DC - Teses de Doutoramento
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- Estudo da função do gene Delta4 na regulação da angiogénesePublication . Benedito, Rui Miguel Santos; Duarte, António José de Freitas; Costa, Luís Filipe Lopes daO sistema vascular é um dos mais importantes nos mamíferos, sendo o primeiro a tornar-se funcional durante o desenvolvimento embrionário. A sua formação requer uma coordenação complexa de diversos mecanismos genéticos e celulares. O estudo destes mecanismos é de grande importância para o desenvolvimento de novas terapias que pretendam inibir ou promover a formação de novos vasos sanguíneos (neoangiogénese). A via de sinalização Notch está envolvida na diferenciação de uma grande variedade de tipos celulares. Esta tese descreve um trabalho de investigação com o objectivo de caracterizar a função de um novo ligando da via Notch (Delta4), no desenvolvimento vascular em mamíferos. Para isso recorreu-se à análise de uma linha de ratinhos com inactivação de um ou dos dois alelos do gene Delta4. A caracterização fenotípica destes mutantes permitiu descobrir que o gene é essencial para a diferenciação e desenvolvimento dos vasos arteriais e que uma diminuição da sinalização Notch induzida por este ligando aumenta a sensibilidade das células endoteliais a estímulos angiogénicos. No seu conjunto os resultados mostram que o gene Delta4 é fundamental para o desenvolvimento normal dos vasos sanguíneos, podendo ter também um papel importante na neoangiogénese do adulto, pelo que constitui um alvo terapêutico a explorar.
- Estudo da regulação da circulação ocular num modelo de olho isolado de coelhoPublication . Delgado, Esmeralda Sofia da Costa; Carvalho, Luís Filipe Sobral da Silva; Luís, José Paulo Pacheco SalesA circulação ocular compreende dois compartimentos: o retiniano possui receptores autonómicos e é autoregulado, enquanto que o coroideu possui inervação adrenérgica e existe controvérsia acerca da presença de autoregulação. Nesta tese criámos e optimizámos um modelo experimental de olho isolado de coelho para ajudar a clarificar os mecanismos de controlo locais da circulação ocular independentemente de outras interferências presentes num modelo in vivo. Surpreendentemente, antes da administração de qualquer fármaco, observámos respostas de vasomotricidade intrínseca em todos os modelos experimentais. Validámos o modelo utilizando fármacos adrenérgicos de efeitos conhecidos. A injecção de fenilefrina (α1-adrenérgico) desencadeou uma resposta vasoconstritora e diminuiu a frequência e aumentou a amplitude das oscilações enquanto que a prazozina (α1-bloqueador), a fentolamina (α1,2 -bloqueador) e o labetalol, (αβ-bloqueador) provocaram vasodilatação e diminuíram a intensidade das oscilações de vasomotricidade intrínseca. Analisámos as variações de pressão decorrentes da injecção intraarterial de outros neurotransmissores: a Endotelina-1, o L-NAME (antagonista do Óxido Nítrico) e o Neuropéptido Y induziram uma resposta vasoconstritora e aumento da frequência e amplitude das oscilações enquanto que a L-Arginina (precursor do Óxido Nítrico), o Péptido Vasointestinal e a amlodipina (bloqueador dos canais de cálcio) desencadearam os efeitos opostos. Estes resultados abrem novas perspectivas para a manipulação terapêutica das doenças oculares isquémicas como sejam a retinopatia diabética e o glaucoma
- Pain evaluation and control after routine interventions in cattlePublication . Stilwell, George Thomas; Lima, Miguel Luís Mendes Saraiva; Broom, Donald MauriceDisbudding and castration are two routine interventions in cattle practice. Both can cause severe pain and cause poor welfare. Through plasma cortisol levels and behaviour evaluation we measured pain caused by different disbudding and castration methods. We also studied the efficacy of several anaesthesia and analgesia protocols. The main conclusions are: - Cortisol together with behaviour assessment is very useful in detecting calves in pain. - Certain behaviours are only shown by very young calves. - Vocalization should not be used as a sign of pain in calves. - Scoop disbudding causes long term pain and local anaesthesia is not efficient. - Hot-iron disbudding causes severe pain during the procedure but does not differ from paste disbudding in the next hours. Local anaesthesia plus analgesia does reduce pain cause by these methods. - Xylazine causes an increase in cortisol even if pain is not induced. - Pain caused by clamp-castration lasts for at least 48 hours and is only controlled by long acting analgesics. - Surgical castration causes intense pain but shorter if two incisions are made instead of just one.
