No description available.

No description available.

Objective markers to identify higher fertility individuals are needed to maximize livestock breeding success. Two heparin-binding proteins, which are reflective of fertility in bulls, have been biochemically identified as fertility-associated antigen (FAA) and tissue inhibitor of metalloproteinases-2 (TIMP-2). These four studies were designed to examine the importance of those proteins in relation to reproduction in bulls and other livestock species. In the first study, indirect immuno-fluorescent microscopy was performed to localize FAA and TIMP-2 to livestock sperm. FAA was localized on spermatozoal acrosomes of bulls and rams, but no cross-reactivity was observed for stallions. TIMP-2 labeling was observed on acrosomes and posterior heads, which was species dependent. Localization patterns for FAA and TIMP-2 were further investigated during heparin-induced capacitation and acrosome reactions of bovine sperm. In study two, an enzyme-linked immunosorbent assay (ELISA) was developed to determine concentrations of FAA in bovine seminal plasma (SP). A commercially available TIMP-2 ELISA was utilized to quantify TIMP-2. Respective mean concentrations of FAA and TIMP-2 in SP were 6.661.487 ug/ml and 1.180.045 mg/ml. Concentrations of FAA in SP did not correspond to bull fertility potential, however, older bulls with higher concentrations of TIMP-2 in SP sired more calves. The third study evaluated utility of an amplified fragment length polymorphism with bovine TIMP-2 gene specific primers to amplify a 700 bp genomic DNA (gDNA) product from sperm. From 53 bulls screened, 22.6% were negative for the 700 bp amplicon. There was a three-fold likelihood for 700 bp negative bulls to not sire a calf compared to 700 bp positive bulls. The product was cloned and sequenced, but no homology to TIMP-2 was detected. Therefore, the product represented novel bovine gDNA sequence. The fourth study identified an equine homologue to the bovine FAA gene. Immuno-based diagnostics had not detected FAA in stallion semen. The equine DNA homologue was 88.5% identical in nucleotide and 86% in amino acid sequences to bovine FAA. Subtle differences in the amino acid sequence are likely responsible for the inability to detect FAA in stallion semen with FAA antibodies to bovine FAA.

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No description available.

