of the obtained teeth. The specimens were divided in 7 groups according with their treatment. Gels containing inhibitors (E-64, specific cathepsin B inhibitor II, chlorhexidine, galardin NaF, placebo), or without treatment, were produced, applied only one time over the surface and made the erosive challenge (90s, 4x a day for 5 days) and made profilometric analysis. The specimens were incubated in a solution containing collagenase of Clostridium histolyticum type VII for 96 hours and then a second profilometric analysis was made to determine the thickness of the MOD. Two specimens were separated for the electronic microscopy scan analysis. B) Subproject 2: Dentine sticks (6 mm X 2 mm X 1 mm) (n=60) were cut from the medium coronary portion of the teeth and completely demineralized by immersion in EDTA 0,5 M (pH7,4) ifor 30 days and washed in deionized water under constant agitation in 4º C for 72 hours. The specimens were divided in 6 groups (divided by inhibitors: E-64, NaF and negative control, pH 5,5 or 7,2) and incubated in artificial saliva containing their respective inhibitors for 24 hours, 7 days and 21 days; by the end of each period, the specimens were weighted to evaluate the loss of mass and analised the presence of CTX. A)Subproject 1: the loss of demineralized tissue (m, média± SD) was : CHX 8,4±1,7b, Gala 8,6±1,9b, IECB 9,6±1,4a, E64 9,9±1,3a, NaF 9,9±1,7a, P 10,9±2,2a, ST 11,0±1,5a. The loss of demineralized tissue was: CHX 15,4±2,2b, Gala 16,0±1,8b, IECB 17,6±2,4a, E64 17,6±2,0a, NaF 17,3±2,8a, P 19,1±2,1a, ST 18,9±2,4a. The MMP inhibitors reduced significantly the loss of organic matrix and demineralized tissue in comparison with other groups (p<0,05). There was no significant difference found between the thickness of the remaining demineralized organic matrix.(p=0,845). B)Subproject: In the loss of mass, there was a significant difference in relation to the inhibitor (F=20,047, p<0,0001) and incubation time (F=222,462, p<0,0001) with significant interaction between these criteria, in the periods of lesser time of incubation, the loss was similar for all the tested groups, in the period of higher time of incubation, the group containing NaF demonstrated the best results. In the analysis of CTX, there was significant difference in relation the inhibitors (F46,543, p<0,0001), pH (F=14,836, p<0,0004) and time of incubation (F=161,438, p<0,0001)with significant interaction between these criteria, as occurred in the mass loss, there was no statistic difference in the period of lesser incubation. In the period of higher time of incubation, once again, the NaF group demonstrated the best results. The CTX accumulated value, the E64 groups and negative control had the greater accumulated values of CTX, the NaF group, regardlessof the pH, demonstrated significant reduction in relation to the other groups. After the analysisof the results of both subprojects, we can indicate that the MMPs are the proteases of greater importance in the progression of the dentin erosion, thus, its inhibition is of graeter importance for the reduction of this pathology. Even the CCs don´t playing the part directly for the progression of erosion, they are effective in the cascade of the activation of other proteases, like the MMPs themselves. In this manner, its inhibition can also be important for the indirect reduction of the progression of the erosion. In this present study, we can prove that the NaF has inhibiting potential over the dentin CCs, thus, suggesting a new inhibitor of CCs. With the results of this study, we can affirm that the MMPs are the main proteases in the progression of the dentin erosion and that the NaF has inhibiting potential in the dentin CCs.
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Boscariol, Rya. "Studies on ovine CD4 : genomic sequence analysis and protein cleavage studies with cathepsin proteases." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81601.
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Here we report the expression and purification of two recombinant Fasciola hepatica enzymes, catL2 and catL5 which were used to perform cleavage studies with substrates potentially encountered by the parasite in vivo; BSA, hIgG3K and the important T cell marker, CD4. We examined the digestion products generated by the cleavage of human CD4 with catL5 using mass spectrometry and predicted candidate cleavage sites by performing a theoretical digest of the protein.Ovine CD4 is also of interest to us as a target of F. hepatica cathepsin L activity. Here we confirm a recently reported ovine CD4 cDNA sequence and the existence of a single nucleotide polymorphism (T/C) within this sequence. The polymorphism translates to a serine-proline switch near the hinge region of the protein. Additionally, we have found that this polymorphism is also present in genomic DNA, suggesting that two alleles of CD4 exist in the ovine genome.
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Jayaraj, Ramamoorthi, and Jayaraj@menzies edu au. "Expression of stage-specific Fasciola proteases and their evaluation in vaccination trials." RMIT University. Applied Science, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081029.100156.
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The liver flukes Fasciola hepatica and F. gigantica cause infectious disease in ruminants and humans. The geographical range of these two parasite species (temperate and tropical respectively) ensures that infection can occur worldwide. Although anthelmintic treatment is effective against disease, emerging drug resistant strains leads to the development of a vaccine. However, despite several decades of research, there is no commercial vaccine available. The main challenge at present is to produce recombinant proteins in an immunologically active form using recombinant DNA technology. This is an essential step in Fasciola vaccine production. Cysteine proteases are probably the most important facilitators of virulence in flukes and are produced by all stages of the fluke life-cycle. Two classes of cysteine protease are found in the excretory and secretory material of liver flukes- these are cathepsin L and cathepsin B. As such, the major aims of this thesis were to investigate the expression and purification of Fasciola recombinant cysteine proteins, and characterisation by SDS-PAGE and immunoblotting using monoclonal and polyclonal antibodies. These studies demonstrate the production of functionally active cathepsin proteins in S. cerevisiae BJ3505 cells which will lead to vaccine candidate analysis. The second aim of this thesis was to determine the protective efficacy of stage specific target antigens against experimental infection. In addressing this issue, the protective efficacy of single and multivalent recombinant protein vaccinations of adult stage F. hepatica cathepsin L5, immature F. gigantica cathepsin L1g and juvenile F. hepatica cathepsin B were analysed in Sprague Dawley rats against F. hepatica infection. This study demonstrates that juvenile fluke target antigen-cathepsin B induces better immune protection than adult fluke antigen-cathepsin L5. Cocktails of juvenile and adult stage fluke recombinant proteins (cathepsin B and L5) elicited the highest protective immunity against experimental infection and this combination showed not only reduction in fluke recovery and size of flukes, but also marked diminution in the intensity of liver lesions in vaccinated rats. In order to assess the immunogenic property of an early infective stage fluke secreting cysteine protease as a vaccine candidate, DNA vaccination vectors encoding cathepsin B were analysed in BALB/c mice. In this study, the ability of four DNA vaccination strategies such as secretory, chemokine-activating, lymph node targeting vectors encoding cathepsin B were assessed by antibody titre, antibody avidity, western blotting and ELIPSOT assay. The results have further validated the immunoprophylactic potential of a cathepsin B vaccine against F. hepatica. In this study, we have expressed and attained high yields of F. gigantica cathepsin L1g from E. coli BL21, and compared this to a yeast-expressed system. This protease was over-expressed and formed insoluble inclusion bodies that were subsequently solubilised with urea or guanidine hydrochloride. In order to purify the urea-solubilised protein, step-wise urea gradient chromatography was used. For refolding of solubilised protein, a dilution and dialysis procedure was utilised. Proteolytic activity was confirmed by gelatin SDS-PAGE analysis. In conclusion, the determination of the immune potential of recombinant stage specific antigens allows the development of effective vaccines against Fasciola infection.
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Florence, William C. "Increased stability of class II MHC-peptide complexes in macrophages infected with Mycobacterium avium and the examination of a novel role for Cathepsin L in the innate immune response to Francisella Novicida infection." The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1173298339.
