Análise comparativa de diferentes metodologias para cultivo de células-tronco mesenquimais derivadas de tecido muscular

Pós-graduação em Biociências - FCLAS

Produção de anticorpos monoclonais murinos dirigidos contra células-tronco mesenquimais de coelho

Mesenchymal stem cells (MSCs) are a heterogeneous population of cells that proliferate in vitro as plastic-adherent cells, have fibroblast-like morphology and can differentiate into bone, cartilage and fat cells. Therapeutic potential of MSCs have been studied in experimental models, such as rabbit, in Laboratory of Cell Engineering of Botucatu. However, no specific markers have been reported for expanded rabbit MSCs, which hampers the isolation of pure MSC populations by immunophenotypic characterization. Thus, the objective of this study was to produce monoclonal antibodies (mAbs) to rabbit MSCs. MSCs derived from rabbit bone marrow (BM) were isolated, cultured, expanded ex vivo, and immunized into three BALB/c mices, and spleen cells subsequently harvested were used to generate hibridoma cell lines secreting antibodies against MSCs. Hybridoma cells were screened by flow cytometry and antibody-producing cells were subjected to subsequent rounds of retests. MSC1-160 obtained the best positivity for IgG expression and was cloned by limiting dilutions and micromanipulation. Ascitic fluid from ten best clones was purified by affinity chromatography in Protein A-sepharose CL-4B column and purification control was performed by electrophoresis in agarose gels. The purified IgG were tested against rabbit MSCs, obtaining high positivity by flow Cytometry. In conclusion, we developed 10 mAbs, MSC1-160 A20, A30, A41, A47, A55, A60, A63, A69, A81, and A82, that recognize rabbit MSC cell surface antigens showing potential for immunophenotypic characterization of rabbit MSC cell lines

Uso de células-tronco para o tratamento de tendinite em eqüinos

Brazil has the fourth largest horse herd in the world, this is due the recognition and appreciation that the different equestrian games are having within the country. Injuries of the tendon, especially in the digital flexor tendon, are the main cause of athletic life reduction among horses. The treatment of tendinitis in horses seeks full recovery of the damage tissue reestablishing the function previously lost, however conventional treatments have proven to be ineffective when considered the quality of the scar tissue and the rate of recurrence. Due to this, the use of adult stem cells to the treatment of musculoskeletal injuries of horses has been studied for some time. This method of treatment consists of aspiration of bone marrow or removal of subcutaneous fat tissue and implantation of these cells in the injured tissue. After obtaining the bone marrow the implantation can be performed with total bone marrow, with the mononuclear fraction of MSC or with cells cultured in vitro. From the fat tissue is used the stromal vascular fraction obtained by collagenase digestion, followed or not by cell culture. According to some studies, cell therapy with material obtained from bone marrow or adipose tissue has shown to be viable, given that these materials are abundant in repair components such as mesenchymal stem cells (MSC), growth factors and other components of the collagen matrix. Several studies using both types of cells have shown great potential and promising clinical results. However, knowledge of the biology and characterization of these cells remain largely unknown, and therefore is needed great care and caution when using stem cells for the treatment of musculoskeletal disorders in horses

Transplante de medula óssea para suporte de células tronco hematopoiéticas em cães acometidos por linfoma

Lymphoma represents the most prevalent hematopoietic malignancy in small animal medicine. It is highly responsible to chemotherapy and therefore several protocols are used as therapeutic tools. For that reason, the bone marrow transplantation, enshrined in human medicine through initial trial in canine patients, has increasingly become the focus of studies in order to make it a reality also in veterinary medicine. First, the treatment with the chosen chemotherapy protocol is made. As complete remission of lymphoma is observed, it must be initiated the bone marrow harvesting. The obtained material is subjected to the processes of erythrocyte depletion, plasma depletion, cryoprotectants addition, total nuclear cells counting, hematopoietic progenitor quantification, analysis of cell viability and freezing. Following that, with radiotherapy or application of cyclophosphamide, the conditioning phase of the patient who is receiving the transplantation is carried out. The bags containing hematopoietic stem cells are then thawed and transplanted into the receptor organism. Support with hematopoietic stem cells allows the use of lethal doses of chemotherapy or radiotherapy and has been shown to considerably raise the disease remission time and survival rate of the canine patients

