Em termos de importância no processo de invasão de tumores e metástases, o desequilíbrio entre a expressão das gelatinases e seus inibidores teciduais resulta em degradação do principal constituinte da matriz extracelular, o colágeno tipo IV, favorecendo os processos de invasão tumoral e metástase. Foram avaliadas 53 lesões de 47 cães submetidos a cirurgia excisional com confirmação diagnóstica de mastocitoma por avaliação histopatológica e imuno-histoquímica para MMP-2, MMP- 9, TIMP-2 e TIMP-1. Os mastocitomas foram classificados em graus I (bem diferenciados), II (moderadamente diferenciados) a III (pouco diferenciados), segundo os critérios estabelecidos por Patnaik, Ehler e Macewen (1984) e as mesmas lesões foram classificadas em alto e baixo grau de malignidade de acordo com o sistema proposto por Kiupel et al. (2011). Os resultados obtidos através da imuno-positividade de mastócitos e leucócitos polimorfonucleraes para as quatro proteínas foram comparados as duas graduações estabelecidas, bem como ao tempo de sobrevida pós-cirúrgica e a mortalidade em função do tumor. Verificou-se que as metaloenzimas e seus inibidores não demonstram nenhum tipo de associação de expressão entre elas. Dentre os marcadores imuno-histoquímicos, a expressão mastocitária de TIMP-1 apresentou associação com o tempo de sobrevida, independente do tratamento quimioterápico adjuvante. Quanto menor a expressão de TIMP-1 nos mastócitos, maiores as taxas de mortalidade e menores os períodos de sobrevida. Enquanto que MMP-2, MMP-9 e TIMP-2 não foram indicadores prognósticos para esta neoplasia. Assim, nossos resultados mostram que o TIMP-1 apresentou-se como bom indicador de sobrevida e, portanto, sugere-se que a utilização deste índice prognóstico adicional a classificação histológica pode aumentar a precisão de prognostico dos mastocitomas em cães auxiliando na adequação de terapias apropriadas / In terms of importance regarding to tumor invasion process and metastasis , the imbalance between the expression of gelatinases and their tissue inhibitors results in degradation of the main constituent of the extracellular matrix, collagen type IV, favoring the processes of tumor invasion and metastasis. Fifty-three lesions, of 47 dogs submitted to surgical excision confirmed the diagnosis of mast cell tumor were evaluated by histopathologic examination and immunohistochemistry for MMP-2, MMP-9, TIMP-2 and TIMP-1. Mast cell tumors were histologically classified as grades I (well differentiated), II (moderately differentiated) to III (poorly differentiated), according to the criteria established by Patnaik, Ehler and MacEwen (1984) and the same lesions were classified as high and low-grade of malignancy according to the system proposed by Kiupel et al. (2011). The results obtained by the immunopositivity of mast cells and polymorphonuclear leukocytes to the four proteins were compared with two established graduations, as well as to post-surgical survival times and mortality due to mast cell disease. It could be verified that metalloenzymes and their inhibitors do not establish any association between the expression of the cell types evaluated. Within the immunohistochemical markers, mast cell expression of TIMP-1 was associated with survival time, independent of adjuvant chemotherapy. The lower the expression of TIMP-1 in mast cells, higher the mortality rates and smaller post-cirurgical survival time. While MMP-2, MMP-9 and TIMP-2 levels were not prognostic indicators for this cancer. Thus, our results show that the TIMP-1 appeared as a good indicator of survival time and therefore it is suggested that the use of this prognostic index as additional histological classification can increase the accuracy of prognosis of mast cell tumors in dogs assisting in the adaptation of appropriate therapies