- Relações anatomotopográficas cranioencefálicas para procedimentos neurocirúrgicos intracranianos em canídeos. A ultra-sonografia como técnica de neuronavegação para cirurgia em tempo realPublication . Carreira, Luis Miguel Alves; Ferreira, António José Almeida; Burilo, Fernando ListeA cirurgia intracraniana não é ainda uma prática corrente em Medicina Veterinária, apresentando limitações associadas à variabilidade morfológica craniana dos doentes, à dificuldade da técnica de intervenção cirúrgica inerente às estruturas envolvidas, ao acesso a sofisticados meios de diagnóstico e constituição de equipas especializadas. Um sólido conhecimento sobre as relações anatomotopográficas cranioencefálicas que apresentem repetibilidade, permite ao neurocirurgião desenhar mentalmente as afinidades existentes entre as estruturas superficiais e as mais profundas, assumindo ainda as que se interpõem ao longo da trajectória cirúrgica traçada, melhorando a orientação topográfica pré e intra-cirurgica em cada doente, diminuindo em muito os riscos da cirurgia e aumentando a probabilidade de êxito. O presente trabalho desenvolvido em 69 crânios e respectivos 138 hemisférios cerebrais (direito e esquerdo) considerando canídeos do tipo braquicéfalos, dolicocéfalos e mesacéfalos, permitiu concluir que o que os parece diferenciar mais é o esqueleto facial de cada um, mais do que a caixa craniana, já que as relações anatomotopográficas cranioencefálicas estudadas apresentaram uma repetibilidade evidente para os pontos craniométricos, suturas cranianas e estruturas/pontos encefálicos seleccionados, quanto à sua localização (distância e posição) em todos eles.
- Estudo de gliomas cerebrais no cão, padrões imagiológicos, estudo das mutações de p53, expressão dos receptores do factor de crescimento epidérmico (EGFR), e marcadores imunohistoquímicosPublication . Jesus, Sandra de Oliveira Tavares de Sousa; Ferreira, António José Almeida; Duarte, António José de FreitasO objectivo deste trabalho foi estudar os padrões imagiológicos em TAC dos tumores cerebrais do cão, com particular interesse nos gliomas, assim como tentar determinar o estatuto dos genes p53 e EGFR nestes tumores, realizando estudos moleculares e de imunohistoquímica. Foram realizadas TACs crânio-encefálicas em 105 cães, tendo-se confirmado em todos a presença de lesão intracraniana. O Boxer foi a raça mais representada com 51,4%, a média de idades foi de 9,44 anos e o sintoma mais frequente foi epilepsia (62,7%). 43,8% dos gliomas eram hipodensos, 62,5% captaram contraste, destes 60% captaram contraste em anel, e 80% revelaram desvio da foice do cérebro. Dos 26 tumores classificados por histopatologia, 61,5% foram gliomas e 26,9% meningiomas. O estudo das mutações do gene p53 foi realizado em 19 casos, incluindo 12 gliomas e 6 meningiomas, não tendo sido detectada qualquer mutação. A expressão relativa do gene EGFR, avaliada por PCR em tempo-real, revelou ser significativa em meningiomas, oligodendrogliomas e astrocitomas (p<0,05) e em gliomas (p<0,01). O estudo imunohistoquímico foi realizado em 24 casos, incluindo 16 gliomas e 8 meningiomas, não tendo sido encontradas diferenças significativas entre as marcações de gliomas e meningiomas para Ki-67 e EGFR.
- Contribution to the staging and immunological study of equine recurrent airway obstruction (RAO)Publication . Tilley, Paula Alexandra Botelho Garcia de Andrade Pimenta; Luís, José Paulo Pacheco Sales; Ferreira, Manuel BrancoRecurrent airway obstruction (RAO) is one of the most frequent lung diseases in horses and is similar to human asthma. We characterized equine RAO differential diagnosis (DD) in horses with long term cough and established a parallel between the DD in equine RAO and in human asthma. We correlated clinical, endoscopic, thoracic X-ray and bronchoalveolar lavage fluid scores in horses with RAO to establish relevance of each factor for the characterization of RAO stages in order to suggest a staging method. Cardiovascular effects of RAO were assessed. We also evaluated the response to skin prick tests (SPT) and in vitro allergy tests with common aeroallergens in horses with RAO and characterized RAO in Portugal by identifying relevant allergic factors. The importance of a thorough diagnosis is emphasized, including BAL and respiratory endoscopy, and a DD parallel is made with vocal cord dysfunction in man. A score model for the characterization of RAO stages is suggested. The first ECG and EcoCG values for Lusitano/Lusitano-cross horses are published with subtle changes in the RAO group. In this highly selected population immediate aeroallergen hypersensitivity was significant, allergy being a probable aetiopathogenic mechanism in all RAO group horses.
