Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and depression in sarcolemmal Na+-K+-ATPase activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, and reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 60 min of global ischemia were associated with augmented activities of both calpain and MMPs and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. However, reperfusion of ischemic heart produced depression in Na+-K+-ATPase activity, no change in the augmented calpain activity, but decreases in augmented MMP-2 activity and Na+-K+-ATPase content. MDL28170, a calpain inhibitor, was more effective in attenuating I/R-induced alterations than doxycycline, an MMP inhibitor. Incubation of control SL preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1, α2 and β2 without changes in β1. These results support the view that activation of calpain is involved in depressing Na+-K+-ATPase activity and degradation of its subunits in hearts subjected to I/R injury.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

An investigation of the expression profile of mRNA encoding Calpain 3, the causative agent in the inherited human muscular disease Limb Girdle Muscular Dystrophy Type 2A, was conducted in two representative mammalian species, human and mouse. Transcripts encoding Calpain 3 were identified from mammalian tissues other than skeletal muscle. In human Peripheral Blood Mononuclear Cells (PBMCs) these transcripts were identified in both the T-cell and B-cell compartments and in a number of human blood cell lines representing different haematopoietic lineages. Calpain 3 transcripts encoding the murine homologue were also described from mouse PBMCs and from murine tissues involved in haematopoiesis. In addition to the confirmation of Calpain 3 expression in non-skeletal muscle tissues in both these species, transcripts were identified with precise and defined deletions, which mapped to known exon-exon boundaries in the Calpain 3 gene from both species. These deletions constituted the removal by alternative splicing of skeletal muscle-specific components of the Calpain 3 protein known to regulate its function in this tissue. Monoclonal antibodies to the Calpain 3 protein were used to confirm the presence of Calpain 3 protein in non-skeletal muscle tissues of both human and mouse. In humans the expression of Calpain 3 protein was confirmed in PBMCs and in the mouse, Calpain 3 expression was confirmed in tissues of the haematopoietic compartment. In both species the Calpain 3 protein expressed correlated with translation from a transcript lacking the skeletal muscle-specific components generated by alternative splicing. An attempt was made using a Yeast Two Hybrid assay to identify potential regulatory molecules of Calpain 3 in human PBMCs, but without a definitive candidate molecule being found. A developmental model of muscle differentiation (murine C2C12 myoblast cells) was used to ascertain the expression profile of Calpain 3 in the early stages of myofibrillogenesis. Using Quantitative Real Time PCR the expression profile of Calpain 3 was assessed in differentiating C2C12 cells. These results showed that the absolute levels of Calpain 3 transcription were elevated during differentiation and that a temporal Calpain 3 isoform shift occurred during this process. This temporal shift in expression was from transcripts having identical deletions to those seen in the haematopoietic tissues, to full length transcripts representative of skeletal muscle-specific Calpain 3. The identification of Calpain 3 expression outside skeletal muscle tissue is novel and the isoforms expressed in these tissues are structurally more analogous to the ubiquitously expressed calpains. This has implications for LGMD2A where a loss of function of Calpain 3 in non-skeletal muscle tissue could be compensated for by the ubiquitous calpains, thus explaining the lack of any non-muscle tissue pathology in LGMD2A patients.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 79-89). Also available on the World Wide Web.

Les calpaïnes 1 et 2 sont des protéases à cystéine ubiquitaires et la calpastatine est leur inhibiteur naturel, également ubiquitaire. Les calpaïnes sont impliquées dans le développement de la réponse inflammatoire via l’activation par protéolyse partielle de plusieurs substrats (activation de NF-κB par le clivage de I-κBα, remodelage du cytosquelette des cellules inflammatoires, clivage de la protéine chaperonne HSP 90…).Il a été précédemment démontré que les calpaïnes favorisent le vieillissement neuronal. Nous avons pu montrer dans un modèle murin que l’inhibition in vivo des calpaïnes par la surexpression de calpastatine limite le vieillissement notamment rénal et vasculaire. L’inflammation liée au vieillissement ou « inflammaging » est considérablement réduite par l’inhibition spécifique des calpaïnes. Ceci est dû, au moins en partie, à l’effet des calpaïnes sur la production de cytokines pro-inflammatoires et sur la maturation de l’interleukine-1.Si les calpaïnes intracellulaires exercent un rôle pro-inflammatoire, les calpaïnes externalisées ont un effet anti-inflammatoire via le clivage de TLR2. Les calpaïnes peuvent en effet être excrétées hors des cellules via les transporteurs ABCA1. Dans le cadre d’un modèle murin de mélanome, nous avons pu montrer que l’inhibition des seules calpaïnes extracellulaires par la surexpression de calpastatine extracellulaire préserve TLR2 et limite ainsi la progression de la tumeur.Les calpaïnes intra- et extracellulaires sont des médiateurs majeurs de la réponse inflammatoire et modulent « l’inflammaging » ainsi que la réponse immune anti-tumorale. / Calpain 1 and 2 are cysteine proteases and calpastatin is their natural inhibitor. Calpains and calpastatin are ubiquitous. Calpains are involved in inflammatory response development via activation by partial proteolysis of several substrates (NF-kappaB activation by I-kappaBalpha cleavage, remodeling of inflammatory cells cytoskeleton, cleavage of chaperone protein HSP90 ... ). It has been previously shown that calpains promote neuronal aging. We have shown in a mouse model that inhibition of calpain by calpastatin overexpression limits renal and vascular aging. The inflammation associated with aging or "inflammaging" is considerably reduced by specific inhibition of calpain. This is due, at least in part, to calpain effect on production of pro-inflammatory cytokines and in maturation of interleukin-1 alpha. If intracellular calpains are pro-inflammatory, secreted calpains have an anti-inflammatory effect via cleavage of TLR2. Calpains can indeed be excreted out of the cells via the transporter ABCA1. In the context of a mouse model of melanoma, we have shown that inhibition of extracellular calpain by only extracellular calpastatin overexpression preserves TLR2 and thus limit the progression of the tumor.Calpains intra- and extracellular are major mediators of inflammatory response and modulate the "inflammaging" and the anti-tumor immune response.

Within the framework of this diploma, a genotypization of 162 beef cattle of the ČESTR commercial breed from was performed in order to analyze the potential influence of gene coding calpain I (CAPNI) on the resulting beef tenderness. CAPN1 present on the BTA29 chromosome, was selected in previous studies as a candidate gen for QTL. Polymorphism of CAPNI gene was studied using PCR/RFLP method and CAPN530 marker. Genotype identification resulted from this procedure. A 341 bp long fragment was present in homozygotes AA, two fragments of 195 and 146 bp were present in homozygotes GG, and three fragments of 341, 195 and 146 bp were detected in heterozygotes AG. The genotypization output was subsequently statistically evaluated. 11 homozygotes AA, 62 homozygotes GG and 89 heterozygotes AG were detected in the analyzed beef cattle population. More frequent G allele occurred in the set with the frequency of 0,657 and A allele with the frequency of 0,343. Warner-Bratzler shear test was employed to determine beef tenderness based on the shear force attribute. Samples of raw and grilled beef aging 1, 14 and 28 days after the slaughter were analyzed. Statistical methods were used to evaluate relationship between genotype and detected amount of shear force. In case of raw beef, only the samples aging 1 day after the slaughter showed significant difference in the shear force. The most positive results for the given parameter were achieved in case of AG genotype. Samples aging 14 and 28 days after the slaughter showed no difference in shear force and related beef tenderness, indicating no genotype influence. In case of grilled beef, no significant difference in sheer force indicating possible genotype influence was detected at any day of analysis. Genotype influence on grilled beef tenderness within the test animal population is minimal and statistically inconclusive.