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Mauricio, Anna Theresa. "Heterocyclic alpha-aminoalkylphosphonate diphenyl esters as inhibitors of serine proteases : Part II: Basic alpha-aminoalkylphosphonate diphenyl esters as inhibitors of cathepsin G." Diss., Georgia Institute of Technology, 1996. http://hdl.handle.net/1853/27157.
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Hooshdaran, Bahman. "DUAL INHIBITION OF CATHEPSIN G AND CHYMASE AFTER ISCHEMIA REPERFUSION: THE ROLE OF INFLAMMATORY SERINE PROTEASES IN ISCHEMIA REPERFUSION INJURY." Diss., Temple University Libraries, 2017. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/475423.
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BioengineeringPh.D.
Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality in the world (4). Restoration of coronary flow to the ischemic myocardium by interventions such as angioplasty, thrombolytic treatment or coronary bypass surgery is the current standard therapy for AMI (5). However, reperfusion of the ischemic myocardium may result in paradoxical cardiomyocyte dysfunction and worsen tissue damage, in a process known as “reperfusion injury” (6). Ischemic reperfusion (IR) injury may intensify pathological processes that contribute to the generation of oxyradicals, disturbances in cation homeostasis, and depletion of cellular energy stores, which may elicit arrhythmias, contractile dysfunction, and ultrastructural damage of the myocardium. These changes can lead to heart failure and ultimately sudden death. The exact mechanisms of IR injury are not fully known (7). Molecular, cellular, and tissue alterations such as cell death, inflammation, neurohumoral activation, and oxidat
Temple University--Theses
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Vargas, Paola Andrea Ortiz. "Genes de cisteíno-proteases de Trypanosoma spp. de mamíferos: polimorfismo e relações filogenéticas." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-22092014-175527/.
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Tripanossomas de mamíferos constituem um dos grupos mais complexos da família Trypanosomatidae, abrangendo parasitas com ciclos de vida e estruturas populacionais heterogêneos. De acordo com a diversidade, filogenias baseadas em genes SSUrDNA e gGAPDH segregaram estes parasitas em 4 Clados principais: T. brucei, T. cruzi, T. theileri e T. lewisi. Catepsinas L e B (CATL e CATB), as principais atividades proteolíticas dos tripanossomas, participam não apenas na degradação de proteínas como também em eventos biológicos como diferenciação, invasão celular, virulência e evasão do sistema imune. Comparamos os perfis proteolíticos de enzimas CATL em tripanossomas patogênicos e não patogênicos e também isolamos e sequenciamos os domínios catalíticos dos genes CATL e CATB em diversas espécies dos principais clados. Os resultados provaram a utilidade destes marcadores no diagnóstico e genotipagem de T. cruzi, T. rangeli, T. theileri e T. congolense, assim como na construção de filogenias robustas da família Trypanosomatidae, congruentes com os marcadores tradicionais.Trypanosomes of mammals comprise one of the most complex groups of the family Trypanosomatidae, including parasites with heterogeneous life cycles and population structures. According to such diversity, phylogenetic analyzes based on SSUrDNA and gGAPDH genes segregate these parasites in 4 major clades: T. brucei, T. cruzi, T. lewisi and T. theileri. Cathepsins L and B (CATL and CATB), the main proteolytic activities of trypanosomes, are not only involved in protein degradation but also in biological events such as cell differentiation, cell invasion, virulence, and evasion from the immune system. We comparatively analysed the CATL proteolytic profiles in pathogenic and non-pathogenic trypanosomes, and isolated and sequenced the catalytic domains of CATB and CATL genes in several species of the major clades. Our results demonstrated the usefulness of both markers in the diagnosis and genotyping of T. cruzi, T. rangeli, T. congolense and T. theileri as well as in the construction of robust phylogenies of the family Trypanosomatidae, congruent with traditional markers.
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Mehrtens, (nee Nikkel) Janna Marie. "The Design, Synthesis and Biological Assay of Cysteine Protease Specific Inhibitors." Thesis, University of Canterbury. Chemistry, 2007. http://hdl.handle.net/10092/3271.
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This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.
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Pimenta, Marcela Valente. "Avaliação da resistência de formas mutantes da enzima L-asparaginase a proteases séricas humanas." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-10092018-173712/.
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O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações.The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications.
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Issa, Najwa. "Détection des protéases microbiennes par la voie immunitaire Toll chez Drosophila melanogaster." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ043.
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Chez la drosophile, l’activation du récepteur Toll menant à une réponse antimicrobienne peut se faire par deux voies différentes. Ces deux voies sont activées soit par des récepteurs dédiés, les Pattern Recognition Receptors (PRRs) reconnaissant des motifs moléculaires microbiens, soit par la coupure d’une molécule circulante appelée Perséphone par des protéases microbiennes extrêmement diverses sécrétées pendant une infection. Cependant, le mécanisme par lequel Perséphone est activée demeurait ambigu. Nous avons identifié une région unique dans Perséphone fonctionnant comme un appât pour les protéases exogènes indépendamment de leur origine, type ou spécificité. Une coupure dans cette région constitue la première étape d’une activation séquentielle de Perséphone ; elle permet de recruter la cathepsine circulante 26-29-p, qui va générer la forme active de Perséphone.Ces travaux montrent comment un récepteur de l’immunité innée, Perséphone, peut être activé par un signal de danger, en l’occurrence des enzymes microbiennes, et non par la détection de motifs moléculaires qui peuvent être présents dans la flore microbienne hébergée par les animauxIn Drosophila, the antimicrobial response against infections can be triggered by two different extracellular mechanisms that both lead to the activation of the Toll receptor. These two mechanisms are activated either by the recognition of specific microbial determinants by Pattern Recognition Receptors (PRRs), or by the cleavage of the circulating serine protease Persephone by a wide range of microbial proteases secreted during infections. However, the molecular mechanism underlying Persephone activation remained ambiguous. We identified a unique region in Persephone pro-domain that functions as a bait for exogenous proteases independently of their origin, type or specificity. Cleavage of Persephone in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous circulating cysteine cathepsin 26-29-p. Our data establish Persephone itself as an immune receptor able to sense a broad spectrum of microbes through the recognition of danger signals rather than molecular patterns
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Vargas, Paola Andrea Ortiz. "Genes de cisteíno proteases (Catepsina L-like) de Trypanosoma rangeli: polimorfismo, relações filogenéticas e alvos para diagnóstico e genotipagem." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-16072009-154538/.
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Nós isolamos e seqüenciamos genes que codificam Catepsina L-like em diversos isolados de T.rangeli de humano, mamíferos silvestres e Rhodnius spp., do centro e sul da América. Análises filogenéticas de seqüências que codificam a proteína madura de T. rangeli, outras espécies de Trypanosoma e Leishmania e duas espécies de bodonídeos, posicionaram T.rangeli próximo a T.cruzi de acordo com a ordem de divergência determinada em filogenias baseadas em SSUrDNA. Uma análise de 17 seqüências do domínio catalítico de CatL-like de isolados representativos da diversidade filogenética e distribuição geográfica de T. rangeli, apoiaram as mesmas linhagens filogenéticas previamente definidas. Seqüências do gene CatL-like também foram usados para padronizar ensaios de PCR para diagnóstico de T. cruzi e T. rangeli. Além disso, um método de genotipagem por PCR multiplex segregou os isolados de T. rangeli nas principais linhagens previamente estabelecidas. Este é o primeiro estudo usando um gene codificador de proteína para comparar isolados de T. rangeli de linhagens distintas.We have isolated and sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of T. rangeli from human, wild mammals and Rhodnius spp., from Central and South America. Phylogenetic analysis of sequences encoding the mature CatL-like enzymes from T. rangeli (Rangelipain), other Trypanosoma and Leishmania species, and two species of bodonids, positioned T. rangeli closest to T. cruzi corroborating the same order of divergence showed in phylogenies based on SSU rDNA. Analysis of 17 sequences of the catalytic domains of CatL-like genes isolates representative of the phylogenetic diversity and geographical range of T.rangeli supported previously defined phylogenetic lineages. Sequences of CatL-like genes were used to standardize PCR assays for the diagnosis of T. rangeli and T. cruzi, and a genotyping method of multiplex-PCR distributed of isolates of T. rangeli in the major phylogenetic lineages previously established. This is the first study using protein-encoding genes to compare isolates from T. rangeli of distinct lineages.