Percepções de profissionais de uma organização de procura de órgãos sobre a entrevista com familiares de potenciais doadores

In the last twenty years, and more expressively in the past decade, the search for improvement in the organ transplantation system has contributed to the achievement of positive results in the Brazilian statistics related to the survival of transplanted individuals. However, for a transplantation to be performed, a number of phases must be followed by professionals working in organ search services. The phase that generates the greatest tension is undoubtedly that of family interviews, as it involves both the feelings of relatives who are facing a recent loss and those of interviewers who need to know how to deal with families at this time. The goal of this study was to apprehend perceptions of professionals working for an Organ Search Organization concerning interviews with relatives of potential donors. The qualitative methodology was used by adopting the Collective Subject Discourse for data organization. Interviews were conducted with six staff members of an organ search service whose experience as interviewers comprised from eight to fourteen years of work. It was possible to apprehend the professionals’ perceptions of family interviews and identify facilitating and hindering factors in this phase of the donation process. Such factors mainly involve: contact with the teams assisting patients, the difficult moment that families are experiencing and the teams’ as well as the institution’s preparation for rendering this type of service. The balance between the offer of and demand for organs in Brazil will only be achieved by means of an efficient and effective structure in transplantation organ search services and increased donation consent rates to be obtained by adequate communication between interviewers and relatives

Fluxo institucional de resíduos de equipamentos elétricos e eletrônicos e identificação de seus potenciais impactos ambientais e na saúde

A preocupação ambiental em relação aos resíduos de equipamentos elétricos e eletrônicos (REEE) vem crescendo muito nos últimos anos devido à presença de substâncias perigosas que podem causar danos ao ambiente e à saúde. O trabalho teve por objetivo investigar o destino de REEE gerados no Campus Quadrilátero da Saúde/Direito (CQS/D) da Universidade de São Paulo (Faculdade de Medicina, Faculdade de Saúde Pública, Escola de Enfermagem, Instituto de Medicina Tropical e Faculdade de Direito), a partir do momento em que os mesmos deixam as dependências da Instituição e identificar potenciais riscos à saúde e ao ambiente, decorrentes das atuais ações de gerenciamento dos REEE nas unidades estudadas. A pesquisa desenvolveu-se em 3 etapas: a) pesquisa bibliográfica e documental; b) visitas às unidades que compõem o CQS/D, e identificação de cinco intituições filantrópicas como principais destinos; c) visitas aos locais identificados para investigar as atividades desenvolvidas para a recuperação de equipamentos, componentes ou materiais recicláveis, com base em um formulário desenvolvido. Adicionalmente, foram realizadas visitas a outros quatro locais de destino de REEE. Foi identificado que as etapas realizadas nas instituições são: recebimento, teste, conserto e desmontagem manual e que o destino dos REEE pode ser considerado problemático, já que os rejeitos acabam sendo descartados como resíduo comum, podendo causar danos ao ambiente e à saúde. Além disso há riscos ocupacionais, pois os trabalhadores, além de não utilizarem equipamentos de proteção individual necessários, nem sempre recebem capacitação para exercer suas funções

Papel reparador e imunomodulador das células-tronco mesenquimais (CTMs) deficientes de receptor para interferon-gamma (IFNy) em modelos renais agudos

