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

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

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance

Análise de polimorfismos dos genes da rota quinureniana em pacientes com meningite bacteriana.

Bacterial meningitis (BM) is still an important infectious disease causing death and disability. Invasive bacterial infections of the central nervous systems (CNS) generate some of the most powerful inflammatory responses known, which contributes to neuronal damage. The DNA microarray technology showed alterations in the kynurenine (KYN) pathway that is induced in BM and other diseases associated with inflammation, leading to brain injury. Our main aim was to search SNPs previously described in the KYN path enzymes to investigate a putative association of this SNPs with imbalanced in this pathway in patients with BM. The patients included in this study were 33 males and 24 females, with ages varying from 02 months to 68 years. SNPs were located inside of the domain conserved in KYNU, IDO, KATI and KATII. Primers were designed for analysis of SNPs already described by PIRA-PCR followed by RFLP. The analysis of KYNU+715G/A SNP found a heterozygous frequency of 0.033. We did not found the variant allele of SNP KYNU+693G/A, KATI+164T/C, KATII+650C/T and IDO+434T/G. Despite of previews studies showing the importance of KYN pathway we did not found one association of these SNPs analyzed with susceptibility or severity of MB in study population.

Associação entre polimorfismos funcionais nos genes da MMP-7 e MMP-9 e o perfil clinicopatológico do carcinoma epidermóide de língua

Matrix metalloproteinase-7 (MMP-7) and -9 (MMP-9) modulate important functions strictly related to the development, invasion and metastasis of several human cancers among them the squamous cell carcinoma of the tongue (SCCT). However, individual genetic factors such as the functional single nucleotide polymorphisms (SNPs) influence the pattern of protein expression of these MMPs and thus may be related to the variability observed in the clinical behavior of patients with SCCT. In this context, the present cross-sectional study aimed to evaluate the association between the frequency of the functional SNPs MMP-7 -181 A/G and MMP-9 -1562 C/T and the clinical (age, gender and metastasis) and pathological (malignancy histological grading and immunohistochemistry expression) features of SCCT cases. Genotyping of these SNPs were performed by PCR-RFLP on DNA samples from 71 cases of SCCT and 60 individuals without cancer who constitute the control group. Among the results of this research, it was observed that the frequency of the polymorphic alleles MMP-7 -181 G and MMP-9 -1562 T in SCCT patients was 28% and 12%, respectively, and the frequency of the heterozygotes A/G (PR = 2.00; p < 0.001) and C/T (PR = 1.54; p = 0.014) were significantly higher in the patient group than in the controls. The prevalence of patients carrying the combination of SNPs studied was significantly associated with SCCT cases (PR = 2.00; p = 0.011) and metastasis (PR = 2.00; p < 0.001). Furthermore, with the frequency of SNPs analyzed, the age, gender, histological grading and immunoreactivity of MMP-7 and MMP-9 formed clinical and pathological parameters relevant to the identification of population subgroups more related to the development of SCCT and metastasis. Based on these results, it is suggested that the protein expression levels of MMP-7 and -9 substantially influence the balance between their pro- and anticancer biological functions and hence the clinicopathological profile of the squamous cell carcinoma of the tongue

Avaliacao de polimorfismos nos genes XPD e XRCC3 em carcinomas orais de celulas escamosas

Oral squamous cell carcinoma (OSCC) is an important cause of morbidity and mortality worldwide despite recent advances in treatment. There are several studies aiming to find markers that may improve the assessment of this disease prognosis. Studies about genetic polymorphisms have gained prominence due to their influence on individual susceptibility to cancer development. The aim of this study was to evaluate the association between the frequency of polymorphisms XPD Lys751Gln and XRCC3 Thr241Met and clinicopathological features of OSCC cases, including age, sex, presence or absence of metastases, and histological grading of malignancy according to Bryne (1998). Sample consisted of 54 cases of OSCC and 40 cases of inflammatory fibrous hyperplasia (IFH). OSCC cases were classified as low or high grade. DNA samples were previously extracted from paraffin blocks. Genotypes for each case were determined through PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism). Results were analyzed by Fisher s exact test and Chi-square test and the odds ratio was calculated considering p < 0.05 to indicate statistical significance. For XPD, Lys/Gln genotype was more common in IFHs (n=28; 70%) than in OSCCs (n=24; 44.4%) (OR: 0.3; p<0.05). Frequency of Gln allele was higher in high-grade lesions when compared to low grade lesions (0.48 and 0.21, respectively) (OR: 3.4; p<0.05). For XRCC3, Met allele was more common in OSCC than in IFH (0.49 and 0.35, respectively) (OR: 2.6; p<0.05). Met/Met genotype was associated with presence of metastases (OR: 8.1; p<0.05). There was no statistically significant association between the genotypes and the age or sex of patients. In the present sample, the higher frequency of XPD Gln allele in IFH reveals a possible protective role of this variant against the development of OSCC. However, its association with high-grade lesions indicates that this allele could influence the tumor progression after the neoplasia development. The presence of XRCC3 Met allele, in turn, seems to contribute to the development of OSCC and metastases

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance