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1
Lourbakos, Afrodite 1972. "Activation of human protease-activated receptors by proteases from a periodontal pathogen." Monash University, Dept. of Biochemistry and Molecular Biology, 2001. http://arrow.monash.edu.au/hdl/1959.1/8876.
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2
Agrotis, Alexander George. "The regulation of human autophagy by ATG4 proteases." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10048926/.
Full textAbstract:
Autophagy is an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane organelles called autophagosomes. Lipidation of ubiquitin- like LC3/GABARAP proteins on the autophagosome membrane is essential for autophagy. The cysteine protease ATG4 executes C-terminal processing (priming) of newly-synthesised LC3/GABARAP to enable subsequent lipidation. ATG4 is also proposed to be important for deconjugating/delipidating LC3/GABARAP from autophagosome membranes, although the ex- act purpose of this is unclear in mammals. Four ATG4 isoforms (ATG4A-D) exist in mammals, however the functional redundancy of these proteins in cells is poorly understood. The aim of this thesis is to characterise the redundancy of human ATG4 proteins and investigate their deconjugation roles in cells. In Chapter 3, I show that human HAP1 and HeLa cells lacking ATG4B exhibit a severe but incomplete defect in LC3/GABARAP processing and autophagy. By further genetic depletion of ATG4 isoforms I uncover that ATG4A, ATG4C and ATGD all contribute to residual priming activity, which is sufficient to enable lipidation of GABARAPL1 on autophagosomes. In Chapter 4, I reveal that delipidation of LC3/GABARAP by ATG4 isoforms is not essential for autophagic degradation of the cargo receptor p62/SQSTM1, arguing that delip- idation by ATG4 has limited importance in mammalian autophagy compared to LC3/GABARAP priming. Finally, I report the discovery of a novel deconjugation function of ATG4 isoforms in the removal of LC3/GABARAP conjugates from endogenous proteins such as ATG3 and ATG7. Thus, I provide the first evidence that LC3/GABARAP can act as a protein modifier and that ATG4 can reverse such modifications, opening up a new avenue of future research to under- stand the functional relevance of this phenomenon.
3
Ranjit, Najju. "Characterisation of proteases involved in proteolytic degradation of haemoglobin in the human hookworm Necator americanus." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/20651/1/Najju_Ranjit_Thesis.pdf.
Full textAbstract:
With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1�Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.
4
Ranjit, Najju. "Characterisation of proteases involved in proteolytic degradation of haemoglobin in the human hookworm Necator americanus." Queensland University of Technology, 2008. http://eprints.qut.edu.au/20651/.
Full textAbstract:
With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.
5
Gonzalez, Santana Bibiana. "Cysteine proteases: potential serodiagnostic reagents for human Schistosomiasis and Fasciolosis." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110691.
Full textAbstract:
Schistosomiasis and fasciolosis are parasitic diseases that affect a great number of people, particularly in developing countries, and cause huge global morbidity. Diagnosis is essential for control, treatment and prognosis of the diseases and yet a simple, cheap, sensitive and specific assay is not readily available for either of them. In the current study, cathepsin B (SmCB) and cathepsin L1 (FhCL1) were investigated as potential diagnostic reagents to detect schistosomiasis and fasciolosis, respectively, in humans. The genes encoding SmCB and FhCL1 were expressed Pichia pastoris and the proteins isolated to homogeneity by affinity chromatography. The SmCB ELISA was optimized for antigen concentration, primary antibody dilution and secondary antibody dilution using a pool of sera obtained from patients that were coprologically-positive or negative for schistosomiasis. A clear distinctive was achieved between these two sera pools. However, when employed to screen a panel of patients from Senegal the test failed to provide satisfactory discrimination between schistosoma-infected and schistosoma-negative individuals. The FhCL1 ELISA was optimized using sera from Fasciola-infected individuals from Cuba, and samples from Cuban and Canadian non-infected patients. We determined the optimal dilution for the primary antibody and also assessed/compared the performance of anti-total IgG, IgG4, IgG1 and IgG2 secondary conjugated antibodies. Total IgG provided the best discrimination between Fasciola- infected and non-Fasciola infected individuals with a 99.99% sensitivity and specificity. Furthermore, by screening sera obtained from patients infected with various worm and protozoan diseases we showed that the FhCL1 ELISA does not cross-react with other diseases commonly found in similar geographical regions as fasciolosis. In conclusion, diagnosis of human schistosomiasis still remains uncertain and more studies need to be performed to improve our diagnostic test using SmCB. On the other hand, we have developed a simple, sensitive, specific and accurate test to detected human fasciolosis by using FhCL1, a major protease released by the parasite. The P. pastoris expression system allowed us to obtain up to 80 mg of FhCL1 enzyme per 4 L culture. Therefore, we not only have developed a standardized test that showed high specificity and sensitivity but we also have the methodology to obtain sufficent quantities of antigen needed future mass screening of human fasciolosis in affected regions.La schistosomiase et la fasciolose sont deux maladies parasitaires qui touchent un grand nombre de personnes, en particulier dans les pays en développement, causant une morbidité élevée. Le diagnostic est essentiel pour le contrôle, le traitement et le pronostic de ces maladies et pourtant aucun essai simple, abordable, sensible et spécifique n'est disponible à ce jour pour l'une d'entre elles. Dans le cadre de la présente étude, cathepsine B (SmCB) et cathepsine L1 (FhCL1) ont fait l'objet d'une investigation sur leur potentiel à être utiliser pour diagnostiquer la schistosomiase et fasciolose, respectivement, chez les humains. Dans la présente étude, les gènes encodant SmCB et FhCL1 ont été exprimés dans Pichia pastoris et les protéines isolées par chromatographie d'affinité. Le test ELISA pour SmCB a été optimisé pour une concentration en antigène et pour une dilution d'anticorps primaire et secondaire en utilisant un pool de sérums provenant de patients qui étaient positifs ou négatifs pour la schistosomiase suivant des examens coprologique. Une distinction claire entre ces deux bassins de sérums a été observée. Toutefois, lorsque le test a été utilisé pour dépister des patients du Sénégal, il a échoué à fournir une discrimination satisfaisante entre les individus infectés et non-infectés par la schistosomiase.Le test ELISA pour FhCL1 a été optimisé à l'aide de sérums provenant de personnes cubaines infectées par la fasciolose et de patients non-infectés de Cuba et du Canada. Nous avons déterminé la dilution optimale pour l'anticorps primaire et également évalué et comparé la performance des anticorps secondaires conjugués contre les IgG totaux, IgG4, IgG1 et IgG2. Les IgG totaux ont fourni la meilleure discrimination entre les personnes infectées et non-infectées par la fasciolose avec une sensibilité et une spécificité de 99.99 %. En plus, en appliquant le test de dépistage sur des patients infectés par d'autres variétés de vers et de protozoaires, nous avons démontré que le test ELISA pour FhCL1 ne réagit pas de façon croisée avec d'autres maladies retrouvées couramment dans les régions géographiques où se trouve la fasciolose.En conclusion, le diagnostic de la schistosomiase humaine reste encore incertain et d'autres études doivent être effectuées pour améliorer notre test de dépistage utilisant SmCB. D'autre part, nous avons développé un test simple, sensible, spécifique et précis pour le dépistage de la fasciolose humaine en utilisant FhCL1, une protéase majeure relâchée par le parasite. Le système d'expression de P. pastoris nous a permis d'obtenir jusqu'à 80 mg de FhCL1 par 4 litres de culture. Dans la présente étude, nous avons non seulement mis au point un test standardisé démontrant une spécificité et une sensibilité élevées, mais également développé une procédure pour produire de grandes quantités d'antigènes nécessaires au dépistage à grande échelle de la fasciolose humaine dans les régions touchées.
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Riddick, Antony C. P. "Expression profiling of proteases and related genes in human prostate tumours." Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398820.
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Gast, Alain. "Proteases et emphyseme pulmonaire : etude des inhibiteurs de proteases recueillis par lavage bronchoalveolaire." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13070.
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Wang, Bingjie. "Novel function of human beta-defensin 2 : protecting epidermal barrier against pathogenic proteases." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28756.
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Atopic Dermatitis (AD) is a common chronic relapsing inflammatory skin disease affecting 15 - 20% of children and 2 - 10% of adults worldwide, with significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics and infections. Another typical feature of AD is skin infections, especially from Staphylococcus aureus (S. aureus), which closely relates with the disease severity. Although not a normal flora, S. aureus is found on 75-100% of AD lesions (< 30% on healthy skin). S. aureus secrete a range of virulence factors, including extracellular toxins and proteases which contribute to disease pathogenesis. S. aureus serine protease A (SspA/V8) is a well-characterised extracellular protease widely expressed among different S. aureus strains. The pathogenic effect of V8 protease has been demonstrated in vivo, damaging murine skin integrity via effects on the stratum corneum (SC), but the targets for this V8-mediated damage remains unclear. The capacity of proteases to induce barrier dysfunction has been proposed as a key driving force in the initiation and exacerbation of AD. Thus, understanding the host factors that maintain barrier function is a priority in developing novel therapeutic approaches. This thesis therefore aimed at detecting host factors which can combat the barrier dysfunction caused by pathogenic proteases, assessing their relevance in vitro and ex vivo and elucidating the underlying mechanisms. Firstly, an in vitro skin barrier integrity model was developed, using both immortalized and primary keratinocytes, to evaluate the barrier damage mediated by pathogenic proteases. The results revealed that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment. In addition, studies demonstrated that V8 protease itself was sufficient to induce barrier disruption, and this phenotype was not dependent on cell death, but rather on breaking down of cell-cell junctions. Key tight junction proteins including claudin-1 and occludin were found to be degraded by V8 protease. Next, a wide range of host and bacterial factors were investigated to determine whether they could promote protection of keratinocytes against V8 damage. Several factors, including IL-1β, TNF-α, heat-killed Staphylococcus epidermidis (which is the main skin normal flora), were found to induce protection against V8 protease, with IL-1β having the strongest effect. In addition, data indicated that this IL-1β-mediated protection was independent of effects on claudin-1 but occurred via secretion of a transferrable host factor. Induction of keratinocyte expression of the antimicrobial/host defence peptide human beta-defensin 2 (hBD2) was found to be the mechanism underpinning this IL-1β- induced protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was also protective. An ex vivo model using human skin tissue was also established to address this novel function of hBD2, and preliminary data indicated that exogenous hBD2 protected against V8-mediated damage in this system. Overall, my data reveal a novel function for the antimicrobial/host defence peptide hBD2. This modulatory property of hBD2, independent of its antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential to prevent S. aureus-mediated aggravation of skin barrier dysfunction in AD.