A insuficiência renal aguda (IRA) é uma patologia que apresenta alta incidência na população e elevada morbimortalidade. Apesar de todos os avanços terapêuticos já obtidos, essas taxas ainda continuam elevadas. Uma possível alternativa, atualmente sugerida, seria o transplante de células-tronco. O processo regenerativo das células-tronco mesenquimais (CTMs) já foi demonstrado em diversos modelos experimentais e em alguns ensaios clínicos. O mecanismo de ação mais sugerido é a ação parácrina das CTMs na área lesada. Ainda, sabe-se que nesse ambiente, citocinas pró-inflamatórias, como TNF-α e IFNγ, ativam as CTMs para seu papel reparador. O presente estudo busca analisar o papel do IFNγ na ativação das CTMs em modelos renais. As CTMs de animais nocautes para receptor de IFNγ (IFNγR KO) e de animais selvagens (controle/ C57/Bl6) foram isoladas do tecido adiposo. Essas células foram caracterizadas por imunofenotipagem e diferenciação em adipócitos e osteócitos. A lesão renal aguda foi obtida através do clampeamento dos pedículos renais de camundongos machos C57/Bl6, por 45 min. Após 4hs da lesão isquêmica, as CTMs IFNγR KO e CTMs controles foram administradas intraperitonealmente, e 24hs após a cirurgia os animais foram sacrificados. O tratamento com CTMs selvagens apresentou significativa redução dos níveis de uréia e creatinina sérica. No entanto, a redução desses níveis séricos com CTMs IFNγR KO foi menos intensa. Com relação à análise da resposta inflamatória do rim, os dados demonstram que a expressão de RNAm de IL-6 é maior nos animais tratados com CTMs IFNγR KO quando comparada ao tratamento com CTMs selvagens; porém, os dois tratamentos apresentam expressão reduzida em comparação aos animais não tratados. Já a expressão de RNAm de IL-10 é maior em animais tratados com CTMs em comparação aos não tratados... (Resumo completo, clicar acesso eletrônico abaixo)

Sintese de Aminochalconas e Nitrochalconas como potenciais inibidores de alfaglicosidases

A descoberta de agentes hipoglicemiantes constitui ainda um grande desafio no tratamento do diabetes, devido ao elevado índice de efeitos adversos e à baixa eficácia dos fármacos existentes. As alfa-glicosidases, enzimas essenciais ao metabolismo dos açúcares, são alvos terapêuticos estratégicos para a descoberta e desenvolvimento de fármacos antidiabéticos. Na busca de novos modelos estruturais com atividade antidiabética, o presente projeto teve como objetivo a síntese de aminochalconas e nitrochalconas como inibidores potenciais de alfaglicosidases. Para atingir o objetivo delineado foi planejada a síntese de 4’-nitrochalconas através da condensação de Claisen-Schmidt, que conduziriam à obtenção das respectivas 4’- aminochalconas na presença de cloreto de estanho II como agente redutor. Ao todo foram sintetizadas 14 substâncias, sendo 6 nitrochalconas e 8 aminochalconas que foram purificadas utilizando-se técnicas cromatográficas tais como Cromatografia em Coluna e Cromatografia em Camada Delgada Preparativa. A identificação espectroscópica foi realizada por Ressonância Magnética Nuclear de 13C e 1H. As chalconas sintetizadas foram avaliadas por meio de ensaios de inibição de alfa-glicosidases de Saccharomyces cerevisiae. As substâncias 10 e 14 apresentaram valores de CI50 (concentração capaz de inibir em 50% a atividade enzimática) de 1,23 e 1,88 μM respectivamente, mostrando-se mais potentes que a acarbose (CI50 4,90 μM), a qual foi empregada como controle positivo. Os dados de CI50 revelaram que a presença de hidroxilas no anel B, bem como a posição e o número desses substituintes é de fundamental importância para a inibição da atividade enzimática. Em contrapartida, a presença de grupos doadores ou receptores de elétrons no anel A é pouco significativa para a inibição de

Potenciais aves dispersoras de sementes das espécies vegetais zoocóricas da Mata da Bica, Botucatu - SP