Nós humanos estamos inevitavelmente expostos a uma mistura de poluentes, e evidências nos mostram que estes poluentes aumentam a incidência de desordens reprodutivas. Já se sabe que camundongos cronicamente expostos a níveis de poluentes ambientais em São Paulo apresentam: mudanças no ciclo estral, alteração no número de folículos ovarianos, aumento das perdas embrionárias pós implantacionais e alterações na morfologia placentária. Nós hipotetizamos que as alterações na resposta uterina a implantação possa ser mediado por mastócitos e células NK uterinas levando ao aumento na incidência de perdas pós implntacionais associadas a poluição do ar. Para testar essa hipótese, camundongos foram expostos a níveis ambientais de poluentes até os 60 dias de vidas usando câmaras de exposição [uma câmara recebendo ar filtrado e a outra ar ambiente (poluído) não]. Ao atingir a idade reprodutiva os animais foram colocados para acasalar, os machos utilizados neste estudo não foram expostos aos poluentes, e no 6° e 8° dia pós-coito a gestação foi terminada. O desempenho reprodutivo foi avaliado e as fêmeas foram subdivididas em 4 grupos de acordo com a idade gestacional (6º ou 8º dpc) e exposição (ar filtrado ou ar não filtrado). Métodos estereológicos foram usados para avaliar o desenvolvimento dos sítios de implantação, contagem das células NK uterinas e mastócitos. A concentração de média de PM2.5 nas câmaras com o ar não filtrado foi de 27.5 g.m-3 e 6.5 g.m-3 na câmara com ar filtrado (P<0.001). A exposição a poluição do ar nos primeiros dias de gestação mostrou há diferenças no desenvolvimento dos compartimentos do sítio de implantação no 8° dia pós-coito: redução do volume endométrio e volume total do sítio.Quando nós avaliamos o número total de NK uterina o 8° dia pós-coito, observamos que há uma redução no número destas células nos animais expostos a poluição do ar. Os mastócitos no 6° dia gestacional também se mostram em menor número nos animais expostos a poluição, mas essa diferença desaparece com o avanço da gestação. Na avaliação histopatológica do endométrio, verificamos que há uma redução do volume de células deciduais, de trofoblasto, e um aumento do volume de glândulas nos sítios implantacionais no 8° dia pós-coito de animais expostos ao ar poluído. Nossos resultados confirmam os achados de estudos prévios que correlacionam a exposição a poluição do ar com aumento na incidência de falhas implantacionais e fertilidade diminuída. A avaliação da morfologia uterina e do número de células NK uterinas e mastócitos sugerem que os compostos presentes no ar interferem ou prejudicam o processo de implantação embrionária por alterações na resposta do sistema imunológico materno / Humans are inevitably exposed to mixtures of environmental contaminants, and a vast body of evidence now links exposure to these chemicals with an increased incidence of reproductive disorders. We have shown that mice chronically exposed to ambient levels of air pollution (AP) in São Paulo city have negative reproductive performance: resulting in changes in estrous cyclicity, number of follicles and increased post implantation loss rate as well defective placentation. We hypothesized that alterations in uterine response to implantation could be mediated by mast and uNKcells leading to increased incidence of pos implantation losses associated with exposure to air pollution. To test this female mice were exposed to ambient levels of AP from birth to 60 days of age using exposure chambers (receiving filtered air or non filtered situated near to a high traffic crossroad). On reaching the reproductive age, estrous cyclicity, was evaluated and females were allowed to mate to non exposed males and exposures continued until 6ºdpc or 8ºdpc when pregnancy was terminated. Reproductive performance was assessed and females, subdivided in to 4 groups according to gestational day (6ºdpc or 8ºdpc ) and exposure (filtered or non filtered air). Stereological methods and immunohistochemical techniques were used to evaluate implantation sites (IS) development, do cell identification (NK cells and Mast cells) and counts. Mean concentration of PM2.5 in the non-filtered chamber was 27.5 g.m-3 and 6.5 g.m-3; in the filtered chambers (P<0.001). Females exposed to air pollution previously and during the first days of pregnancy showed borderline differences in the development of the compartments of the IS on day 8 of gestation: volume of the endometrium is reduced as well as the IS total volume. When we evaluated the total number of uNk cells (8ºdpc), we observed that there is a decrease in those animals exposed to air pollution The number of mast cells (6º dpc) are also reduced when compared to females exposed to filtered air, but as pregnancy progress this differences in the number of mast cells disappear. The histopathological evaluation was observe the decrease in the volume occupied by decidual and trophoblast cells, and increased the volume of glands, showing development delay in implantation sites in 8dpc.Our data confirm results from previous studies that link exposure to AP and decreased fertility and increased implantation failure. Evaluation of the uterine morphology and the number of mast call and uNkcell suggests that components present in AP interfere or impair embryonic implantation trought changes in maternal immune system responses