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West, Andrew. "Investigations by mass spectrometry of the interactions of novel serine protease inhibitors with herpes simplex virus type 2 and human cytomegalovirus proteases." Thesis, University of Warwick, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343830.
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Mulloy, Rory. "Identification of Transmembrane and Extracellular Host Proteases that Promote Human CoV Entry and Syncytium Formation." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42673.
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LOMBARDI, ANNALIA. "Glucocorticoids and proteases in the pathophysiology of pregnancy and parturition." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1005638.
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Understanding the physiology of pregnancy and parturition enables effective management of pregnancy complications that could otherwise be life threatening for both mother and fetus. A large body of evidence suggests that glucocorticoids (GC) and matrix metalloproteinases (MMPs) play a key role in the pathophysiology of pregnancy and parturition. Glucocorticoids influence several aspects of embryo implantation process including the maternal immune response, embryo attachment and fetal development. Prolonged exposure to high levels of glucocorticoids in pregnancy is dangerous for both placental and fetal development indicating that tight control of GC levels at the fetal-maternal interface is essential for establishment and maintenance of a healthy pregnancy. GC exert their actions via the glucocorticoid receptor, an ubiquitously expressed nuclear receptor comprised of 9 exons. Previous studies have demonstrated that differential promoter usage and alternative splicing-generated GR mRNA transcripts result in differential cellular GC responses. Matrix metalloproteinases are involved in the extracellular matrix remodeling and they contribute to the success of a pregnancy from embryo implantation to labour and postpartum involution. A misregulation of their expression or their activity is observed in adverse outcomes of pregnancy such as preterm labour. The aim of this thesis was to investigate 1) the expression of GR transcripts and the effect of inflammatory cytokines on the control of GR transcripts expression in cultured first trimester human decidua cells; 2) the expression of the two major groups of MMPs (stromelysins and gelatinases) and their inhibitors TIMP-1 and TIMP-2 in a) the mouse uterus throughout normal gestation, at labour and during inflammation-induced preterm birth and b) the human myometrium at term and preterm, both non-labouring and labouring.
This study demonstrated that first trimester human decidua cells are characterized by a complex pattern of GR mRNA transcripts expression. Among the several GR isoforms expressed, GRβ, GRγ, GRP and GRα-D might be correlated with the development of the glucocorticoid resistance. Moreover, the significant increase in GR isoforms mRNA expression and protein levels observed in IL-1β or TNF-α- treated FT-DCs suggest a role for inflammatory cytokines in regulating decidual GR isoforms abundance.
Furthermore, this study demonstrated that the majority of MMPs (Mmp-3, Mmp-2, Mmp-9, Mmp-10) studied in the mouse uterus displayed higher expression in early gestation compared to late gestation and labour; only Mmp-10 exhibited increased expression at labour suggesting a role in
parturition. In addition, Mmp-3 and Timp-1 increased in the uterus of mice destined to undergo premature labour due to LPS-induced intrauterine inflammation, in contrast to Timp-2, which decreases. These observations may suggest a role of MMP-3 and TIMP-1 in preterm labor. Finally, this work reported a decreased expression of MMP-10 in the human myometrium with the onset of term, but notably not preterm labour. On the other hand, human TIMP-1 is dramatically increased with the onset of term and preterm labour.
In conclusion, this study provide key novel findings in the pathophysiology of pregnancy and parturition highlighting 1) the importance of GCs and their receptors GRs in the establishing of pregnancy and that the identification, characterization and regulation of the several GR isoforms involved in the modulation of glucocorticoid action may lead to potential strategies for the treatment or prevention of a variety of pregnancy-related disorders; 2) the important involvement of MMPs and TIMPs in uterine remodeling during pregnancy and labour and that the balance between MMP and TIMP expression may be critical to controlling the timing of normal labour, alterations in MMP/TIMP ratio lead to multiple pregnancy complications such as preterm birth.
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Zhu, Huijun. "Involvement of different proteases in the execution and regulation of apoptosis in human monocytic THP.1 cells." Thesis, University of Leicester, 1997. http://hdl.handle.net/2381/30787.
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Ced-3, a cysteine protease, is a key effector of apoptotic cell death in Caenorhabditis elegans. However the role of the Ced-3 mammalian homologues, caspases, and other classes of proteases in apoptosis has not been well understood. This study investigated the involvement of proteases in apoptosis induced by different mechanisms using human monocytic THP.1 cells as a model. Apoptosis, as assessed by morphological and biochemical changes, including nuclear condensation and fragmentation, internucleosomal DNA cleavage and poly-(ADP-ribose) polymerase (PARP) degradation, was induced by cycloheximide (25 M), thapsigargin (100 nM), etoposide (25 M) and staurosporine (0.5 M). The induction of apoptosis by all these stimuli was enhanced by N-tosyl-L-lysinyl chloromethyl ketone (TLCK) (100 M), a trypsin-like protease inhibitor, except for etoposide, where apoptosis was inhibited. Staurosporine also induced necrotic cell death, which was prevented by TLCK. N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) (50-75 M), a chymotrypsin-like protease inhibitor, induced all the characteristic apoptotic changes except the fully nuclear condensation and fragmentation, although it inhibited internucleosomal DNA cleavage induced by other stimuli. Caspase-2, caspase-3, caspase-6, and caspase-7 were processed/activated during the induction of apoptosis. Caspase inhibitors either with low sensitivity or with higher selectivity for caspase-3 both proved to be potent in the inhibition of apoptosis. However staurosporine-induced necrosis was resistant to the inhibition of a caspase inhibitor. This study demonstrated that apoptosis can be induced or modified by different protease inhibitors in a single cell line, implying tat proteases are involved in the regulation of apoptosis at multiple stages. TLCK and TPCK inhibitable proteases may control "upstream" events, whereas caspases have a fundamental role in the execution of apoptosis. This study also provides evidence that a apoptosis and necrosis involve different protease activities.
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Kennett, Craig Nader. "Comparative histochemical, immunocytochemical and biochemical studies of proteases and their inhibitors in human gingival tissue and crevicular fluid." Thesis, King's College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336540.
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Vyas, Ishan. "IDENTIFICATION OF PEPTIDASES IN HIGHLY-PATHOGENIC VERSUS WEAKLY-PATHOGENIC NAEGLERIA FOWLERI AMEBAE." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3524.
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Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly-pathogenic mouse-passaged amebae (Mp) and weakly-pathogenic axenically-grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium from Mp and Ax amebae were the presence of an activity band of approximately 58kDa and 100 kDa bands susceptible to the action of cysteine and metallopeptidase inhibitors, respectively. Further roles of the peptidases during the invasion process were examined by in vitro invasion assays in the presence of inhibitors and Cysteine and metallopeptidase inhibitors were found to greatly reduce invasion through the ECM. This study establishes a functional linkage of the expressed peptidases to the invasion process, and these peptidases may serve as a candidate target for therapeutic management of N. fowleri infection.
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Menou, Awen. "Implication des protéases à sérine de la famille des Type II Transmembrane Serine Proteases dans la Fibrose Pulmonaire Idiopathique." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC020/document.
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La Fibrose Pulmonaire Idiopathique (FPI) est une pathologie pulmonaire chronique, progressive, irréversible et mortelle, dont les thérapeutiques sont insuffisantes à ce jour. L'activation de la cascade de la coagulation et des protéases à sérine, délétère dans la progression des maladies pulmonaires chroniques, est une caractéristique de la pathologie. Récemment, un lien a été démontré entre Protease-Activated Receptor-2 (PAR-2), un récepteur cellulaire ubiquitaire, et la progression de la fibrose pulmonaire chez l'homme et la souris. Outre certains facteurs de la coagulation, PAR-2 semble aussi pouvoir être activé par des protéases appartenant à la famille récemment identifiée des Type II Transmembrane Serine Proteases (TTSPs), dont la matriptase et la Human Airway Trypsin-like protease (HAT). Leur rôle dans la fibrogénèse pulmonaire humaine et expérimentale est cependant encore inconnu.Nos travaux montrent pour la première fois qu'il existe une dérégulation de l'expression et de l'activité de ces protéases de la famille des TTSPs chez le patient FPI. In vitro, la matriptase induit des réponses pro-fibrosantes dans les fibroblastes pulmonaires primaires humains via l'activation de PAR-2, tandis que la HAT induit des réponses anti-fibrosantes dans ces cellules et une activation de la voie de la prostaglandine E2. Ces deux TTSPs sont ainsi différemment impliquées dans la fibrogénèse pulmonaire : in vivo, l'inhibition génétique et pharmacologique de la matriptase atténue la fibrose dans le modèle murin de fibrose pulmonaire induite par la bléomycine, et des résultats similaires sont observés suite à la surexpression de la HAT médiée par adénovirus dans ce modèle animal. L'ensemble des résultats obtenus dans ce travail de thèse permet de documenter l'implication de deux protéases à sérine, la matriptase et la HAT, dans la pathogénèse de la FPI et de définir des axes de recherche thérapeutique potentielsIdiopathie Pulmonary Fibrosis (IPF) is a chronic, progressive, irreversible and mortal disease. Therapeutics options that improve the clinical outcome of IPF are limited. Coagulation proteinases and coagulation signaling deregulation, which influences several key inflammatory and fibroproliferative responses, is essential in IPF. Recently, Protease-Activated Receptor-2 (PAR-2) was shown to be involved in pulmonary fibrogenesis, both in Human and in mice. In addition to coagulation factors, PAR-2 can be activated by serine proteases of the emerging Type II Transmembrane Serine Proteases (TTSPs) family, including matriptase and the Human Airway Trypsin-like protease (HAT). Herein we explored the role of matriptase and HAT in the progression of human and experimental pulmonary fibrosis.Our data show that TTSPs matriptase and HAT pulmonary expression and activity are deregulated in patients with IPF. In vitro, matriptase induces PAR-2 dependent pro-fibrotic responses in primary human lung fibroblasts, whereas HAT induces anti-fibrotic effects in these cells, through the activation of prostaglandin E2 pathway. These TTSPs are differently involved in pulmonary fibrogenesis: in vivo, genetic and pharmacological inhibition of matriptase reduces fibrosis in the bleomycin induced lung fibrosis model, while an adenovirus-mediated HAT overexpression in the murine model leads also to a limited lung fibrosis. Here, we highlight the involvement of matriptase and HAT in the pathogenesis of IPF and explore potential therapeutics for lung fibrosis
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Bayraktar, Eda. "Effects Of Ph On Human Growth Hormone Production By Pichia Pastoris Considering The Expression Levels Of Regulatory Genes." Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12610882/index.pdf.