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Mazouni, Chafika. "Evaluation et validation de marqueurs pronostiques et prédictifs dans la prise en charge des patientes présentant un cancer du sein." Thesis, Aix-Marseille 2, 2010. http://www.theses.fr/2010AIX20701.
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L’identification de marqueurs pronostiques et prédictifs du cancer du sein est un facteur important pour une meilleure compréhension du processus évolutif et le développement de thérapies ciblées. Les récepteurs des oestrogènes (RE) représentent ainsi à la fois un marqueur pronostique mais aussi prédictif du traitement par le tamoxifène ou les anti-aromatases. Cependant, un certain nombre de patientes vont évoluer en dépit de traitements anti-hormonaux adaptés. L’objectif de notre travail, a été d’évaluer la méthode de mesure des RE, l’apport des protéases dans la distinction de profils tumoraux pronostiques et prédictifs. Nous avons démontré l’influence du mode de mesure des RE et en particulier de l’expression quantitative sur l’interprétation pronostique et sur une meilleure détermination du bénéfice du traitement en fonction du niveau d’expression des RE. Nous avons montré l’intérêt de l’évaluation des protéases tissulaires uPA, PAI-I et cathépsine-D, pour caractériser l’hétérogénéité des tumeurs en complément des RE. Particulièrement, chez les patientes RE+, des taux élevés de cathépsine-D et de PAI-1 étaient un indicateur de mauvais pronostic. Nous avons développé un nomogramme combinant RE et le statut ganglionnaire à 3 types de protéases : PAI-1, cathépsine-D et la thymidine kinase, pour déterminer la probabilité de survie à 2 et 5 ans. De plus, ces protéases évaluées dans les tumeurs infectées par l’Epstein-Barr virus (EBV), témoignaient de tumeurs biologiquement agressives avec des taux plus élevés de thymidine kinase. Notre travail a contribué à améliorer l’identification de profils des tumeurs en fonction des RE et des protéases et de caractériser les tumeurs viro-induitesThe identification of prognostic and predictive markers is important for a better understanding of the evolutionary process and the development of targeted therapies. Thus estrogen receptors (ER) represent both an important prognostic marker but also predictive of therapies using tamoxifen or aromatase inhibitors. However, a number of patients will evolve despite hormonotherapy. The objective of our work was to evaluate the method for measuring ER, the contribution of proteases in the distinction of prognostic and predictive tumor profiles. In our work, we demonstrated the influence of the mode of measure of ER and in particular its quantitative expression on the prognostic interpretation and a better determination of benefit of treatment depending on the level of expression of ER. We show the interest of the evaluation of tissue proteases uPA, PAI-I and cathepsin-D, to characterize the heterogeneity of tumors in addition to ER. Specifically, in ER + patients, high levels of cathepsin-D and PAI-1 are an indicator of poor prognosis. We developed a nomogram combining ER and nodal status, to 3 types of proteases: PAI-1, cathepsin-D and thymidine kinase, to determine the probability of survival at 2 and 5 years. In addition, these proteases are evaluated in tumors infected with the Epstein-Barr virus (EBV) and shows high rates of thymidine kinase in EBV + BC, reflecting biologically aggressive tumors. Our work has helped to improve the identification of profiles of tumors according to ER and proteases and characterize virus-associated tumors
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Arispe, Angulo Wara Milenka Trawick Mary Lynn. "Inhibitors of human cathepsin L and cruzain as therapeutic agents." Waco, Tex. : Baylor University, 2008. http://hdl.handle.net/2104/5290.
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Cunha, Rodrigo Luiz Oliveira Rodrigues. "Novos aspectos e aplicações da química de teluranas e de teluretos orgânicos." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/46/46135/tde-27082008-073450/.
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A primeira parte desta tese aborda estudos sobre a reatividade de compostos de telúrio eletrofílicos, principalmente tetracloreto de telúrio e tricloretos aromáticos de telúrio. Novos aspectos da reatividade de TeCl4 frente a alcinos e algumas acetofenonas foram observados e, a partir da elucidação estrutural dos compostos obtidos, por cristalografia, uma racionalização mecanística foi proposta para cada caso. As proposições apresentadas encontraram respaldo com a detecção de intermediários transientes por estudos de espectrometria de massas com ionização por electron-spray (ESI-MS/MS). Além de novos aspectos da química do Telúrio, os compostos preparados encontraram aplicação como potentes e seletivos inibidores de cisteíno proteases. Com esta aplicação estabelecida, foram sintetizadas ambos os enantiômeros de uma telurana e a atividade inibitória destas frente a Catepsina B mostrou dependência da estereoquímica devido a dependência estereoquímica da interação entre a enzima e o inibidor. A segunda parte deste trabalho trata do desenvolvimento da reação de abertura de anel de aziridinas por reagentes organometálicos de cobre derivados de teluretos vinílicos e arílicos que resultaram em derivados de aminas homoalílicas ou homobenzílicas. Em seguida, a reatividade de aziridinas alílicas foi estudada frente a uma série de reagentes organometálicos de lítio, magnésio, cobre e zinco que mostraram influenciar a regio- e estereosseletividades das reações de abertura.The first part of this thesis deals with the study of the reactivity of electrophilic tellurium compounds, mainly tellurium tetrachloride and aromatic tellurium trichlorides. New aspects of the reactivity of TeCl4 towards alkynes and some acetophenones were disclosed. A mechanistic rationale for each of the processes studied was possible by the determination of the stereochemistry for each product by monocrystal X-ray diffraction analysis. The proposition of the formation of cationic intermediates in the addition reaction of TeCl4 to alkynes was corroborated by the detection and characterization of transient intermediates by ESI-MS/MS experiments. Besides the new aspects of the Tellurium chemistry found, the prepared compounds showed a high and selective activity as inhibitors of cysteine proteases. A pair of enantiomers of a tellurane showed different activities against Human Catepsin B due to a stereochemical dependence in the enzyme/inhibitor interaction. The second part of the present work deals with the development of the ring opening reaction of aziridines by organometallic reagents of copper prepared from vinylic and arylic tellurides. These reactions led to homoallylic and homobenzylic amine derivatives. Finally, the reactivity of 2-alkenyl aziridines was studied towards a series of organometallic reagents of lithium, magnesium, copper and zinc which biased the regio- and stereoselectivities of the ring opening reactions.
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Maury, Frédéric. "Etude de l'activite synoviale des cysteine-proteases au cours de la polyarthrite rhumatoide." Lille 2, 1993. http://www.theses.fr/1993LIL2M235.
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Verity, Christiana Kelsick. "Cathepsin D-like aspartic protease from Schistosoma japonicum : developmental, enzymological and immunological studies /." [St. Lucia, Qld.], 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16289.pdf.
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Piovan, Leandro. "Síntese e avaliação de compostos de selênio(IV) e telúrio(IV) como inibidores de cisteíno e treonino proteases." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/46/46136/tde-13102011-081714/.
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Neste trabalho está descrito a síntese e avaliação biológica de uma série de compostos de selênio(IV) e telúrio(IV). Esta série foi planejada para que diferentes fatores estruturais pudessem ser avaliados e as possíveis relações entre a estrutura e atividade biológica dos compostos pudessem ser determinadas. Para tanto, selênio, telúrio, cloro e bromo foram diferentemente combinados em um esqueleto carbônico simples, contendo ou não um centro assimétrico. Os compostos de interesse foram sintetizados empregando metodologias quimio-enzimáticas, quando necessário, e reações clássicas da química do selênio e telúrio, levando aos compostos de interesse em poucas etapas e com bons rendimentos. No caso dos ensaios biológicos, parâmetros como potência relativa, constante de inibição de segunda-ordem, mecanismo de inibição, CI50 e viabilidade celular foram determinados dentro das possibilidades experimentais envolvendo cada enzima. As possíveis combinações deram origem a 12 compostos que foram avaliados como inibidores de cisteíno catepsinas B, K, V e S onde a potência relativa dos mesmos pode ser determinada. Para as cisteíno catepsinas V e S, as constantes de inibição de segunda-ordem foram determinadas e ficou evidenciado que a combinação entre telúrio e bromo leva aos compostos mais potentes para estas proteases, enquanto que a combinação entre selênio e cloro origina os inibidores menos potentes. A combinação, selênio e bromo, ou telúrio e cloro forneceu inibidores com potências intermediárias. Este foi o primeiro estudo descrevendo compostos de selênio(IV) como inibidores de proteases. Os mesmos compostos também foram avaliados como inibidores do proteassomo 20S, uma treonino protease, onde se pode observar pela primeira vez que compostos de selênio e telúrio atuam como inibidores desta protease. Os valores de CI50 dos compostos foram determinados e novamente os compostos de telúrio mostraram-se mais potentes do que seus congêneres de selênio. Por outro lado, ensaios em células demonstraram que os compostos de telúrio são direcionados a outro alvo biológico, diferentemente dos compostos de selênio que continuaram a inibir o proteassomo em um lisado celular. Em ensaios de viabilidade celular ficou evidenciado que os compostos de selênio foram mais citotóxicos do que os de telúrio, o que se mostrou muito interessante para desenvolvimento de um agente anticancer onde a resposta biológica desejada é a morte celular.The synthesis and biological evaluation of a series of selenium(IV) and tellurium(IV) compounds have been described in this research. This series was designed to allow different structural factors to be evaluated, and the possible strutucture-activity relationships determinated. Selenium, tellurium, chlorine and bromine were differently combined in a carbon backbone with or without an asymmetric center. The compounds were synthetized by using both chemo-enzymatic methodology and classical selenium and tellurium chemistry. From biological assays, relative potency, second-order inactivation constant, inhibition mechanism, IC50 and cell viability were determinated according to the experimental possibilities involving each enzyme protocol. The 12 compounds synthesized from the possible combinations among selenium, tellurium, chlorine and bromine were evaluated as cysteine cathepsins B, K, V and S inhibitors, and their relative potencies were determined. By determining the second-order inactivation constant for cysteine cathepsins V and S, it was shown that a tellurium and bromine combination led to most powerfull inhibitors. Selenium and chlorine combination led to less potent inhibitiors, while selenium and bromine, and tellurium and chlorine led to inhibitors with intermediate potency. Those compounds were also evaluated as 20S proteasome inhibitors, a threonine protease. We first observed selenium and tellurium-containing compounds acting as inhibitors of 20S proteasome. The IC50 values were determinate and tellurium compounds were more potent again. On the other hand, tellurium compound did not inhibit proteasome in cells, while selenium-containing compound does it. By cell viability assays it was verified that selenium-containing compounds were more cytotoxic than their tellurium analogs. This data is interesting for someone that wishes to develop an anti-cancer agent where the biological response desired is death of cancerous cells.
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Hassanein, Mohamed. "Biochemical and functional characterization of a novel placental protease, cathepsin P, in rat trophoblasts." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 148 p, 2007. http://proquest.umi.com/pqdweb?did=1654487491&sid=6&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Dornbush, Padraick J. "Compound discovery and expression of a putative cathepsin D-like protease in Trichomonas vaginalis." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/181.
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Trichomonas vaginalis is a sexually-transmitted parasite that is the causative agent in the disease trichomoniasis. Resistance to the only FDA-approved medication to this disease, metronidazole, has been on the increase giving rise to the need for finding targets for new inhibitors to exploit. New inhibitors can target enzymes such as 4-coumarate:CoA ligase and S-adenosylhomocysteine hydrolase. Another potential target is a cathepsin D-like protease found in T. vaginalis . This aspartic protease in humans is responsible for degrading proteins in the lysosome, and degrading hemoglobin in P. falciparum as the homologue plasmepsin. Searching the gene database, only one cathepsin-D like protease was discovered throughout the organism's genome. Utilizing RT-PCR, this gene is found to be expressed in two different strains of the organism. Transfection of an epitope-tagged version of this cathepsin D-like protease into T. vaginalis was accomplished, and subsequent immunofluorescence of this tagged version shows it to be localized in intracellular compartments, which can be colocalized using the SNARE and VAMP proteins found in T. vaginalis .
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Beers, Courtney. "The role of cysteine proteases in MHC class II antigen processing and presentation /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/8321.
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Kasabova, Mariana. "Rôle des cathepsines à cystéine et leurs inhibiteurs naturels, les cystatines lors de la fibrose pulmonaire." Thesis, Tours, 2013. http://www.theses.fr/2013TOUR4052.
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Lors de la fibrose pulmonaire idiopathique (FPI), la différenciation fibroblastique s’accompagne d’une accumulation excessive des composants de la matrice extracellulaire ainsi qu’à un dérèglement de la balance protéases / antiprotéases. Nous avons étudié le rôle des cathepsines à cystéine dans la myofibrogenèse et leur contribution potentielle à la physiopathologie de la fibrose pulmonaire chez l’Homme. Pour cela, le profil d’expression des cathepsines ainsi que de leurs inhibiteurs naturels a été évalué dans un modèle cellulaire expérimental, puis dans des myofibroblastes primaires et enfin dans des liquides de lavage broncho-Alvéolaires (LBA) de patients atteints de FPI. Nos résultats montrent que lors de la FPI la cystatine C (inhibiteur naturel des protéases à cystéine) régule les activités protéolytiques des cathepsines extracellulaires et pourrait ainsi contribuer à l’accumulation de collagènes. Elle serait un biomarqueur potentiel de la FPI. D’autre part la cathepsine B participe à la différentiation fibroblastique et son inhibition retarde la myofibrogenèseDuring idiopathic pulmonary fibrosis (IPF), fibroblast differentiation is accompanied by an excessive accumulation of extracellular matrix components as well as an imbalance between proteases and theirs inhibitors. We evaluated the role of human cysteine cathepsins in myofibrogenesis and their potential contribution to the pathogenesis of IPF. Expression of cathepsins and their natural inhibitors have been studied in an experimental cell model, but also in primary myofibroblasts and in bronchoalveolar lavage fluids (BALF) of patients suffering from IPF. Our results show that cystatin C (a natural inhibitor of cysteine proteases) regulates the extracellular proteolytic activities of cathepsins and could contribute to the accumulation of collagens. Cystatin C could also be a potential biomarker of IPF. On the other hand, cathepsin B participates in fibroblast differentiation and its inhibition delays myofibrogenesis
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Roberts, Ladeidra Monet. "ANTIRETROVIRAL DRUGS EFAVIRENZ AND TENOFOVIR AND THEIR EFFECTS ON ARTERIAL REMODELING AND PROTEASE ACTIVITY." Thesis, Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/53731.
