CD81 interacting proteins

Fertilization is a multistep and complex process culminating in the merge of gamete membranes, cytoplasmic unity and fusion of genome. CD81 is a tetraspanin protein that participates in sperm-oocyte interaction, being present at the oocyte surface. CD81 has also been implicated in other biological processes, however its specific function and molecular mechanisms of action remain to be elucidated. The interaction between CD81 and its binding partner proteins may underlie the CD81 involvement in a variety of cellular processes and modulate CD81/interactors specific functions. Interestingly, in a Yeast two Hybrid system previously performed in our lab, CD81 has emerged as a putative interactor of the Amyloid Precursor Protein (APP). In the work here described, bioinformatics analyses of CD81 interacting proteins were performed and the retrieved information used to construct a protein-protein interaction network, as well as to perform Gene Ontology enrichment analyses. CD81 expression was further evaluated in CHO, GC-1 and SH-SY5Y cell lines, and in human sperm cells. Additionally, its subcellular localization was analyzed in sperm cells and in the neuronal-like SH-SY5Y cell line. Subsequently, coimmunoprecipitation assays were performed in CHO and SH-SY5Y cells to attempt to prove the physical interaction between CD81 and APP. A functional interaction between these two proteins was accessed thought the analyses of the effects of CD81 overexpression on APP levels. A co-localization analysis of CD81 and some interactors proteins retrieved from the bioinformatics analyses, such as APP, AKT1 and cytoskeleton-related proteins, was also performed in sperm cells and in SH-SY5Y cells. The effects of CD81 in cytoskeleton remodeling was evaluated in SH-SY5Y cells through monitoring the effects of CD81 overexpression in actin and tubulin levels, and analyzing the colocalization between overexpressed CD81 and F-actin. Our results showed that CD81 is expressed in all cell lines tested, and also provided the first evidence of the presence of CD81 in human sperm cells. CD81 immunoreactivity was predominantly detected in the sperm head, including the acrosome membrane, and in the midpiece, where it co-localized with APP, as well as in the post-acrosomal region. Furthermore, CD81 co-localizes with APP in the plasma membrane and in cellular projections in SH-SY5Y cells, where CD81 overexpression has an influence on APP levels, also visible in CHO cells. The analysis of CD81 interacting proteins such as AKT1 and cytoskeletonrelated proteins showed that CD81 is involved in a variety of pathways that may underlie cytoskeleton remodeling events, related to processes such as sperm motility, cell migration and neuritogenesis. These results deepen our understanding on the functions of CD81 and some of its interactors in sperm and neuronal cells.

APP RERMS enriched matrix as an adhesion substrate for neuritic outgrowth

Specific domains can determine protein structural functional relationships. For the Alzheimer’s Amyloid Precursor Protein (APP) several domains have been described, both in its intracellular and extracellular fragments. Many functions have been attributed to APP including an important role in cell adhesion and cell to cell recognition. This places APP at key biological responses, including synaptic transmission. To fulfil these functions, extracellular domains take on added significance. The APP extracellular domain RERMS is in fact a likely candidate to be involved in the aforementioned physiological processes. A multidisciplinary approach was employed to address the role of RERMS. The peptide RERMS was crosslinked to PEG (Polyethylene glycol) and the reaction validated by FTIR (Fourier transform infrared spectrometry). FTIR proved to be the most efficient at validating this reaction because it requires only a drop of sample, and it gives information about the reactions occurred in a mixture. The data obtained consist in an infrared spectra of the sample, where peaks positions give information about the structure of the molecules, and the intensity of peaks is related to the concentration of the molecules. Subsequently substrates of PEG impregnated with RERMS were prepared and SH-SY5Y (human neuroblastoma cell line) cells were plated and differentiated on the latter. Several morphological alterations were clearly evident. The RERMS peptide provoked cells to take on a flatter appearance and the cytoskeletal architecture changed, with the appearance of stress fibres, a clear indicator of actin reorganization. Given that focal adhesions play a key role in determining cellular structure the latter were directly investigated. Focal adhesion kinase (FAK) is one of the most highly expressed proteins in the CNS (central nervous system) during development. It has been described to be crucial for radial migration of neurons. FAK can be localized in growth cones and mediated the response to attractive and repulsive cues during migration. One of the mechanisms by which FAK becomes active is by auto phosphorylation at tyrosine 397. It became clearly evident that in the presence of the RERMS peptide pFAK staining at focal adhesions intensified and more focal adhesions became apparent. Furthermore speckled structures in the nucleus, putatively corresponding to increased expression activity, also increased with RERMS. Taken together these results indicate that the RERMS domain in APP plays a critical role in determining cellular physiological responses. Here is suggested a model by which RERMS domain is recognized by integrins and mediate intracellular responses involving FAK, talin, actin filaments and vinculin. This mechanism probably is responsible for mediating cell adhesion and neurite outgrowth on neurons.