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In this study, the aim was to investigate the effects of pH on therapeutically important protein, recombinant human growth hormone (rhGH), production by Pichia pastoris considering the expression levels of regulatory genes. In this frame, firstly the host microorganism was selected between two different methanol utilization phenotypes of P. pastoris, Mut+ and MutS on media containing glycerol/methanol or sorbitol/methanol. The highest rhGH production, 120 g L-1, and hGH gene expression, 9.84x109 copies mg-1 CDW, were achieved in the medium containing 30 g L-1 sorbitol and 1% (v/v) methanol by P. pastoris hGH-Mut+ strain. Thereafter, effects of pH on rhGH production and stability were investigated in laboratory scale bioreactors. RhGH was more stable at pH 5.0. Throughout the production, it is seen that medium of pH decreased.
Thereafter, effects of pH on rhGH were investigated in pH controlled pilot-scale bioreactor. In addition to rhGH concentration, AOX intracellular enzyme activity, extracellular proteases concentrationsexpression levels of hGH, AOX, pep4, prb1 and prc1 genes were determined. The highest cell concentration was obtained as 53 g L-1 at pH 6.0 but hGH concentration was found as 24 mg L-1 at t=24 h. The highest rhGH concentration was obtained as 271 g L-1 with 42 g L-1 cell density at pH 5.0 in medium containing sorbitol at t=24 h. At this condition, the overall product and cell yield on total substrate were found as 2.08 mg g-1 and 0.15 g g-1. Furthermore, the highest expression levels of hGH and AOX were attained at pH 5.0. Moreover, by keeping pH at 5.0, expression levels of three types of vacuolar proteases were minimized.
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Peterle, Daniele. "Molecular Mechanism in the Alteration of Hemostasis." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3426350.
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Hemostasis is a finely tuned physiological process that, through the concerted action of several blood cells and proteins, maintains the integrity of the vascular system. This stepwise process begins after a vessel wall injury and includes: an initial vasospasm, a platelet plug formation (primary hemostasis), an assembly and activation of the coagulation factors that results in fibrin deposition at the site of injury (secondary hemostasis), and a final dissolution of the fibrin clot that restores the blood vessel patency (fibrinolysis) (Chapter 1). Alterations affecting one or more of these delicate processes lead to a large number of pathological manifestations, commonly referred to as cardiovascular diseases (CVD). Nowadays, CVD are the major cause of mortality and morbidity worldwide. Despite the social and economic burden of CVD, the currently available pharmaceutical repertoire is relatively limited to a few classes of molecules (heparins, platelet antiaggregants, vitamin-K antagonists, direct thrombin inhibitors) which, however, display important side effects and need to be employed with careful dose adjustments. These difficulties stem primarily from: i) the intrinsically complex nature of the procoagulant and anticoagulant biochemical mechanisms leading to physiological hemostasis, which renders external intervention very risky and unpredictable; ii) the inadequate knowledge of the biochemical mechanisms linking blood coagulation to other vital physio-pathological processes.
The general aim of this Ph.D. project was to investigate some of the molecular mechanisms underlying hemostatic disorders. To address this relevant question, we proceeded by studying selected pathologies for which association with hemostatic complications has either been long-established (i.e., Antiphospholipid Syndrome (APS), infectious diseases) or has just been hypothesized (Parkinson’s disease (PD), Transthyretin-related Amyloidosis (ATTR)), focusing our attention on the physio-pathological proteins involved in the onset of these disorders. In a first stage, our attention was focused on the study of novel interactions between α-thrombin (αT), the key enzyme of the coagulation cascade, with other plasma proteins (i.e., β2-glycoprotein-I, α-synuclein). In a second stage, we investigated an alternative mechanism of activation of prothrombin, the precursor of αT, by a bacterial protease (subtilisin from B. subtilis). Finally, some selected proteases were tested against human transthyretin, whose proteolyzed form is a key factor in the onset of ATTR.
In its traditional pathway, blood coagulation culminates with the FXa-mediated conversion of prothrombin zymogen into active αT, through the formation of the prothrombinase complex on the platelet surface. Mature αT is a 36.7 kDa serine protease with a chymotrypsin-like fold. αT plays a pivotal role in blood coagulation, being able to exert both procoagulant (platelets aggregation, fibrin generation) and anticoagulant (protein C activation) functions. The equilibrium between such different activities is regulated by the interaction of αT with other proteins through its active site and two positively charged regions, called exosites (exosite I and exosite II), which flank the catalytic cleft. In addition, αT is a multifunctional protease that, beyond blood coagulation, plays important roles also in other physiological processes such as inflammation, innate immune system, and nervous systems.
In Chapter 2 we mapped the interaction between αT and β2-Glycoprotein I (β2GpI). β2GpI is a heavily glycosylated 45 kDa protein that resides in human plasma at a physiological concentration of 4 µM (0.25 mg/ml). Since the early 90's, β2GpI has been identified as the major autoantigen in the antiphospholipid syndrome (APS), a severe autoimmune disease clinically characterized by hemostatic alterations such as venous and arterial thrombosis, fetal loss and thrombocytopenia. Despite its involvement in the pathogenesis of APS, the physiological roles of β2GpI remain unclear and both pro- and anti-coagulant functions have been reported for this protein. In a recent work, we have shown that β2GpI selectively inhibits the procoagulant functions of human α-thrombin (i.e. prolongs fibrin clotting time, tc, and inhibits α-thrombin-induced platelets aggregation) without affecting the unique anticoagulant activity of the protease (i.e. the proteolytic generation of the anticoagulant protein C). Here, combining molecular modeling with biochemical/biophysical techniques, we provided a coherent structural model of αT-β2GpI complex. The model has allowed us to understand at the molecular level our previous in vitro results. In particular, our findings suggested that β2GpI may function as an anticoagulant protein, acting as a scavenger of αT for the binding to GpIbα receptor, thus impairing platelets aggregation while enabling normal cleavage of fibrinogen and protein C.
Chapter 3 was dedicated to the role of bacterial proteases in inducing blood coagulation by direct proteolytic activation of prothrombin. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies, and, in about 35% of sepsis cases, by disseminated intravascular coagulopathies (DIC). Here, we show that addition of subtilisin (50 nM–2 µM), a serine protease secreted by the nonpathogenic bacterium Bacillus subtilis, to human plasma induces clotting by proteolytically converting prothrombin into active σPre2, a nicked Pre2 derivative with a single cleaved Ala470–Asn471 bond. Notably, we found that this non-canonical cleavage at Ala470–Asn471 is instrumental for the onset of catalytic activity in σPre2, which was however reduced of about 100-200 fold compared with natural αT. Of note, σPre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of prothrombin by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.
The study object presented in Chapter 4 is the interplay between αT and α-synuclein (αSyn). αSyn is a small (14.6 kDa) presynaptic protein mainly synthesized in the brain and whose aggregation has been shown to trigger the onset of different neurodegenerative diseases, commonly referred to as synucleinopathies (i.e., Parkinson disease). As for β2GpI, the exact physiological role of αSyn is still elusive. Intriguingly, αSyn is also synthesized by platelets and was found to inhibit the Ca2+-dependent release of procoagulant α-granules after αT stimulation. Moreover, clinical evidences clearly indicate that patients affected by neurodegenerative disorders have lower risks of ischemic attack. The collateral effects of αSyn in the pathogenesis and its localization on platelet surfaces prompted us to investigate a possible role of it in the hemostatic system. Here, we studied the effects of αSyn on fibrin generation and platelet activation. Furthermore, we mapped the interaction sites on αSyn and αT. Briefly, our results indicate that the negatively charged C-terminal tail of αSyn binds to the electropositive exosite-2 of thrombin, thus impairing αT-mediated platelet activation in whole blood. At variance, αSyn does not alter the rate of fibrin generation, resulting only in a minor change in the ensuing fibrin structure.
In Chapter 5 we attempted to correlate the onset of systemic transthyretin amyloidosis to an altered activation of blood coagulation. Human transthyretin (hTTR) is an abundant homo-tetrameric plasma protein (0.2 mg/ml) involved in the transport of thyroxine and retinol through the binding to retinol binding protein. Beyond its physiological roles, hTTR is known as an amyloidogenic protein whose aggregation is responsible for several amyloid diseases, including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC). From a mechanistic point of view, the proteolytic cleavage of hTTR represents an important step in fibril formation. In particular, after cleavage around position 50, hTTR C-terminal fragments have been found to aggregate far more efficiently than the full-length hTTR. Nowadays, the protease(s) responsible for this cleavage is yet to be identified although it is predicted to be a serine protease with a trypsin-like fold. Since all coagulation factors are trypsin-like serine proteases, we decided to probe them for the proteolytic cleavage of hTTR. In addition, we also probed some selected bacterial proteases, as well as some digestive apparatus and immune system proteases. hTTR was resistant to all proteases tested except to subtilisin from B. subtilis, which was able to cleave hTTR at pH 7.4, generating in high yields the amyloidogenic fragment hTTR(59-127). Since the hTTR(59-127) fragment was identified in amyloid deposits, these new insights might have relevant implications in hTTR-based amyloidosis.
