Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-09-01T13:05:01Z No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-15T13:46:23Z (GMT) No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-15T13:46:23Z (GMT). No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioides spp. are thermodymorphic fungi that when inhaled by humans, these conidia find a favorable environment, changing to the yeast phase and becoming pathogenic causing paracoccidioidomycosis (PCM), one of the most prevalent systemic mycoses in Brazil. Some antifungals are used in the treatment of PCM. Treatment depends on the patient's progression and tolerability of each drug, but their treatment may be for long periods and cause various side effects in the patient. The chiquimate pathway is coordinated by 7 enzymes that perform consecutive steps to convert erythrose-4-phosphate and phosphoenol pyruvate (PEP) into chorismate. In microorganisms, this pathway is involved in the production of the amino acids phenylalanine, tyrosine and tryptophan; These amino acids are essential to the maintenance of these organisms. In this work, pGEX4T3 vector cloning and heterologous expression of Pb18 EPSP synthase belonging to the chiquimate pathway were performed. This protein was expressed in E. coli (DE3) strain and purified. Antibodies were produced for expression analysis of the protein in Western blot. The modeling of EPSP synthase was performed aiming to identify the amino acids involved in the active site. The pull down-GST assay with soluble Pb18 proteins allowed the identification of 40 proteins that interact with EPSP synthase. These proteins belong to different functional categories, which are involved with the availability of phosphoenol pyruvate, the substrate necessary for the functioning of the chiquimate pathway. / Paracoccidioides spp. são fungos termodimórficos que ao serem inalados pelo ser humano, esses conídios encontram um ambiente propício, mudando para a fase de levedura e tornando-se patogênico causando a paracoccidioidomicose (PCM), umas das micoses sistêmicas de maior prevalência no Brasil. Alguns antifúngicos são empregados no tratamento da PCM. O tratamento depende do avanço da doença e da capacidade de tolerância do paciente a cada medicamento, mas o seu tratamento pode ser por longos períodos e causando diversos efeitos colaterais no paciente. A via do chiquimato é coordenada pela ação de 7 enzimas que realizam passos consecutivos para transformar a eritrose-4-fosfato e fosfoenol piruvato (PEP) em corismato. Em micro-organismos, esta via está envolvida com a produção dos aminoácidos fenilalanina, tirosina e triptofano; estes aminoácidos são essenciais à manutenção desses organismos. Neste trabalho foi realizado a clonagem em vetor pGEX4T3 e expressão heteróloga da EPSP–sintase de Pb18 pertencente à via do chiquimato. Essa proteína foi expressa em linhagem E. coli (DE3) e purificada. Os anticorpos foram produzidos para análise da expressão da proteína em Western blot. A modelagem da EPSP-sintase foi realizada visando identificar os aminoácidos envolvidos no sítio ativo. O ensaio de pull down-GST com proteínas solúveis de Pb18 possibilitou a identificação de 40 proteínas que interagem com EPSP-sintase. Essas proteínas pertencem a diferentes categorias funcionais, as quais estão envolvidas com a disponibilidade de fosfoenol piruvato, substrato necessário para o funcionamento da via do chiquimato.

<p>Dendritic spines are mushroom-shaped appendages on the dendritic branches of neurons. They are invaluable to the function of the brain as they form the major site for excitatory signal transmission in the mammalian brain. These ubiquitous structures have several invaluable and unique characteristics – namely that their morphological and functional characteristics are activity-dependent and undergo remodeling as the spine experiences stimulation. This activity-dependent regulation then in turn modulates the excitatory postsynaptic potential that propagates into the adjacent parent dendrite, and which ultimately reaches the somatic compartment. The mediation of this modulatory effect on the postsynaptic signal by dendritic spines renders them invaluable to the brain’s ability to change neuronal circuits as it learns. The relationship between the structural and functional change in dendritic spines as plasticity is induced remains poorly understood; while efforts have been made to examine the morphology of dendritic spines during plasticity as well as the change to receptor insertion on the postsynaptic density, a comprehensive methodology to interrogate the concomitant changes to several aspects of dendritic spine structure and function as plasticity occurs has not been established. In this study, such a methodology was developed in order to facilitate future study of how a dendritic spine’s diffusional neck resistance, head volume, calcium-sensitive channels (on the postsynaptic density), and excitatory postsynaptic potential amplitude change concurrently as the spine undergoes activity-dependent regulation. This activity-dependent regulation also occurs in groups of spines called “clusters” <em>in vivo</em>, and the structural and functional dynamics of spines as these groups are formed also remains unknown. In order to to facilitate future <em>in vivo</em> studies on how clustered dendritic spines may change dynamically in both structure and function, a methodology for surgically accessing and recording calcium-based activity from the primary auditory cortex was developed, as the frequency-specific tuning of dendritic spines in this cortical area forms a compelling environment in which to study the relationship between spine form and function. </p>