Made available in DSpace on 2014-07-29T15:07:41Z (GMT). No. of bitstreams: 1 Dissertacao_Denise C Toledo.pdf: 1395118 bytes, checksum: 592dff1a4d2ff80089be7e8691c93893 (MD5) Previous issue date: 2012-03-08 / The canine gland has drawn interest for research due to its similarities with the human prostate and the great incidence of lesions. Moreover, the canine prostate shows high incidence of diseases. The main lesions that affect the prostate are prostatitis, benign prostatic hyperplasia (BPH), cysts and adenocarcinoma. Recently attention has been given to lesions considered premalignant such as prostatic intraepithelial neoplasia (PIN) and proliferative inflammatory atrophy (PIA), both studied in the human gland and also found in the canine prostate. In order to evaluate the development of prostate cancer starting as premalignant lesions, some immunohistochemical markers are employed, such as tissue inhibitors of metalloproteinases (TIMP), which have a key role in regulating the catalytic action of metalloproteinases (MMP), and transforming growth factor-β (TGF-β), that induces angiogenesis and inhibits cell proliferation and is considered a mediator of prostate growth. The objective of this study was to investigate the expression of TIMP-1, TIMP-2 and TGF-β in canine normal prostate tissue and with proliferative lesions. For this, 150 adult canine prostates were obtained from postmortem examinations. After microscopic evaluation 54 glands, compatible with normal, epithelial BPH, stromal BPH, PIA, PIN and adenocarcinoma were selected and used to make tissue microarray block (Tissue Microarray - TMA). TMA slides were subjected to immunohistochemistry with anti-TIMP-1, anti-TIMP-2 and anti-TGF-β, to assess staining intensity of epithelial cells and stromal cells.Cytoplasmatic staining of canine prostate cells by TIMP-1, TIMP-2 and TGF-β was observed, with TIMP-1 and TIMP-2 being more expressed in premalignant and malignant lesions, while TGF-β was expressed mainly by normal tissue and BPH. Furthermore, there were differences in the expression between epithelial and stromal cells. / A próstata canina, além das similaridades com a próstata humana, apresenta grande incidência de afecções. As principais lesões que acometem a próstata são as prostatites, a hiperplasia prostática benigna (HPB), os cistos e o adenocarcinoma, sendo que, recentemente, se tem dado atenção às lesões consideradas pré-malignas, como a neoplasia intraepitelial prostática (PIN) e a atrofia inflamatória proliferativa (PIA), ambas estudadas na glândula humana, e também verificadas na próstata do cão. Para avaliar o desenvolvimento de neoplasias prostáticas a partir das lesões pré-malignas, alguns marcadores imunoistoquímicos são empregados, como os inibidores teciduais de metaloproteinases (TIMP), que apresentam importante função na regulação da ação catalítica das metaloproteinases (MMP), e o fator de crescimento transformador β (TGF-β), que induz a angiogênese e inibe a proliferação celular, sendo considerado um mediador do crescimento prostático. O objetivo deste estudo foi verificar a expressão de TIMP-1, TIMP-2 e TGF-β no tecido prostático canino normal e com lesões proliferativas. Para isso foram colhidas, em exames necroscópicos, 150 próstatas de cães adultos e idosos. O material foi avaliado histologicamente e selecionadas amostras de 54 próstatas com predominância de histomorfologia normal, HPB epitelial, HPB estromal, PIA, PIN e adenocarcinoma, que foram utilizadas para a confecção de um bloco de microarranjo tecidual (Tissue Microarray - TMA). As lâminas de TMA foram submetidas à imunoistoquímica com os anticorpos anti-TIMP-1, anti-TIMP-2 e anti-TGF-β, sendo avaliada a intensidade de marcação das células epiteliais e estromais. Verificou-se que há marcação citoplasmática das células prostáticas caninas para TIMP-1, TIMP-2 e TGF-β, sendo as proteínas TIMP-1 e TIMP-2 mais expressas nas lesões proliferativas pré-malignas e malignas, enquanto TGF-β foi expresso principalmente pelo tecido normal e com HPB epitelial e estromal. Ainda, houve diferença de marcação entre células epiteliais e estromais.