Nós humanos estamos inevitavelmente expostos a uma mistura de poluentes, e evidências nos mostram que estes poluentes aumentam a incidência de desordens reprodutivas. Já se sabe que camundongos cronicamente expostos a níveis de poluentes ambientais em São Paulo apresentam: mudanças no ciclo estral, alteração no número de folículos ovarianos, aumento das perdas embrionárias pós implantacionais e alterações na morfologia placentária. Nós hipotetizamos que as alterações na resposta uterina a implantação possa ser mediado por mastócitos e células NK uterinas levando ao aumento na incidência de perdas pós implntacionais associadas a poluição do ar. Para testar essa hipótese, camundongos foram expostos a níveis ambientais de poluentes até os 60 dias de vidas usando câmaras de exposição [uma câmara recebendo ar filtrado e a outra ar ambiente (poluído) não]. Ao atingir a idade reprodutiva os animais foram colocados para acasalar, os machos utilizados neste estudo não foram expostos aos poluentes, e no 6° e 8° dia pós-coito a gestação foi terminada. O desempenho reprodutivo foi avaliado e as fêmeas foram subdivididas em 4 grupos de acordo com a idade gestacional (6º ou 8º dpc) e exposição (ar filtrado ou ar não filtrado). Métodos estereológicos foram usados para avaliar o desenvolvimento dos sítios de implantação, contagem das células NK uterinas e mastócitos. A concentração de média de PM2.5 nas câmaras com o ar não filtrado foi de 27.5 g.m-3 e 6.5 g.m-3 na câmara com ar filtrado (P<0.001). A exposição a poluição do ar nos primeiros dias de gestação mostrou há diferenças no desenvolvimento dos compartimentos do sítio de implantação no 8° dia pós-coito: redução do volume endométrio e volume total do sítio.Quando nós avaliamos o número total de NK uterina o 8° dia pós-coito, observamos que há uma redução no número destas células nos animais expostos a poluição do ar. Os mastócitos no 6° dia gestacional também se mostram em menor número nos animais expostos a poluição, mas essa diferença desaparece com o avanço da gestação. Na avaliação histopatológica do endométrio, verificamos que há uma redução do volume de células deciduais, de trofoblasto, e um aumento do volume de glândulas nos sítios implantacionais no 8° dia pós-coito de animais expostos ao ar poluído. Nossos resultados confirmam os achados de estudos prévios que correlacionam a exposição a poluição do ar com aumento na incidência de falhas implantacionais e fertilidade diminuída. A avaliação da morfologia uterina e do número de células NK uterinas e mastócitos sugerem que os compostos presentes no ar interferem ou prejudicam o processo de implantação embrionária por alterações na resposta do sistema imunológico materno / Humans are inevitably exposed to mixtures of environmental contaminants, and a vast body of evidence now links exposure to these chemicals with an increased incidence of reproductive disorders. We have shown that mice chronically exposed to ambient levels of air pollution (AP) in São Paulo city have negative reproductive performance: resulting in changes in estrous cyclicity, number of follicles and increased post implantation loss rate as well defective placentation. We hypothesized that alterations in uterine response to implantation could be mediated by mast and uNKcells leading to increased incidence of pos implantation losses associated with exposure to air pollution. To test this female mice were exposed to ambient levels of AP from birth to 60 days of age using exposure chambers (receiving filtered air or non filtered situated near to a high traffic crossroad). On reaching the reproductive age, estrous cyclicity, was evaluated and females were allowed to mate to non exposed males and exposures continued until 6ºdpc or 8ºdpc when pregnancy was terminated. Reproductive performance was assessed and females, subdivided in to 4 groups according to gestational day (6ºdpc or 8ºdpc ) and exposure (filtered or non filtered air). Stereological methods and immunohistochemical techniques were used to evaluate implantation sites (IS) development, do cell identification (NK cells and Mast cells) and counts. Mean concentration of PM2.5 in the non-filtered chamber was 27.5 g.m-3 and 6.5 g.m-3; in the filtered chambers (P<0.001). Females exposed to air pollution previously and during the first days of pregnancy showed borderline differences in the development of the compartments of the IS on day 8 of gestation: volume of the endometrium is reduced as well as the IS total volume. When we evaluated the total number of uNk cells (8ºdpc), we observed that there is a decrease in those animals exposed to air pollution The number of mast cells (6º dpc) are also reduced when compared to females exposed to filtered air, but as pregnancy progress this differences in the number of mast cells disappear. The histopathological evaluation was observe the decrease in the volume occupied by decidual and trophoblast cells, and increased the volume of glands, showing development delay in implantation sites in 8dpc.Our data confirm results from previous studies that link exposure to AP and decreased fertility and increased implantation failure. Evaluation of the uterine morphology and the number of mast call and uNkcell suggests that components present in AP interfere or impair embryonic implantation trought changes in maternal immune system responses