Fucana ativa via das Map quinases e inibe a proliferação de células de ovário de hamster chinês (CHO)

Fucan is a term used to denominate L-fucose rich sulfated polysaccharides. The fucans have been studied due their pharmacological activities like antithrombotic, antiproliferative and antioxidant. We have extracted three fucan fractions from the brown seaweed Spatoglossum schröederi. These fucans were denominated Fuc B 1, Fuc B 1.5 and Fuc B 2. The chemical analyzes show that the fucans have very similar composition as demonstrated by agarose electrophoresis gel, sugar and sulfate content. The antiproliferative effect was determined by MTT and BrdU methodologies in CHO cells. The inhibition of proliferation effect of the three fractions was about 40%. Therefore this we proceed just with the Fuc B 2 due the higher yield. There is no apoptosis indication using the anexin V/propidium iodide test. We found a cell cycle phase G1 arrest. The western blotting show that the PKC; pFAK; pERK 1/2 are activated when the cells were treated with fucans. The treatement with inhibitor of MAPK PD98059 extinguished the fucan effect. These results indicates that fucan act by the ERK pathway inducing the cell death.

Estudo do estresse oxidativo e expressão da aromatase e receptor relacionado ao estrógeno gama na placenta humana

 Introdução: Durante a gravidez, devido ao aumento da exigência metabólica placentária há um aumento na produção das espécies reativas de oxigénio (ROS) que podem causar, por exemplo, oxidação de ácidos graxos poli-insaturados na placenta, além disso, nesta fase ocorre um aumento na expressão da aromatase e do receptor relacionado ao estrógeno gama (ERRgama) na placenta humana. Objetivo: O objetivo do estudo foi avaliar os parâmetros de estresse oxidativo e imunomarcação da aromatase e do ERRgama na placenta humana. Métodologia: A capacidade antioxidante total (ACAP), atividade da glutamato cisteína ligase (GCL), concentração glutationa (GSH), peroxidação lipídica e imunomarcação da aromatase e do ERRgama foram analisados em tecido placentário de 58 parturientes. Estas análises foram relacionadas com os dados socio-demográficos das participantes. Resultados: Os recém-nascidos de mães fumantes nasceram com menor peso (p=0,001). A concentração de GSH diminuiu a produção da peroxidação lipídica (p<0,05), por outro lado, a atividade de GCL teve efeito oposto (p<0,001). Encontramos uma diminuição na capacidade antioxidante total e aumento da peroxidação lipídica (p<0,05) na placenta. A placenta de mães fumantes tinham menos marcação da aromatase (p=0,037) já, as mães mais velhas tiveram menos marcação do ERRgama (p=0,009) na placenta. A GSH teve efeito positivo na imunomarcação de ERRgama (p=0,001). Conclusões: A expressão da aromatase e do ERRgamma na placenta são alterados tanto por fatores exógenos, tais como o fumo do cigarro, como por fatores endógenos, tais como a concentração de GSH e a idade da mãe. Os marcadores de estresse oxidativo na placenta são mais elevados em mães mais velhas e em placenta com menor capacidade antioxidante total. 

Exploração de escalonamentos óptimos na gestão de exames médicos

Uma das áreas de aplicação da optimização é a Engenharia Biomédica, pois a optimização intervém no estudo de próteses e implantes, na reconstrução tomográfica, na mecânica experimental, entre outras aplicações. Este projecto tem como principal objectivo a criação de um novo programa de marcação de exames médicos a fim de minimizar o tempo de espera na realização dos mesmos. É efectuada uma breve referência à teoria da optimização bem como à optimização linear e não-linear, aos algoritmos genéticos, que foram usados para a realização deste trabalho. É também apresentado um caso de estudo, formulado como um problema de optimização não linear com restrições. Com este estudo verificou-se que o escalonamento de exames médicos nunca poderá ser optimizado a 100por cento devido à quantidade de variáveis existentes, sendo que algumas delas não são passíveis de prever com antecedência.

Preparo de amostras citológicas de efusões, lavados (broncoalveolar e peritoneal) e líquor pelo método “plasma-tromboplastina”

Dissertação (mestrado)—Universidade de Brasilia, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.