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Donnelly, P. K. "Protease and human immunity." Thesis, University of Newcastle Upon Tyne, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371254.
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Sanchez, Eduardo Milton Ramos. "Avaliação do modelo de hamster para detecção das alterações lipídicas e cardiotoxicidade associadas à terapia contra o vírus da imunodeficiência humana." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-25032010-153939/.
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Com a introdução de uma nova classe de antiretrovirais integrantes da terapia anti-retroviral altamente ativa (HAART) para o tratamento das infecções pelo vírus da imunodeficiência humana, começaram a ser descritos inúmeros efeitos secundários.Na tentativa de se estabelecer um modelo animal para o estudo destes efeitos buscou-se uma espécie com similaridade no perfil e metabolismo lipídico. Iniciou-se estudo em Mesocricetus auratus. Foram avaliados o perfil lipídico e glicêmico,função hepática e renal, níveis de auto-anticorpos anti ox-LDL, perfil eletrocardiográfico, alterações histopatológicas renais e cardíacas nos animais sob dieta hiperlipídica e normal,tratados com Indinavir, inibidor de protease utilizado na HAART. Observou-se uma diminuição da sobrevida nos animais tratados com indinavir, aumento do nível sérico de triglicérides e glicose, redução de auto-anticorpos anti ox-LDL,aumento do segmento QRS no eletrocardiograma, presença de fibrose renal e cardíaca, hipercelularidade glomerular nos animais tratados com a droga com ou sem dieta hiperlipídica quando comparados com os controles. Concluimos que Mesocricetus auratus se apresenta como um bom modelo para o desvendamento dos mecanismos patológicos observados na HAART.With the introduction of a new antiretroviral class use, integrants of highly active anti-retroviral therapy (HAART) for the treatment of infections by human immunodeficiency virus, several side effects started to be described.To establish an animal model for the study of these side effects, was chosen specie that have similarities in the lipidic profile and metabolism. A study in Mesocricetus auratus was started. It was evaluated the lipidic and glicemic profile ,hepatic and renal function, the levels of auto-antibodies against ox-LDL, electrocardiographic profile and renal and cardiac histopathological alterations in these animals under hyperlipidic and normal diets,treated with Indinavir, a protease inhibitor used in HAART.It was observed a decrease in the survival rate in the animals treated with Indinavir; an increase of the triglycerides and glucose serum level; reduction of anti ox-LDL auto-antibodies; increased QRS segment in the electrocardiogram; presence of renal and cardiac fibrosis; glomerular hypercellularity in the animals treated with the drug, with or without hyperlipidic diet when compared with the controls. We conclude that the Mesocricetus auratus is a good model for disclosure of the pathological mechanisms generated by HAART.
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Cunha, Joel da. "Estudo da atividade e polimorfismos da Paraoxonase-1 em indivíduos infectados pelo vírus da imunodeficiência humana tipo-1 (HIV-1) tratados com inibidores de protease." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-10052013-095130/.
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A enzima Paraoxonase-1 (PON1) possui atividades paraoxonase, arilestearase e lactonase, entre outras. É a mais estuda da família das PONs que é composta pela PON1, PON2 e PON3. Sugere-se, que todas atuam inibindo o processo de peroxidação lipídica de moléculas como a lipoproteína de baixa densidade (LDL) e alta densidade (HDL), caracterizando assim um possível papel anti-aterogênico. O gene da PON1 apresenta dois sítios polimórficos, com a troca de uma Gln192Arg (Q/R) e Met55Leu, que estão associados com diferenças na atividade e concentrações séricas da enzima. Por sua vez, indivíduos soropositivos para o HIV-1 apresentam alterações do metabolismo lipídico, que poderiam estar associados a alterações na atividade da PON1 e a terapia antirretroviral (TARV) com inibidores de protease (IP). O objetivo do estudo foi determinar as atividades séricas da PON1 e da arilestearase (ARE), e as freqüências alélicas dos polimorfismos genéticos da PON1 192QR e 55LM, e ainda, avaliar a correlação destes parâmetros com as alterações lipídicas em indivíduos soropositivos para o HIV-1 tratados com IP. No período de Setembro de 2009 até Junho de 2012, 174 indivíduos soropositivos e 46 soronegativos para o HIV-1 foram estudados. Foi realizada a genotipagem dos polimorfismos da PON1 192QR e 55LM através de PCR-RFLP. A atividade sérica da PON1/ARE foi avaliada por espectrofotometria empregando-se como substratos o paraoxon e o fenilacetato, respectivamente. O RNA-HIV-1 foi quantificado pelo método NASBA, e os linfócitos T-CD4+ e T-CD8+ por citometria de fluxo. Os níveis séricos de colesterol total, HDL, LDL, triglicérides (TG), ApoA1 e ApoB100 foram determinados e os anticorpos IgG anti-oxLDL por ELISA. A atividade sérica da PON1 foi inferior nos grupos de soropositivos, p<0,05, porém, a atividade ARE não apresentou diferenças entre os grupos estudados, p>0,05. Ambas as atividades não apresentaram relação com os genótipos PON1 192QR e 55LM, e estes genótipos apresentaram uma freqüência alélica semelhante ao grupo de soronegativos. Os níveis séricos de TG foram superiores nos grupos de soropositivos com TARV, p<0,05, enquanto o grupo tratado com IP apresentou níveis séricos de HDL e Apo-A1 inferiores aos demais grupos, p<0,05. Níveis séricos de Apo-B100, IgG anti-oxLDL, e o índice de risco aterogênico foram superiores no grupo tratado com IP, p<0,05. Concluí-se, que indivíduos soropositivos para o HIV-1 apresentaram alterações no metabolismo lipídico, principalmente nos tratados com IP, que adicionalmente apresentaram um maior índice de risco aterogênico e maiores níveis de anticorpos IgG anti-oxLDL. Estas alterações não apresentaram relação com os polimorfismos PON1 192QR e 55LM da PON1, e demonstraram que a atividade da enzima PON-1 esta diminuída em indivíduos soropositivos para o HIV-1The enzyme Paraoxonase-1 (PON1) has paraoxonase (PON), arylesterase (ARE) and lactonase activities, among others. It is the most studied member of PON family which is composed of PON1, PON2 and PON3. It is suggested that all members acts by inhibiting the peroxidation of lipid molecules as the low-density lipoprotein (LDL) and high-density lipoprotein (HDL), characterizing a potential anti-atherogenic effect. The PON1 gene has two mainly polymorphic sites, with an exchange of Gln192Arg (Q/R) and Met55Leu (L/M), which are associated with differences in activity and serum concentrations of the enzyme. In turn, seropositive individuals for HIV-1 show changes in lipid metabolism, which could be associated with changes in the activity of PON1 and highly active antiretroviral therapy (HAART) with protease inhibitors (PI). The aim of this study was to determinate the serum PON and ARE activities of PON1, the allele frequencies of PON1 192QR and PON1 55LM genetic polymorphisms and evaluate the correlation between these parameters and lipid abnormalities in seropositive patients for HIV-1 treated with IP. In the period from September 2009 until June 2012, 174 seropositive individuals and 46 soronegative individuals for HIV-1 were studied. We performed PON1 192QR and 55LM genotyping by PCR-RFLP. Serum activities PON and ARE of PON1 were evaluated by spectrophotometry using paraoxon and phenylacetate, respectively, as substrates. The HIV-1-RNA was quantified by the NASBA method, and lymphocytes T-CD4+ and T-CD8+, by flow cytometry. Serum levels of total cholesterol, HDL, LDL, triglycerides (TG), apoA1 and ApoB100 were determined. IgG anti-oxLDL antibodies were quantified by ELISA. The serum PON1 activity was lower in the seropositive group, p<0.05, however, ARE activity did not differ between groups, p>0.05. Both activities had no relation with the PON1 192QR and PON1 55LM genotype, and these individuals showed an allele frequency similar to the seronegative group. Serum levels of TG were higher in groups of HIV-positive with HAART, p<0.05, while the IP-treated group showed serum levels of HDL and ApoA1 lower than other groups, p <0.05. Serum levels of ApoB100, IgG anti-oxLDL antibodies, and atherogenic risk indices were higher in the group treated with PI, p<0.05. It was concluded that individuals HIV-1-infected showed changes in lipid metabolism, especially in those treated with IP, which additionally showed a higher rate of atherogenic risk and higher levels of IgG anti-oxLDL antibodies. These changes did not correlated with PON1 192QR and 55LM polymorphisms and demonstrated that the activity of PON1enzyme is decreased in individuals seropositive for HIV-1
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DI, FEDE MARTINA. "Dissecting the role of Neisseria Heparin Binding Antigen cleavage during adaptation of Neisseria meningitidis to mucosal surface." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1009815.
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Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and is one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, including lactoferrin and kallikrein, cleave NHBA protein upstream or downstream a conserved Arg-rich motif, respectively. The cleavage results in the release of the C-terminal portion of the protein. C-terminal fragment originating from the processing of meningococcal proteases, referred as C2 fragment, exerts a toxic effect on endothelial cells altering their permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistently with previous findings showing that N. meningitidis traverses the epithelial barrier without disrupting the junctional structures. Unexpectedly, epithelial cells constantly secreted proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich motif. This cleavage might inactivate the toxic effect of C2 fragment by eliminating its docking domain. Epithelial cell proteases processed also the NHBA full-length protein, and we demonstrated it on live bacteria. Moreover, looking for the epithelial cell protease responsible for this processing, we identified the C3-convertase of alternative complement pathway as a novel human protease able to cleave NHBA during meningococcal infection. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. This cleavage occurs at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with.
