Resumo: A traça-das-crucíferas (Plutella xylostella) é uma praga que ataca culturas de brássicas, causando enormes prejuízos econômicos aos agricultores. O aparecimento de populações resistentes a inseticidas tem dificultado seu manejo, fazendo-se necessário a busca por alternativas de controle que viabilizem a economia do custo de produção, preservando o ambiente de inseticidas químicos em altas doses. As plantas possuem um mecanismo constitutivo de resistência que atua na sua defesa frente às diferentes adversidades bióticas e abióticas, o que representa altos gastos metabólicos. O mecanismo de resistência pode ser melhorado com a indução de resistência por um ou mais elicitores, como é o caso das bactérias promotoras de crescimento. O objetivo deste estudo foi detectar sequências gênicas expressas em resposta à indução de resistência de repolho à traça das crucíferas. A bactéria utilizada como indutora das respostas de defesa foi a Kluyvera ascorbata devido à ação já descrita anteriormente com repolho e traça-das-cruciferas, onde ficou evidente sua influência na defesa contra essa praga. Por meio do sequenciamento de cDNA foi possível identificar uma significativa alteração no metabolismo de plantas que abrigavam esta bactéria, suprimindo da ação de muitos genes, durante o processo de defesa da planta contra o estresse causado pela injúria de lagartas de P. xylostella / Abstract: The diamondback moth Plutella xylostella is a pest that attacks brassics causing great economic losses to the growers. The rise of resistant populations to the chemical insecticides imposes difficulties on this pest management, so the search of other ways to overcame this situation became quite relevant so as to help to reduce production costs and at the same time avoiding the use of high dosage of the commonly used pesticides. Plants exhibit a constitutive resistance mechanism that acts favoring their defense towards biotic and abiotic stress situations, which involves high metabolic energetic challenges. The defense mechanisms may be improved with the induction of resistance by a number of different elicitors, such as the use of plant growth promoting bacteria. The aim of the present work was to detect plant gene sequences in response to cabbage resistance induction against the diamondback moth. Kluyvera ascorbata was used to induce the plant's growth promotion elicitor due to previously described influence. Analyzing DNA sequencing procedures from cDNA library samples, it was possible to observe drastic differences on the plants metabolism when such bacteria was used, either by suppressing the action of several genes while the plant's defense mechanisms was in action against injuries caused by P. xylostella larvae / Orientador: Manoel Victor Franco Lemos / Coorientador: Robson Thomaz Thuler / Banca: Janete Apparecida Desidério Sena / Banca: João Roberto Spotti Lopes / Mestre

Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-25Bitstream added on 2014-06-13T19:54:05Z : No. of bitstreams: 1 crialesi_pcb_me_jabo.pdf: 717305 bytes, checksum: ddf9d322f3798935db316f769791eac7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A traça-das-crucíferas (Plutella xylostella) é uma praga que ataca culturas de brássicas, causando enormes prejuízos econômicos aos agricultores. O aparecimento de populações resistentes a inseticidas tem dificultado seu manejo, fazendo-se necessário a busca por alternativas de controle que viabilizem a economia do custo de produção, preservando o ambiente de inseticidas químicos em altas doses. As plantas possuem um mecanismo constitutivo de resistência que atua na sua defesa frente às diferentes adversidades bióticas e abióticas, o que representa altos gastos metabólicos. O mecanismo de resistência pode ser melhorado com a indução de resistência por um ou mais elicitores, como é o caso das bactérias promotoras de crescimento. O objetivo deste estudo foi detectar sequências gênicas expressas em resposta à indução de resistência de repolho à traça das crucíferas. A bactéria utilizada como indutora das respostas de defesa foi a Kluyvera ascorbata devido à ação já descrita anteriormente com repolho e traça-das-cruciferas, onde ficou evidente sua influência na defesa contra essa praga. Por meio do sequenciamento de cDNA foi possível identificar uma significativa alteração no metabolismo de plantas que abrigavam esta bactéria, suprimindo da ação de muitos genes, durante o processo de defesa da planta contra o estresse causado pela injúria de lagartas de P. xylostella / The diamondback moth Plutella xylostella is a pest that attacks brassics causing great economic losses to the growers. The rise of resistant populations to the chemical insecticides imposes difficulties on this pest management, so the search of other ways to overcame this situation became quite relevant so as to help to reduce production costs and at the same time avoiding the use of high dosage of the commonly used