Estudo da maturação eritróide com base no CD35 e no CD44 em síndromes mielodisplásicas

As Síndromes Mielodisplásicas (SMD) representam um grupo de doenças clonais da célula pluripotencial hematopoiética caracterizadas por hematopoiese ineficaz e aumento do risco de progressão para Leucemia Mieloblástica Aguda. Considerando que a linha eritróide está muitas vezes afectada em SMD e a avaliação da displasia desta linha representa um desafio na análise imunofenotípica de medulas mielodisplásicas, o objectivo deste trabalho foi estudar a expressão de CD44 e de CD35 na maturação eritróide normal e a forma como essa expressão é afectada em SMD. Este estudo foi efectuado em 16 amostras de medula óssea (MO) normais/reactivas e em 48 amostras de MO de indivíduos diagnosticados com SMD e sem tratamento. De acordo, com a Organização Mundial de Sáude (OMS) os doentes com SMD foram classificados em CRDM (n=20), AREB-1 (n=15) e AREB-2 (n=13) e de acordo com o International Prognostic Scoring System (IPSS) em Baixo risco (n=5), Intermédio-1 (n=8) e Intermédio-2 e Alto risco (n=14). A caracterização imunofenotípica foi efectuada através de um protocolo de imunofluorescência directa de marcação-lise-lavagem, utilizando os anticorpos monoclonais: anti-CD34; anti-HLA-DR; anti-CD117; anti-CD44; anti-CD35; anti-CD45, anti-CD123 e anti-CD133. Para a análise, procedeu-se à identificação e quantificação das células de linha eritróide em MO total e nos diferentes estádios de maturação e à avaliação da expressão de CD44 e de CD35. Os resultados deste estudo revelaram o CD35 como um marcador precoce na diferenciação eritróide normal e um aumento da expressão de CD44 e da percentagem de eritroblastos em SMD, sendo este aumento mais evidenciado nos estádios mais avançados da doença.

Síntese verde de nanopartículas de prata a partir de extrato aquoso do tubérculo de Curcuma longa associadas à quitosana e avaliação da atividade antitumoral in vitro em câncer de pele não melanoma (linhagem A431)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação Nanociência e Nanobiotecnologia, 2016.

Otimização da caracterização de fibras musculares estriadas em cão (Canis familiaris) por histoquímica e imunohistoquímica com potencial aplicação ao diagnóstico

Dissertação de Mestrado Integrado em Medicina Veterinária

Estudo morfológico e imunohistoquímico da expressão de PCNA e p63 no carcinoma espinocelular oral em gatos

Dissertação de Mestrado Integrado em Medicina Veterinária

Epigenetics and alternative splicing

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Contribution to the study of prognostic factors in canine oral squamous cell carcinoma

Tese de Doutoramento em Ciências Veterinárias, na Especialidade de Clínica

Caracterização química e biológica de peptídeos com propriedades antiproliferativas isolados da secreção cutânea do anuro Hypsiboas raniceps (Cope, 1862)

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2016.

Toxicidade in vitro e in vivo de quantum dots de carbono recobertos com boronato

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2016.

Projeto raia de fogo: aspectos populacionais da raia de fogo (Dasyatis marianae Gomes, Rosa & Gadig, 2000) e pesca de elasmobrânquios no complexo recifal do Parracho de Maracajaú

The species Dasyatis marianae inhabits coastal areas associated with coral reefs, considered endemic to the northeast of Brazil, occurring from the State of Maranhão to the south of Bahia. Specimens of this species are commonly sighted by divers and fishermen in the area of Maracajaú reefs, a complex reef that is part of the Environmental Protection Area of Coral Reefs (EPACR), which was developed in this study about the ecology and biology of the D. marianae, in order to characterize aspects of population structure in the area of the reef complex of Parracho de Maracajaú. We analyzed 120 specimens caught by artisanal fishing site of the size, weight, sex, stage of maturity and stomach contents. Most subjects were adult males (1.7:1) and was more abundant for rays with lengths between 25 and 29cm of LD, where females reach larger sizes, a feature common to other rays. The largest specimens were captured in the area of seagrass, which is preferred for the species. The distribution of species in the area showed an ontogenetic and sexual segregation, where the youthful occur near the beach, which is a likely area for nursery and growth of the adult females prevail in the seagrass, which apparently has a high prey availability, and Adult males are more distant, a higher proportion occurring in outlying areas, suggesting a habit more exploratory than the females. The evaluation of the reproductive system indicated 3 reproductive cycles per year, one young per pregnancy, and showed that the mature males were smaller than females. The cubs of D. marianae size at birth 12 to 15cm LD. As for diet, the species was characterized as carnivorous crustacean specialist. The performance of visual censuses in different localities allowed to evaluate the density of D. marianae in different environments of the complex. The species occurs in greater numbers in seagrass, environment very important for the conservation of the species. 100 individuals of D. marianae marked in reef complex area enrolled in a recapture rate of 3%. Some behavioral aspects were evaluated, as diurnal pattern of activity, interaction with cleaning and fish Pomacanthus paru followers as Lutjanus analis and Carangoides bartholomaei. Overall, much of the information obtained should be used for management of the species