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Murray, Ewan Hector. "Investigating structure and function relationships in human Stefin A." Thesis, University of Sheffield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274964.
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Boffito, Marta. "Clinical pharmacology of human immunodeficiency virus-1 protease inhibitors." Thesis, University of Liverpool, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403237.
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Boucaut, Kerry Jane. "Function and regulation of the human serine protease Testisin." Thesis, Queensland University of Technology, 2002.
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Fraga, Tatiana Rodrigues. "Identificação de proteases de Leptospira envolvidas com mecanismos de escape do sistema complemento humano." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-27112014-094144/.
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A leptospirose é uma zoonose causada por leptospiras patogênicas. Para estabelecer a infecção, estas bactérias desenvolveram estratégias de escape ao sistema complemento. Neste trabalho demonstramos que o sobrenadante de cultura de leptospiras patogênicas é capaz de inibir as três vias do complemento. Observamos que esse sobrenadante possui atividade proteolítica sobre C3, C3b e iC3b, além do FB (via alternativa), C2 e C4b (via clássica e das lectinas). As proteínas C3, C4, C2 e FB também foram clivadas quando soro humano normal (SHN) foi utilizado como fonte de complemento. Demonstramos que as proteases atuam em conjunto com os reguladores do hospedeiro Fator I e Fator H na clivagem de C3b. As clivagens foram inibidas pela 1,10-fenantrolina, sugerindo a participação de metaloproteases. Metaloproteases de leptospira da família das termolisinas foram produzidas como proteínas recombinantes e clivaram C3 no SHN. Concluímos que proteases de leptospiras patogênicas podem desativar moléculas do complemento e são potencias alvos para novas terapias em leptospirose.Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. To establish the infection, these bacteria have developed strategies to escape the complement system. In this work, we demonstrate that culture supernatant from pathogenic Leptospira is capable of inhibiting the three complement pathways. We observe that this supernatant possess proteolytic activity under C3, C3b and iC3b, FB (alternative pathway), C2 and C4b (classical and lectin pathways). The proteins C3, C4, C2 and FB were also cleaved when normal human serum (NHS) was used as a source of complement. We demonstrate that these proteases act together with the host regulators Factor I and Factor H in C3b cleavage. The cleavages were inhibited by 1,10-phenanthroline, suggesting the involvement of metalloproteinases. Leptospira metalloproteinases from the thermolysin family were produced as recombinant proteins and cleaved C3 in NHS. We concluded that proteases from pathogenic Leptospira can inactivate complement molecules and are potential targets for new therapies in leptospirosis.
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Fletcher, Jean Margaret. "Proteolytic mechanisms involved in the metastasis of human melanoma cells." Master's thesis, University of Cape Town, 1994. http://hdl.handle.net/11427/27116.
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The metastatic process requires that tumour cells are capable of traversing various micro-environmental barriers, such as the basement membrane. There are various proteolytic mechanisms which could contribute to the process, plasminogen activation by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is one such mechanism. Extensive reports in the literature (reviewed in the introduction) indicate that most tumour cells synthesize uPA and that it is this enzyme, particularly when receptor-bound, which plays a role in invasion. UCT-Mel 3 is a human malignant melanoma cell line which was established in our laboratory, and has been shown to be highly metastatic in the nude mouse. This cell line is typical of many melanomas in that it synthesizes only tPA and not uPA. In part 1 of this thesis I further investigated the plasminogen activator production by these cells (at the level of mRNA as well as activity) as well as expression of plasminogen activator inhibitor PAl-1 and receptors for tPA and uPA (uPAR). UCT-Mel 3 cells expressed uPAR although uPA was not detected. I also examined cells cultured from two metastatic deposits. Interestingly, the metastatic cells produced PAl-1 which was undetected in the parent cells. After confirming that UCT-Mel 3 do not express detectable levels of uPA, I attempted (in part 2) to determine whether tPA could play a comparable role to that of uPA in the invasive process. My strategy was to inhibit the expression of tPA via two different methods, namely the use of antisense RNA and ribozyme. I then hoped to isolate clones producing no tPA, which would have been injected into nude mice in order to assay for metastasis. Unfortunately, neither of these methods proved to be successful in abrogating tPA expression. I was thus unable to achieve the ultimate aim of the project. However, during the course of the study a number of unforeseen problems arose. Firstly, the clonal variation within the cell population, and secondly, my inability to obtain antisense transfectants. I have speculated that a possible reason for the latter may be that the cells are in fact unable to grow in the absence of tPA.
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Bosnic, Anthony Martin. "Human antibody responses to a chlamydia-secreted protease factor : a thesis /." San Antonio : UTHSC, 2005. http://learningobjects.library.uthscsa.edu/cdm4/item%5Fviewer.php?CISOROOT=/theses&CISOPTR=14&REC=5.
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Winston, Alan. "The clinical implications of human immunodeficiency virus-1 protease inhibitor pharmacokinetics." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444601.
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Guðmundsdóttir, Ingibjörg Jóna. "The role of protease-activated receptor-1 in the human vasculature." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/29130.
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Background: Thrombin is a powerful cardiovascular agonist and a vital link between thrombosis and inflammation. In addition to its role in the coagulation cascade, it directly activates platelets, inflammatory cells, endothelium and vascular smooth muscle. Proteaseactivated receptor-1 (PAR-1) has been proposed as the principal thrombin receptor in man although its actions in vivo have not been defined. The aim of this thesis was to determine the direct vascular actions of PAR-1 agonism in the human venous and arterial circulations. Objectives: The effects of PAR-1 activation on dorsal hand vein diameter were measured by the Aellig technique in healthy volunteers, compared with activation of the trypsin receptor PAR-2, and further assessed in the presence or absence of norepinephrine, the glycoprotein (GP)IIb/IIIa antagonist tirofiban, and endothelial denudation. In the arterial circulation, forearm blood flow was measured by venous occlusion plethysmography. Intra-arterial PAR-1 activating peptide was co-infused with tirofiban, and compared with PAR-2 activation and bradykinin infusion. Platelet-monocyte binding (a sensitive measure of platelet activation) and tissue plasminogen activator release (t-PA) were measured throughout. In subsequent studies, effects of inhibition of the endothelium-dependent vasodilators nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarisation factor (EDHF) on PAR-1 activation were assessed, as well as comparing the effects of PAR-1 activation in smokers and non-smokers. Methods: Activation of PAR-1 caused dose-dependent venoconstriction (P < 0.001) that was unaffected by norepinephrine or tirofiban co-infusion and endothelial denudation. In forearm resistance vessels, arterial PAR-1 activation increased forearm blood flow (P < 0.001), t-PA release (P < 0.001) and platelet-monocyte binding (PO.OOOl). Activation of PAR-2 caused venous (P < 0.001) and arterial (P < 0.01) dilatation without t-PA release or platelet activation. Although blockade of prostacyclin production had no effect, PAR-1 induced arterial vasodilatation was attenuated by inhibition of NO synthesis (PO.OOOl) and EDHF (PO.05), and abolished by their combination (PO.Ol). Smokers had impaired PAR-1 mediated vasodilatation and t-PA release. Results: We have, for the first time, demonstrated that PAR-1 agonism in vivo in man causes arterial dilatation, venoconstriction, platelet activation and t-PA release that is mediated through endothelium-dependent and independent pathways, and impaired in smokers. These unique and contrasting effects are of major physiological relevance to the regulation and resolution of intravascular thrombosis. These findings have implications for the development and therapeutic use of thrombin receptor antagonists and direct thrombin inhibitors.
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Byrne, Emma Jane. "The activity of human rhinovirus 14 3C protease in artificial polyproteins." Thesis, University of St Andrews, 1999. http://hdl.handle.net/10023/14310.
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HRV14 3C acts as a protease and has a role in RNA replication in vivo, interacting with a cloverleaf structure in picornaviral genomic RNA. Picornaviral 3C proteases are able to cleave both N- and C-terminally producing 3CDpro, or, 3Cpro and 3DPol, respectively. In order to investigate the mechanisms whereby these alternative processing pathways are adopted an artificial polyprotein system was constructed, composed of viral sequences from the P3 region of the viral polyprotein flanked by reporter genes. Two antibiotic resistance genes (KanR, TetR) were cloned to act as reporter genes flanking the viral region of interest. Analysis of the cleavage products in a coupled TnT system showed whether N- or C-terminal cleavage had occurred. 3Cpro cleaved preferentially at its N-terminus in [KanR3CproTetR] with a lesser degree of cleavage at its C-terminus. When 3ABC was used as the viral component of the system cleavage at the N-terminus of 3Cpro was also observed. The use of 3CDpro as the viral component also had a regulatory effect on the site (N- or C-terminal) of cleavage by 3Cpro. With 3CDpro as the viral component of the artificial reporter polyprotein cleavage occurred at both the N-and C-termini of 3Cpro as well as at the C-terminus of 3Dpol. This surprising result has led to comparisons with the proteolytic action of viral proteases in the caliciviruses and some plant viruses and the proposal of possible evolutionary links between these viruses. The use of the antibiotic resistance genes as reporters allowed investigation into the use of antibiotic resistance phenotypes in E.coli for monitoring cleavage of the artificial polyprotein. Preliminary results indicated that the system may be useful as a genetic screen to quantify large numbers of mutants.
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Andersen, Henrik. "Human protease activated receptor 4 and its role in platelet activation /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9235.
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Parry, Christopher Marc. "Molecular identification, characterisation and processing of the Human Herpesvirus-6 Protease." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557278.
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Silva, Ludmila Bezerra da. "Identificação de proteases de Leptospira envolvidas na degradação de proteínas da matriz extracelular e do plasma humano." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-31012018-112506/.
