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Made available in DSpace on 2016-12-08T16:24:14Z (GMT). No. of bitstreams: 1 PGCA13MA112.pdf: 807586 bytes, checksum: b293744892841538c2ea9e0a5ae8325a (MD5) Previous issue date: 2013-07-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The in vitro production of pig embryos progressed significantly in the last decade. However, the embryo growing rates remain variable and with low embryonic quality. The chromosomal abnormalities as a result of high polyspermy rates and the inefficient culture systems are pointed as the main problems. Thus, the aim of this study was to improve the rates of monospermy, and also the hatching rates of in vitro produced porcine embryos. The first study investigated decreasing sperm concentrations combined with different incubation periods during in vitro fertilization (IVF). A total of 829 oocytes were matured and allocated in two experiments. In the first, oocytes were IVF using different sperm concentrations (62.5, 125, 187.5 or 250 x 103 sperm / mL). In the second experiment the oocytes were IVF using 62.5 x 103 sperm / mL pointed in experiment 1, for 2, 3 or 4 hours incubation time. Then the zygotes were cultured during twelve hours to evaluate the fertilization parameters, or during seven days to evaluate embryonic development. A third experiment was performed to evaluate the sperm acrosome reaction according to the incubation period. The higher sperm concentration reduced embryo production from 33.9% to 13.4%. The reduction of the incubation period from 4 to 3 or 2 hours significantly increased the rates of cleavage (42.1 to 71.6% and 73.3%), embryo development (14 to 34.7 and 38.9%) and hatching (25 to 66.7 and 65.7%. Increased sperm concentration and incubation period significantly altered the parameters related to fertilization, substantially impairing the monospermic embryos production. The number of sperm with reacted acrosome increased significantly as incubation time was increased. In the second study we evaluated the assisted hatching of porcine embryos IVP by zona pellucid incision or treatment with pronase. Oocytes were matured and in vitro fertilized with 62.5 x 103 spermatozoa / mL concentration for three hours. The zygotes were cultured for seven days, and then embryonic development and hatching rates evaluated. It was also determined the cell density and cell apoptosis rates of hatched embryos. Both the manual incision or pronase digestion were effective in weakening the ZP of embryos with high cell density. However, it was observed a reduction in the survival rates, and higher apoptosis rates in hatched embryos treated with Pronase. With basis on the data we concluded that a reduction in sperm concentration and incubation period decreases the incidence of polyspermy and improves the embryo development rates. This is probably due to reduction of capacitated /reacted sperm available for fertilization. Also, the weakening of the zona pellucid by incision improves significantly the number and quality of embryos hatched at the end of embryo culture / A produção in vitro de embriões suínos avançou significativamente na última década. Entretanto, os índices de desenvolvimento embrionário continuam inconstantes e a qualidade dos embriões extremamente baixa. A incidência de anormalidades cromossômicas decorrentes das altas taxas de polispermia e a ineficiência dos sistemas de cultivo são apontados como os principais impasses. Desta forma, este estudo buscou melhorar os índices de monospermia, e as taxas de eclosão dos embriões suínos produzidos in vitro. O primeiro estudo investigou a utilização de diferentes concentrações espermáticas e períodos de incubação dos gametas durante a fecundação in vitro (FIV). Para tanto, um total de 829 oócitos foram maturados e destinados à dois experimentos. No primeiro, os oócitos foram FIV utilizando diferentes concentrações espermáticas (62.5, 125, 187.5 ou 250 x 103 espermatozoides/mL). No segundo, os oócitos foram FIV utilizando 62.5 x 103 espermatozoides/mL durante duas, três ou quatro horas. Após a FIV os zigotos foram cultivados por doze horas para análise dos parâmetros da fertilização ou por sete dias para o estudo do desenvolvimento embrionário. Um terceiro experimento avaliou o número de espermatozóides com acrossoma reagido de acordo com o período de incubação. A máxima concentração de espermatozóides reduziu a produção embrionária (de 33.9% para 13.4%). Já a redução do período de incubação de quatro para três ou duas horas, aumentou significativamente as taxas de clivagem (42.1% para 71.6 e 73.3%), de desenvolvimento embrionário (14% para 34.7 e 38.9%) e de eclosão (25% para 66.7 e 65.7%). Tanto o aumento da concentração espermática, como o aumento período de incubação alteraram significativamente a maioria dos parâmetros relacionados a fecundação, prejudicando substancialmente a produção de embriões monospérmicos. O número de espermatozoides com acrossoma reagido aumentou significativamente com o pronlongamento da fecundação. No segundo estudo, investigou-se a indução da eclosão assistida de embriões suínos PIV através da incisão da zona pelúcida ou pelo tratamento com pronase. Os oócitos foram maturados e fecundados in vitro com uma concentração de 62.5 x 103 espermatozóides/mL por três horas. Os zigotos foram cultivados durante sete dias, então, avaliados quanto ao desenvolvimento embrionário e a incidência de eclosão. Determinamos também a densidade celular e os índices de apoptose celular dos embriões eclodidos. Tanto a incisão da zona como a digestão com pronase foram efetivos na fragilização da ZP dos embriões com alta densidade celular. Todavia, constatou-se uma redução da sobrevivência e aumento do índice de apoptose celular dos embriões eclodidos pelo tratamento com pronase. Com base nos dados obtidos concluímos que a redução da concentração de espermatozóides e do período de incubação diminuem a incidência de polispermia e melhoram o desenvolvimento embrionário. Isto é provavelmente devido a redução da disponibilidade de espermatozóides capacitados/reagidos para a fecundação. Da mesma forma, a fragilização da zona pelúcida através da incisão, melhora significativamente o número e a qualidade dos embriões eclodidos ao final do cultivo embrionário

Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans ce travail, nous nous sommes intéressés au développement de différents biocapteurs se basant sur l'immobilisation d'enzymes ou de bactéries sur des microélectrodes en vue d’une détection électrochimique. Nous avons montré les potentialités d’application de deux biocapteurs conductimétriques à base de protéinase K ou de protéinase K et de pronase à la détection des modifications de conformation de la myoglobine et de l’albumine de sérum bovin au cours de leur relargage à partir de microsphères de poly (ε-caprolactone). Nous avons également mis au point un biocapteur conductimétrique à base de catalase et d’alcool oxydase pour une détection rapide et sensible des alcools ainsi que deux biocapteurs à catalase pour la détection impédimétrique et conductimétrique du cyanure et l’étude des interactions catalase-cyanure. Nous avons enfin élaboré des biocapteurs bactériens à base de Pseudomonas putida F1 pour la détection du trichloroéthylène dans les eaux souterraines. Pour cela, une voie originale d’immobilisation des cellules, basée sur la fonctionnalisation du transducteur à l’aide de couches autoassemblées et d’anticorps, ainsi que l’utilisation de nanotubes de carbone, a été explorée / The development of biosensors is an expanding research area. Indeed, biosensors are rapid, selective and cost-effective analytical tools which find applications in various fields (environment, health, food,…). They are constituted of a sensitive biological element (antibody, enzyme, microorganism, DNA…), which can selectively recognize one analyte or a group of analytes, associated to an electrochemical, optical or thermal transducer. In this work, we developed different biosensors based on enzymes or bacteria immobilised onto microelectrodes in view of electrochemical detection. First, we demonstrated the potentialities of two conductometric biosensors based on proteinase K or proteinase K and pronase for the detection of myoglobin and bovine serum albumine conformation changes during their release from poly (ε-caprolactone) microspheres. Then, we elaborated a bi-enzymatic conductometric biosensor with catalase and alcohol oxidase as sensing elements, for a rapid and sensitive detection of alcohols. Catalase impedimetric and conductometric biosensors were also developed for cyanide detection and used for the study of catalase-cyanide interactions. Finally, we prepared Pseudomonas putida F1 whole cell biosensors for the determination of trichloroethylene in groundwaters. For that, an original route, including the functionalisation of the transducer with a self-assembled-monolayer and antibodies, and the use of single-wall carbon nanotubes, was investigated for cell immobilisation

The first project in this dissertation describes the total synthesis of yaku’amide A analogues. Natural product yaku’amide A possesses potent anticancer activity and exhibits a novel mode of action. However, due to its complex asymmetrical isoleucine dehydroamino acids, the synthesis of this polypeptide poses a unique challenge. Despite the efficient synthesis developed in our lab, the total synthesis of this natural product remains lengthy. In order to simplify the overall synthesis, symmetrical dehydroamino acids were incorporated to replace the dehydroisoleucine residues yielding two analogues of yaku’amide A that closely resembles the conformation of the natural product. Biological testing of the simplified analogues disclosed similar potency to that of yaku’amide A. The second part of this dissertation focuses on the influence that dehydroamino acids have on secondary structures. Peptides have an important role in medicine despite their limitations due to poor bioavailability and stability. Therapeutic use of peptides can be enhanced by designing new strategies to improve the proteolytic stability of these compounds. Attempts to increase peptide stability using trisubstituted and tetrasubstituted dehydroamino acids (ΔAAs) have been reported. Similarly, modified ΔAAs should also help tune the electronic and steric properties of peptides, while improving proteolytic stability. However, studies of peptides containing modified ΔAAs and are scarce. This project describes the synthesis and studies of incipient 310 helical tetrapeptides containing dehydroamino acids. Bulky and cyclic ΔAAs were demonstrated to alter the conformation of these tetrapeptides and impart greater stability against proteolysis and thiol additions. We believe these results can be a powerful tool to design peptide drug candidates with high proteolytic resistance and stability. tetrasubstituted dehydroamino acids (ΔAAs) have been reported. Similarly, modified ΔAAs should also help tune the electronic and steric properties of peptides, while improving proteolytic stability. However, studies of peptides containing modified ΔAAs and are scarce. This project describes the synthesis and studies of incipient 310 helical tetrapeptides containing dehydroamino acids. Bulky and cyclic ΔAAs were demonstrated to alter the conformation of these tetrapeptides and impart greater stability against proteolysis and thiol additions. We believe these results can be a powerful tool to design peptide drug candidates with high proteolytic resistance and stability.