<p> The Notch signalling pathway is a lateral inhibition pathway that serves to limit the number of cells in a proneural cluster (a group of equipotent cells) that will adopt a neural cell fate during neurogenesis in Drosophila. The proper segregation of neural and epidermal progenitor cells during neurogenesis requires the expression of both the proneural genes and the neurogenic genes. Expression of proneural genes, such as achaete, gives cells the potential to commit to a neural cell fate. The neurogenic genes encode proteins that act in the Notch signalling cascade and are required for cell fate determination during Drosophila neurogenests. Notch and Delta are neurogenic genes that encode large transmembrane proteins. Interaction between the extracellular domains of Notch and Delta is thought to transmit a signal to the nucleus by way of the DNAbinding Suppressor of Hairless protein. In response to Notch activation Suppressor of Hairless is translocated to the nucleus where it activates the transcription ofthe neurogenic genes ofthe Enhancer of split complex (E(spl)-C). The products of the E(spl)-C are bHLH transcription factors. They possess a Cterminal tryptophan-arginine-proline-tryptophan (WRPW) motif that interacts with the product of another neurogenic gene, groucho. The groucho gene product encodes a protein containing a WD40 repeat element. When bound to Groucho, E(spl) bHLH proteins are able to repress transcription of proneural genes, such as achaete, thereby directing the cell to adopt a non-neural cell fate.</p> <p> A number of murine groucho homologues have been identified and named Grg's (Groucho related genes). Three full length Grg proteins have been identified which contain all five domains found in the Drosophila Groucho protein. Two short Grg proteins have also been identified which only contain one of the domains found in the full-length Grg proteins. A number of murine homologues of the Drosophila E(spl)-C have also been identified and named Hes (Hairy/Enhancer of split) proteins. Like the gene products of the Drosophila E(spl)-C, the Hes proteins are bHLH proteins containing a C-terminal WRPW motif. One of the Hes proteins, Hes3, is lacking a basic domain and therefore lacks the DNA-binding activity possessed by the other Hes proteins. </p> <p> Attempts were made to detect interactions between Grg and Hes proteins using co-immunoprecipitation techniques. The anti-WD40 antibody, which recognizes the long WD40-containing Grg proteins, was able to specifically immunoprecipitate 35S-labelled Grgl . This antibody was also able to recognize WD40-containing Grg proteins present in Pl9 cell extracts. However, attempts to co-immunoprecipitate radiolabelled Hesl and AMLlb proteins with Grg proteins present in P19 cell extract were unsuccessful due to the low affinity of the antiWD40 antibody and the background caused by the binding of the test proteins to Sepharose. A second method of co-immunoprecipitation was attempted using an HA-tagged Grgl fusion protein and a commercially available anti-HA antibody. The attempt to co-immunoprecipitate 35S-labelled Hesl with radiolabelled HAtagged Grg 1 was unsuccessful due to a high degree of background caused by Hesl binding to protein G Agarose. Using the Yeast Two-Hybrid interaction assay, the WD40-containing Grg proteins, Grgl and Grg4, were found to interact with Hesl. However, using the same assay WD40-containing Grg proteins were found not to interact with Hes3, which lacks DNA-binding activity. A Western blot was performed to determine if the Hes3 fusion proteins were being expressed in transformed yeast but none were detected. This may have been due to the poor affinity of the anti-GAL4 activation domain antibody. A similar Western blot demonstrated that the Grg proteins, fused to the GAL4 DNA binding domain, were being expressed in transformed yeast extract. The WD40-containing Grg proteins, Grgl and Grg4, were also found not to interact with AMLlb, a protein which contains a C-terminal VWRPY domain which is reminiscent of the Cterminal WRPW interaction domain found in Hes proteins and Drosophila E(spl) proteins. However, WD40-containing Grg proteins were able to interact with an AML 1 b mutant in which the VWRPY motif was mutated to VWRPW in the Yeast Two Hybrid assay. </p> / Thesis / Master of Science (MSc)