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Leptospiras são bactérias espiroquetas altamente móveis dotadas de estratégias que possibilitam grande eficiência nos processos de invasão e disseminação no hospedeiro. Nosso grupo demonstrou previamente que leptospiras patogênicas secretam proteases capazes de clivar e inativar moléculas-chave do sistema complemento humano, o que confere a essas bactérias a capacidade de driblar os mecanismos de defesa do sistema imune inato. Dada a rápida disseminação das leptospiras durante o processo de infecção, aventou-se a hipótese de que essas proteases secretadas pudessem alvejar uma gama maior de moléculas do hospedeiro. No presente estudo, a atividade proteolítica de proteínas secretadas por leptospiras sobre um painel de moléculas da matriz extracelular e do plasma foi avaliada. O sobrenadante de cultura da estirpe virulenta L. interrogans sorovar Kennewicki Fromm degradou fibrinogênio humano, fibronectina plasmática, colágeno Tipo I, e as proteoglicanas decorina, biglicam e lumicam. A atividade proteolítica foi inibida por 1,10-fenantrolina, sugerindo o envolvimento de metaloproteases. Laminina, matrigel, plasminogênio e trombina não foram clivados por proteases presentes nos sobrenadantes. Ainda, os dados indicam que a produção de proteases deve ser um determinante de virulência importante, uma vez que os sobrenadantes de estirpes saprófitas ou patogênicas atenuadas em cultura não apresentaram atividade proteolítica sobre componentes da matriz ou do plasma. A análise dos genomas de leptospiras disponíveis nos levou à identificação de quatro termolisinas, metaloproteases presentes apenas nas espécies patogênicas. Uma delas, codificada pele LIC13322, foi produzida na forma recombinante e apresentou atividade proteolítica sobre fibrinogênio, biglicam e decorina. Em paralelo, foram também realizadas análises comparativas dos exoproteomas das estirpes Fromm e Patoc I. Algumas metaloproteases que podem estar envolvidas na degradação de moléculas do hospedeiro foram identificadas. A capacidade de clivar moléculas do tecido conjuntivo e proteínas da cascata de coagulação pode certamente contribuir para a invasão e a destruição tecidual observadas durante a infecção por Leptospira.Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the human complement system, allowing these bacteria to circumvent host´s innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm degraded human fibrinogen, plasma fibronectin, collagen Type 1, and the proteoglycans decorin, biglycan, and lumican. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Laminin, matrigel, plasminogen and thrombin were not cleaved by proteases present in the supernatants. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic acticity against ECM or plasma components. A search against Leptospira genomes allowed identification of four thermolysins, which are metalloproteases found exclusively in pathogenic species. One of them, encoded by LIC13322, was produced in the recombinant form and displayed proteolytic activity against fibrinogen, biglycan and decorin. Comparative exoproteomic analyses using Fromm and Patoc I strains were also performed and allowed identification of a few metalloproteases that could be involved in the degradation of host components. The ability to cleave connective tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.
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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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Ding, Yan Shirley. "Expression, purification and charaterization of recombinant human T-cell leukemia virus type I protease." Diss., Georgia Institute of Technology, 1998. http://hdl.handle.net/1853/29992.
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Ni, Hao II. "Expression of Human Protein C in Transgenic Tobacco." Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/33367.
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Human Protein C (hPC) is a vitamin K-dependent serine protease that has a critical role in the naturally-occurring anticoagulant pathway. Upon activation of the zymogen by thrombin at the endothelial cell surface, the active form of hPC has anticoagulant activity in hemostasis due to its ability to inactivate factors Va and VIIIa. For biological activity, hPC requires several post-translational modifications including proteolytic cleavage, disulfide bond formation, b-hydroxylation, g-carboxylation, and N-linked glycosylation. Plants have the eukaryotic protein modifying mechanisms required for many human proteins and may provide a safe, cost-effective system for producing hPC on a large-scale basis. Tobacco (Nicotiana tabacum L.) is particularly well suited for use as a bioreactor for high-value recombinant proteins. Tobacco is one of the easiest plants to transform, it is an excellent biomass producer and can produce up to a million seeds from a single genetically engineered plant. Previous attempts to produce hPC in tobacco were limited by expression levels.
The overall goal of the research was to develop transgenic plants that express hPC at higher levels. A cDNA encoding hPC was fused to an enhanced constitutive 35S promoter (35SDE ) and introduced into a plant transformation vector. The hPC construct was introduced into tobacco leaf disks using Agrobacterium tumefaciens-mediated transformation, and 30 transgenic plants were generated.
Stable integration of the hPC gene construct into the tobacco genome and transgene copy number were determined by genomic Southern hybridization and segregation analyses. The majority of transgenic plants expressed the hPC transgene based on RNA analyses by northern hybridization. Plants utilizing the enhanced 35S promoter had equivalent levels of expression to previously generated hPC-containing plants. A variety of polyclonal and monoclonal antibodies raised against hPC were tested for detection of hPC standards and tobacco-synthesized hPC by western immunoblotting. Novel proteins in the size range of hPC heavy chain cross-reacted with anti-heavy chain hPC antibodies in 35SDE:hPC plants. Thus, plants may be capable of synthesizing hPC and proteolytically processing it to light and heavy chains. Although further experiments will be required to confirm the identity of these putative hPC proteins in tobacco, these result suggest that analyses of hPC expressed in plants have been limited by effective tools for detecting the hPC gene product rather than expression levels determined by the transgene promoter.Master of Science
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Thomas, James A. "A protease cascade regulates egress of Plasmodium falciparum from the human erythrocyte." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10045224/.
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Malaria parasites invade erythrocytes and replicate inside a parasitophorous vacuole (PV). Invasive merozoites eventually egress in a process that involves sequential rupture of first the PV membrane (PVM) then the erythrocyte membrane. Egress is protease-dependent, with both cysteine and serine proteases implicated. The parasite serine protease SUB1 is stored in merozoite secretory organelles that are discharged into the PV 10 minutes before erythrocyte membrane rupture. Pharmacological inhibition of SUB1 activity or discharge blocks egress, but the mechanism by which SUB1 regulates egress is unclear. In the PV, SUB1 cleaves multiple substrates including SERA6, a putative cysteine protease. In asexual blood stages of Plasmodium falciparum, the agent of the most dangerous form of malaria, SERA6 is believed to be essential but its function and whether this depends on SUB1 is unknown. Here it is shown that conditional disruption of the P. falciparum SUB1 or SERA6 genes produces two distinct, lethal phenotypes. SUB1-null parasites undergo none of the morphological changes that precede egress and fail to rupture the PVM. In contrast, PVM rupture and the typical erythrocyte membrane poration occur normally in SERA6-null parasites but erythrocyte membrane rupture does not occur. Complementation studies demonstrate that SERA6 is an enzyme and that processing by SUB1 is required for its function. This study concludes that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes leading to egress.
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Martucci, Morena <1983>. "Aging in human liver and skeletal muscle: studies on proteasomes." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6292/1/Martucci_Morena_tesi.pdf.
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Aging is a complex phenomenon that affects organs and tissues at a different rate. With advancing age, the skeletal muscle undergoes a progressive loss of mass and strength, a process known as sarcopenia that leads to a decreased mobility and increased risk of falls and invalidity. On the other side, another organ such as the liver that is endowed with a peculiar regenerative capacity seems to be only marginally affected by aging. Accordingly, clinical data indicate that liver transplantation from aged subjects has, in specific conditions, function and duration comparable to those achievable with grafts of liver from young donors. The molecular mechanisms involved in these peculiar aging patterns are still largely unknown, but it is conceivable that protein degradation machineries might play an important role, as they are responsible for the maintenance of cellular homeostasis. Indeed, it has been suggested that alteration of proteostasis may contribute to the onset and progression of several age-related pathological conditions, including skeletal muscle wasting and sarcopenia, as well as to the aging phenotypes.
The ubiquitin-proteasome system (UPS) is one of the most important cellular pathways for intracellular degradation of short-lived as well as damaged proteins. To date, studies on the age-related modifications of proteasomes in liver and skeletal muscle were performed prevalently in rodents, with controversial results, while only preliminary observations have been obtained in human liver and skeletal muscle. In this scenario, we want to investigate and characterize in humans the age-related modifications of proteasomes of these two different organs.
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Martucci, Morena <1983>. "Aging in human liver and skeletal muscle: studies on proteasomes." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6292/.
Full textAbstract:
Aging is a complex phenomenon that affects organs and tissues at a different rate. With advancing age, the skeletal muscle undergoes a progressive loss of mass and strength, a process known as sarcopenia that leads to a decreased mobility and increased risk of falls and invalidity. On the other side, another organ such as the liver that is endowed with a peculiar regenerative capacity seems to be only marginally affected by aging. Accordingly, clinical data indicate that liver transplantation from aged subjects has, in specific conditions, function and duration comparable to those achievable with grafts of liver from young donors. The molecular mechanisms involved in these peculiar aging patterns are still largely unknown, but it is conceivable that protein degradation machineries might play an important role, as they are responsible for the maintenance of cellular homeostasis. Indeed, it has been suggested that alteration of proteostasis may contribute to the onset and progression of several age-related pathological conditions, including skeletal muscle wasting and sarcopenia, as well as to the aging phenotypes.
The ubiquitin-proteasome system (UPS) is one of the most important cellular pathways for intracellular degradation of short-lived as well as damaged proteins. To date, studies on the age-related modifications of proteasomes in liver and skeletal muscle were performed prevalently in rodents, with controversial results, while only preliminary observations have been obtained in human liver and skeletal muscle. In this scenario, we want to investigate and characterize in humans the age-related modifications of proteasomes of these two different organs.
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Mulley, John Charles. "Genetic marker studies in humans /." Title page, contents and summary only, 1985. http://web4.library.adelaide.edu.au/theses/09PH/09phm958.pdf.
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Amamura, Thaís Akemi. "Clivagem de proteínas do complexo de ataque à membrana do sistema complemento humano por proteases de leptospiras patogênicas." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-09032017-134114/.