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Highly antiretroviral therapies (HAART) have been implemented to slow the progression of the human immunodeficiency virus (HIV). Although these new advances in the medications for HIV-positive patients have contributed in longer life expectancy, comorbidities, such as cardiovascular disease, still cause higher number of deaths among HIV-positive patients than in the regular population. Because of the intrinsic inflammation caused by the HIV virus, atherogenesis is more likely to occur and is driven by infected macrophages. These macrophages are known to secrete cathepsins, but infection causes the macrophages not to perform their function properly as an immune agent. I hypothesize that antiretroviral drugs play an important role in arterial remodeling by affecting cells within the artery and causing an alteration of cathepsin activity, leading to an increased risk of atherosclerosis in HIV patients. To test this hypothesis, we incubated THP-1 monocytes with antiretroviral drugs efavirenz and tenofovir individually to observe any changes in cathepsin activity. These lysates were analyzed through multiplex cathepsin zymography and quantified through densitometry. We found that our hypothesis held true for efavirenz and tenofovir in THP-1 monocytes, which caused decreased cathepsin K activity compared to vehicle controls. Still, stimulation of peripheral blood mononuclear cells (PBMCs) with efavirenz and tenofovir caused differential effects. Together, our data suggest that the HAART interaction with monocytes that are physiologically relevant to our system possibly contributes to the advancement of atherogenesis in HIV+ patients.
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Concha, Menéndez María Carolina. "Etude du rôle de la SpH protéase dans la restructuration de la chromatine mâle post-fécondation et dans l'initiation des cycles cellulaires embryonnaires chez l'oursin." Paris 6, 2004. http://www.theses.fr/2004PA066384.
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Park, Keon-Young. "Predicting patient-to-patient variability in proteolytic activity and breast cancer progression." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/53479.
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About one in eight women in the United States will develop breast cancer over the course of her lifetime. Moreover, patient-to-patient variability in disease progression continues to complicate clinical decisions in diagnosis and treatment for breast cancer patients. Early detection of tumors is a key factor influencing patient survival, and advancements in diagnostic and imaging techniques has allowed clinicians to spot smaller sized lesions. There has also been an increase in premature treatments of non-malignant lesions because there is no clear way to predict whether these lesions will become invasive over time. Patient variability due to genetic polymorphisms has been investigated, but studies on variability at the level of cellular activity have been extremely limited. An individual’s biochemical milieu of cytokines, growth factors, and other stimuli contain a myriad of cues that pre-condition cells and induce patient variability in response to tumor progression or treatment.
Circulating white blood cells called monocytes respond to these cues and enter tissues to differentiate into monocyte-derived macrophages (MDMs) and osteoclasts that produce cysteine cathepsins, powerful extracellular matrix proteases. Cathepsins have been mechanistically linked to accelerated tumor growth and metastasis. This study aims to elucidate the variability in disease progression among patients by examining the variability of protease production from tissue-remodeling macrophages and osteoclasts. Since most extracellular cues initiate multiple signaling cascades that are interconnected and dynamic, this current study uses a systems biology approach known as cue-signal-response (CSR) paradigm to capture this complexity comprehensively.
The novel and significant finding of this study is that we have identified and predicted donor-to-donor variability in disease modifying cysteine cathepsin activities in macrophages and osteoclasts. This study applied this novel finding to the context of tumor invasion and showed that variability in tumor associated macrophage cathepsin activity and their inhibitor cystatin C level mediates variability in cancer cell invasion.
These findings help to provide a minimally invasive way to identify individuals with particularly high remodeling capabilities. This could be used to give insight into the risk for tumor invasion and develop a personalized therapeutic regime to maximize efficacy and chance of disease free survival.
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Larminie, Christopher Geoffrey Carson. "Isolation and characterisation of four cathepsin B-like cysteine protease genes from the free living nematode Caenorhabditidis elegans." Thesis, University of Glasgow, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294196.
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Friedrich, Beate. "Cathepsine B, H, L und ihre Inhibitoren im Gewebe und in Zellkulturen der Prostata." Doctoral thesis, [S.l.] : [s.n.], 1999. http://deposit.ddb.de/cgi-bin/dokserv?idn=957675186.
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Specker, Edgar. "De-novo-Design und Synthese neuer Leitstrukturen als Übergangszustandsmimetika zur selektiven Inhibition der HIV-1-Protease und Cathepsin D." [S.l.] : [s.n.], 2004. http://archiv.ub.uni-marburg.de/diss/z2004/0511/.
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Pierre, Ophélie. "Etude in vitro des mécanismes impliqués dans les effets sensoriels des ciguatoxines et brévétoxines - Focus sur la signalisation de PAR2 F.HuetabM.Severino-FreirecJ.ChéretaO.GouinaJ.PraneufdO.PierreaL.Miseryab1C.Le Gall-Ianotto." Thesis, Brest, 2019. http://www.theses.fr/2019BRES0111.
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La ciguatéra est une intoxication consécutive à l’ingestion de poissons contaminés par des neurotoxines marines appelées ciguatoxines (CTXs) et est caractérisée par des symptômes sensoriels intenses et persistants comme des paresthésies, une allodynie au froid et un prurit. Les CTXs, non commercialisées, et leur analogue structural et fonctionnel appelé brévétoxine-1 (PbTx-1) ont pour cible primaire le canal sodium sensible au potentiel de membrane (Nav), particulièrement exprimé par les neurones sensoriels. Afin d’identifier les mécanismes moléculaires des symptômes sensoriels, nous avons étudié sur neurones sensoriels, kératinocytes et cocultures l’effet de la ciguatoxine-2 (P-CTX-2) et de la PbTx-1. A l’aide des techniques d’imagerie calcique, d’immunocytochimie, de dosages immunologiques et de microscopie confocale nous avons montré le rôle majeur de la cathepsine S et de PAR2 dans la signalisation induite par la PbTx-1 et la P-CTX-2. Des explorations plus approfondies ont montré l’internalisation de PAR2 signant l’activation de la voie canonique en aval du récepteur. Nous avons également montré l’activation de la voie biaisée en aval de PAR2. De plus, ce travail a permis de mettre en évidence une sensibilisation des récepteurs du prurit (TRPA1, TRPV1, TRPV4, MrgprA3, PAR2 et Nav TTX-r) par ces neurotoxines pouvant contribuer à expliquer les symptômes sensorielsCiguatera fish poisoning is consecutive to ingestion of contaminated fishes by marine neurotoxins called ciguatoxins (CTXs). This intoxication is characterized by persistent sensory disorders such as paresthesia, cold dysesthesia, pain and an intense pruritus. CTXs, not commercialized, and their analogous called brevetoxin-1 (PbTx-1) target the sodium voltage gated channels (Nav), particularly expressed on sensory neurons. In order to identify molecular mechanisms of sensory disorders by those neurotoxins we studied the effect of the ciguatoxine-2 (P-CTX-2) and the PbTx-1 on sensory neurons, keratinocytes and co-culture.Using calcium imaging, immunochemistry, immunoassay and confocal microscopy we showed the critical role of cathepsin S and PAR2 in the P-CTX-2/PbTx-1 signaling. Further explorations highlight PAR2 internalization, canonical and biased signaling of PAR2 involvement. In addition, this work bring to light sensory receptor sensitization including TRPA1, TRPV1, TRPV4, MrgrprA3, PAR2 and TTX-r Nav likely participating to sensory disturbances
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Büth, Heiko [Verfasser]. "Contribution of the lysosomal cysteine protease cathepsin B to extracellular matrix remodeling during keratinocyte migration and wound healing / Heiko Büth." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2008. http://d-nb.info/1034895893/34.
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Lindmark, Anders. "On the biosynthesis and processing of cathepsin G, leukocyte elactase, and azurocidin neutrophil granule members of a hematopoietic serine protease superfamily /." Lund : Dept. of Hematology, Lund University, 1997. http://catalog.hathitrust.org/api/volumes/oclc/38985787.html.
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GUINEC, NATHALIE. "Les proteases lysosomiales humaines : action des cathepsines b, h, l normales et b tumorale sur une matrice extracellulaire intacte." Paris 6, 1993. http://www.theses.fr/1993PA066378.