The semidecidual forests, as tropical forests, presents zoochoric dispersal syndrome in the most of their plant species, with approximately one third of frugivorous species responsible for the seeds dispersal are birds. The bills width is a limiting factor, it must be equal or bigger than the seeds width, because the viable seeds dispersal depends on leaving the digestive system after been swallowed hole. Birds and plants inventorys were realized in a semidecidual Forest fragment, in Botucatu – SP, called Mata da Bica. After this, the birds consumers of fuits/seeds were selected (frugivorous, granivorous and onivorous) and also the zoochoric plants, both for building a possibilities matrix, to verify the potential seed dispersers according to the seeds and bills width. Going to the matrix end we can observe a decrease in the interaction possibillities, because the seeds width increases and the bills width decreases. It has been observed that 50,7% seeds could be dispersed by all listed birds, because the seeds were narrower than the narrowest bill. In the other way large seeded species, as Holocalix balansae and Hymemaea courbaril has shown only two possible dispersers: Trogon surrucura and Piaya cayana. There are no possible dispersers to Syagrus oleraceae in the matrix. The need for large birds or other animals that works as large seed disperses demand conservation efforts, since most of these animals need big and conserved areas for their life habits

Identificação de áreas potenciais para implantação de aterro sanitário no distrito de floresta do sul em Presidente Prudente/SP

O crescimento das sociedades urbanas tem gerado um aumento significativo na produção dos resíduos sólidos urbanos, tornando o tema de disposição final amplamente discutido nas gestões públicas. Recentemente, a partir da criação da Lei Federal nº 12.305/2010, que institui a Política Nacional dos Resíduos Sólidos, foi estabelecido que a disposição final ambientalmente adequada dos rejeitos deve ser feita exclusivamente em aterros. O presente trabalho teve então como objetivo a avaliação de áreas potenciais para instalação de aterro sanitário no Distrito de Floresta do Sul no município de Presidente Prudente/SP. Para tanto, utilizou-se o SIG, como ferramenta do geoprocessamento, para análise espacial. Inicialmente, construiu-se um banco de dados geográficos da região de estudo com os dados de geomorfologia, hidrologia, declividade, malha urbana e rede viária. Em seguida, foram geradas representações na categoria MNT, onde os valores foram normalizados em intervalos [0,1] através da lógica booleana e fuzzy... (Resumo completo, clicar acesso eletrônico abaixo)

Marcadores de superfície de Células-tronco: estudo de reação cruzada inter-espécies

Mesenchymal stem cells (MSCs) are adult multipotent cells with fibroblastoid morphology and adherent to plastic. Furthermore, they can be obtained from different sources. Besides bone marrow, these cells are taken from umbilical cord blood, umbilical vein, saphenous vein, peripheral blood, arteries, liver and fetal pancreas, placenta, dental pulp and adipose tissue. MSCs derived from adipose tissue are important because of the abundant number of cells that can be obtained from this tissue, easy access and little discomfort to the patient. This study compared two techniques for obtaining MSCs from adipose tissue: mechanical dissociation (MD) and enzymatic digestion (ED). We also analyzed the inter-species cross-reactions using commercial monoclonal antibodies directed against surface antigens of stem cells from different species: mouse, horse, rabbit, monkey and human. We found that MD technique is favorable in relation to ED within 15 days of culture, and ED is more efficient in the first days of culture. The data also showed that MD causes less damage to cellular DNA. About inter-species cross-reactions, the monoclonal antibody A69 directed against stem cells from rabbits, which can be used in veterinary medicine, particularly in research involving horses

Seleção de áreas potenciais para implantação da zona de processamento e exportação de Fernandópolis - SP utilizando geotecnologia

The implementation of an Export and Processing Zone (ZEPs) brings several benefits to the local, state and federal economy, but often, only socioeconomic factors are considered, apart from several other factors that should be analyzed, such as the environment. In this context of industrialization and the struggle for sustainable development, this work propose to incorporate the environmental variable in the decision process for establishing industrial areas, in particular, the ZPE in the city of Fernandópolis, São Paulo state, Brazil, by examining several physical and environmental factors such as slope intervals, geological features, pedological factors and land use. Developed using a multicriteria analysis, a model has been elaborated, where these factors have received a proportional value according with their importance, supported by a GIS tool (Geographical Information System) and remote sensing products, such as images from CBERS satellite and SRTM radar, showing the suited areas for industrial activities, considering environmental conditions. This model may assist to take better decision about the ZPE implementation area and to reduce the negative environmental impacts that would result of poorly planned locations