Submitted by Jéssica Moraes Malheiros (jessicamalheiros@yahoo.com.br) on 2018-05-03T18:53:41Z No. of bitstreams: 1 TESE MALHEIROS, J. M..pdf: 1363679 bytes, checksum: 33291bc89815d196826e037eeda5b237 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-05-04T18:11:25Z (GMT) No. of bitstreams: 1 malheiros_jm_dr_jabo.pdf: 1400936 bytes, checksum: bf9a223495b522a13300edf9c4026a2e (MD5) / Made available in DSpace on 2018-05-04T18:11:25Z (GMT). No. of bitstreams: 1 malheiros_jm_dr_jabo.pdf: 1400936 bytes, checksum: bf9a223495b522a13300edf9c4026a2e (MD5) Previous issue date: 2018-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente trabalho teve como objetivo avaliar a associação da expressão gênica e proteômica com a maciez da carne de bovinos da raça Nelore. A partir de uma população de 90 animais foram selecionados três grupos experimentais por meio da análise de força de cisalhamento (FC) e índice de fragmentação miofibrilar (MFI), sendo: carne moderadamente macia, carne moderadamente dura e carne muito dura. A expressão dos genes foi avaliada por meio da análise de PCR em tempo real e a análise proteômica foi realizada com base na separação de proteínas por meio da eletroforese bidimensional (2D-PAGE) e caracterizção por espectrometria de massas com ionização eletrospray (ESI-MS/MS). A expressão da isoforma da calpastatina (CAST2) mostrou-se up regulated (P<0,05) nos grupos de carne moderadamente dura e muito dura. Os genes HSP90AA1, DNAJA1 e HSPB1, os quais representam as proteínas de choque térmico Hsp90, Hsp40 e Hsp27, respectivamente, mostraram expressão down regulated (P<0,05) no grupo de carne moderadamente macia em relação ao grupo de carne muito dura. Na análise proteômica, a expressão do spot protéico das enzimas metabólicas TPI e PGM1, proteína estrutural PFN1 e aminiopeptidase LAP3 se mostraram up regulated (P<0,05) no grupo de carne moderadamente macia, enquanto que a expressão das proteínas estruturais (ACTA1, ACTB, ACTG1 e MLC1), estresse oxidativo (PRDX6, PRDX2, PRDX1 and PARK7), proteínas de choque térmico (HSP90AA1, HSP90AB1, HSPA1A, HSPA1B, HSPA1L, HSPD1 e HSPB1), e co-chaperonas e regulação celular (CD37, STIP1 e ARHGDIA) se mostraram down regulated (P>0,05) no mesmo grupo experimental. Estes resultados fornecem uma visão importante de novos possíveis marcadores biológicos atuantes no processo de amaciamento da carne, o que pode colaborar para melhor entender e gerar novas estratégias de seleção nos programas de melhoramento genético de bovinos Nelore. / The objective of this study was to evaluate the association of gene expression and proteomics with meat tenderness in Nellore cattle. From population of 90 animals three experimental groups were selected by shear force (SF) and/or myofibrillar fragmentation index (MFI): moderately tender meat, moderately tough meat and very tough meat. Gene expression was evaluated by real-time PCR and proteomics analysis was performed based on protein separation by two-dimensional gel electrophoresis (2D-PAGE) and characterisation by eletrospray ionisation mass spectrometry (ESI-MS/MS). Expression of the calpastatin isoform (CAST2) was up-regulated (P<0.05) in the moderately tough and very tough meat groups. Expression of the HSP90AA1, DNAJA1 and HSPB1 genes, wich represent the heat shock proteins Hsp90, Hsp40 and Hsp27, respectively, were down-regulated (P<0.05) in the moderately tender meat in relation to the very tough group. In the proteomics analysis, the expression of the protein spots of metabolism TPI1 and PGM1, structural protein PFN1, and aminopeptidase LAP3 were up regulated (P<0.05) in the moderately tender meat, while the expression of structural proteins (ACTA1, ACTB, ACTG1 and MLC1), oxidative stress (PRDX6, PRDX2, PRDX1 and PARK7), heat shock protein (HSP90AA1, HSP90AB1, HSPA1A, HSPA1B, HSPA1L, HSPD1 and HSPB1) and co-chaperones and cellular regulatory (CD37, STIP1 and ARHGDIA) were down regulated (P>0.05) in the same experimental group. The present results suggest an important view of possible new biological markers in the meat tenderization process, wich permit to unsderstand and generate new strategies for selection in Nellore cattle breeding programs. / FAPESP: 15/13021-1