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

Tecnologia mais limpa para produção de mel seco de cana e sua inclusão em rações de frangos de corte

This study analyzed the effects of adding dry sugar cane molasses (MSC) to the feed of broiler chickens, and determining the economic feasibility of use of this type of diet; 240 male Ross race broiler chickens, one day in age, were utilized in this study. The experimental desing was a completely randomized whit 6 treatments and 4 replications, in 24 portions of 10 birds per parcel. The treatments corresponded to 6 rations (T1-T6) in phase initial (1-21 days) and 6 rations (T1-T6) in phasem finish (22-42 days) characterized by substitution of corn meal in levels increase 0, 5, 10, 15, 20 e 25% by the molasse dried sugar-cane. The birds received water and free ration during the whole creation phase, being the iso-proteins and iso-calories rations. The variance analysis showed the 1 a 21 days significant differences for average gain weight (P<0,05), average consumption of ration (P<0,05) and average alimentary conversion (P<0,05) and the 22 a 42 days, the analysis of variance showed significant differences for gain in weight (P<0,01) and average alimentary conversion (P<0,05). There no difference significant on average consumption of ration (P>0,05) the 22 a 42 days of age. Results showed out that is possible to use molasse dried sugar-cane up to 8,3 % in broilers ration. It was concluded the level of 8,3 % of addition gave the best economical returns in the experimental conditions

Fontes primárias da história do vácuo e da pressão atmosférica na sala de aula: cartas e jornais históricos em articulação com o livro-texto do ensino médio

The use of the History and the Philosophy of Science (HPS) for the teaching of science and scientific subjects has been advocated in recent decades. It has been pointed out that the History of Science could make for a deeper learning of scientific concepts, since it would promote a contact with the problems which that knowledge has set out to solve. Furthermore, historical episodes would serve to put the discussions about the nature of scientific knowledge into context. With a view to explore those potentialities, the literature in the field has sought to identify the challenges and obstacles for the didactic transposition of subjects from the History of Science. Amongst many aspects, the deficiencies in the training of teachers, so that they can work with the insertion of HPS in the classroom have been highlighted. Another aspect that has been mentioned to be a challenge has to do with the didactic transposition of the Primary Sources, that is, of the original texts on the History of Science. The Primary Sources have significant potentialities: making a connection possible between scientists and concepts, showing the difficulties faced during scientific endeavors, perceiving the role of mistakes as obstacles to be surpassed, not as defeat, etc. On the other hand, there has been little exploration of these concepts in an educational context, due to their own peculiarities. The original texts are often hard to understand and their interpretation demands knowledge of the historical and scientific context in which they were written, as well as skills pertaining to the conduction of research in the field of the History of Science. With this scenario in mind, the research towards this Professional MSc degree starts from the challenge of elaborating and discussing proposals which could enable the didactical transposition of the Primary Sources. We have worked specifically with Primary Sources on the History of the Vacuum and of the Atmospheric Pressure, because of the insertion of these subjects in the Brazilian High School curriculum, in connection with the didactical textbooks. "Historic Journals" were made up from clippings of the original historical texts, as was a Didactical Unit, which takes the usual textbooks as a basis and contemplates using the Journals and the entire Primary Sources in High School. At last, we have elaborated and implemented a course designed for the preparation of teachers and for being an opportunity for the discussion of the feasibility of putting these kinds of proposal into practice

Construção de modelos multivariados para determinação de lipídios totais e unidade em leite em pó comercial utilizando espectroscopia no infravermelho próximo