Em termos de importância no processo de invasão de tumores e metástases, o desequilíbrio entre a expressão das gelatinases e seus inibidores teciduais resulta em degradação do principal constituinte da matriz extracelular, o colágeno tipo IV, favorecendo os processos de invasão tumoral e metástase. Foram avaliadas 53 lesões de 47 cães submetidos a cirurgia excisional com confirmação diagnóstica de mastocitoma por avaliação histopatológica e imuno-histoquímica para MMP-2, MMP- 9, TIMP-2 e TIMP-1. Os mastocitomas foram classificados em graus I (bem diferenciados), II (moderadamente diferenciados) a III (pouco diferenciados), segundo os critérios estabelecidos por Patnaik, Ehler e Macewen (1984) e as mesmas lesões foram classificadas em alto e baixo grau de malignidade de acordo com o sistema proposto por Kiupel et al. (2011). Os resultados obtidos através da imuno-positividade de mastócitos e leucócitos polimorfonucleraes para as quatro proteínas foram comparados as duas graduações estabelecidas, bem como ao tempo de sobrevida pós-cirúrgica e a mortalidade em função do tumor. Verificou-se que as metaloenzimas e seus inibidores não demonstram nenhum tipo de associação de expressão entre elas. Dentre os marcadores imuno-histoquímicos, a expressão mastocitária de TIMP-1 apresentou associação com o tempo de sobrevida, independente do tratamento quimioterápico adjuvante. Quanto menor a expressão de TIMP-1 nos mastócitos, maiores as taxas de mortalidade e menores os períodos de sobrevida. Enquanto que MMP-2, MMP-9 e TIMP-2 não foram indicadores prognósticos para esta neoplasia. Assim, nossos resultados mostram que o TIMP-1 apresentou-se como bom indicador de sobrevida e, portanto, sugere-se que a utilização deste índice prognóstico adicional a classificação histológica pode aumentar a precisão de prognostico dos mastocitomas em cães auxiliando na adequação de terapias apropriadas / In terms of importance regarding to tumor invasion process and metastasis , the imbalance between the expression of gelatinases and their tissue inhibitors results in degradation of the main constituent of the extracellular matrix, collagen type IV, favoring the processes of tumor invasion and metastasis. Fifty-three lesions, of 47 dogs submitted to surgical excision confirmed the diagnosis of mast cell tumor were evaluated by histopathologic examination and immunohistochemistry for MMP-2, MMP-9, TIMP-2 and TIMP-1. Mast cell tumors were histologically classified as grades I (well differentiated), II (moderately differentiated) to III (poorly differentiated), according to the criteria established by Patnaik, Ehler and MacEwen (1984) and the same lesions were classified as high and low-grade of malignancy according to the system proposed by Kiupel et al. (2011). The results obtained by the immunopositivity of mast cells and polymorphonuclear leukocytes to the four proteins were compared with two established graduations, as well as to post-surgical survival times and mortality due to mast cell disease. It could be verified that metalloenzymes and their inhibitors do not establish any association between the expression of the cell types evaluated. Within the immunohistochemical markers, mast cell expression of TIMP-1 was associated with survival time, independent of adjuvant chemotherapy. The lower the expression of TIMP-1 in mast cells, higher the mortality rates and smaller post-cirurgical survival time. While MMP-2, MMP-9 and TIMP-2 levels were not prognostic indicators for this cancer. Thus, our results show that the TIMP-1 appeared as a good indicator of survival time and therefore it is suggested that the use of this prognostic index as additional histological classification can increase the accuracy of prognosis of mast cell tumors in dogs assisting in the adaptation of appropriate therapies