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A leptospirose é causada por bactérias que pertencem ao gênero Leptospira. Em um estudo realizado por nosso grupo, observou-se que as proteases secretadas por leptospiras patogênicas foram capazes de clivar a molécula C3 do Complemento e seus fragmentos C3b e iC3b, além de Fator B, C4b e C2. Neste trabalho expandimos a análise da atividade proteolítica sobre os componentes do Complexo de Ataque à Membrana (MAC): C6, C7, C8 e C9. Nós observamos que essas proteases clivam todos os componentes do MAC inclusive o complexo solúvel formado e que essas clivagens ocorrem de modo tempo-dependente. Além disso, as clivagens dessas moléculas ocorrem de modo seletivo, pois mesmo utilizando quantidades reduzidas de sobrenadantes ainda foi possível observar produtos de clivagem. A atividade proteolítica foi inibida pela 1,10fenantrolina, indicando a participação de metaloproteases. O reconhecimento de quais moléculas do MAC são clivadas por proteases de leptospiras patogênicas pode contribuir para o desenvolvimento de estratégias terapêuticas na infecção por estes patógenos.Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. In a previous study, our group observed that the proteases secreted by Pathogenic Leptospira were capable of cleaving C3 of Complement, as well as the fragments C3b and iC3b, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Membrane Attack Complex (MAC). We observed that the protease cleaves all MAC components including soluble complex formed and that these cleavages occur in a time-dependent manner and in a selective way, since even when reduced quantities of supernatants were used, the cleavage products were still observed. The proteolytic activity was inhibited by 1,10phenanthroline, indicating the participation of metalloproteinases. The recognition that MAC molecules are cleaved by proteases of pathogenic leptospires can contribute to the development of therapeutic strategies for the infection by these pathogens.
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Maurice, Kelly. "Propriétés biochimiques, enzymatiques et physiologiques de la Human Airway Trypsin-like protease (HAT)." Mémoire, Université de Sherbrooke, 2010. http://savoirs.usherbrooke.ca/handle/11143/4046.
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La fibrose kystique (FK) est caractérisée par une inflammation pulmonaire chronique. Cette dernière est définie entre autres par une augmentation importante de la production de mucus. Celui-ci s'accumule dans les bronches empêchant ainsi l'éventuel passage de l'air. De plus, le mucus forme une barrière protectrice pour les bactéries qui va causer l'inflammation des poumons. La protéase human airway trypsin-like (HAT) a été retrouvée dans les expectorations de patients atteints d'asthme et bronchite chronique, pathologies similaires à la FK. Elle semble jouer un rôle dans l'inflammation car elle augmente entre autres la production de MUC5AC, protéine composant majoritairement le mucus. Le but de cette étude était de synthétiser la HAT dans une conformation active dans un système eucaryote pour étudier ses propriétés biochimiques, enzymatiques ainsi que son potentiel pro-inflammatoire dans la lignée cellulaire pulmonaire Calu-3. L'ADNc codant pour la partie soluble de la HAT a été inséré dans le vecteur pMT-BiP V5-His. Cet ADN recombinant fut ensuite transfecté dans les cellules de Drosophile Schneider 2 (S2). La HAT recombinante a par la suite été purifiée et son activité protéolytique testée avec un substrat fluorogénique. Ses propriétés biochimiques et enzymatiques ont été étudiées ainsi que son effet sur la production d'IL-8 et sa détection dans les expectorations de patients. La HAT purifiée était «enzymatiquement» pure, donc l'effet protéolytique observé avec le substrat fluorogénique est dû à la protéase. Les analyses de ses propriétés biochimiques montrent que l'activité protéolytique de la HAT est inhibée par un pH acide (<6.5), certains inhibiteurs synthétiques (l'aprotinine, la leupeptine et le PEFABLOC) et endogène (i.e: alpha-2 anti-plasmine (a2-AP)) de protéases à sérine. D'un autre côté, les essais enzymatiques révèlent que la HAT a une préférence pour les substrats ayant une arginine en P4, P3 et Pl plutôt que ceux ayant un glutamate en P4, P2 et P1'. Finalement, l'étude sur l'implication physiologique de l'enzyme démontre que la protéase ne semble pas avoir d'effet ou au plus peu d'effet sur la production d' IL-8. [Symboles non conformes]
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Paskas, Svetlana. "Expression and function of protease-activated receptors in human monocyte-derived dendritic cells." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-55899.
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Forney, John Russell. "Interaction of the Human Serine Protease Inhibitor Alpha-1-antitrypsin with Cryptosporidium parvum." DigitalCommons@USU, 1997. https://digitalcommons.usu.edu/etd/3961.
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The human serine protease inhibitor (serpin) alpha-1-antitrypsin (AAT) was studied for potential interaction with components of the protozoan parasite Cryptosporidium parvum. A homogenate prepared from C. parvum oocysts was incubated with purified human AAT, and complexes formed between the serpin and components of the homogenate were detected using an enzyme-linked immunosorbent assay (ELISA). Serpin:parasite infections were effectively blocked by preincubating AAT with a cognate target enzyme, porcine pancreatic elastase, prior to performing the ELISA on the homogenate. Incubation of a mixture of C. parvum oocysts and sporozoites with AAT demonstrated preferential fluorescence labeling of the sporozoite surface membrane by indirect immunofluorescence assay. Localization of serpin complexes on sporozoites was confirmed by immunogold electron microscopy.
AAT was evaluated for in vitro anticryptosporidial activity in a bovine fallopian tube epithelial (BFTE) cell culture system using both oocysts and filter purified sporozoites as inocula. Serial dilutions of AAT were mixed with oocysts (or sporozites) and used to inoculate BFTE cell monolayers. Inoculted cells were maintained at 37ºC/5% CO2 and collected at 24-,48-,72-, and 96-hr post-inoculation intervals. The addition of AAT and other select protease inhibitors (i.e.,antipain, aprotinin, leupeptin, soybean trypsin inhibitor, and phenylmethylsulfonyl floride) significantly inhibited parasite infection (P<0.01) in a concentration- and time-dependent manner when bleach-decontaminated oocysts were used in the inoculum.
The anticryptosporidial activity of AAT is postulated to be linked to an antagonistic effect on oocyst excystation and, putatively, the forced expenditure of bioenergetic reserves prior to host cell invasion. This postulate was supported by the observations that serpin activity had no statistically significant effect on reducing established in vitro infections (i.e., 24 hr post-inoculation prior to addition of AAT) and did not inhibit infection of BFTE cells when inoculted with sporozoite preparations. The combined application of AAT and the aminoglycoside paromomycin demonstrated a synergistic anticryptosporidial effect on in vitro infection and suggested the basis for a multi-agent therapeutic protocol in preventing cryptosporidosis. These studies collectively demonstrated an inticryptosporidial potential for serine protease inhibitors, in particular for AAT, and suggest an alternative approach to conventional therapeutic strategies.
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Reyskens, Kathleen Maria Simone Elise. "The maladaptive effects of HIV protease inhibitors (Lopinavir/Ritonavir) on the rat heart." Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/85782.
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Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Although antiretroviral treatment decreases HIV-AIDS morbidity/mortality, long-term effects include onset of insulin resistance and cardiovascular diseases. Increased oxidative stress and dysregulation of the ubiquitin-proteasome system (UPS) are implicated in protease-inhibitor (PI)-mediated cardio-metabolic pathophysiology. We hypothesized that PI treatment (Lopinavir/Ritonavir) elevates myocardial oxidative stress and concomitantly inhibits the UPS, thereby attenuating cardiac function. Lopinavir/Ritonavir was dissolved in 1% ethanol (vehicle) and injected into mini-osmotic pumps that were surgically implanted into Wistar rats for eight weeks vs. vehicle and sham controls. Subsequently, we evaluated metabolic parameters and heart function (ex vivo and in vivo methods) at baseline and following ischemia-reperfusion. PI-treated rats exhibited weight gain, increased serum LDL-cholesterol, higher tissue triglycerides (heart, liver), but no evidence of insulin resistance. It also upregulated hepatic gene expression of acetyl-CoA carboxylase β and 3-hydroxy-3-methylglutaryl-CoA-reductase, key regulators of fatty acid oxidation and cholesterol synthesis, respectively. Further, PI-treated hearts displayed impaired UPS, increased superoxide dismutase (SOD) activity and unaltered superoxide levels, and elevated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) peptide levels. Perfusion data revealed contractile dysfunction at baseline and following ischemia-reperfusion, while post-ischemic hearts exhibited decreased ATPase specific activity vs. matched controls. Early changes initiated by PI treatment resemble the metabolic syndrome and reflect a pre-atherogenic profile. Moreover, the effects of PIs on cardiac contractile function may in part be triggered by impaired UPS activity together with strain on the mitochondrial energetic system. Our study alerts to cardio-metabolic side effects of PI treatment and raises the question of the most appropriate co-therapies for patients on chronic antiretroviral treatment.