- Embryo-maternal interactions leading to embryonic development and survival in the bovine : role of progesterone and prostaglandinsPublication . Torres, Ana Catarina Belejo Mora; Costa, Luís Filipe Lopes daThe objectives of this thesis were to evaluate steroidogenic and prostanoid embryo-maternal interactions leading to embryonic development and survival in cattle, and to evaluate therapeutic strategies at embryo transfer (ET) designed to enhance embryo survival. In vitro experiments (three experimental chapters) - bovine early (Day 7) embryos i) had transcription of genes coding for enzymes progesterone (P4) and of prostaglandins (PGs) synthesis pathways (StAR, P450scc,3β-HSD, PTGS2, PGFS, PGES); ii) produced these mediators (P4, PGF2α, PGE2) into culture medium; iii) had a significant increase in transcription levels of the above genes (except StAR) associated to first embryonic cellular differentiation; iv) derived from pre-pubertal oocyte donors had transcription levels of the above genes similar to those of embryos derived from post-pubertal cyclic heifers (except for 3β-HSD, which tended to be higher in embryos from cyclic heifers). Additionally, v) in a developed luteal cells (LC) co-culture model, LC induced an embryotrophic effect, significantly increasing blastocyst yield and quality; however, this embryotrophic effect was not associated with an increase in embryonic gene transcription or production of P4, PGF2α, PGE2; vi) Embryos co-cultured with LC did not exert a luteotrophic effect upon the cells; and vii) oil overlaying of culture wells exerted an embryotrophic effect, but absorbed P4 from culture medium. In vivo experiments (two experimental chapters) - novel in-vivo models considering poor developmental competence embryos (demi-embryos) and either sub-normal fertility recipients (lactating high-yielding dairy cow) or high fertility recipients (virgin dairy heifers) were used to evaluate the effect of hCG and carprofen treatment at embryo transfer on embryo survival and on plasma P4 and PSPB concentrations of recipients. Conclusions were that: i) treatment with hCG induced formation of secondary CL, increased plasma P4 concentrations, survival rate of demi-embryos and pregnancy rate of recipients (only in cows). Embryos were rescued beyond maternal recognition of pregnancy (MPR), but later embryonic survival, growth until implantation and placental PSPB secretion until Day 63 of pregnancy (only tested in cows) were not affected; ii) embryonic survival following MRP is not under direct dependency of maternal P4 concentrations; iii) treatment with carprofen had no significant effect on plasma P4 concentrations and embryonic survival, but decreased the luteotrophic effect of hCG.
- Notch signaling, genital remodeling and reproductive functionPublication . Murta, Daniel José de Moura Carita Dinis; Costa, Luís Filipe Lopes da; Duarte, António José de FreitasThe objective of this thesis is to evaluate the association between Notch signaling, genital cellular remodeling and reproductive function, in the mouse model. Five experimental chapters are included in this thesis. In experiments 1 and 2, we evaluated transcription and expression patterns of Notch component and effector genes in testis post-natal development, along the spermatogenic cycle and the epididymis. In experiment 3, we evaluated the male reproductive phenotype of in vivo Notch blockade by DAPT. In experiments 4 and 5 we evaluated transcription and expression patterns of Notch component and effector genes in the ovary, oviduct and uterus along the estrous cycle. Results indicate that Notch signaling is active and associated to male and female genital cellular remodeling. In the male, results prompt for a regulatory role of Notch signaling in spermatogonia pool maintenance, onset of spermatogenesis, in the pace of the spermatogenic cycle, germ cell identity, and epididymis spermatozoa maturation. In the female, results prompt for a regulatory role in ovarian follicle and corpus luteum development, and oviduct and uterus epithelial cell turnover and function. Notch signaling is operating in the testis and ovarian cellular interstitium, and in luminal and glandular epithelia of genital tract, probably regulating intercellular communication.