In this work calibration models were constructed to determine the content of total lipids and moisture in powdered milk samples. For this, used the near-infrared spectroscopy by diffuse reflectance, combined with multivariate calibration. Initially, the spectral data were submitted to correction of multiplicative light scattering (MSC) and Savitzsky-Golay smoothing. Then, the samples were divided into subgroups by application of hierarchical clustering analysis of the classes (HCA) and Ward Linkage criterion. Thus, it became possible to build regression models by partial least squares (PLS) that allowed the calibration and prediction of the content total lipid and moisture, based on the values obtained by the reference methods of Soxhlet and 105 ° C, respectively . Therefore, conclude that the NIR had a good performance for the quantification of samples of powdered milk, mainly by minimizing the analysis time, not destruction of the samples and not waste. Prediction models for determination of total lipids correlated (R) of 0.9955, RMSEP of 0.8952, therefore the average error between the Soxhlet and NIR was ± 0.70%, while the model prediction to content moisture correlated (R) of 0.9184, RMSEP, 0.3778 and error of ± 0.76%

Implicações tectônicas na hidrologia do aqüífero Barreiras e sistema lacustre do Bonfim, Nísia Floresta-RN

This MSc dissertation presents the results of a research carried out in a 500 km2 area in the Nísia Floresta county. The main goal of the research was to evaluate fault influence on hidrology features of aquifers and lakes, mainly in the Barreiras Group and in the Bonfim lake cluster respectively. The Precambrian crystalline basement is made of Caicó Complex rocks. They are capped by cretaceous sedimentary rocks and by cenozoic sedimentary rocks. Only the latter outcrop in the study area, wheareas the former are described in boreholes. Faults cut across all stratigraphic units and their main trends are NW, NE and E-W, which have been generated by E-W compression. Subordinate N-S trending faults also take place and have been generated by N-S oriented compression. Fault controlled hydrologic features are observed throughout the study area. There are sudden changes in saturated thicknesses of the Barreiras Aquifer due to vertical displacement of the Barreiras Group. The most important underground water source of the Bonfim Lake is related to abrupt thickness changes of the aquifer. In addition, the main faults control the underground drainage network and, probably, change in direction of equipotential surfaces seen on the potenciometric map. Regarding the surface hydrologic features, faults also control river and stream channels, as well as lake origin and shapes. The Bonfim Lake, in particular, has its peculiar shape, which follows NW and NE lineaments, and origin related to faulting and probably underground carstics processes

Correlação entre contexto morfoestrutural e sismicidade nas regiões de João Câmara e São Rafael (RN)

This MSc thesis describes brittle deformation in two seismic zones located in north-eastern Brazil: João Câmara and São Rafael, Rio Grande do Norte State. Both areas show seismogenic faults, Samambaia and São Rafael, indicated by narrow zones of epicentres with a strike of 040o, a lenght of 30 km and 4 km, and a depth of 1-12 and 0,5-4 km, respectively. The first seismological and geological studies suggested blind faults or faults that were still in the beginning of the nucleation process. The region is under E-W-oriented compression and is underlain by Precambrian terrains, deformed by one or more orogenic cycles, which generated shear zones generally marked by strong pervasive foliation and sigmoidal shapes. The crystalline basement is capped by the Cretaceous Potiguar basin, which is also locally capped by Pliocene continental siliciclastic deposits (Barreiras Formation), and Quaternary alluvium. The main aim of this study was to map epicentral areas and find whether there are any surface geological or morphotectonic expression related to the seismogenic faults. A detailed geological map was carried out in both seismic areas in order to identify brittle structures and fault-related drainage/topographic features. Geological and morphotectonic evidence indicate that both seismogenic faults take place along dormant structures. They either cut Cenozoic rocks or show topographic expression, i.e., are related to topographic heights or depressions and straight river channels. Faults rocks in the Samambaia and São Rafael faults are cataclasite, fault breccia, fault gouge, pseudotachylyte, and quartz veins, which point to reactivation processes in different crustal levels. The age of the first Samambaia and the São Rafael faulting movement possibly ranges from late Precambrian to late Cretaceous. Both fault cut across Precambrian fabric. They also show evidence of brittle processes which took place between 4 and 12 km deep, which probably have not occurred in Cenozoic times. The findings are of great importance for regional seismic hazard. They indicate that fault zones are longer than previously suggested by seismogenic studies. According to the results, the methodology used during this thesis may also be useful in other neotectonic investigation in intraplate areas

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