Submitted by ANA PAULA BARBOSA RODRIGUES DE FREITAS null (anapaula_b22@yahoo.com.br) on 2016-08-02T23:34:18Z No. of bitstreams: 1 Tese Doutorado - Ana Paula Barbosa Rodrigues de Freitas Final.pdf: 2474072 bytes, checksum: 89629b7295e607d20073a144561101a1 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-05T13:02:51Z (GMT) No. of bitstreams: 1 freitas_apbr_dr_guara.pdf: 2474072 bytes, checksum: 89629b7295e607d20073a144561101a1 (MD5) / Made available in DSpace on 2016-08-05T13:02:51Z (GMT). No. of bitstreams: 1 freitas_apbr_dr_guara.pdf: 2474072 bytes, checksum: 89629b7295e607d20073a144561101a1 (MD5) Previous issue date: 2016-07-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste trabalho foi a aplicação de técnicas de otimização envolvendo múltiplas respostas em problemas da área ambiental, mais especificamente no tratamento de efluentes fenólicos. O efluente é oriundo da Brasquip Ambiental, que é uma empresa de engenharia e prestação de serviços. A otimização foi realizada pelos métodos GRG, Desirability, Simulação Monte Carlo Estocástica. Neste trabalho o efluente foi degradado por meio de processos alternativos; os Processos Oxidativos Avançados. No estudo desenvolvido, as variáveis respostas utilizadas foram: % remoção de Carbono Orgânico Total, Demanda Química de Oxigênio e Fenóis Totais. A função Compromisse Programming foi aplicada como um método de aglutinação, sendo também, comparada com os resultados obtidos pelos métodos Desirabilty. As variáveis de decisão, ou variáveis entrada, obtidas na Simulação Monte Carlo Estocástico, nos métodos Desirability e GRG foram validados experimentalmente. Logo, as remoções obtidas para o Arranjo Ortogonal de Taguchi e do Método de Superfície de Resposta (MSR) foram significativas para as variáveis respostas obtidas, mas a remoção de fenóis totais foi a mais significativa. No estudo estocástico, os valores experimentais obtidos do Taguchi e do MSR também apresentaram-se relevantes para a mineralização do efluente fenólico. Os resultados obtidos experimentalmente da Simulação Monte Carlo Estocástica apresentaram próximos dos obtidos pelo software, sendo que o algoritmo utilizado foi o Optquest. A função CP apresentou valores relevantes para a degradação do efluente, sendo que para este trabalho a Simulação Monte Carlo Estocástica e GRG foram mais significativos se comparado com método Desirability. / The objective of this work was the application of multiple optimization techniques involving responses from environmental problems, specifically in the treatment of phenolic wastewater. The effluent comes from the Environmental Brasquip, which is an engineering company and services. The optimization was performed by the methods GRG, Desirability, Monte Carlo Simulation Stochastic. In this work the effluent was degraded by alternative processes; the Advanced Oxidation Processes. In the study, the variables used responses were:% removal of Total Organic Carbon, Chemical Oxygen Demand and Total Phenols. The Compromisse Programming function has been applied as an agglutination method, also being compared with the results obtained by Desirabilty methods. The decision variables, or variables input obtained in Monte Carlo simulation Stochastic in Desirability methods and GRG were validated experimentally. So removals obtained for Orthogonal Arrangement Taguchi and Response Surface Method (RSM) were significant to the variable responses obtained, but the removal of total phenols was the most significant. In stochastic study, the experimental values and the Taguchi MSR also showed themselves relevant for the mineralization of the phenolic effluent. The experimental results of Monte Carlo Simulation of Stochastic presented near obtained by software, whereas the algorithm used was OptQuest. The CP function showed significant values for degradation of effluent, and for this work to Monte Carlo Simulation Stochastic and GRG were more significant compared with Desirability method.