- Recombinant feline interferon omega therapy in cats naturally infected with Feline Immunodeficiency Virus : clinical, viral and immunological relevancePublication . Leal, Rodolfo Assis Oliveira; Gil, Solange Judite Coelho Alves; Niza, Maria Manuela Grave Rodeia Espada; Tavares, Luís Manuel MorgadoType-I Interferons are well-known cytokines which among their main functions are key components of the host immune response against viral infections. Due to its immune modulation properties, they are commonly used in the therapeutic approach of various diseases such as retroviral infections. Recombinant feline interferon omega (rFeIFN-ω) is the first interferon licensed for use in veterinary medicine. Although it is commonly administered in retroviral infections, namely in Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infected cats, few studies reported its clinical benefits and mechanisms of action. This thesis aims to clarify the main properties of the licensed rFeIFN-ω protocol (3 cycles of 5 daily subcutaneous administrations of 1MU/kg beginning on days 0, 14 and 60) in naturally retroviral infected cats living in an animal shelter, evaluating its effect not only on clinical improvement but also on concurrent viral excretion, viremia/proviral load and various immune biomarkers such as acute phase proteins and cytokine profile. Recognizing the non specific and subtle clinical presentation of the majority of FIV-infected cats, this work also presents and evaluates an alternative oral rFeIFN-ω protocol (0.1MU/cat during 90 days) to be used in client-owned FIV-infected cats. Results showed that the licensed rFeIFN-ω protocol induces a significant clinical improvement, with a concurrent reduction of opportunistic viral infections and an increase on acute phase proteins (APP) profile. The alternative protocol also revealed an important clinical improvement but without significant changes on opportunistic viral infections (which were of low level in the tested group) or on APP profile. In both protocols, no changes were remarked on viremia neither on T-helper 1/T-helper 2 cytokine profiles meaning that this compound may lack an anti-viral activity for retroviruses in vivo and do not act on the acquired immune response of FIV-positive cats. However, there was a significant reduction of the interleukin-6 plasma levels (pro-inflammatory cytokine) in cats treated with the licensed protocol and a decrease on its mRNA expression in cats treated orally. This shows that rFeIFN-ω can have anti-inflammatory properties, which are more evident in the higher doses of the licensed protocol. More than contributing for a better knowledge of rFeIFN-ω, this thesis explores its immune modulation properties and validates a new oral protocol which can be included on future FIV-guidelines.
- Structure and function relationships in novel cohesin-dockerin complexesPublication . Cameron, Kate; Fontes, Carlos Mendes Godinho de Andrade; Najmudin, ShabirCohesin-dockerin interactions orchestrate the assembly of carbohydrate degrading multi-enzyme complexes produced by anaerobic bacteria termed cellulosomes. Type I dockerins typically display a dual binding mode which has been suggested to allow increased flexibility for cellulosome assembly. In contrast, structural work on type II dockerins suggests that they display a single binding mode. In this work structure function studies were developed in the cellulosomal systems of Clostridium thermocellum, Bacteroides cellulosolvens, and Acetivibrio cellulolyticus. The data provides novel structural and dynamic insights into the mechanism of substrate recognition by cellulosomes (Chapter 2). In addition, to understand the mechanism of cellulosome assembly in more elaborate cellulosomal systems, structural studies of novel type I and type II cohesin-dockerin complexes of B. cellulosolvens and A. cellulolyticus were developed. The crystal structure of a type I cohesin from B. cellulosolvens cell surface anchoring scaffoldin ScaB is reported (Chapter 3). This type I cohesin is highly similar to the type I cohesins from C. thermocellum and C. cellulolyticum and its cognate type I dockerin displays a dual binding mode. In Chapter 4, the structure of the type II X-dockerin from A. cellulolyticus in complex with a type II adaptor cohesin in two distinct orientations is described. The dockerin displays structural symmetry which is reflected by the presence of two essentially identical cohesin binding surfaces, suggesting that flexibility modulated by the dockerin dual binding mode is extended to type II complexes. In Chapter 5, the structure of a A. cellulolyticus type I cohesin-dockerin complex involved in cell surface attachment is described. Typical of type I cohesin-dockerin interactions this dockerin displays a dual binding mode, with a complex interface much more extensive than that observed in other type I complexes, resulting in an extremely tight interaction (Ka ~ 1012 M). Furthermore, data reveal that residues located at dockerin positions 12, 14 and 19 modulate the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. In conclusion, this work demonstrates the importance of the dockerin dual binding mode to incorporate additional flexibility to cellulosome assembly and also polycellulosome assembly and cell surface attachment.