Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The conservation of forest species requires the prior knowledge of genetic parameters belonging to the population, to help to design strategies for this purpose. This study aimed to evaluate genetic parameters and suggest indicators of sustainability in populations of Schinus terebinthifolius Raddi. aiming the conservation and maintenance of diversity in this species. In analysis of the study area, were selected 20 descriptors, and these suggested 20 indicators of sustainability for the area. Tender vegetal leaves were sampled in five populations located on the shores and islands of São Francisco River, and the material collected from 15 individuals for each population. In the DNA extraction it was used tender leaves of individuals and 2% CTAB method. We applied 20 primers of 10 bases of arbitrary sequence in amplification, the products were separated in agarose gel at 1% submitted to horizontal electrophoresis, stained with ethidium bromide and visualized in ultraviolet light. The presence and absence of bands were used to construct a binary matrix for the analysis of genetic parameters. In each population the percentage of polymorphic loci ranged from 32.92% to 45.34%. The average gene diversity of Nei was 0.37. The total genetic variation observed, 63.60% corresponded to variation among populations, and 36.40% within populations. The gene flow (Nm) estimated was 0.28. Thus the conclusion is the populations are genetically isolated, and analyzed the genetic parameters can be used as indicators of sustainability of the area. / A conservação de espécies florestais requer o conhecimento prévio de parâmetros genéticos pertencentes às populações, para se traçar estratégias para este fim. Assim, este trabalho teve como objetivo avaliar parâmetros genéticos e sugerir indicadores de sustentabilidade em populações de Schinus terebinthifolius Raddi. visando à conservação e manutenção da diversidade nesta espécie. Em análise da área de estudo, foram selecionados 20 descritores, e destes sugeridos 20 indicadores de sustentabilidade da área. As amostragens foram realizadas em cinco populações localizadas nas margens e ilhas do Rio São Francisco, sendo coletado material de 15 indivíduos para cada uma das populações. Na extração do DNA, empregou-se folhas tenras dos indivíduos e o método CTAB 2%. Foram usados 20 oligonucleotídeos de 10 bases de sequência arbitrária nas amplificações, cujos produtos foram separados em gel de agarose 1% submetidos à eletroforese horizontal, corados com brometo de etídio e visualizados em luz ultravioleta. A presença e a ausência de bandas foi usada para a construção de uma matriz de binária para a análise dos parâmetros genéticos. Em cada população a porcentagem de locos polimórficos variou de 32,92 a 45,34%. A diversidade média gênica de Nei foi de 0,37. Da variação genética total observada, 63,60% correspondeu à variação entre as populações; e, 36,40% dentro das populações. O fluxo gênico (Nm) estimado foi de 0,28. Desta forma conclui-se que as populações encontram-se isoladas geneticamente e os parâmetros genéticos analisados podem ser utilizados como indicadores de sustentabilidade da área.

O relógio biológico é um mecanismo molecular autossustentado gerador de ritmos. Ele integra vias de percepção das condições ambientais com um oscilador central para gerar respostas fisiológicas rítmicas em escalas diária e sazonal. Nas plantas, o relógio biológico está associado a vias metabólicas e fisiológicas importantes, como fotossíntese. Na cana-de-açúcar, uma gramínea de grande interesse econômico, estudos realizados em condições circadianas mostraram que o relógio biológico tem uma influência superior àquela vista em outras plantas. Assim, este trabalho visa a compreender os mecanismos de funcionamento do oscilador central do relógio biológico da cana-de-açúcar crescida em campo. Para tanto, foram investigados o transcriptoma de diferentes órgãos da cana-de-açúcar; a expressão de isoformas alternativas e de múltiplos alelos dos genes do relógio biológico da cana; e o efeito do sombreamento mútuo das plantas em campo sobre o funcionamento do relógio biológico. Os resultados obtidos sugerem que o relógio biológico é funcional e sincronizado entre os diferentes órgãos da cana-de-açúcar analisados. Os transcritos regulados sinergicamente pelo relógio biológico e pelo ambiente flutuante pertencem a vias metabólicas, fisiológicas e de regulação gênica e epigenéticas todas essenciais à produtividade da cana-de-açúcar. O sombreamento mútuo observado em campo parece alterar a fase de expressão de genes do relógio biológico da cana-de-açúcar. Além disso, eventos de splicing alternativo foram observados nos genes do relógio biológico em condições de baixa temperatura e múltiplos alelos dos genes do relógio biológico são expressos e a regulação de sua expressão parece ser sazonal. / The circadian clock is a self-sustaining molecular mechanism that generates rhythms. It perceives the environmental conditions and connects this pathway with its central oscillator, generating daily and seasonal rhythms of physiological responses. In plants, the circadian clock is associated with major metabolic and physiological pathways. In sugarcane, an economically important grass, previous studies showed that the circadian clock has the largest influence on plants seen so far under circadian conditions. This work aims to understand how the central oscillator of the circadian clock works in field-grown sugarcane. Thus, the transcriptome from different sugarcane organs; the expression of alternative isoforms and multiple alleles of circadian clock genes; and the effect of mutual shading in the field on the circadian clock function were analyzed. The results suggest that there is a functional and synchronized circadian clock in the different sugarcane organs. The transcripts regulated synergistically by the circadian clock and the variable environment are related to metabolic, physiological, genetic or epigenetic pathways, all important to sugarcane productivity. Mutual shading observed in the field seems to change the phase of expression of the sugarcane circadian clock. Besides, alternative splicing events have been reported for circadian clock genes under low temperature conditions and multiple alleles of circadian clock genes are expressed and their expression is likely to be seasonally regulated.

Orientador: Mônica Barbosa de Melo / Dissertação (mestrado - Universidade Estadual de Campinas, Intituo de Biologia / Made available in DSpace on 2018-08-20T18:09:19Z (GMT). No. of bitstreams: 1 Braghini_CarolinaAyumi_M.pdf: 2316733 bytes, checksum: 1031a8585bbf2541b2b39db621d9f45d (MD5) Previous issue date: 2012 / Resumo: O glaucoma compreende um grupo heterogêneo de neuropatias ópticas, caracterizadas pela escavação do disco óptico e perda progressiva do campo visual, representando uma das maiores causas mundiais de perda irreversível da visão. Em 1997, o gene Myocilin (MYOC) foi descoberto, e mutações neste gene foram envolvidas no desenvolvimento do glaucoma primário de ângulo aberto (GPAA) e do GPAA do tipo juvenil (GPAA-J). No Brasil, 35,7% e 3,85% dos pacientes com GPAA-J e GPAA, respectivamente, são portadores de mutações no gene MYOC. O gene citocromo P450, família 1, subfamília B, polipeptídeo 1 (CYP1B1), primeiramente associado ao glaucoma congênito primário, tem sido apontado como modulador do fenótipo do GPAA na presença de alterações no gene MYOC. Recentemente, observaram-se mutações no gene CYP1B1 associadas ao GPAA e GPAA-J, independentemente da presença de alterações estruturais no gene MYOC, em diferentes populações. Este projeto se propôs a avaliar mutações nos genes MYOC e CYP1B1, utilizando técnicas de PCR e sequenciamento direto, em 100 indivíduos pertencentes a famílias com GPAA ou GPAA-J e 43 pacientes não relacionados portadores de GPAA-J, bem como avaliar a possível modulação do gene CYP1B1 no fenótipo da doença. Uma nova mutação no gene MYOC, c.1187_1188insCCCAGA, foi identificada segregando com a doença em três gerações de uma família. De acordo com análises in silico, esta mutação pode alterar os contatos internos da proteína, além de comprometer um sítio de fosforilação de caseína quinase II. Nas demais três famílias foi detectada a mutação C433R, já descrita anteriormente. Como os membros das famílias apresentavam fenótipos bastante variados, foi realizada a análise da possível modulação do fenótipo da doença pelo gene CYP1B1. Contudo, as análises mostraram a não associação de variantes deste gene na modulação do fenótipo do glaucoma nestas famílias. Nos casos não relacionados de GPAA-J, foram observadas as mutações P370L, Q368X e C433R. Nenhuma mutação no gene CYP1B1 foi identificada nestes casos, mas somente polimorfismos: R48G, A119S, V243V, V432L, A443G, D449D e N453S. De acordo com este e outros estudos, é possível concluir que mutações no gene MYOC tem papel importante no desenvolvimento do GPAA e GPAA-J, sendo a mutação C433R a mais frequente na população brasileira. Além disso, o gene CYP1B1 parece ter menor contribuição no desenvolvimento destes tipos de glaucoma em nossa população / Abstract: Glaucoma comprises a group of heterogeneous optic neuropathies characterized by excavation of the optic disc and progressive loss of visual field, representing a major global cause of irreversible blindness. In 1997, the Myocilin gene (MYOC) was discovered, and mutations in this gene were involved in the development of primary open-angle glaucoma (POAG) and juvenile-onset of POAG (JOAG). In Brazil, 35.7% and 3.85% of patients with POAG and JOAG, respectively, are carriers of mutations in the MYOC gene. The gene cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), primarily associated with primary congenital glaucoma, has been related to phenotypic modulation of POAG in the presence of MYOC gene mutations. Recently, mutations in the CYP1B1 gene associated with POAG and JOAG were observed, regardless the presence of structural alterations in the MYOC gene in different populations. This project proposed to evaluate mutations in the MYOC and CYP1B1 genes using PCR and direct sequencing, in 100 individuals belonging to families with JOAG or POAG and 43 unrelated JOAG patients, and assess the possible role of the CYP1B1 gene in modulating the disease phenotype. A novel mutation in the MYOC gene, c.1187_1188insCCCAGA, was detected, segregating with the disease in three generations of one family. According to in silico analysis, this mutation can change the internal contacts of the protein, and has altered a phosphorylation site of casein kinase II. In the other three families the C433R mutation was detected, as described earlier. As family members presented with very different phenotypes, the CYP1B1 gene was evaluated as a possible modulator of the disease. However, the analysis showed no association of variants in CYP1B1 gene in modulating the glaucoma phenotype in these families. In unrelated cases of JOAG, the mutations P370L, Q368X and C433R were detected. No mutation in the CYP1B1 gene was observed in these cases, but only polymorphisms: R48G, A119S, V243V, V432L, A443G, D449D, and N453S. According to the present and other studies, we conclude that mutations in the MYOC gene play an important role in the development of POAG and JOAG, and the C433R mutation is the most frequent MYOC alteration in the Brazilian population. Furthermore, the CYP1B1 gene seems to have less contribution to the development of these types of glaucoma in our population / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Made available in DSpace on 2014-08-20T14:22:07Z (GMT). No. of bitstreams: 1 Dissertacao_Ciane_ Xavier_ Goncalves.pdf: 6437123 bytes, checksum: b433321a77238daeb8ea8ae563d42e6b (MD5) Previous issue date: 2008-07-07 / Pear is a fruit tree that belongs to the family Rosaceae, subfamily Pomoideae and genus Pyrus. In Brazil, the pear production lays around 20 thousand ton, and this fact occurs mainly due to the lack of adapted cultivars to the climate conditions.The present work was developed with the objective to study the pollen viability and compatibility of different cultivars, selections and rootstocks for the pear culture. This work was divided into five batches: in the experiment 1 it was evaluated the in vitro germination of 14 genotypes of pear at different incubation times (2, 4 and 6 hours) in BOD incubator and in lightless condition at 25°C. In the experiment 2, the incubation time was set for each genotype according the best results obtained in the previous experiment and it was assessed different temperatures of incubation (20, 30 and 40 °C) on in vitro germination of 14 pear genotypes. The basic germination medium consisted of 100 g L-1 sucrose and 10 g L-1 agar. In the third experiment, time and temperature of in vitro incubation of the pollen grains were chosen conform to the previous experiments. It was evaluated the germination of ten pear genotypes under the influence of the increase of different combinations of boric acid and calcium nitrate to the basic germination medium. In the experiment 4 it was assessed the in vitro germination of pollen grains of pear genotypes (varying temperature and time of incubation) in BOD incubator, lightless and boric acid in three concentrations added to the basic germination medium. Experiment 5 evaluated the gametic compatibility in pear genotypes through pollen tube growth in pistil of flower 120 hours after pollination. Therefore, to the assessed genotypes it was evaluated the self-pollination either in the field or laboratory, cross-pollination or open-pollination. The germination percentage of the pear genotypes studied in the first experiment depends on time and sample. Among the pear genotypes assessed in the second experiment the temperature of incubation at 20°C is recommended and at 40°C is damaging for germination. In the third experiment, in general, either basic culture medium or enriched with 200 mg L-1 boric acid promote higher rates of in vitro germination of pollen grains of the pear genotypes; the germination percentage decreased whether enriched with 1200 mg L-1 of calcium nitrate, independently of boric acid concentration. For the pear genotypes evaluated in the fourth experiment it is recommended to germinate the pollen grains in either basic culture medium or enriched with 100 mg L-1 of boric acid. In the fifth experiment the ovule fecundation did not occur for most genotypes and pollination type; therefore, cross-pollination benefits fecundation. / A pereira é uma frutífera que pertencente à família Rosaceae, subfamília Pomoideae e ao gênero Pyrus. No Brasil, a produção de peras é somente de, aproximadamente, 20 mil toneladas e este fato ocorre, principalmente, pela falta de cultivares adaptadas às condições climáticas. O presente trabalho foi desenvolvido com o objetivo de verificar a viabilidade e compatibilidade de pólen de diferentes genótipos de pereira no Rio Grande do Sul. Este trabalho foi dividido em cinco experimentos: No experimento 1, avaliou-se a germinação in vitro de 14 genótipos de pereira, em diferentes tempos de incubação (2, 4 e 6 horas) em incubadora tipo BOD, em ausência de luz, a 25°C. Para o experimento 2, o tempo de incubação foi fixado para cada genótipo conforme os melhores resultados obtidos no experimento anterior, avaliando-se as diferentes temperaturas de incubação (20, 30 e 40°C), na germinação in vitro de 14 genótipos de pereira. O meio de germinação básico foi composto de 100 g L-1 de sacarose e 10 g L-1 de ágar. No terceiro experimento, o tempo e a temperatura de incubação dos grãos de pólen in vitro foram fixados, conforme os experimentos anteriores; avaliou-se a germinação de dez genótipos de pereira sob a influência do acréscimo de diferentes combinações de ácido bórico e nitrato de cálcio ao meio de germinação básico. No experimento de número 4 avaliou-se a germinação in vitro de grãos de pólen de genótipos de pereira (variando-se a temperatura e o período de incubação) em estufa tipo BOD, em ausência de luz, e ainda, o ácido bórico em três concentrações adicionado ao meio de germinação básico. No quinto experimento, avaliou-se a compatibilidade gametofítica em genótipos de pereira, através do desenvolvimento do tubo polínico no pistilo da flor, 120 horas após a polinização; para os genótipos em estudo avaliou-se a autopolinização de campo, autopolinização de laboratório, polinização cruzada e a polinização aberta. Conclui-se que, para os genótipos de pereira estudados no primeiro experimento a percentagem de germinação depende do tempo e da amostra. Para o segundo experimento, entre os genótipos de pereira estudados, a temperatura de incubação a 20°C é recomendável e a 40°C é prejudicial para a germinação. Para o terceiro experimento, de maneira geral, em meio de cultura básico ou acrescido de 200 mg L-1 de ácido bórico promove elevados índices de germinação in vitro de grãos de pólen dos genótipos de pereira; e a percentagem de germinação é dimunuída quando acrescenta-se a concentração de 1200 mg L-1 de nitrato de cálcio, independente da concentração de ácido bórico. No quarto experimento, para os genótipos de pereira estudados, é recomendável a germinação de grãos de pólen in vitro em meio de cultura básico ou acrescido de 100 mg L-1 de ácido bórico. Para o quinto experimento, a fecundação dos óvulos não ocorreu na quase totalidade de genótipos e tipos de polinização; de modo geral, a polinização cruzada beneficia a fecundação.

No description available.

<p>In this thesis, we explore several stochastic models associated withthe two-parameter Poisson-Dirichlet distribution and population genetics.The impacts of mutation, selection and time onthe population evolutionary process will be studied by focusing on two aspects of the model:equilibrium and non-equilibrium. In the first chapter, we introduce relevant background on stochastic genetic models, andsummarize our main results and their motivations. In the second chapter, the two-parameter GEM distribution is constructedfrom a linear birth process with immigration. The derivationrelies on the limiting behavior of the age-ordered family frequencies. In the third chapter, to show the robustness of the sampling formula we derive the Laplace transform of the two-parameterPoisson-Dirichlet distribution from Pitman sampling formula. The correlationmeasure of the two-parameter point process is obtained in our proof. We also reverse this derivationby getting the sampling formula from the Laplace transform. Then,we establish a central limit theorem for the infinitely-many-neutral-alleles modelat a fixed time as the mutation rate goes to infinity.Lastly, we get the Laplace transform for the selectionmodel from its sampling formula. In the fourth chapter, we establisha central limit theorem for the homozygosity functions under overdominant selectionwith mutation approaching infinity. The selection intensity is given by a multiple of certain powerof the mutation rate. This result shows an asymptotic normality for the properly scaled homozygosities,resembling the neutral model without selection.This implies that the influence of selection can hardly be observed with large mutation. In the fifth chapter, the stochastic dynamics of the two-parameter extension of theinfinitely-many-neutral-alleles model is characterized by the derivation of its transition function,which is absolutely continuous with respect to the stationary distribution being the two-parameter Poisson-Dirichlet distribution.The transition density is obtained by the expansion of eigenfunctions.Combining this result with the correlation measure in Chapter 3, we obtain the probability generatingfunction of a random sampling from the two-parameter model at a fixed time. Finally, we obtain two results based on the quasi-invariance of the Gamma processwith respect to the multiplication transformation group.One is the quasi-invariance property of the two-parameter Poisson-Dirichletdistribution with respect to Markovian transformation group.The other one is the equivalence between the quasi-invarianceof the stationary distributions of aclass of branching processes and their reversibility.</p> / Doctor of Philosophy (PhD)

X-linked myopathy with excessive autophagy (XMEA) is a skeletal muscle disorder inherited in recessive fashion, affecting boys and sparing carrier females. Onset is in childhood with weakness of the proximal muscles of the lower extremities, progressing slowly to involve other muscle groups. Pathological analysis of skeletal muscle biopsies shows no inflammation, necrosis or apoptosis. Instead, forty to 80% of fibers exhibit giant autophagic vacuoles with heterogeneous degradative content. Numerous critical functions of all cells are compartmentalized in particular pH environments established by the intracellular transmembrane V-ATPase proton pump complex. Assembly of this complex, directed by the Vma21p chaperone, is well-studied in yeast but completely unknown in other organisms. The aim of my project was a better understanding of XMEA pathogenesis, with a focus on finding the disease-causing gene. In this thesis, I identify mutations in XMEA patients in a novel, previously uncharacterized gene, which we name VMA21. Most of the mutations are located in splicing-relevant positions and decrease splicing efficiency. After establishing that XMEA is caused by hypomorphic alleles of the VMA21 gene, I show that VMA21 is the diverged human orthologue of the yeast Vma21p protein, and that like Vma21p, it is an essential assembly chaperone of the V-ATPase. Decreased VMA21 reduces V-ATPase activity, resulting in altered lysosomal pH and a blockage at the degradative step of autophagy. Towards understanding disease pathogenesis, I show evidence of compensatory autophagy upregulation consecutive to the impaired clearance. Accumulated autolysosomes due to increased autophagy continue to face the degradative block and are slow to disappear. Instead, they merge to each other and form the characteristic giant XMEA vacuoles. These results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy. This work describes the clinical outcome at the cusp of tolerable reduction in V-ATPase, with implications on common diseases like osteoporosis and cancer metastasis, where increased V-ATPase activity is an important component. Our XMEA patients show that the safety margin of reducing V-ATPase activity in humans is wide, increasing the potential to utilize chemical or biological V-ATPase inhibitors as possible therapies.

O sistema sanguíneo ABO é o mais importante grupo sanguíneo na medicina transfusional. Atualmente, a determinação do tipo sanguíneo dos doadores de sangue é feita através de testes sorológicos rotineiros de laboratório, porém outros testes realizados com o DNA humano obtido de amostra de sangue, tornam-se complementos valiosos para a determinação correta do grupo sanguíneo do doador e do receptor, aumentando a segurança transfusional. Estudos realizados com o grupo sanguíneo A3 e A3B demonstraram que este subgrupo possui um alto grau de heterogeneidade, uma vez que diversos eventos moleculares foram associados a ele, embora apenas um pequeno número de amostras tenha sido testado até hoje. Nesse trabalho, foi investigada a frequência e os tipos de eventos moleculares do grupo sanguíneo A3 e A3B em um grupo de 100 doadores de sangue. Para seleção desse grupo foram analisadas 12.283 amostras de doadores de sangue saudáveis de ambos os sexos do grupo sanguíneo A e AB obtidas na Fundação Pró-Sangue/Hemocentro de São Paulo, pelos métodos teste em tubo e gel teste. Obtiveram-se 13 amostras A3 e 87 amostras A3B. Após extração e quantificação do DNA genômico das amostras utilizou-se amplificação do DNA pela técnica de reação da polimerase em cadeia alelo específico (PCR-ASP) para as regiões 467C>T, 829G>A, 871G>A e 1060C>A (del C) do exon 7 do gene ABO. Os resultados de amplificação das regiões estudadas apontam para um novo genótipo, aqui denominado A30*/ , presente em 30,7% das amostras A3 e em 58,6% das amostras A3B (A30*/B10*) (necessária a confirmação por sequenciamento); detectou-se também o genótipo A302/A301 em 30,7% das amostras A3 e o genótipo A302/B104 em 17,1% das amostras A3B; outros genótipos já descritos para subgrupo A3 (A301/A301, A302/A302, uma amostra de cada) foram também verificados bem como presença da mutação na região 871G>A em 9 amostras A3 ; verificou-se também que mutações nas regiões 467 C>T e 1060 C>A (del C), anteriormente descritas somente em indivíduos A2 e A2B, são muito frequentes em indivíduos A3 e A3B. / ABO blood system is the most important blood group in transfusional medicine. Actualy, the blood group type in blood donors and receptors is determined by serological laboratorial tests, complemented by human DNA blood tests that assure the correct blood group determination for the blood donor and receptor, optimizing transfusional safety. Studies on blood subgroup A3 e A3B demonstrated a large subgroup heterogenity, with various associated molecular changes in small sample groups tested. At present study, it was proposed investigation about the frequency and the types of molecular alterations occuring among 100 blood donors samples phenotyped as A3 e A3B. These samples are selected after tub and gel tests analysis of 12.283 A and AB samples of healthy blood donors of both sex from Fundação Pró-Sangue/Hemocentro de São Paulo. Thirteen A3 and 87 A3B samples were selected, each sample genomic DNA was extracted and quantified and it was performed DNA amplification by alellic specific polimerase chain reaction (PCR-ASP). It was studied exon 7 ABO gene mutations 467C>T, 829G>A, 871G>A and 1060C>A (del C). Amplification results pointed that a new genotipe is present at these A3 and A3B subgroup, named in this study as the A30*/ genotipe (sequencing confirmation needed); these genotipe is present in 30.7% A3 samples and in 58.6% A3B samples (A30*/B10*). However, it was detected the ulterior decrived genotipes: A302 present in 30.7% A3 samples (A302/A301) and in 17.1% A3B samples (A302/B104); for subgroup A3 it was observed the genotipes A301/A301, A302/A302 (one sample each) and the presence of 871G>A mutation at 9 A3 samples; 467 C>T and 1060 C>A (del C) exon 7 ABO gene mutations descrived at A2 e A2B samples, are very frequents at these A3 e A3B samples tested.

Developments within the field of genetic analysis have during the last decade become enormous. Advances in DNA sequencing technology have increased throughput from a thousand bases to over a billion bases in a day and decreased the cost thousandfold per base. Nevertheless, to sequence complex genomes like the human is still very expensive and efforts to attain even higher throughputs for less money are undertaken by researchers and companies. Genotyping systems for single nucleotide polymorphism (SNP) analysis with whole genome coverage have also been developed, with low cost per SNP. There is, however, a need for genotyping assays that are more cost efficient per sample with considerably higher accuracy. This thesis is focusing on a technology, based on competitive allele-specific extension and microarray detection, for genetic analysis. To increase specificity in allele-specific extension (ASE), a nucleotide degrading enzyme, apyrase, was introduced to compete with the polymerase, only allowing the fast, perfect matched primer extension to occur. The aim was to develop a method for analysis of around twenty loci in hundreds of samples in a high-throughput microarray format. A genotyping method for human papillomavirus has been developed, based on a combination of multiplex competitive hybridization (MUCH) and apyrase-mediated allele-specific extension (AMASE). Human papillomavirus (HPV), which is the causative agent in cervical cancer, exists in over a hundred different types. These types need to be determined in clinical samples. The developed assay can detect the twenty-three most common high risk types, as well as semi-quantifying multiple infections, which was demonstrated by analysis of ninety-two HPV-positive clinical samples. More stringent conditions can be obtained by increased reaction temperature. To further improve the genotyping assay, a thermostable enzyme, protease, was introduced into the allele-specific extension reaction, denoted PrASE. Increased sensitivity was achieved with an automated magnetic system that facilitates washing. The PrASE genotyping of thirteen SNPs yielded higher conversion rates, as well as more robust genotype scoring, compared to ASE. Furthermore, a comparison with pyrosequencing, where 99.8 % of the 4,420 analyzed genotypes were in concordance, indicates high accuracy and robustness of the PrASE technology. Single cells have also been analyzed by the PrASE assay to investigate loss of alleles during skin differentiation. Single cell analysis is very demanding due to the limited amounts of DNA. The multiplex PCR and the PrASE assay were optimized for single cell analysis. Twenty-four SNPs were genotyped and an increased loss of genetic material was seen in cells from the more differentiated suprabasal layers compared to the basal layer. / QC 20100714

Hemos analizado la variabilidad genética de una zona no codificante autosómica, el pseudogén homólogo al gen de la glucocerebrosidasa (psGBA). Parte del análisis se ha realizado desde la perspectiva de la genética de poblaciones humanas. Desde un punto de vista más genómico hemos establecido la dinámica de la región, a fin de entender las causas del espectro de variación. Hemos analizado el papel de la mutación, recombinación, conversión génica y, especialmente, selección. Por otra parte, psGBA es importante en la producción de alelos complejos GBA-psGBA, que provocan los tipos más severos de la enfermedad de Gaucher. Mostramos cómo el conocimiento de la variabilidad en psGBA ayuda al reconocimiento de estos alelos complejos. Finalmente, con los datos de variabilidad de dos regiones parálogas situadas en la misma región cromosómica (gen GBA / pseudogen psGBA) hemos comparado los patrones de mutación que presenta una misma secuencia bajo diferentes presiones selectivas. / We have analyzed the genetic variability in a non-coding autosomal region, the pseudogene homologous to the glucocerebrosidase gene (psGBA).Part of the analysis has been performed from the human populations point of view.From a more genomic perspective, we have established the region dynamics in order to understand the causes of the variability pattern. We have analyzed the role of mutation, recombination, gene conversion and, especially, selection.On the other hand, psGBA is important in the production of complex alleles GBA-psGBA, that lead to the most severe types of Gaucher disease. We show how the knowledge of psGBA variability helps to the identification of those complex alleles.Last, from the variability data from two paralogous regions located in the same chromosomal region (GBA gene /psGBA pseudogene) we have compared the mutation patterns shown by the same sequence under different selective pressures.