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Les cellules cancereuses ont la particularite de secreter des proteases normalement lysosomiales. Ces proteases pourraient etre responsables de la lyse de la matrice extracellulaire et des membranes basales observee dans l'environnement tumoral. Parmi ces enzymes, les cysteine-proteases de la super-famille de la papaine ont ete decrites. Cette these etudie la digestion d'une membrane basale intacte, la capsule de cristallin de buf par les cathepsines b, b tumorale, h et l humaines. Dans un premier chapitre, la capacite de digestion est mise en evidence et les produits de digestion sont identifies, caracterises et quantifies par des methodes immunochimiques. Le deuxieme chapitre etudie les activites proteolytiques associees aux polypeptides issus de la digestion de la fibronectine. Ce sont des activites dirigees contre les composants des membranes basales et qui apparaissent sous l'action d'une protease exogene, elles s'integrent dans une cascade proteolytique qui contribue a l'efficacite de la lyse. Un autre facteur explique l'importance de ce phenomene, c'est la fixation des cysteine-proteases sur/dans la structure de la membrane basale. L'etude de ses parametres fait l'objet du troisieme chapitre. En complement, la presence de polypeptides issus des composants des membranes basales a ete recherchee dans le plasma de malades souffrant de differents types de cancer
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Smith, Eliot T. "Bioengineering the Expression of Active Recombinant Human Cathepsin G, Enteropeptidase, Neutrophil Elastase, and C-Reactive Protein in Yeast." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etd/1198.
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The yeasts Pichia pastoris and Kluyveromyces lactis were used to express several recombinant human proteins for further biochemical characterization. Two substitution variants of recombinant human enteropeptidase light chain (rhEPL) were engineered to modify the extended substrate specificity of this serine protease. Both were secreted as active enzymes in excess of 1.7 mg/L in P. pastoris fermentation broth. The substitution variant rhEPL R96Q showed significantly reduced specificities for the preferred substrate sequences DDDDK and DDDDR; however, the rhEPL Y174R variant displayed improved specificities for these substrate sequences relative to all other reported variants of this enzyme. The neutrophil serine proteases human cathepsin G (hCatG) and human neutrophil elastase (HNE) were expressed in P. pastoris and HNE was also expressed in K. lactis. The recombinant variants rhCatG and rHNE, with intact C-terminal extensions, were expressed as fusion proteins with the soluble heme-binding domain of cytochrome B5 (CytB5) and an N-terminal hexahistidine (6xHis) tag for purification. The CytB5 domain was linked to the native N-termini of active rhCatG and rHNE by the EPLcleavable substrate sequence DDDDK~I, where ~ is the sessile bond. These fusion proteins were directed for secretion. The yeast P. pastoris expressed up to 3.5 mg/L of EPL-activable rHNE in fermentation broth; however, only 200 μg/L of rhCatG could be produced by this method. Recombinant expression in K. lactis never surpassed 100 μg/L of activable rHNE. The CytB5 fusion domain was present in the heme-bound form, conferring a red color and 410 nm absorbance peak to solutions containing the fusion proteins. This absorbance pattern was most readily visible during the purification of CytB5-rHNE from P. pastoris. Human C-reactive protein (hCRP) and the substitution variant CRP E42Q were expressed in recombinant form and secreted by P. pastoris. Both products were found to bind phosphocholine (PCh) in the same manner as native hCRP. Difficulties encountered during purification revealed that wild type recombinant CRP (rCRP) was produced at 2 different molecular masses. The P. pastoris recombinant expression system yielded better results than K. lactis. Bioreactor-scale fermentation in a 5 L vessel facilitated expression and characterization of these recombinant proteins.
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Moura, Alexandre Santos de. "Catepsinas B vitelolíticas de Culex quinquefasciatus." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-18072014-105711/.
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Apesar de Culex quinquefasciatus ser um eficiente vetor de doenças tais como a filariose linfática, febre do Nilo Ocidental e outras várias neuroviroses, poucas pesquisas sobre sua fisiologia têm sido conduzidas. Como em todos os animais ovíparos, o desenvolvimento embrionário dos mosquitos depende da degradação dos nutrientes armazenados no ovo, sendo a catepsina B uma protease que tem sido identificada e caracterizada em vários insetos como envolvida nesta função. Neste trabalho identificamos, por espectrometria de massas, duas catepsinas B de Culex quinquefasciatus, parcialmente purificadas por autoproteólise de extrato total de ovos. Segundo a anotação de suas sequências no banco de dados específico para vetores, o VectorBase, elas são enzimas parálogas e suas sequências apresentam 77% de homologia. Denominadas neste trabalho como CatB1 e CatB2, ambas são expressas simultaneamente no corpo gorduroso de todas as fêmeas vitelogênicas de nossa colônia e sua atividade pode ser detectada nos ovários vitelogênicos, sugerindo sua origem materna. A transcrição de ambas as enzimas se inicia após o repasto sanguíneo (ARS), alcançando o pico de expressão às 36 h ARS, de forma bastante semelhante à da vitelogenina.Despite Culex quinquefasciatus be an efficient vector of diseases such as lymphatic filariasis, West Nile fever and other various neurotrophic viruses, little research on its physiology have been conducted. As in all oviparous animals, embryonic development of mosquitoes depends on the degradation of the nutrients stored in the egg. Cathepsin B is a protease that has been identified and characterized in a number of insects as involved in this function. In this work we have identified, by mass spectrometry, two cathepsins B of Culex quinquefasciatus, partially purified by self-proteolysis of total egg extract. According to the annotation of their sequences in the specific vector database, the VectorBase, they are paralogue enzymes and their sequences have 77% homology. Named in this work as CatB1 and CatB2, both are expressed simultaneously in the fat body of all vitellogenic females of our colony and its activity can be detected in vitellogenic ovaries, suggesting a maternal origin. The transcription of both enzymes starts post blood meal (PBM), reaching their peak of expression at 36 h PBM, quite similar to vitellogenin.
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Mokhawa, Gaone. "Using bioinformatics tools to screen for trypanosomal cathepsin B cysteine protease inhibitors from the SANCDB as a novel therapeutic modality against Human African Trypanosomiasis (HAT)." Thesis, Rhodes University, 2016. http://hdl.handle.net/10962/3304.
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Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a fatal chronic disease that is caused by flagellated protozoans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. HAT is spread by a bite from an infected tsetse fly of the Glosina genus. Up to 60 million people in 36 countries in sub-Saharan Africa are at a risk of infection from HAT with up to 30 000 deaths reported every year. Current chemotherapy for HAT is insufficient since the available drugs exhibit unacceptable side effects (toxicity) and parasite resistance. Novel treatments and approaches for development of specific and more potent drugs for HAT are therefore required. One approach is to target vital proteins that are essential to the life cycle of the parasite. The main interest of this study is to explore Trypanosoma brucei cathepsin B-like protease (TbCatB) structural and functional properties with the primary goal of discovering non peptide small molecule inhibitors of TbCatB using bioinformatics approaches. TbCatB is a papain family C1 cysteine protease which belongs to clan CA group and it has emerged as a potential HAT drug target. Papain family cysteine proteases of Clan CA group of Trypanosoma brucei (rhodesain and TbCatB) have demonstrated potential as chemotherapeutic targets using synthetic protease inhibitors like Z-Phe-Ala-CHN2 to kill the parasite in vitro and in vivo. TbCatB has been identified as the essential cysteine protease of T. brucei since mRNA silencing of TbCatB killed the parasite and resulted in a cure in mice infected with T. brucei while mRNA silencing of rhodesain only extended mice life. TbCatB is therefore a promising drug target against HAT and the discovery and development of compounds that can selectively inhibit TbCatB without posing any danger to the human host represent a great therapeutic solution for treatment of HAT. To understand protein-inhibitor interactions, useful information can be obtained from high resolution protease-inhibitor crystal structure complexes. This study aims to use bioinformatics approaches to carry out comparative sequence, structural and functional analysis of TbCatB protease and its homologs from T. congolense, T, cruzi, T. vivax and H. sapien as well as to identify non-peptide small molecule inhibitors of TbCatB cysteine proteases from natural compounds of South African origin. Sequences of TbCatB (PDB ID: 3HHI) homologs were retrieved by a BLAST search. Human cathepsin B (PDB ID: 3CBJ) was selected from a list of templates for homology modelling found by HHpred. MODELLER version 9.10 program was used to generate a hundred models for T. congolense, T, cruzi and T. vivax cathepsin B like proteases using 3HHI and 3CBJ as templates. The best models were chosen based on their low DOPE Z scores before validation using MetaMQAPII, ANOLEA, PROCHECK and QMEAN6. The DOPE Z scores and the RMSD (RMS) values of the calculated models indicate that the models are of acceptable energy (stability) and fold (conformation). Results from the different MQAPs indicate the models are of acceptable quality and they can be used for docking studies. High throughput screening of SANCDB using AutoDock Vina revealed nine compounds, SANC00 478, 479, 480, 481, 482, 488, 489, 490 and 491, having a strong affinity for Trypanosoma spp. cathepsin B proteases than HsCatB. SANC00488 has the strongest binding to Trypanosoma spp. cathepsin B proteases and the weakest binding to HsCatB protease. Molecular dynamics (MD) simulations show that the complexes between SANC00488 and TbCatB, TcCatB, TcrCatB and TvCatB are stable and do not come apart during simulation. The complex between this compound and HsCatB however is unstable and comes apart during simulation. Residues that are important for the stability of SANC00488-TbCatB complex are Gly328 of the S2 subsite, Phe208, and Ala256. In conclusion SANC00488 is a good candidate for development of a drug against HAT.