Η HARP (Heparin Affin Regulatory Peptide) είναι ένας αυξητικός παράγοντας με Μ.Β. 18 kDa που ανήκει στην οικογένεια των αυξητικών παραγόντων που έχουν συγγένεια με την ηπαρίνη. Eμπλέκεται στην ανάπτυξη των νευριτών, την επούλωση πληγών και φαίνεται να παίζει σημαντικό επαγωγικό ρόλο στις διαδικασίες της ογκογένεσης, καθώς επάγει την αγγειογένεση και εμφανίζεται σε υψηλές συγκεντρώσεις τόσο σε καρκινικούς ιστούς, όσο και σε κυτταρικές σειρές καρκινικών κυττάρων. Στο πλαίσιο μελέτης της σχέσης δομής/δράσης του αυξητικού αυτού παράγοντα, χρησιμοποιούνται τόσο συνθετικά πεπτίδια, όσο και ανασυνδυασμένες τροποποιημένες μορφές του αυξητικού αυτού παράγοντα. Σε φυσιολογικές συνθήκες, η εκκρινόμμενη HARP πέπτεται από ένζυμα του κυτταρικού μικροπεριβάλλοντος και προκύπτουν πεπτίδια που παρουσιάζουν βιολογικές δράσεις παρόμοιες ή και αντίθετες από αυτές της HARP. Φαίνεται λοιπόν πως η δράση του αυξητικού αυτού παράγοντα ρυθμίζεται τόσο στο επίπεδο βιοσύνθεσης και έκκρισης, όσο και από τη δράση ενζύμων του εξωκυττάριου χώρου. Στην παρούσα εργασία μελετήθηκε η δράση ενός συνθετικού πεπτιδίου το οποίο αντιστοιχεί στα αμινοξέα 65-97 που εντοπίζονται στην ΤSR περιοχή προς το καρβοξυτελικό άκρο της HARP. Με δεδομένο ότι τι πεπτίδιο αυτό εμφανίζει αντιαγγειογενετική δράση, πραγματοποιήθηκαν χρονοεξαρτώμενα και δοσοεξαρτώμενα πειράματα, με σκοπό τη μελέτη της δράσης του στον πολλαπλασιασμό, τη μετανάστευση και την επούλωση πληγών. Στο πλαίσιο αυτών των μελετών, ελέγξαμε τη δράση του στην έκφραση των μεταλλοπρωτεϊνασών ΜΜP-2 και ΜΜP-9, των αναστολέων τους ΤΙMP-1 και ΤΙMP-2 καθώς και του κολλαγόνου και της ελαστίνης σε πρωτογενείς καλλιέργειες ενδοθηλιακών κυττάρων από ομφάλιο λώρο (HUVEC cells). Τα αποτελέσματα έδειξαν πως το συνθετικό αυτό πεπτίδιο καταστέλλει τον πολλαπλασιασμό, την μετανάστευση αλλά και την επούλωση πλήγών των κυττάρων HUVEC με δοδοεξαρτώμενο και στατιστικώς σημαντικό τρόπο. Επιπλέον από τα πειράματά μας δεν παρατηρήθηκε μεταβολή στα πρωτεϊνικά επίπεδα έκφρασης των μεταλλοπρωτεϊνασών ΜΜP-2 και ΜΜP-9 καθώς και των αναστολέων τους ΤΙMP-1 και ΤΙMP-2. Ωστόσο, παρατηρήθηκε στατιστικώς σημαντική μεταβολή στα επίπεδα γονιδιακής έκφρασης των αναστολέων ΤΙMP-1 και ΤΙMP-2 όπως επίσης και της ελαστίνης και του κολλαγόνου IV. / Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. HARP was originally described as a neurite outgrowth promoting molecule, which appears to increases during recovery from injury and is thought to be involved in angiogenesis expression, playing a major role in the cell growth and differentiation that are associated with regeneration in several tissues. HARP is expressed in several human tumors and tumor cell lines and is also indicated in high serum levels of patients with different types of cancer. HARP contains two random coiled clusters of basic residues (N- and C-terminal) and two b-sheet domain. Each b-sheet domain contains a thrombospondin repeat I (TSR-I) motif, which have been suggested to be responsible for the interaction of HARP with heparin. Our project is based on C-TSR-I domain, corresponding to amino acids 65–97 of HARP peptide, respectively, required for the neurite outgrowth activity of HARP. In this study, we investigate the impact of C-TSR on basic biological functions of endothelial cells (HUVEC) such as proliferation, migration, the expression of MMP-2 and MMP-9 and their inhibitors (TIMP-1, TIMP-2) that contribute to the ECM remodeling. Time course and dose-response experiments revealed that CTSR reduces proliferation, migration and wound healing, without affecting the protein levels of MMP-2 and MMP-9 and their inhibitors (TIMP-1, TIMP-2). Moreover, CTSR inhibits the expression of TIMP-1 and TIMP-2 contributing to the ECM remodeling. Concluding, HARP could act as pro- or anti-angiogenic factor, depending on the system used and the cell microenvironment.

No description available.