pesticides. Plants exhibit a constitutive resistance mechanism that acts favoring their defense towards biotic and abiotic stress situations, which involves high metabolic energetic challenges. The defense mechanisms may be improved with the induction of resistance by a number of different elicitors, such as the use of plant growth promoting bacteria. The aim of the present work was to detect plant gene sequences in response to cabbage resistance induction against the diamondback moth. Kluyvera ascorbata was used to induce the plant’s growth promotion elicitor due to previously described influence. Analyzing DNA sequencing procedures from cDNA library samples, it was possible to observe drastic differences on the plants metabolism when such bacteria was used, either by suppressing the action of several genes while the plant’s defense mechanisms was in action against injuries caused by P. xylostella larvae

Orientador: Nídia Alice Pinheiro / Banca: Elaine Sbroggio de Oliveira Rodini / Banca: Daniel Araki Ribeiro / Resumo: O câncer gástrico é o quarto tipo mais comum e o segundo em mortalidade. Apesar da diminuição do número de casos nos últimos tempos ainda é um grande problema mundial. E mais, sua associação com o Helicobacter pylori (HP) está cada vez mais preocupante, devido à alta prevalência de cepas patogênicas dessa bactéria. Além disso, mutações em genes que agem no ciclo celular podem levar ao câncer. O gene que codifica a proteína p53 é responsável pelo reparo de lesões no DNA durante o ciclo celular funcionando como um regulador transcricional. Quando o reparo não é viável a proteína p53 induz a entrada da célula danificada em apoptose. Mutações mais freqüentes ocorrem nos exons 5, 6, 7 8 e 9. No atual trabalho investigou-se a possível associação da mutação no gene p53, correlação com presença da cepa patogênica CagA+ e estadiamento do tumor. Foram estudadas 55 amostras de câncer gástrico extraídas por biópsia durante gastrectomia. PCR foi utilizada para os exons 5 ao 9, análise de mutação por sequenciamento automático e dados clínicos foram coletados incluindo infecção por Helicobacter pylori/CagA+. Trinta e dois casos apresentaram mutações em p53 sendo que 6 deles apresentaram mutação em mais de um exon. 53% apresentaram-se infectados por HP CagA+. 87% dos pacientes estavam em estadiamento avançado do tumor (II, III e IV) e 80% eram do tipo intestinal. Quanto a correlação de p53 e cagA+ a casuística é relativamente pequena, e assim a correlação de p53 e cagA não foi vista. Os resultados confirmam a relevância das mutações em p53 e da presença da cepa patogênica CagA nos casos de câncer gástrico, contudo estudos devem ser realizados com um maior número amostral. Neste caso, o uso da p53 mutada pode ser utilizada em diagnóstico indicando, assim um melhor tratamento, desde que há casos em que sua alteração impõe resistência... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The gastric cancer is the fourth most common type and the second in mortality. Despite the decrease in the number of cases in recent times is still a major problem worldwide. Moreover, its association with Helicobacter pylori (HP) is increasingly worrying, because of the high prevalence of pathogenic strains of this bacterium. Furthermore, mutations in genes that act in the cell cycle can lead to cancer. The gene encoding the p53 protein is responsible for repair of lesions in DNA during cell cycle working as a transcriptional regulator. When the repair is not viable in the p53 protein induces the entry of the cell into apoptosis damaged. Change frequently occur in exons 5, 6, 7 8 and 9. In the current work investigated is the possible association of the mutation in the p53 gene, correlation with the presence of pathogenic strain CagA + and staging of the tumor. We studied 55 samples of gastric cancer extracted by biopsy taken during gastrectomy. PCR was used to the exons 5 to 9, analysis of mutation by sequencing automatic and clinical data were collected including infection by Helicobacter pylori / CagA +. Thirty-two patients had mutations in p53 is that 6 of them had more than one mutation in exon. 53% showed up infected HP CagA +. 87% of the patients were in advanced staging of the tumor (II, III and IV) and 80% were of the intestinal type. As the correlation of p53 and cagA + the casuistic is relatively small, and thus the relationship of p53 and cagA was not seen. The results confirm the relevance of mutations in p53 in the presence of pathogenic strain CagA in cases of gastric cancer, but studies must be conducted with greater sample. In this case, the use of p53 mutant can be used in diagnosis indicating thus a better treatment, since there are cases where its amendment requires resistance to certain types of treatment, regardless of the presence of H pylori... (Complete abstract click electronic access below) / Mestre

