Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Avaliação do efeito de um adoçante comercial com sucralose na marcação de constituintes sanguíneos com tecnécio-99m, na morfologia das hemácias e na biodisponibilidade dos radiofármacos pertecnetato de sódio e ácido dietilenotriaminopentacético-tecnécio-99m em ratos Wistar.

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Identificação e avaliação de propriedades de polissacarídeos sulfatados de diferentes fontes naturais que possibilitem sua aplicabilidade biotecnológica

Sulfated polysaccharides (SP) are widely distributed in animals and seaweeds tissues. These polymers have been studied in light of their important pharmacological activities, such as anticoagulant, antioxidant, antitumoral, anti-inflammatory, and antiviral properties. On other hand, SP potential to synthesize biomaterials like as nanoparticules has not yet been explored. In addition, to date, SP have only been found in six plants and all inhabit saline environments. However, the SP pharmacological plant activities have not been carrying out. Furthermore, there are no reports of SP in freshwater plants. Thus, do SP from marine plants show pharmacological activity? Do freshwater plants actually synthesize SP? Is it possible to synthesize nanoparticles using SP from seaweed? In order to understand this question, this Thesis was divided into tree chapters. In the first chapter a sulfated polysaccharide (SPSG) was successfully isolated from marine plant Halodule wrightii. The data presented here showed that the SPSG is a 11 kDa sulfated heterogalactan contains glucose and xylose. Several assays suggested that the SPSG possessed remarkable antioxidant properties in different in vitro assays and an outstanding anticoagulant activity 2.5-fold higher than that of heparin Clexane® in the aPTT test; in the next chapter using different tools such as chemical and histological analyses, energy-dispersive X-ray analysis (EDXA), gel electrophoresis and infra-red spectroscopy we confirm the presence of sulfated polysaccharides in freshwater plants for the first time. Moreover, we also demonstrate that SP extracted from E. crassipes root has potential as an anticoagulant compound; and in last chapter a fucan, a sulfated polysaccharide, extracted from the brown seaweed was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution for hydrophobic chains of 1H NMR was approximately 93%. SNFfuc-TBa125 in aqueous media had a mean diameter of 123 nm and zeta potential of -38.3 ± 0.74 mV, measured bydynamic light scattering. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0 43.7% at SNFuc concentrations of 0.05 0.5 mg/ mL and RAEC non-tumor cell line proliferation displayed inhibition of 8.0 22.0%. On the other hand, nanogel improved CHO and RAW non-tumor cell line proliferation in the same concentration range. Flow cytometric analysis revealed that this fucan nanogel inhibited 786 cell proliferation through caspase and caspaseindependent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle

Efeitos ambientais e genéticos sobre escores visuais e ganho de peso à desmama de animais formadores da raça Brangus

Estudaram-se os efeitos da idade da vaca ao parto e da idade do animal à desmama, bem como os efeitos genéticos aditivo direto e materno e da heterozigose individual, sobre os escores visuais de conformação, precocidade e musculatura e ganho de peso do nascimento à desmama, de animais formadores da raça Brangus. Foram analisados 53.683, 45.136, 52.937 e 56.471 dados de conformação, precocidade e musculatura à desmama e ganho de peso do nascimento à desmama, respectivamente, de animais nascidos entre 1986 e 2002, provenientes do arquivo zootécnico da empresa Gensys Consultores Associados S/C Ltda. Os efeitos de ambiente e genéticos sobre as características em estudo foram analisados pelo método de quadrados mínimos usando modelos matemáticos que incluíram grupo de contemporâneos como variável classificatória e a idade da vaca ao parto, a idade do animal à desmama e os efeitos aditivo direto e materno e da heterozigose individual como co-variáveis. Todos os efeitos incluídos nos modelos afetaram significativamente as características avaliadas, com exceção do efeito da idade da vaca ao parto sobre o ganho de peso do nascimento à desmama e do efeito aditivo materno sobre todas as características estudadas. Os efeitos ambientais e genéticos revelaram-se importantes fontes de variação para as características estudadas e devem, pois, ser considerados na distinção e comparação dos animais para seleção.

Efeitos genéticos e ambientais sobre o perímetro escrotal de animais da raça Caracu

Neste trabalho foram avaliados os efeitos de idade (IDS) e peso aos 15 meses (P15) sobre medidas de perímetro escrotal ao sobreano de 1.892 machos da raça Caracu e estimada a herdabilidade desta característica. Utilizaram-se dois modelos de análise: em um dos modelos, foram incluídos o efeito de grupo de contemporâneos (GC) e, como covariável, a idade ao sobreano (efeitos linear e quadrático); e, no outro, o efeito de GC e, como covariável, o peso ao sobreano (efeitos linear e quadrático). A idade não teve efeito significativo no perímetro escrotal, o que está relacionado à homogeneidade dos animais nos grupos de contemporâneos, enquanto os efeitos linear e quadrático de P15 foram significativos sobre o perímetro escrotal, indicando grande influência do peso sobre a variação desta característica. A herdabilidade do perímetro escrotal foi estimada pelo método Bayesiano utilizando-se um modelo animal. O modelo incluiu os efeitos de GC e P15 (linear e quadrático) e os efeitos genéticos aditivo direto e residual. O valor médio estimado de herdabilidade do PE ao sobreano foi 0,38 e comprova que esta característica pode ser utilizada como critério de seleção para precocidade sexual em programas de melhoramento genético de animais da raça Caracu.

Sclerocorneal limbal stem cell autograft transplantation in dogs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Perfil eletroforético de proteínas séricas do sangue do cordão umbilical de cães

A importância do estudo do sangue do cordão umbilical (SCU) tem sido verificada pela presença de células-tronco hematopoéticas no SCU humano. O modelo experimental em cão tem propiciado informações importantes nos transplantes em humanos. Entretanto, apesar da importância do SCU e do modelo experimental em cães, não existem estudos sobre a eletroforese de proteínas séricas do cordão umbilical canino. No presente protocolo experimental, foi feita a colheita de SCU de 20 neonatos caninos, o qual foi utilizado para o fracionamento eletroforético de suas proteínas. Os valores médios absolutos obtidos para proteínas séricas totais, albumina, alfa, beta e gamaglobulinas no sangue do cordão umbilical de cães ao nascimento foram 3,09±0,59; 1,51±0,38; 0,87±0,17; 0,68±0,12 e 0,03±0,01, respectivamente. Todos os resultados apresentaram-se abaixo dos níveis reportados para animais adultos, devido à passagem das proteínas e suas frações ocorrerem principalmente através do colostro e pela imaturidade hepática. em todos os traçados eletroforéticos do SCU de cães, foi observada uma pequena curva entre alfaglobulina 2 e betaglobulina 1, não relatada no soro de cães adultos. Portanto, neste experimento, foram observadas diferenças quantitativas no traçado eletroforético das proteínas séricas do SCU de cães, ao nascimento, diferindo-os, neste particular, de cães em diferentes fases da vida pós-colostral.