The Drosophila heart is a tubular vessel surrounded by a dynamic scaffold of extracellular matrix (ECM) proteins. Heart development and function rely upon protease-mediated remodelling and turnover of the ECM, and changes in ECM composition correlate with age and cardiac disease. Previous research has shown that a family of proteases called matrix metalloproteinases (MMPs), and their inhibitors (TIMPs), are necessary for normal cardiac cell migration and lumenogenesis. The Drosophila heart expands considerably throughout growth, but the role of MMP activity has not been elucidated at this time. I examine the role of the two Drosophila MMPs, MMP1 and MMP2, as well as TIMP, in defining larval heart structure and ECM protein distribution. I observe heart phenotypes via immunofluorescence labelling and confocal microscopy using loss-of-function mutants, gene over-expression, and gene knock-down techniques. Reduced MMP1 function during embryogenesis correlates with myofibrillar disorganisation, whereas reduced MMP2 function or TIMP over-expression both result in cardia bifida as well as increased density and ectopic localisation of Collagen-IV and Pericardin. Post-embryonic MMP reduction compromises cardiac structural integrity but does not affect Pericardin localisation. Live imaging of the larval heart with optical coherence tomography (OCT) and light microscopy reveals that reduced MMP2 function correlates with decreased heart rate but not impaired dilation or contraction. These data suggest that MMP2 activity during embryogenesis is critical for larval heart development. In contrast, post-embryonic protease function appears to have a less pronounced effect on ECM protein distribution throughout larval development. / Thesis / Master of Science (MS) / The fruit fly (Drosophila) heart undergoes significant changes in organisation and size throughout development and growth. The heart is surrounded and supported by a network of extracellular matrix (ECM) proteins, which is regulated by proteases, including matrix metalloproteinases (MMPs). Previous research has shown that MMPs are required for normal heart formation. I demonstrate that a reduction in MMP activity during embryonic development results in larval heart defects and an increase in the disorganisation of ECM proteins around the heart, whereas reduction during larval development results in less pronounced protein mislocalisation. These findings are corroborated via over-expression of an MMP inhibitor.

A endometriose é uma doença de origem multifatorial, caracterizada por presença de tecido endometrial fora da cavidade uterina, responsável por sintomas álgicos com grande impacto na qualidade de vida da paciente, além de ser um dos principais fatores de infertilidade. Muitos estudos já foram realizados no intuito de explicar a etiopatogenia da endometriose, assim como muito tem sido estudado para encontrar novas estratégias de tratamento. Várias linhas de medicamentos têm sido estudadas com este intuito, agindo em diferentes pontos da etiopatogênese da doença, uma delas na inibição de metaloproteinases da matriz extracelular, que tem papel no remodelamento do mesotélio do peritônio e angiogênese. O objetivo deste estudo foi avaliar a influência de uma droga (doxiciclina) de baixo custo, com ação conhecida na inibição das metaloproteinases, em endometriose peritoneal induzida em ratas. Para isso, foram usadas 30 ratas adultas Wistar com lesão induzida de endometriose, divididas em três grupos, um grupo controle (C, n=10) sem tratamento, um grupo onde foi administrado doxiciclina em baixa dose (BD, n=10) e um grupo onde foi realizado doxiciclina em alta dose (AD, n=10). Foi realizada avaliação da área das lesões de cada rata e estudo imunohistoquímico para positividade de anticorpo primário de metaloproteinase de matriz 9 (MMP9) e de inibidor de metaloproteinase de matriz 2 (TIMP2). A doxiciclina atuou reduzindo a área das lesões nos grupos BD e AD (p=0,0052) em relação ao grupo C e reduzindo a expressão do TIMP2 no grupo AD (p=0,0009) em relação aos grupos BD e C. Não houve resultado significativo na expressão da MMP9. / Endometriosis is a multifactorial origin disease, characterized by the presence of endometrial tissue outside the uterine cavity, responsible for painful symptoms with important impact on the life quality of the patient, besides being one of the main factors of infertility. Many studies have already been carried out to explain the etiopathogenesis of endometriosis, and much has been studied to find new treatment strategies. Several lines of drugs have been studied for this purpose, acting at different points in the etiopathogenesis of the disease, one of them in the inhibition of extracellular matrix metalloproteinases, which plays a role in the remodeling of the peritoneum mesothelium and angiogenesis. The purpose of this study was to evaluate the influence of a low-cost drug (doxycycline), with known action on the inhibition of metalloproteinases, in induced peritoneal endometriosis in rats. Thirty adult Wistar rats with endometriosis-induced lesions were divided into three groups: one untreated control group (C, n = 10), one group receiving low dose doxycycline (BD, n = 10) and a group where high dose doxycycline (AD, n = 10) was performed. An evaluation of the lesion area of each rat and immunohistochemical study for primary antibody to matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase inhibitor 2 (TIMP2) was performed. Doxycycline worked by reducing the area of lesions in the BD and AD groups (p = 0.0052) in relation to the C group and reducing the expression of TIMP2 in the AD group (p = 0.0009) in relation to the BD and C groups. There was no significant effect on MMP9 expression in the present study.