Soft tissue sarcomas (STS) are rare human malignant neoplasms, arising mostly from stem cells within non-skeletal connective tissues. They account for approximately 1% of all human malignancies. Matrix metalloproteinases (MMPs) are enzymes involved in degradation of the extracellular matrix and their expression by cancer cells allows the cells to penetrate basement membranes and tissue matrix, thereby invading and metastasising. The most studied malignant tumours from the perspective of MMP expression and its relationship to malignant behaviour are epithelial-derived carcinomas. MMPs role in invasion and metastasis of sarcomas has been very little investigated. This is in part because of the difficulty in accumulating sufficient tumour tissue to enable statistically relevant analysis of sufficient tumours. The purpose of this thesis was to examine the expression of key MMPs - MMP-2, MMP-7, MMP-9, and MMP-14 and their inhibitors (TIMP-1 and TIMP-2) at the invasive/subcapsular edge of human malignant and benign connective tissue tumours using immunohistochemistry, a technique that allows a very high level of reaction product localisation within tumours. In three different STS types and appropriate benign equivalents, the expression of MMPs -2, -7, -9, and -14 and their inhibitors (TIMPs -1 and -2) were measured using intensity of staining and the percentage area of staining by image analysis. The results were compared between tumour types and against histological grading that is widely used as a prognostic factor. The findings from this research indicated that metalloproteinases were commonly expressed in STS and benign equivalents. There were differences in expression of some benign versus malignant neoplasms of the same group. No uniform pattern of expression of any of MMPs was observed across the tumours, but some of the data, most notably that for expression of MMP-2 and -9 indicate, a role for MMPs in malignant behaviour and some showed (e.g. MMPs -7 and -14) change in expression with the grade of malignant tumours in the same broad category. There is some evidence of an inverse relationship between MMP and appropriate TIMP expression suggesting that a failure of inhibition, as much as increased expression, is a feature of malignancy.

Matrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating lesions in both experimental autoimmune encephalopathy (EAE) and multiple sclerosis patients suggesting its possible role in pathological events associated with autoimmune neurodegenerative processes. In addition, MMP-28 has been linked to modulation of immune response and activation of macrophages which presents another role of this enzyme in autoimmune pathologies. In the study described herein, MMP-28 has been shown to affect myelin composition and appearance, mitochondrial protein content, and vesicular transport proteins. Moreover, the decrease in myelin basic protein quantity observed in healthy MMP-28KO animals affected the myelin staining intensity in various brain regions including corpus callous. Cellular energetic studies did not reveal differences in mitochondrial function in MMP-28KO animals and no difference in reactive oxygen species was observed. In the EAE model, MMP-28 deletion increased the occurrence of atypical form of EAE characterized by increased inflammation of arbor vitae of the brain. In addition, MMP-28 deletion decreased the inflammatory infiltrates present in brains obtained from EAE animals. Lastly, MMP-28 has been shown to affect cellular energetics and activation of bone marrow derived macrophages during the initial stages and after 24 h activation. In addition, MMP-28 deletion increased proinflammatory cytokines and receptors CD86 and iNOS found in M1 polarized macrophages. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

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Bacteroides fragilis is a gram negative, anaerobic rod, that is a member of the normal colonic microflora of most mammals, and it is the anaerobe most commonly isolated from human soft tissue infections. During the past decade, strains of B. fragilis that produce an enterotoxin have been implicated as the cause of diarrhea in a number of animals, including humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (Mr~ 20,600) that causes rapid morphological changes in human colon carcinoma cell lines, particularly, HT-29. This dissertation research began in 1993 with the purpose of determining how this enterotoxin, termed fragilysin, causes diarrhea. The deduced amino acid sequence revealed a signature zinc binding consensus motif (His-Glu-Xx-Xxx-His-Xxx-Xxx-Gly-Xxx-Xxx-His/Met) characteristic of metalloproteinases. Sequence analysis showed close identity with metalloproteinases within the zinc-binding and Met-turn regions. Purified fragilysin contained 1 gram atom of zinc per molecule, and it hydrolyzed a number of proteins, including gelatin. Optimal proteolytic activity occurred at 37° C and pH 6.5. Activity was inhibited by metal chelators but not by inhibitors of other classes of proteinases. When fragilysin is injected into ligated ileal and colonic loops of animals, there is significant tissue damage and a subsequent dose dependent fluid response. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; and, to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to chelated toxin. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). Furthermore, there is a direct effect on the tight junction proteins. Fragilysin appears to cause diarrhea by proteolytically degrading the paracellular barrier of epithelial cells. Fragilysin is a recently discovered virulence factor that could contribute to the pathogenesis of B. fragilis in both intestinal and soft tissue infections. This research was supported by a Public Health Service grants AI 322940 and AI 32940-03 from the National Institute of Allergy and Infectious Diseases, and by the Commonwealth of Virginia project 6127250 / Ph. D.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extracellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMPactivated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents.