La mastocytose est une maladie hétérogène, caractérisée par une accumulation de mastocytes dans l’organisme. Les enfants et les adultes ont des mutations différentes de c-Kit. Dans un premier travail, nous avons montré que seules les formes adultes sont associées à la réactivation de la télomérase, alors que les formes pédiatriques ne sont pas. Cela semble être lié aux différences de mutations de c-Kit observées entre adultes et enfants et pourrait expliquer pourquoi seules les formes pédiatriques de mastocytose régressent spontanément et non les formes adultes. Ces résultats aident à mieux comprendre la physiopathologie de la mastocytose. Dans un second travail, nous a étudié le lien entre la longueur des télomères et les troubles psychologiques des adultes atteints de mastocytose. Nous avons montré que réactions émotionnelles négatives sont corrélées au raccourcissement de la longueur des télomères des leucocytes et que l’érosion télomérique est fortement prédite par les défauts de régulation des émotions. Nous émettons l'hypothèse qu’au cours des troubles neuropsychologiques, le mastocyte pourrait être impliqué dans le raccourcissement de la longueur des télomères en périphérie. / Mastocytosis is a heterogeneous disease characterized by an accumulation of mast cells. Children and adults hold different c-Kit mutations. In a first work, we showed that only adult forms are associated with reactivation of telomerase whereas pediatric forms are not. This seems to be linked to the differential c-Kit mutations observed between adults and children and could explain why only pediatric mastocytosis spontaneously regress in comparison with adult forms. These results help to better elucidate the pathophysiology of mastocytosis. In a second work, we studied the link between the telomere length and the psychological features of adults with mastocytosis and showed that negative emotionality correlated negatively to telomere length and that telomere shortening was strongly predicted by emotion regulation deficits. We hypothesize that in psychological disorders, mast cell may represent the link between brain and periphery and induce telomere shortening.

Myeloid Derived Suppressor Cells (MDSC) represent a significant hurdle to cancer immunotherapy because they dampen anti-tumor cytotoxic T cell responses. Previous studies have reported on the myelo-depletive effects of certain chemotherapies. Using guadecitabine, a next-generation DNA methyltransferase inhibitor (DNMTi), we observed significantly reduced tumor burden in the 4T1 murine mammary carcinoma model. Guadecitabine treatment prevents excessive tumor-induced myeloid proliferation and systemic accumulation, and skews remaining MDSCs toward a beneficial antigen-presenting phenotype. Together, this alters the splenic environment to improve T cell activation and interferon-gamma (IFNg) production. Additionally, guadecitabine enhances the therapeutic effect of adoptively transferred antigen-experienced lymphocytes to diminish tumor growth and improve overall survival. Based on these findings, the immune-modulatory effects of guadecitabine can help rescue the anti-tumor immune response and could contribute to the overall effectiveness of current cancer immunotherapies. Allergies and asthma are common ailments that are on the rise around the world. Mast cells play a direct role in the signs and symptoms characteristic in allergic patients. The family of A Disintegrin And Metalloproteinases (ADAMs) are involved in regulating many cellular processes by cleaving surface receptors, ligands, and signaling molecules. We sought to determine the role of ADAM17 in mast cell activity. In studies using ADAM17-deficient mast cells, percent degranulation and cytokines released by IgE-mediated activation were significantly reduced. Interestingly, ionomycin-activation was unchanged, suggesting ADAM17 may be involved in IgE-mediated mast cell activation upstream of calcium release. Additionally, ADAM17MC-/- mice showed protection from IgE-, but not histamine-, mediated passive systemic anaphylaxis (PSA). The underlying mechanism behind the reduced degranulation occurs through signaling deficiencies downstream of Lyn phosphorylation. Together, the data suggest that ADAM17 is required for proper mast cell signaling through its interaction with the Src family kinase, Lyn.

<p>It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease.</p><p>The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (<6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma. </p>

<p>Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. </p><p>In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an <i>in vivo</i> substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.</p>

It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease. The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (&lt;6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.

Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an in vivo substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.