AFRIKAANSE OPSOMMING: Alhoewel anti-retrovirale behandeling MIV-VIGS morbiditeit/mortaliteit verlaag, bestaan daar langtermyn effekte soos die aanvang van insulienweerstandigheid en kardiovaskulêre siektes. Verhoogde oksidatiewe stres en wanregulering van die ubikwitien-proteosoomsisteem (UPS) word geïmpliseer met protease-inhibeerder (PI) gemediëerde kardio-metaboliese patofisiologie. Ons hipotetiseer dat PI behandeling (Lopinavir/Ritonavir) miokardiale oksidatiewe stres verhoog, en gevolglik die UPS inhibeer waardeur dit kardiale funksie verander. Lopinavir/Ritonavir is in 1% etanol (draer) opgelos en in ‘n mini-osmotiese pomp ingespuit wat chirurgies in Wistar rottes ingeplant is vir agt weke vs. draer en valskontroles. Gevolglik het ons die metabolise parameters en hartfunksie (ex vivo en in vivo metodes) op basislyn en na afloop van ischemie-reperfusie ondersoek. PI-behandelde rotte het ‘n toename in massa getoon asook verhoogde serum LDL-cholesterol, hoër weefseltrigliseriede (hart, lewer), maar geen bewys van insulienweerstandigheid nie. Dit het ook hepatiese asetielko-ensiem A karboksilase β en 3-hidrokise-3-metielglutariel KoA reduktase geenuidrukking opwaarts gereguleer, wat sleutel reguleerders van vetsuuroksidasie en cholesterolsintese onderskeidelik is. Verder, het PI-behandelde harte ingeperkte UPS, verhoogde SOD aktiwiteit en onveranderde superoksiedvlakke vertoon, asook verhoogde peroksisoomproliferator-geaktiveerde reseptor-γ ko-aktiveerder 1-α (PGC-1α) peptiedvlakke. Perfusie data toon kontraktiele wanfunskionering gedurende basislyn en na afloop van ischemie-reperfussie, terwyl post-ischemiese harte verlaagde ATPase spesifieke aktiwiteit vs gepaarde kontrole vertoon. Vroeë veranderinge wat deur PI behandeling veroorsaak word, kom ooreen met die metabolise sindroom en reflekteer op ‘n pre-aterogeniese profiel. Bowendien kan die effekte van PIs op kardiale kontraktiele funksie deels veroorsaak word deur die ingeperkte UPS aktiwiteit tesame met die las op die mitochondriale energie sisteem. Ons studie waarsku teen kardio-metaboliese newe effekte met PI behandeling en rig die vraag; wat die mees gepaste ko-behandeling vir pasiënte op chroniese anti-retrovirale behandeling is.
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Tung, Kwok-kwan. "Epigenetic inactivation and tumor suppressive roles of hepatocyte growth factor activator inhibitors(HAIs) in human hepatocellular carcinoma." View the Table of Contents & Abstract, 2007. http://sunzi.lib.hku.hk/hkuto/record/B3971164X.
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Fonseca, Iêda Maria Rocha Lima Vieira da. "Avaliação da concentração de proteínas totais na saliva humana frente a diferentes protocolos de tratamento da saliva." reponame:Repositório Institucional da UFC, 2015. http://www.repositorio.ufc.br/handle/riufc/14015.
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FONSECA, Iêda Maria Rocha Lima Vieira da. Avaliação da concentração de proteínas totais na saliva humana frente a diferentes protocolos de tratamento da saliva. 2015. 40 f. Dissertação (Mestrado em Odontologia) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2015.Submitted by denise santos (denise.santos@ufc.br) on 2015-11-19T12:39:00Z No. of bitstreams: 1 2015_dis_imrlvfonseca.pdf: 763869 bytes, checksum: 7ff734931eddb02f3b4465dc7f77d0f5 (MD5)
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The need to preserve the stability of saliva samples during and/or after collection has been considered a factor that might influence its analytical results, compromising the reliability and reproducibility of these analytical methods. The main challenges related to saliva preservation include the complexity of saliva composition, and its inherent elevated proteolitic activity. Thus, collection and storage of saliva requires specific precautions to preserve its components. The present study aimed to evaluate the protein concentration of saliva samples obtained from 10 healthy adults, aged 23 – 65 years, with a mean age of 31 years, subject to different pre-analytical sample preparation. Following collection, the salivary flow rate was calculated, and saliva samples from each volunteer were fractioned and divided in six different groups, in which each of these groups corresponded to a different type of pre-analytical sample preparation. The groups were as follows: G1- immediate centrifugation, no addition of protease inhibitor, room temperature during 24 hours; G2- immediate centrifugation, no addition of protease inhibitor, -80oC during 30 days; G3- immediate centrifugation, protease inhibitor added during collection, -80oC during 30 days; G4- immediate centrifugation, protease inhibitor added during analysis, -80oC during 30 days; G5- centrifugation 30 days after collection, no addition of protease inhibitor, -80oC during 30 days; G6- centrifugation 30 days after collection, protease inhibitor added during analysis, -80oC during 30 days. Total protein concentration was analyzed in duplicates, through the bicinchoninic acid assay. After analysis, the total protein concentration in each group was statistically correlated through Pearson and Spearman correlation tests and compared using repeated measure ANOVA (p<0.05). The mean total protein concentrations showed a negative correlation with salivary flow rate in G1 (P= 0,020), G4 (P= 0,027) and G5 (P= 0,05). Total protein concentration and age were only statistically correlated in G3 (P= 0,01). The mean total protein concentrations did not significantly differ between groups, F(5,45)= 1,132, P= 0,358. These results were also observed when comparing the mean total protein concentrations normalized by each individual’s salivary flow rate, F(5,45) = 2,068, P= 0,087. In conclusion, the methodological alterations proposed for the preparation of saliva samples before analysis did not generate significant quantitative alterations in total protein concentration within these samples.
A necessidade de preservar a estabilidade da saliva durante e/ou depois da coleta, tem sido considerado um fator que pode influenciar os resultados obtidos na análise desse fluido, comprometendo a confiabilidade e reprodutibilidade de tais métodos analíticos. Os principais desafios relacionados à preservação da saliva referem-se à complexidade da sua composição e da sua elevada atividade proteolítica inerente. Consequentemente, coleta e armazenamento da saliva exigem precauções especiais para preservação de seus componentes. O presente estudo se propôs a avaliar a concentração proteica de amostras de saliva total de dez voluntários adultos, saudáveis, com idades variando de 23 a 65 anos, com média de 31 anos submetidas a alterações metodológicas no preparo pré-analítico da amostra. Após coletadas, os fluxos salivares foram calculados e as amostras de saliva de cada indivíduo foram fracionadas e divididas em seis diferentes grupos, onde cada um desses grupos correspondeu a um tipo diferente de preparo pré-analítico da amostra. Os grupos foram conforme segue: G1- centrifugação imediata, inibidor ausente, temperatura ambiente por 24 horas; G2- centrifugação imediata, inibidor ausente, -80oC por 30 dias; G3- centrifugação imediata, inibidor no ato da coleta, -80oC por 30 dias; G4- centrifugação imediata, inibidor no ato da análise, -80oC por 30 dias; G5- centrifugação após 30 dias, inibidor ausente, -80oC por 30 dias; G6- centrifugação após 30 dias, inibidor no ato da análise, -80oC por 30 dias. As concentrações de proteínas totais foram avaliadas pelo método do ácido bicinconínico, em duplicatas. Após análise a concentração de proteínas totais em cada grupo foi estatisticamente correlacionada pelos testes de Pearson e Spearman, e comparações feitas realizadas por meio do teste ANOVA para medidas repetidas (p<0.05). As concentrações médias de proteínas totais demonstraram uma correlação negativa significativa com o fluxo salivar em G1 (P= 0,020), G4 (P= 0,027) e G5 (P= 0,05). Proteínas totais e idade só demonstraram correlação significativa em G3 (P= 0,01). As concentrações de proteínas totais médias não diferiram de forma significante entre os grupos, F(5,45)= 1,132, P= 0,358. Esses resultados foram, também, observados ao se comparar as médias de proteínas totais com base no fluxo salivar dos voluntários, F(5,45) = 2,068, P= 0,087. Em conclusão, as alterações metodológicas ora propostas no tratamento das amostras de saliva não redundaram em alterações quantitativas significantes nas concentrações de proteínas totais presentes nesse fluido.
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Richardson, Susanne. "Effect of human kallikreins HK2 and HK3 on the anti-protease system of the cervical mucus of the human female." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0018/MQ38181.pdf.
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Phenix, Barbara N. "A new role for human immunodeficiency virus (HIV)-1 protease inhibitors: Suppression of apoptosis." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/29000.
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Protease inhibitor (PI)-induced improvements in CD4 T cell counts may be in part independent of PI effects on HIV replication. Since HIV-associated CD4 T cell depletion occurs by apoptosis, we analysed the effect of Pis on apoptosis in peripheral blood lymphocytes (PBLs) from HIV-infected patients and in an uninfected T cell line. The in vivo effects of Pis were monitored in an animal model of stroke and in HIV negative patients taking anti-retroviral therapy (ART) in the context of post-exposure prophylaxis (PEP).
Patient PBLs and Jurkat T cells were cultured with Pis. Following stimulation, apoptosis was measured by annexin-V, and loss of mitochondrial membrane permeability (Deltapsim) was assessed in cells and isolated mitochondria using DiOC6(3). The mechanism of inhibition was determined at the level of caspase activity, and of protein and messenger ribonucleic acid (mRNA) synthesis of various pro- or antiapoptotic factors. For in vivo experiments, mice were given Pis by gastric lavage at various time points prior to and following transient forebrain ischaemia. Histological analyses were performed on hippocampal sections. Apoptosis of ex vivo peripheral blood mononuclear cells (PBMCs) of patients taking PEP (AZT, lamivudine, nelfinavir) was assessed prior to, on, and post-therapy in response to a variety of stimuli.
Results revealed that Pis reduced spontaneous and anti-Fas induced apoptosis both in CD4 and CD8 T cells from HIV patients. Jurkat T cell apoptosis, Deltapsi m, cytochrome c release, and caspase 8 cleavage were also inhibited by Pis. The mechanism responsible for inhibition of apoptosis does not involve modification of caspase activity, protein, or mRNA synthesis. Mitochondrial involvement was confirmed following inhibition of viral protein R (Vpr)- and atractyloside (Atr)-induced Deltapsim of isolated mitochondria. Apoptosis in hippocampal sections of mice having undergone transient forebrain ischaemia was inhibited by PI treatment, as was camptothecin-induced apoptosis in PBMCs from patients taking PEP.
In conclusion, Pis inhibit apoptosis in PBLs from HIV-infected patients and in uninfected Jurkat T cells. The mechanism appears to involve mitochondria, as inhibition of Vpr- and Atr-induced Deltapsim of isolated mitochondria was observed. Stroke-induced apoptosis may be inhibited in vivo by Pis, and importantly, survival following anti-Fas challenge may be positively influenced.
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Bredin, Cecilia G. "Studies of cell migration and matrix protease production in human lung cancer cell lines /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-969-2/.
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