Orientador: Vera Lúcia Gil da Silva Lopes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T15:18:27Z (GMT). No. of bitstreams: 1 Araujo_TaniaKawasakide_M.pdf: 2044257 bytes, checksum: 88ecc1c41da8ad27c2eef91360a2d656 (MD5) Previous issue date: 2011 / Resumo: A patogênese das fendas orais envolve tanto fatores genéticos como ambientais. Mutações que causam fenda labiopalatal (FL±P) em camundongos apontam diretamente para genes candidatos em humanos. Por exemplo, as FL±P foram descritas em modelos animais deficientes ou com mutações nos genes TFAP2A e BMP4, sendo que mutações neste último foram detectadas em casos de FL±P não sindrômica na população chinesa. Os dois genes citados também estão associados, em modelos animais, a alterações da morfogênese de outros órgãos. Objetivou-se investigar o envolvimento dos genes BMP4 e TFAP2A na etiologia da FL±P em uma amostra de pacientes brasileiros por meio de reação em cadeia da polimerase (PCR), digestão com enzima HphI (PCR-RFLP) e sequenciamento direto. A casuística foi composta por 45 indivíduos, classificada, clinicamente, de acordo com a International Clearinghouse for Birth Defects and Surveillance and Ressearch (ICBDSR 2007). Com a finalidade de comparar com a literatura, utilizou-se a divisão em dois grupos: FL±P sindrômica (20 casos), FL±P não sindrômica (25 casos). A análise estatística foi feita com o teste do qui-quadrado. A substituição 538T®C (rs17563) no gene BMP4, anteriormente descrita como associada à FLP na população chinesa e em alguns indivíduos com microforma de FLP, foi encontrada em 34 (75,5%) indivíduos. Utilizou-se grupo controle de 169 indivíduos normais, sem casos de fendas orofaciais em três gerações e sem ascendência oriental. Não houve diferença significativa na freqüência dos genótipos e alelos do polimorfismo rs17563 no gene BMP4 entre os casos com FL±P e o grupo controle (p=0,3347, OR=0,718255, 95%CI, 0,4-1,27). Entretanto, para que a análise estatística seja conclusiva, é necessário aumento do número amostral. Alterações de sequência no gene TFAP2A, descritas como polimorfismos em base de dados genômicos, foram identificadas em cinco casos. Deste modo, não existem evidências de que os genes TFAP2A e BMP4 estejam envolvidos na gênese de FL±P nesta amostra. / Abstract: Information regarding research throughout the years shows that the pathogenesis of cleft lip and palate (CL±P) involves genetic and environmental factors. Mutations that cause CL±P in mice point directly to candidate genes in humans, as TFAP2A and BMP4 gene. The phenotype of animal models with mutations in these genes included clefts. Mutations in BMP4 have also been detected in cases of nonsyndromic CL±P in the Chinese population. Considering this, the aim of our study is to investigate the involvement of BMP4 and TFAP2A genes in the etiology of CL±P in the Brazilian population. Mutation screening by direct sequence and enzyme digestion with HphI (PCR-RFLP) were applied. Casuistry was composed by 45 patients, clinically classified by International Clearinghouse for Birth Defects and Surveillance and Ressearch (ICBDSR 2007). To compare with the literature, they were divided in syndromic CL±P (20 cases) and nonsyndromic CL±P (25 cases). Statistical analysis was performed using the chi square. The sample included 45 individuals, separated into two groups: The substitution 538T ® C (rs17563) in the BMP4 gene, previously described as associated with CL±P in the Chinese population and in some individuals with microforms of CLP was found in 34 (75.5%) subjects. A control group of 169 normal subjects, which had no cases of orofacial clefts in three generations and no Oriental ascendancy, was used. There was no significant difference in frequency of genotypes and alleles of rs17563 polymorphism in the gene BMP4 between cases with CL ± P and the control group (p = 0.3347, OR = 0.718255, 95% CI, 0.4 to 1.27). However, further studies with larger samples are necessary to elucidate this aspect. At TFAP2A gene was detected two single nucleotide polymorphisms. Thus, there is no evidence of involvement of TFAP2A and BMP4 genes in the CL±P in this sample. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientadores: Carlos Eduardo Steiner, Carmen Silvia Bertuzzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T18:18:42Z (GMT). No. of bitstreams: 1 Carlin_MarceloPaschoalete_M.pdf: 1614647 bytes, checksum: 62e5b428719069b2b173ab2baf51970f (MD5) Previous issue date: 2011 / Resumo: A transformação de glicogênio em glicose acontece através de reações químicas realizadas por enzimas específicas e uma deficiência em uma delas leva ao acúmulo de glicogênio, resultando em distúrbios hereditários conhecidos como doenças de depósito de glicogênio (GSD, da sigla em inglês), ou glicogenoses. A glicogenose tipo I (GSDI), responsável por mais de 90% dos casos, é causada pela deficiência de G6Pase, enzima chave na homeostase da glicose sanguínea. Seu complexo enzimático é constituído por duas subunidades (catalítica e translocase) que