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Souza-Rodrigues, Renata Duarte de. "Análise do efeito de substâncias liberadas por adesivos dentinários sobre a atividade e a expressão gênica de proteases da matriz extracelular (MMPs e CTs) em células-tronco da polpa dentária humana." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/23/23134/tde-18032015-173726/.
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Adesivos dentinários aplicados diretamente sobre dentina aumentam a atividade de enzimas endógenas deste tecido que degradam colágeno, colocando em risco a integridade da camada híbrida de restaurações estéticas. Estes adesivos podem também alcançar a polpa dentária indiretamente através do fluído dos túbulos dentinários por substâncias liberadas pelos mesmos. Desta forma, a polpa dentária poderia responder a estas substâncias por meio de síntese e/ou aumento da atividade de colagenases, o que poderia colaborar na degradação da camada híbrida. Sendo assim, o objetivo desse trabalho foi avaliar o efeito das substâncias liberadas por sistemas adesivos dos tipos autocondicionante e condicione e lave sobre a atividade e a expressão gênica de metaloproteinases (MMPs) e cisteíno-catepsinas (CTs) em células-tronco da polpa dentária humana. Foram aplicados meios de cultura condicionados por adesivos do tipo autocondicionante e condicione e lave polimerizados e não polimerizados sobre culturas celulares por 24 horas. O meio de cultivo fresco foi usado como controle. Depois de 24, 48, 72 e 96 horas, as atividades gelatinolíticas de MMP-2 e de MMP-9 foram avaliadas por meio da técnica de zimografia em gel de gelatina. Nos mesmos tempos experimentais, a modulação da expressão gênica das MMPs (1, 2, 3, 7, 9, 13 e 14) e das CTs (B e K) foi analisada por meio de reação de transcriptase reversa quantitativa em tempo real (qRT-PCR). Os resultados obtidos dos dois experimentos foram avaliados por meio do teste estatístico ANOVA, complementado pelo teste de Tukey (p<0.05). Todos os grupos mostraram atividade gelatinolítica aumentada de MMP-2 e MMP-9. Até 72 horas, as atividades foram similares em todos os grupos experimentais. Diferenças significativas apareceram somente em 96 horas. De forma geral, as maiores atividades de MMPs foram observadas nas culturas celulares tratadas com o adesivo autocondicionante. Para a MMP-2, o grupo do adesivo autocondicionante polimerizado mostrou atividade intermediária, enquanto o grupo não polimerizado mostrou a maior atividade. Os dois grupos do adesivo condicione e lave polimerizado e não polimerizado mostraram atividade de MMP-9 intermediária, enquanto o grupo autocondicionante polimerizado mostrou maior atividade que o grupo controle. O qRT-PCR revelou que a maioria das MMPs e CTs analisadas tiveram a expressão gênica positivamente modulada em 24 e 48 horas. MMP-7 e MMP-9 não foram expressos em nenhum grupo experimental. Baseados nas limitações deste estudo in vitro, concluímos que substâncias liberadas por sistemas adesivos são capazes de influenciar células-tronco de polpa dentária humana levando ao aumento da atividade de MMP-2 e MMP-9 e também à modulação positiva de genes das MMPs e CTS estudadas.Adhesive systems directly applied to dentin increase the activity of endogenous collagen degrading proteinases of the dentin, which jeopardizes the integrity of the hybrid layer of aesthetic restorations. These adhesives can also reach the dental pulp through the dentinal fluid indirectly by substances leached from them. Then, the dental pulp tissue could respond by synthetizing and/or increasing the activity of collagen proteases, which in turn could collaborate to the hybrid layer degradation. Then, the aim of this study was to evaluate the effect of substances leached from self-etch and etch-and-rinse adhesive systems on the expression and activities of matrix metalloproteinases (MMPs) and cysteine cathepsins (CT-B and CT-K) in human dental pulp stem cells. Culture media conditioned by polymerized or non-polymerized self-etch and etch-and-rinse adhesive systems were applied to the cultures for 24 hours. Fresh medium was used as control. After 24, 48, 72 and 96 hours, the gelatinolytic activities of MMP-2 and MMP-9 were assessed by zymography technique. At the same experimental time gene expression of MMPs (1, 2, 3, 7, 9, 13 e 14) and CTs (B e K) were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data was compared by ANOVA complemented by the Tukey´s test (p<0.05). All experimental groups showed increased gelatinolytic activity for MMP-2 and MMP-9. Until 72 hours, the activities were similar regardless the group. Significant differences appeared only after 96 hours. Overall, the highest activities of MMPs were observed in the cultures treated with the self-etch adhesive. For MMP-2, the group of polymerized self-etch adhesive showed intermediary activity, while the group of non-polymerized adhesive showed the highest activity. Both polymerized and non-polymerized etch-and-rinse adhesive groups showed intermediary MMP-9 activity, while the group of polymerized self-etch adhesive showed higher activity than control. The qRT-PCR revealed that most of MMPs and CTs analyzed presented the gene expression positively modulated at 24 and 48 hours. MMP-7 and MMP-9 were not expressed in any experimental group.Based on the limitations of this in vitro study, it was concluded that substances leached from adhesive systems are able to influence human dental pulp stem cells leading to the increase of the activity of MMP-2 and MMP-9 along with positive modulation of MMPs and CTS studied genes.
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Sudau, Daniela [Verfasser]. "Charakterisierung des Protease-aktivierten Rezeptors Typ 4 (PAR4) und Cathepsins G in der Ösophagusmukosa von Patienten mit gastroösophagealer Refluxerkrankung (GERD) und funktionellem Sodbrennen (FH) / Daniela Sudau." Magdeburg : Universitätsbibliothek, 2017. http://d-nb.info/1151571547/34.
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Vandooren, J., G. Opdenakker, Paul M. Loadman, and D. R. Edwards. "Proteases in cancer drug delivery." 2015. http://hdl.handle.net/10454/10005.
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NoWhereas protease inhibitors have been developed successfully against hypertension and viral infections, they have failed thus far as cancer drugs. With advances in cancer profiling we now better understand that the tumor “degradome” (i.e. the repertoire of proteases and their natural inhibitors and interaction partners) forms a complex network in which specific nodes determine the global outcome of manipulation of the protease web. However, knowing which proteases are active in the tumor micro-environment, we may tackle cancers with the use of Protease-Activated Prodrugs (PAPs). Here we exemplify this concept for metallo-, cysteine and serine proteases. PAPs not only exist as small molecular adducts, containing a cleavable substrate sequence and a latent prodrug, they are presently also manufactured as various types of nanoparticles. Although the emphasis of this review is on PAPs for treatment, it is clear that protease activatable probes and nanoparticles are also powerful tools for imaging purposes, including tumor diagnosis and staging, as well as visualization of tumor imaging during microsurgical resections.
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Tsao, Shin-Mei, and 曹士梅. "Molecular characterization of two mosquito cathepsin B-like proteases involved in vitellogenesis." Thesis, 1998. http://ndltd.ncl.edu.tw/handle/90463249683067407392.
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Gewies, Andreas [Verfasser]. "Investigation of the ubiquitin-specific protease UBP41 and of the lysosomal cysteine proteases cathepsin-L and cathepsin-B as potential mediators of proapoptotic signalling / submitted by Andreas Gewies." 2003. http://d-nb.info/970053924/34.
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"Expression, Purification, and Characterization of the Mast Cell Proteases Chymase and Cathepsin G." East Tennessee State University, 2008. http://etd-submit.etsu.edu/etd/theses/available/etd-0103108-102414/.
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Váchová, Jana. "Rekombinantní aspartátové proteasy krev sajících parazitů." Master's thesis, 2010. http://www.nusl.cz/ntk/nusl-285198.
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The blood fluke Schistosoma mansoni and the hard tick Ixodes ricinus produce an aspartic protease cathepsin D which initiates degradation of hemoglobin, their key nutrient. First, in the presented work, the protocol for refolding and activation of the zymogen of cathepsin D from I. ricinus (IrCatD) was developed and optimized. In acidic pH the propeptide of IrCatD zymogen was removed by an auto-activation mechanism. Further, a kinetic assay with fluorogenic substrates was employed to study functional properties of IrCatD including pH optimum, substrate and inhibition specificities. Second, two isoforms of cathepsin D from S. mansoni (SmCatD) were produced using recombinant expression in E. coli. These recombinant proteases were isolated from inclusion bodies using affinity chromatography under denaturating conditions, and protocol for their refolding was developed and optimized. The studied aspartic proteases are pharmacological targets: inhibitors of SmCatD represent potential chemotherapeutics for the treatment of schistosomiasis, and IrCatD is a candidate antigen for the development of novel anti-tick vaccines.
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Talacko, Pavel. "Katepsin L z klíštěte obecného: analýza proteolytické aktivity a její regulace." Master's thesis, 2012. http://www.nusl.cz/ntk/nusl-308293.
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The hard tick Ixodes ricinus is an important blood-feeding parasite that transmits tick- borne diseases, such as tick-borne encephalitis and Lyme disease. Ticks employ a battery of proteolytic enzymes, including cathepsins, to digest their bloodmeal. These proteins are potential targets for the development of anti-tick vaccines. This work is focused on cathepsin L from I. ricinus (IrCL), namely its isoenzymes IrCL1 and IrCL3. IrCL1 was expressed in Pichia pastoris and chromatographically purified. Its substrate specifity was determined by the cleavage of (a) peptide fluorogenic substrates and (b) protein substrates analyzed by mass spectrometry. The proteolytic activity of IrCL1 was modulated by its interaction with glycosaminoglycans, which affected the pH optimum value. Futhermore, a proteolytically active mutant of IrCL1 with reduced number of N-glycosylation sites was prepared; this form will be used for crystallization experiments. IrCL3 was expressed in Escherichia coli, refolded and activated to its active form. The proteolytic activity of IrCL3 is in many ascpects similar to that of IrCL1, including substrate specifity, acidic pH optimum and activity modulation by glycosaminoglycans. Key words: cysteine proteases, cathepsin L, hard tick I. ricinus, substrate specifity, proteolytic activity...
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KONVIČKOVÁ, Jitka. "Factors regulating the expression and activity of digestive enzymes in the tick \kur{Ixodes ricinus}." Master's thesis, 2015. http://www.nusl.cz/ntk/nusl-187610.
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The intracellular proteolysis of ingested meal plays an essential role in tick development. The thesis focuses on the factors influencing the expressions and activities of digestive enzymes in Ixodes ricinus females during the feeding and post-feeding period. We have revealed the effect of fertilization on blood feeding and digestion. The females cannot reach the rapid engorgement phase without being fertilized. The rate of mated females in the nature proved the presumption that mating can occur even off the host. Implementation of in vitro feeding technique further extended our current knowledge about tick digestive apparatus. Adult females were fed on hemoglobin-rich and hemoglobin-poor diet and the mRNA expression levels of digestive proteases were determined. In line with obtained data, we assumed that albuminolysis is conducted by the same or similar pathway as hemoglobinolysis. The gene silencing method and protein immuno-detection were used to unequivocally identify the isoforms of 'early expressed' IrCL1 and 'late expressed' IrCL3 isoform of cathepsin L.
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史德芬. "Investigation of Novel Cathepsin S Cysteine Protease inhibitors." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/06127062984467729475.
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McDowall, Jaclyn. "Protease distribution in J774 macrophages." Thesis, 2007. http://hdl.handle.net/10413/4188.
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Cathepsin, matrix metalloproteinase (MMP) enzyme and tissue inhibitor of MMP (TIMP) distribution in J774 mouse macrophages has not been comprehensively studied. The distribution and vesicle regulation, trafficking and release of these is important, possibly suggesting drug targets for the therapeutic regulation of inflammatory disease and phagosomal killing of pathogenic organisms in J774 and other macrophages. Percentage
immunofluorescence and ultrastructural enzyme and inhibitor distribution, together with LysoTracker (acidity) and lysosome-associated membrane proteins (LAMPs) colocalisation (both indicating late endosome or “lysosomal” association), western blot estimates of percentage processed- and unprocessed intracellular and secreted enzyme and inhibitor, and vesicle size was used to assign enzyme and inhibitor to “classical” vesicle types. Antibodies against TIMP-1 and TIMP-2 were raised and all antibodies characterised for this purpose.
Together these were used to assign cathepsins H, S, D, B and L to possible secretory vesicles (±20 nm, non-acidic, LAMPs-negative, containing precursor enzymes) and identify at least 6 other endosome-“lysosome-like” vesicles. Cathepsin H appears to be present in classical early endosomes (±100 nm, non-acidic, LAMPs-negative) and cathepsin S in late endosomes(±50 nm, acidic, LAMPs-positive) and possibly “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive). Both cathepsins H and S, however, seem only reliable markers if used together with additional markers. Cathepsin D appearsmainly associated with “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic,
LAMPs-positive), possibly consisting of further subpopulations which requires furtherinvestigation e.g. labelling for LAMP-1 and LAMP-2 and cathepsin D. Cathepsins B and Lmay occur in late endosomes and/or hybrid organelles and “secretory lysosomes” containing cathepsins B, D and L may also exist (±30-50 nm, acidic, LAMPs-positive).
The distribution of MMP-9, TIMP-1 and -2 in vesicles (non-acidic, LAMP-2-negative) thatappear novel and distinct from late endosome-“lysosome” vesicles were also demonstrated.
In LPS-stimulated cells, the identity of the large (±450 nm), possible recycling endosomes (Rab11-positive, LAMPs-negative), containing colocalised MMP-9 and TIMP-1, needs investigation i.e., requires further verification with triple labelling and EM. Possible cell membrane and recycling endosome localisation of TIMP-2 needs confirmation with labelling
of non-permeabilised cells and labelling for MT1-MMP and proMMP-2, respectively.Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.
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DeWitt, Christina A. Mireles. "Recovery and utilization of catheptic proteases from surimi wash water." Thesis, 2000. http://hdl.handle.net/1957/27537.
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