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Two hundred and forty three fungal isolates were obtained from roots of four Woollsia pungens (Cav.) F. Muell plants collected from a field site in New South Wales, Australia. 175 sterile isolates were slow growing and dark-coloured on 2 percentage malt agar and were selected for further analysis. Microsatellite-primed PCR using the primers (GACA)4 and (GTG)5 separated these isolates into 50 genets. Isolates representative of 43 genets (including 168 isolates in total) formed typical ericoid mycorrhizal structures when inoculated onto roots of Vaccinium macrocarpon Ait (Ericaceae), confirming their status as mycorrhizal endophytes. It was estimated that a minimum of 43 genetically-distinct mycorrhizal mycelial genets were present in the root systems of the sampled W. pungens population with 7 to 15 distinct endophytic genets identified in each host plant, indicating that considerable genetic diversity exists within the endophyte population. While most genets were represented by less than 8 isolates, 3 genets contained up to 41 isolates, suggesting that root system colonisation by some mycelia may be extensive. While most fungal genets were shown to be confined to individual plants, 2 genets (genets 32 and 33), however, were present within the root systems of 2 adjacent plants (plants C and D), suggesting that the two root systems were interconnected by the endophyte mycelia. The ITS region of 13 mycorrhizal endophytes and a non-mycorrhizal isolate selected from the endophyte population were sequenced and compared to the sequence of Hymenoscyphus ericae as well as sequences from the GenBank database. A phylogenetic tree was generated from the nucleotide sequence data. This analysis revealed that 6 putative taxa were present in the root systems of 4 host plants. No isolates were positively identified to genus or species level. Closest matches with fungal sequences in the database indicated that most isolates probably belonged to the order Leotiales. Cluster analysis on the basis of the ITS sequences indicated that H. ericae was not clustered together with any endophytes from W. pungens, suggesting that endophytes of W. pungens are not identical to the known ericoid mycorrhizal fungus H. ericae. H. ericae had a low degree of sequence similarity with isolates from W. pungens, with similarities ranging from 68.3-80.6%. Cluster analysis based on DNA sequences of the ITS region did not fully support the groupings inferred from microsatellite-based fingerprinting. / Master of Science (Hons)

Metastasis is a feared aspect of cancer and little is known about the underlying mechanisms. It is proposed that metastasis is caused by cell fusion between tumour and immune active phagocyte cells, for example macrophages. Such hybrid cells could then develop immortality and chemo tactic mobility. In two different systems it was examined whether it is possible to detect variation in cancer cells that would explain an initial fusion between tumour cells and leukocyte cells. Both systems included use of STR markers. Human colon carcinoma cells, which originally had been grown in nude mice, were investigated with mouse specific primers. These showed no trace of mouse DNA, which they most probably would have if cell fusion had occurred. Human breast cancer cells grown in nude mice, that had received injection of stem cell from male blood, showed no presence of Y-chromosomes. Blood, which was analyzed from one of the mice, showed a weak presence of something else than just mouse DNA. The result was however vague and hard to evaluate, and tries to reproduce the positive outcome failed. No evidence, which indicated that cell fusion occurred, was possible to demonstrate. On the other hand, there are previous studies that show how metastases can express macrophage specific properties, which gives all reason for further investigations.