determinam os subtipos Ia e Ib. A GSDIa, também conhecida como doença de von Gierke, é a mais comum das GSDI com 80 a 90% dos casos e corresponde a uma deficiência na sub-unidade catalítica da G6Pase. A GSDIb é a segunda forma mais prevalente e mais grave. É resultante da deficiência da glicose-6-fosfato translocase que transporta a glicose-6-fosfato para o lúmen do retículo endoplasmático, onde a unidade catalítica da G6Pase está situada. Em ambas, a deficiência enzimática é o resultado de mutações genéticas nos genes que codificam estas enzimas, conhecidos respectivamente como G6PC e G6PT1. O diagnóstico bioquímico é recomendável caso se queira desvendar e recomendar modalidades de tratamento, porém não fornece informação suficiente para a determinação do subtipo envolvido. Para essa diferenciação é necessário análise enzimática da G6Pase. Como essa enzima não é expressa em tecidos como fibroblastos ou linfócitos, sua aferição só é possível por procedimento cirúrgico, através de biópsia hepática. Nesse contexto, a clonagem do cDNA do G6PC e G6PT1 possibilitou o rastreamento de mutações responsáveis pelos subtipos Ia e Ib, o que permite a alternativa de um diagnóstico menos invasivo baseado em técnicas de biologia molecular através de amostras de sangue. No presente estudo, treze pacientes com sintomas clínicos sugestivos de GSDIa e Ib foram investigados através do sequenciamento genético. Foram detectadas para o gene G6PC cinco alterações, incluindo, três mutações de ponto (G68R, R83C e Q347X) e dois polimorfismos (c.511G>A e c.1176T>C), todos previamente descritos. Já para o gene G6PT1 foram encontradas quatro alterações: uma mutação de ponto conhecida (G149E), uma inserção do tipo frameshift inédita na literatura especializada (c.1338_1339insT) e dois polimorfismos (c.1287G>A e c.1076-28C>T). A frequência das mutações em nosso meio é semelhante à observada na literatura, na qual a mutação R83C também é a mais frequente. Além disso, o presente estudo acrescentou a descrição de uma nova mutação. A pesquisa de ambos os genes deve ser considerado na investigação dessa condição para definir os subtipos envolvidos, pois no caso de ausência de alterações no gene 6PC, sugere-se o rastreamento no gene G6PT1. O estudo molecular dessa condição abre a possibilidade do diagnóstico precoce que é importante para estabelecer um tratamento correto aos pacientes, evitando o surgimento de complicações tardias e melhorando a qualidade de vida. Além disso, contribui para o aconselhamento genético adequado do casal podendo confirmar a estimativa do isco entre os próximos filhos e, eventualmente, permitir diagnóstico pré-natal por nálise de mutação / Abstract: Glucose transformation into glycogen is mediated by specific enzymes and a deficiency in one of them may cause glycogen accumulation, resulting in hereditary disorders known as glycogen storage diseases (GSD), or glycogenosis. Glycogenosis type I (GSDI), responsible for more than 90% of cases, is caused by deficiency of the glucose-6-phosphatase (G6Pase), the key enzyme in blood glucose homeostasis. Its enzyme complex consists of two subunits (catalytic and transporter) that determine subtypes Ia and Ib. GSDla, also known as von Gierke disease, is the most common GSDI responsible for 80 to 90% of cases and corresponds to a deficiency in the catalytic subunit of G6Pase. GSDIb is the second most prevalent but also the most severe, resulting from deficiency of lucose-6- phosphate translocase that transports glucose-6-phosphate into the lumen of the endoplasmic reticulum, where the catalytic unit of G6Pase is located. In both types, enzymatic deficiency results from genetic mutations in the genes that codify these enzymes, known as G6PC and G6PT1. Biochemical essay for GSDI is useful to confirm the diagnosis and to recommend treatment, however it does not allow the determination of the disease subtype. For this differentiation, enzymatic analysis of G6Pase is necessary. Since this enzyme is not expressed in tissues such as fibroblasts or lymphocytes, activity determination is only possible by liver biopsy. In this context, the cDNA cloning of G6PC and G6PT1 allowed the screening of mutations responsible for subtypes Ia and Ib, which gives the alternative of a less invasive diagnosis based on molecular biology techniques, using blood samples. In this study, thirteen patients with clinical symptoms suggestive of GSDIa and Ib were investigated through genetic sequencing. Five changes were detected in G6PC, including three known point mutations (G68R, R83C and Q347X) and two polymorphisms (c.511G> A and c.1176T>C). Concerning the G6PT1 gene, four changes were found: a known point mutation (G149E), a novel frameshift insertion (c.1338_1339insT) and two polymorphisms (c.1287G>A and c.1076-28C>T). The frequency of mutations in this population is similar to that observed in the literature, in which R83C is also the most frequent. Additionally, this