Initialement, la vision restreinte des MMPs en tant que de simples « destructeurs » de la matrice extracellulaire avait largement sous-estimé limportance et la diversité de leurs contributions aux différentes étapes de la progression tumorale et de la dissémination métastatique. Ces dernières années, les fonctions complexes des MMPs dans la progression tumorale ont été renforcées par la découverte de nombreux nouveaux substrats dégradés, clivés ou activés par les MMPs, incluant non seulement des composants de la matrice extracellulaire mais aussi des récepteurs, des molécules dadhésion et de nombreuses molécules potentiellement bioactives. La complexité et la diversité de laction des MMPs dans la progression tumorale fournissent certaines explications quant à « léchec thérapeutique » de linhibition des MMPs dans les études cliniques de cancers humains. Une compréhension complète et spécifique de la biologie de chaque MMP est néanmoins requise avant de les considérer à nouveau comme des cibles thérapeutiques contre le cancer ou dautres pathologies. Les travaux réalisés dans le cadre de ce doctorat ont contribué à la compréhension des mécanismes moléculaires impliquant la MT1-MMP dans la croissance et langiogenèse tumorale et ont identifié, pour la première fois, une implication de la MT4-MMP dans la croissance tumorale et la dissémination métastatique du cancer du sein. Malgré sa faible activité de dégradation de la matrice extracellulaire, cette protéase à lien glycosylphosphatidyl inositol pourrait promouvoir la progression tumorale en affectant la structure des vaisseaux sanguins intratumoraux.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Orientador: Jose Mauro Granjeiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T17:09:02Z (GMT). No. of bitstreams: 1 Paiva_KatiuciaBatistadaSilva_D.pdf: 3483379 bytes, checksum: 7c286d954d5465463a66555914cbdafc (MD5) Previous issue date: 2007 / Resumo: O objetivo deste trabalho foi delinear o perfil de expressão temporal e espacial das MMP-2 e -9, TIMP-1 e -2 e RECK durante os eventos de formação de tecidos mineralizados (osso, esmalte e dentina) em camundongos em fase embrionária, recém nascidos e indivíduos adultos através de imunohistoquímica e hibridização in situ. Durante a amelogênese em incisivos de rato adulto, na fase de secreção, as MMPs e RECK foram imunocoradas na região infracelular dos ameloblastos de secreção e RECK foi ainda detectado difuso no citoplasma destas células. MMP-9 foi localizada nas células do retículo estrelado e RECK nas células do epitélio externo. Na fase de transição, detectados uma fraca imunomarcação ao nível da membrana dos ameloblastos de transição para as MMPs e RECK esteve difuso no citoplasma destas células. Na camada papilar, as MMPs e RECK foram imunomarcadas nos macrófagos ou células dendríticas. Do início ao final da fase de maturação, a expressão de RECK foi aumentando nos ameloblastos de maturação e nas células da camada papilar, enquanto que a expressão das MMPs foi diminuindo nestas células. AS TIMPs foram detectadas somente nos ameloblastos na fase de maturação. Nós observamos RECK e a MMP-9 no citoplasma do odontoblastos, provavelmente, no complexo de Golgi ou na rede do retículo endoplasmático rugoso. Durante a ossificação intramembranosa da mandíbula e maxila, os osteoblastos foram imunocorados pelas MMPs, TIMPs e RECK. Na degradação da cartilagem de Meckel, MMPs, TIMPs e RECK foram imunomarcadas nas células do pericôndrio, bem como o mRNA de RECK. Durante a odontogênese, RECK foi imunocorado nas células do epitélio oral migrando ao ecto-mesênquima, na fase de broto, no epitélio interno do órgão do esmalte, na fase de capuz e em odontoblastos e ameloblastos na fase final de campânula. Transcritos de RECK foram localizados em todo o germe dentário na fase de capuz, mais concentrado no nó-do-esmalte secundário, na fase de campânula inicial e nos odontoblastos e ameloblastos, na fase final de campânula. O osso alveolar foi marcado em todos os períodos. Durante a ossificação endocondral, os condrócitos foram imunopositivos para as MMPs, TIMPs e RECK, na fase de diferenciação dos condrócitos (E13). Na fase de molde cartilaginoso (E14) os condrócitos hipertróficos foram imunocorados para as MMPs e RECK. RECK e TIMPs foram também encontradas no pericôndrio. Na fase de invasão vascular e celular (E15), MMPs, TIMPs e RECK foram expressos por células que migram do colar ósseo para o centro da diáfise, bem como por osteoclastos/condroclastos próximos ao septo transverso. Os condrócitos hipertróficos continuam imunocorados. De E16 a PN1, as MMPs, TIMPs e RECK foram expressas por osteoblastos e condrócitos hipertróficos na placa de crescimento e pelas células do periósteo e pericôndrio. Os resultados obtidos apontam para a expressão diferenciada de MMPs, TIMPs e RECK nos diversos eventos estudados, sugerindo que a atividade biológica destas proteínas regula a degradação da matriz extracelular tanto durante o desenvolvimento dos tecidos como sua manutenção. Além disso, pela primeira vez, demonstra-se a expressão de RECK pelas células formadoras de tecido ósseo e dentário. / Abstract: Our objective was to analyse the spatial-temporal distribution of MMP-2, MMP-9, TIMP-1, TIMP-2 and RECK during development of mineralized tissue (bone, enamel, and dentine) in embryos, newborn, and adult mice by immunohistochemistry and in situ hybridization. During rat amelogenesis, at the secretion phase, MMPs and RECK were immunostained in the ameloblast infracelular region and RECK was also detected in the cytoplasm of these cells. MMP-9 was localized in the stellated cells and RECK in the outer enamel epithelium cells. At the transition phase, a weak immunostaining was observed at the ameloblast membranes for MMPs and RECK. RECK was also detected in the cytoplasm of these cells. At the papillary layer, MMPs and RECK have been observed in macrophages and/or dendritic cells. At early and late maturation phases, MMPs and Reck profiles were similar to the transition phase, but the immunostaining was less pronounced. TIMPs were identified exclusively in maturation ameloblasts throughout the maturation phase. We also observed that the cytoplasm of odontoblasts, probably at the Golgi apparatus and/or the RER network were immunostained for Reck and MMP-9. During mandible and maxillae intramembranous ossification, osteoblasts were immunostained for MMPs (early stage), TIMPs (late stage) and RECK. In Meckel cartilage degradation, MMPs, RECK and TIMPs mRNA and protein were found in perichondrial cells. During odontogenesis migrating epithelial cells in bud stage, enamel inner epithelial cells in cap stage, and ameloblasts and odontoblasts in bell stage were immunostained for RECK. Also, RECK mRNA was found difuse in all tooth germ in cap stage, mainly localized in primary enamel knout in early bell stage, and in ameloblasts and odontoblasts in late bell stage. The alveolar bone was immunolabelled in all periods. During endochondral ossification, chondrocytes were immunopositive for MMPs, RECK, and TIMPs during chondrocyte differentiation (E13). At the cartilaginous template (E14), the hypertrophic chondrocytes (HC) were immunostained for MMPs and RECK. RECK and TIMPs immunopositive cells were found in the perichondrium. At the vascular and cellular invasion (E15), MMPs, RECK and TIMPs were expressed by migrating cells from bone collar as well as by osteoclasts/chondroclasts close to the transverse septum. HC remained immunostained. From E16 to PN1, MMPs, TIMPs, and RECK were expressed by osteoblasts and HC in the growth plate and by cells in the perichondrium and periosteum. The results show the differential expression of MMPs, TIMPs, and RECK during the processess studied, sugesting that the biological activity these proteins regulates the MEC degradation and its maintenance in tissue development. Our results show for the first time that RECK is expressed by bone-forming and tooth-forming cells during mouse endochondral and intramembranous and in embryonic and adult odontogenesis, even if these cells are from different embryonic origins. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Abstract To investigate the turnover of type IV collagen in skeletal muscle in conditions where muscle function is impaired, type IV collagen and proteins regulating its degradation were studied during 1, 3 and 7 days of immobilization, 3- and 10-day glucocorticoid treatment and in neuromuscular diseases. In addition, fibrillar type I and III collagens were studied during immobilization and in neuromuscular diseases. The mRNA levels of type I, III and IV collagens were decreased during immobilization and during 10-day dexamethasone treatment. Gene expression and quantity of (pro)MMP-2 was increased during immobilization but decreased during dexamethasone treatment. The expression of TIMP-2 was decreased both during immobilization and dexamethasone treatment. Decreased gene expression and increased degradation caused decreased concentration of type IV collagen, suggesting net degradation of type IV collagen during immobilization. While the gene expression and degradation were decreased during dexamethasone treatment, the amount of type IV collagen was not changed. Dexamethasone thus seemed to slow down the turnover of type IV collagen. Decreased mRNA levels of collagens and prolyl 4-hydroxylase suggest decreased biosynthesis of collagens during immobilization. The mRNA levels of collagens I, III and IV were increased in polyneuropathy and polymyositis. The concentration and staining intensity of type IV collagen was increased in polyneuropathy, as was also the quantity and staining intensity of (pro)MMP-9. The results suggest accumulation of type IV collagen in the basement membranes of muscle cells and capillaries in polyneuropathy muscles. Lengthened position during immobilization partly prevented the atrophy and changes in collagen metabolism in plantarflexors. Endurance running was effective in preventing muscle atrophy during dexamethasone treatment, but exercise did however fail to prevent the changes observed in type IV collagen synthesis and degradation.