Graduation date: 1992

<p>Metastasis is a feared aspect of cancer and little is known about the underlying mechanisms. It is proposed that metastasis is caused by cell fusion between tumour and immune active phagocyte cells, for example macrophages. Such hybrid cells could then develop immortality and chemo tactic mobility. In two different systems it was examined whether it is possible to detect variation in cancer cells that would explain an initial fusion between tumour cells and leukocyte cells. Both systems included use of STR markers. Human colon carcinoma cells, which originally had been grown in nude mice, were investigated with mouse specific primers. These showed no trace of mouse DNA, which they most probably would have if cell fusion had occurred. Human breast cancer cells grown in nude mice, that had received injection of stem cell from male blood, showed no presence of Y-chromosomes. Blood, which was analyzed from one of the mice, showed a weak presence of something else than just mouse DNA. The result was however vague and hard to evaluate, and tries to reproduce the positive outcome failed. No evidence, which indicated that cell fusion occurred, was possible to demonstrate. On the other hand, there are previous studies that show how metastases can express macrophage specific properties, which gives all reason for further investigations.</p>

In order to study sperm competition and mating dynamics in the snow crab Chionoecetes opilio, a genomic library was established with the goal of identifying highly polymorphic microsatellite markers. Six pairs of DNA primers were designed to amplify markers Cop3-4, Cop4-1, Cop5, Cop10, Cop24-3 and Cop111 by the polymerase chain reaction (PCR). All markers produced patterns as expected from single loci inherited in a mendelian fashion, except for Cop5 which revealed a multi-locus banding pattern. The cross-amplification of the six loci in seven additional crabs species revealed DNA polymorphisms at one or more loci for each species. Markers Cop3-4 and Cop24-3 were used to determine paternity of larvae of primiparous females both from the wild and from multiple mating experiments under laboratory settings. The two markers were also used to genotype the contents of female spermathecae in order to determine the number of number of male genotypes present. Spermathecal contents of wild-caught females were cut into several cross-sections and each section genotyped individually. Histological analysis of spermathecae was carried out to complement genetic data in order to elucidate patterns of sperm competition. Single paternity was observed for the progeny of all females. The analysis of laboratory females showed displacement was the mechanism by which single paternity was obtained by the last males to mate. The analysis of wild females revealed that their spermathecae contained on average the sperm of at least 3.7 males. Larvae appeared to be sired by males whose genotypes were found in the spermathecal cross-sections toward the blind-end of the spermathecae. This suggested that they were the first males to mate with females they guarded until oviposition, and females remated with other males thereafter. Also, a comprehensive account of the mating dynamics was carried out in a wild population of the Northwest Gulf of Saint Lawrence (Eastern Canada) and demonstrated the e

Made available in DSpace on 2014-08-13T14:50:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-12Bitstream added on 2014-08-13T18:01:36Z : No. of bitstreams: 1 000741724_20141201.pdf: 197926 bytes, checksum: ac91e5d8eb4d509b8ea2984eab3e732a (MD5) Bitstreams deleted on 2014-12-02T13:27:21Z: 000741724_20141201.pdf,Bitstream added on 2014-12-02T13:27:59Z : No. of bitstreams: 1 000741724.pdf: 972060 bytes, checksum: 9c1d21f7846078e519f3d9100e012d54 (MD5) / Nas últimas décadas, a incidência de indivíduos com sobrepeso ou obesos vem aumentando exponencialmente. Hoje, a obesidade é considerada pela Organização Mundial da Saúde como uma epidemia mundial, com graves consequências que podem levar à morte. A obesidade é uma desordem multifatorial que envolve fatores hereditários, ambiente e estilo de vida, e suas consequências não são apenas sociais ou psicológicas, mas estão principalmente relacionadas à presença de co-morbidades como a hipertensão arterial, diabetes tipo II, doenças cardiovasculares e vários tipos de câncer. Portanto, o controle da obesidade é um desafio para a manutenção da saúde humana, atraindo o interesse de inúmeros pesquisadores que buscam o entendimento dos mecanismos associados ao seu desenvolvimento, bem como novos métodos terapêuticos e de prevenção. Com base nessas premissas, o presente estudo objetivou avaliar a associação entre alterações genéticas e a obesidade, com especial foco para a presença de danos no DNA e para o perfil de expressão e polimorfismos gênicos. A casuística do estudo incluiu 300 mulheres cadastradas na lista de espera para a realização de cirurgia bariátrica e 300 mulheres saudáveis, eutróficas, pareadas por idade. O teste do cometa foi utilizado para avaliação de danos primários no DNA de células sanguíneas; os polimorfismos dos genes da grelina (GHRL)e leptina (LEP)e dos seus receptores (GHSR e LEPR), da interleucina 6(IL-6) e da serotonina (5-HT2C) foram analisados pela técnica de RT-PCR; o perfil de expressão gênica em linfócitos foi avaliado pela metodologia do DNA microarray e a expressão dos genes adiponectina (ADIPOQ) e leptina (LEP) em adipócitos foi avaliada pela técnica de qRT-PCR. Os resultados mostraram que a frequência dos polimorfismos dos genes GHRL(rs26802), GHRS(rs572169), LEP(rs7799039), LEPR(rs 1137101), IL-6 (rs1800796) e 5-HT2C ... / In the last decades, the incidence of overweight and obesity has increased worldwide. Nowadays, obesity is considered by the World Health Organization as a global epidemic with severe consequences that can lead to death. Obesity is a multifactorial disorder which involves different factors such as genetic, environment and life style, and its consequences are not only social or psychological, but are also related to the presence of comorbidities such as hyperthension, type 2 diabetes, heart diseases and many types of cancer. Therefore, obesity control has become a challenge for the human health maintenance, catching the attention of researchers that are trying to understand the mechanisms associated to its development, as well as therapeutical and preventive methods. Based on the information above, the present study aimed to evaluate the association between genetic alterations and obesity, with special focus on the presence of DNA damage, gene expression profiling and genetic polymorphisms. Our study included 300 morbid obese women registered for the bariatric surgery and 300 healthy eutrophic women, matched by age. The comet assay was used to assess primary DNA damage in blood cells; the genetic polymorphisms of ghrelin (GHRL), leptin (LEP) and their receptors (GHSR and LEPR), interleukin-6 (IL-6) and serotonin receptor (5-HT2C) were evaluated by the RT-PCR; gene expression profiling in lymphocytes was assessed by DNA microarrays; and adiponectin (ADIPOQ) and leptin (LEP) gene expression in adipocytes were evaluated by qRT-PCR. Our results showed that the frequencies of GHRL (rs26802), GHRS (rs572169), LEP (rs7799039), LEPR (rs 1137101), IL-6 (rs1800796) and 5-HT2C (rs 3813929) polymorphisms were not different between obese and control groups. However, obese presented higher levels of primary DNA damage (single and double strand breaks, alkali-labile sites and oxidative damage) than eutrophic women, indepedent on ...

It is common practice for DNA STR profiles to be analyzed using an analytical threshold (AT), but as more low template DNA (LT-DNA) samples are tested it has become evident that these thresholds do not adequately separate signal from noise. In order to confidently examine LT-DNA samples, the behavior and characteristics of the background noise of STR profiles must be better understood. Thus, the background noise of single source LT-DNA STR profiles were examined to characterize the noise distribution and determine how it changes with DNA template mass and injection time. Current noise models typically assume the noise is independent of fragment size but, given the tendency of the baseline noise to increase with template amount, it is important to establish whether the baseline noise is randomly found throughout the capillary electrophoresis (CE) run or whether it is situated in specific regions of the electropherogram. While it has been shown that the baseline noise of negative samples does not behave similarly to the baseline noise of profiles generated using optimal levels of DNA, the ATs determined using negative samples have shown to be similar to those developed with near-zero, low template mass samples. The distinction between low-template samples, where the noise is consistent regardless of target mass, and standard samples could be made at approximately 0.063 ng for samples amplified using the Identifiler^TM Plus amplification kit (29 cycle protocol), and injected for 5 and 10 seconds. At amplification target masses greater than 0.063 ng, the average noise peak height increased and began to plateau between 0.5 and 1.0 ng for samples injected for 5 and 10 seconds. To examine the time dependent nature of the baseline noise, the baselines of over 400 profiles were combined onto one axis for each target mass and each injection time. Areas of reproducibly higher noise peak heights were identified as areas of potential non-specific amplified product. When the samples were injected for five seconds, the baseline noise did not appear to be time dependent. However, when the samples were injected for either 10 or 20 seconds, there were three areas that exhibited an increase in noise; these areas were identified at 118 bases in green, 231 bases in yellow, and 106 bases in red. If a probabilistic analysis or AT is to be employed for DNA interpretation, consideration must be given as to how the validation or calibration samples are prepared. Ideally the validation data should include all the variation seen within typical samples. To this end, a study was performed to examine possible sources of variation in the baseline noise within the electropherogram. Specifically, three samples were prepared at seven target masses using four different kit lots, four capillary lots, in four amplification batches or four injection batches. The distribution of the noise peak heights in the blue and green channels for samples with variable capillary lots, amplifications, and injections were similar, but the distribution of the noise heights for samples with variable kit lots was shifted. This shift in the distribution of the samples with variable kit lots was due to the average peak height of the individual kit lots varying by approximately two. The yellow and red channels showed a general agreement between the distributions of the samples run with variable kit lots, amplifications, and injections, but the samples run with various capillary lots had a distribution shifted to the left. When the distribution of the noise height for each capillary was examined, the average peak height variation was less than two RFU between capillary lots. Use of a probabilistic method requires an accurate description of the distribution of the baseline noise. Three distributions were tested: Gaussian, log-normal, and Poisson. The Poisson distribution did not approximate the noise distributions well. The log-normal distribution was a better approximation than the Gaussian resulting in a smaller sum of the residuals squared. It was also shown that the distributions impacted the probability that a peak was noise; though how significant of an impact this difference makes on the final probability of an entire STR profile was not determined and may be of interest for future studies.

Made available in DSpace on 2015-10-06T13:03:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-20. Added 1 bitstream(s) on 2015-10-06T13:18:39Z : No. of bitstreams: 1 000849919.pdf: 1764719 bytes, checksum: cc1c24d4e6a06602b8ef15a77cbefe66 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / No presente estudo, foram analisadas citogeneticamente cinco espécies de peixes pertencentes ao gênero Trichomycterus: T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha e Trichomycterus sp., provenientes de diferentes bacias hidrográficas brasileiras. Foram utilizadas as técnicas de citogenética clássica (coloração com Giemsa, localização das RONs pela marcação com nitrato de Prata e bandamento C) e cito-moleculares (Hibridação Fluorescente in situ - FISH com sondas de DNAr 5S e 18S e o snDNA U2). Todas as espécies apresentaram número diploide de 2n=54 cromossomos com variações em sua fórmula cariotípica. Trichomycterus diabolus, T. iheringi, T. zonatus e Trichomycterus cf. mimonha apresentaram seu cariótipo com 34m + 12sm + 8st e Trichomycterus sp. apresentou 36m + 10sm + 8st, além da ocorrência de cromossomos supranumerários. As espécies também revelaram diferenças em relação ao tamanho dos dois primeiros pares cromossômicos do cariótipo, onde Trichomycterus sp. e T. diabolus apresentaram o primeiro e segundo metacêntrico de tamanho semelhante e maiores em relação aos demais cromossomos, enquanto em T. zonatus, Trichomycterus cf. mimonha e T. iheringi apenas o primeiro metacêntrico foi considerado o maior par. O bandamento C evidenciou blocos heterocromáticos instersticiais nos pares 2, 3, 7, 8, 19 e 23 de T. iheringi e no par 18 de T. diabolus, sendo que as demais espécies apresentaram blocos centroméricos de diferentes tamanhos espalhados por quase todo o cariótipo. As RONs foram identificadas em apenas um par cromossômico e foram coincidentes com a hibridação fluorescente in situ realizada com a sonda para DNAr 18S, com exceção de T. zonatus e Trichomycterus sp. que apresentaram marcações centroméricas no primeiro par e em um pequeno metacêntrico, respectivamente. A hibridação com a sonda para DNAr 5S revelou resultados mais diversos, sendo detectado um par para... / In the present study five species of fish from the genus Trichomycterus were analyzed cytogenetically, including T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha and Trichomycterus sp., collected at different Brazilian basins. The analyses involved classical (Giemsa conventional staining, localization of NORs for silver nitrate marking and C-banding) and molecular cytogenetic techniques (fluorescent in situ hybridization with ribosomal 5S and 18S, and U2 snDNA probes). All species showed diploid number of 2n = 54 chromosomes with variations in karyotype formula. Trichomycterus diabolus, T. iheringi, T. zonatus and Trichomycterus cf. mimonha presented their karyotype with 34m + 12sm + 8st and Trichomycterus sp. presented 36m + 10sm + 8st, besides the occurrence of supernumerary chromosomes. The species also reveals differences in relation to the size of the first two chromosome pairs of the karyotype, where Trichomycterus sp. and T. diabolus presented the first and second metacentric with similar size and larger than the other chromosomes, while in T. zonatus, Trichomycterus cf. mimonha and T. iheringi only the first metacentric was considered the largest pair. The constitutive heterochromatin showed interstitial blocks in pairs 2, 3, 7, 8, 19 and 23 in T. iheringi and the par 18 in T. diabolus and the other species presented centromeric blocks with different sizes spread throughout the greater part of the karyotype. NORs were identified in only one chromosome pair and were coincident with the fluorescent in situ hybridization using probes of 18S rDNA, except for T. zonatus and Trichomycterus sp. that showed additional centromeric signals on the first pair and other small metacentric, respectively. FISH using the probes of 5S rDNA was differentially distributed in the different species, with one chromosome pairs detected in T. diabolus, two pairs in T. iheringi, T. zonatus and three ...

Orientador: Daisy Maria Fávero Salvadori / Banca: Elza Tieme Sakamoto Hojo / Banca: Raquel Alves dos Santos / Banca: Camila Correa / Banca: Débora Damasceno / Resumo: Nas últimas décadas, a incidência de indivíduos com sobrepeso ou obesos vem aumentando exponencialmente. Hoje, a obesidade é considerada pela Organização Mundial da Saúde como uma epidemia mundial, com graves consequências que podem levar à morte. A obesidade é uma desordem multifatorial que envolve fatores hereditários, ambiente e estilo de vida, e suas consequências não são apenas sociais ou psicológicas, mas estão principalmente relacionadas à presença de co-morbidades como a hipertensão arterial, diabetes tipo II, doenças cardiovasculares e vários tipos de câncer. Portanto, o controle da obesidade é um desafio para a manutenção da saúde humana, atraindo o interesse de inúmeros pesquisadores que buscam o entendimento dos mecanismos associados ao seu desenvolvimento, bem como novos métodos terapêuticos e de prevenção. Com base nessas premissas, o presente estudo objetivou avaliar a associação entre alterações genéticas e a obesidade, com especial foco para a presença de danos no DNA e para o perfil de expressão e polimorfismos gênicos. A casuística do estudo incluiu 300 mulheres cadastradas na lista de espera para a realização de cirurgia bariátrica e 300 mulheres saudáveis, eutróficas, pareadas por idade. O teste do cometa foi utilizado para avaliação de danos primários no DNA de células sanguíneas; os polimorfismos dos genes da grelina (GHRL)e leptina (LEP)e dos seus receptores (GHSR e LEPR), da interleucina 6(IL-6) e da serotonina (5-HT2C) foram analisados pela técnica de RT-PCR; o perfil de expressão gênica em linfócitos foi avaliado pela metodologia do DNA microarray e a expressão dos genes adiponectina (ADIPOQ) e leptina (LEP) em adipócitos foi avaliada pela técnica de qRT-PCR. Os resultados mostraram que a frequência dos polimorfismos dos genes GHRL(rs26802), GHRS(rs572169), LEP(rs7799039), LEPR(rs 1137101), IL-6 (rs1800796) e 5-HT2C ... / Abstract: In the last decades, the incidence of overweight and obesity has increased worldwide. Nowadays, obesity is considered by the World Health Organization as a global epidemic with severe consequences that can lead to death. Obesity is a multifactorial disorder which involves different factors such as genetic, environment and life style, and its consequences are not only social or psychological, but are also related to the presence of comorbidities such as hyperthension, type 2 diabetes, heart diseases and many types of cancer. Therefore, obesity control has become a challenge for the human health maintenance, catching the attention of researchers that are trying to understand the mechanisms associated to its development, as well as therapeutical and preventive methods. Based on the information above, the present study aimed to evaluate the association between genetic alterations and obesity, with special focus on the presence of DNA damage, gene expression profiling and genetic polymorphisms. Our study included 300 morbid obese women registered for the bariatric surgery and 300 healthy eutrophic women, matched by age. The comet assay was used to assess primary DNA damage in blood cells; the genetic polymorphisms of ghrelin (GHRL), leptin (LEP) and their receptors (GHSR and LEPR), interleukin-6 (IL-6) and serotonin receptor (5-HT2C) were evaluated by the RT-PCR; gene expression profiling in lymphocytes was assessed by DNA microarrays; and adiponectin (ADIPOQ) and leptin (LEP) gene expression in adipocytes were evaluated by qRT-PCR. Our results showed that the frequencies of GHRL (rs26802), GHRS (rs572169), LEP (rs7799039), LEPR (rs 1137101), IL-6 (rs1800796) and 5-HT2C (rs 3813929) polymorphisms were not different between obese and control groups. However, obese presented higher levels of primary DNA damage (single and double strand breaks, alkali-labile sites and oxidative damage) than eutrophic women, indepedent on ... / Doutor