Diferenciação miogênica “in vitro” de células tronco mesenquimais murinas de diferentes origens

Muscular dystrophy refers to a group of more than 30 genetical disorders characterized by progressive weakness and degeneration of the skeletal muscle. No effective therapy is available at present. Recent studies have reported that the transplantation of stem cells can offer an important potential therapy for genetic diseases. Adult bone marrow mesenchymal stem cells have been identified as a nonhematopoietic stem cell population capable of self-renewal with the ability to differentiate into many cell lineages, including bone, fat, cartilage and connective tissue. Because of their similarity with muscle progenitor cells, when they are injected in affected individuals, they are able to migrate into areas of skeletal muscle degeneration and participate in the regeneration process. The adipose tissue represents an alternative source of MSCs that, as the MSCs derived from bone marrow, are capable of in vitro differentiation into osteogenic, adipogenic, myogenic and chondrogenic lineages. The objective of this project is to investigate the “in vitro” myogenic potential of mesenchymal stem cells derived from murine bone marrow and adipose tissue. Four experimental groups were analyzed: mice from lineages Lama2dy-2J/J and C57black and, C2C12 lineage cells and transformed C2C12 expressing the eGFP protein. MSCs cultures were obtained by flushing the bone marrow femurs and tibials with α-MEM or by the subcutaneous and inguinal fat from the mice. Their characterization was done by flow cytometry and in vitro differentiation. Muscle differentiation was studied through the analysis of the expression of transcriptional factors involved in muscle differentiation and/or the presence and amount of specific proteins from muscle differentiated cell. The pluripotency from bone marrow MSCs of the two lineages was evidenced and, in the muscular differentiation... (Complete abstract click electronic access below)

Potenciais singulares na equação de Schrodinger unidimensional via transformada de Laplace

The present work has as its goal to treat well known and interesting unidimensional cases from quantum mechanics through an unusual approach within this eld of physics. The operational method of Laplace transform, in spite of its use by Erwin Schrödinger in 1926 when treating the radial equation for the hydrogen atom, turned out to be forgotten for decades. However, the method has gained attention again for its use as a powerful tool from mathematical physics applied to the quantum mechanics, appearing in recent works. The method is specially suitable to the approach of cases where we have potential functions with even parity, because this implies in eigenfunctions with de ned parity, and since the domain of this transform ranges from 0 to ∞, it su ces that we nd the eigenfunction in the positive semi axis and, with the boundary conditions imposed over the eigenfunction at the origin plus the continuity (discontinuity) of the eigenfunction and its derivative, we make the odd, even or both parity extensions so we can get the eigenfunction along all the axis. Factoring the eigenfunction behavior at in nity and origin, we take the due care with the points that might bring us problems in the later steps of the solving process, thus we can manipulate the Schrödinger's Equation regardless of time, so that way we make it convenient to the application of Laplace transform. The Chapter 3 shows the methodology that must be followed in order to search for the solutions to each problem

Fibras naturais: características químicas e potenciais aplicações

This study has as objective determining the chemical properties that serve as the basis for potential scientific and technological applications of seven species of fibrous plants cultivated in Brazil, which are: Banana” (Musa spp.), “Coco” (Cocos nucifera), Curauá (Ananas erectifolius), Fique (Furcraea andina), Piteira” (Furcraea gigantea), Sisal(Agave sisalana) and “Taboa” (Typha domingensis). The tests for determining the percent dry completely, extractives soluble in cold water, in hot water, in sodium hydroxide 1% and in ethanol-toluene, and the percentages of ash, hydrophobicity, lignin, holocellulose and cellulose were performed at the Department of Environmental Sciences, Agronomy College (FCA) of UNESP – Botucatu, São Paulo State. It also presents a literature review about these fibers and their potential applications. Differences between the results obtained and those found by other authors are possibly explained by variations on the origin of plant material, the harvest season, the climate and soil where they were grown. However, the results and the methodology used, serve as a basis for further studies with natural fibers