study added a description of a new mutation. As result of this study, molecular analysis of both genes should be considered in he investigation of individuals with this GSDI in order to define the subtypes involved. Molecular analysis of G6PC and G6PT1 genes enable the achievement of positive diagnosis of GSDIa and Ib, securely without the need for liver biopsy. It also allows the differentiation of types and subtypes, which is not possible by the biochemical diagnosis. Finally, the identification of the mutation provides an additional tool for the genetic counseling (and eventually prenatal diagnosis) of the parents and other family members / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Domingos da Silva Leite / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T15:47:58Z (GMT). No. of bitstreams: 1 Tiba_MoniqueRibeiro_D.pdf: 3934673 bytes, checksum: 211302f7129afd8376ba9096064a21c4 (MD5) Previous issue date: 2009 / Resumo: Escherichia coli tem sido isolada, com certa freqüência, apresentando antígenos flagelares (H) que não são reconhecidos por nenhum dos anti-soros disponibilizado pelo mais importante centro de referência de E. coli, The International Escherichia and Klebsiella Centre (WHO) do Statens Serum Institut, Copenhague, Dinamarca. Atualmente são reconhecidos 53 antígenos "H" e, nos últimos 29 anos, nenhuma modificação ocorreu na lista dos antígenos flagelares associados à Escherichia coli. Isto posto, os objetivos deste trabalho foram identificar os antígenos flagelares das cepas de E. coli que expressam H não tipável (HNT) e que apresentam fatores de virulência associados à diferentes enteropatias. Esta identificação foi realizada inicialmente, pela reação em cadeia da polimerase (PCR) do gene fliC, responsável pela proteína flagelina, das 53 amostras padrões para os antígenos H e das 20 amostras HNT (H não-tipável). Em seguida, os amplicons foram digeridos por enzimas de restrição e daquelas amostras que apresentaram perfis de restrições distintos daqueles observados para as amostras padrões de antígeno H, foram produzidos soros em coelhos. Foram realizados testes de titulação frente aos 53 antígenos padrões, frente ao antígeno homólogo e frente aos antígenos das amostras HNT. As seqüência gênicas das amostras HNT, obtidas na reação de sequenciamento, foram comparadas aos diferentes genes de fliC armazenados no banco de dados do "National Center for Biotecnology Information" (NCBI) através do sistema BLAST, e o programa ClustalW foi utilizado para alinhamento das seqüências. Os resultados demonstraram que estas amostras apresentaram similaridade com antígenos padrões, entretanto, elas não possuem a mesma seqüência nucleotídica e também não reagiram fenotipicamente com o anti-soro esperado. Os dados obtidos permitem concluir que no conjunto de amostras estudado, treze amostras apresentaram antígeno flagelar diferente daqueles já descritos na literatura, quando utilizado as técnicas de PCR e/ou sorologia. / Abstract: Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Centre (WHO) of the Statens Serum Institute, Copenhagen, Denmark. Are currently recognized 53 H antigens and in the last 29 years, no change occurred in the list of flagellar antigens associated with Escherichia coli. The objectives of this study were to identify the flagellar antigens of E. coli that do not express non-typeable H antigens and presenting the virulence factors associated with different diseases. This identification was performed initially by gene amplification of the fliC, (flagellin protein) by the polymerase chain reaction (PCR) in all 53 standards E. coli strains for the H antigens and 20 non-typeable H-antigens E. coli strains, being then, the amplicons were digested by restriction enzymes. Anti-sera were produced in rabbits, those strains that showed different restriction profiles of these patterns observed for the nontypeable H antigens E. coli strains. Agglutination testes were carried out against the 53 antigens standards, against the homologous antigen and H antigens of the non-typeable strains. DNA sequences were compared to different fliC genes stored in the database of the National Center for Biotecnology Information (NCBI) through the BLAST, and ClustalW program was used to align the sequences. The results showed that although these strains have homology with a standard H-antigen, they do not have the same nucleotide sequence and did not phenotypically reacted with the antiserum expected. The data obtained showed that thirteen strains had a different H antigen those already described in the literature when used the techniques of PCR-RFLP and/or serology. / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Purpose: Current DNA detection techniques require complicated procedures, specialized training, expensive equipment, invasive samples and significant amount of sample collection and processing time. The purpose of this research was to develop a rapid, accurate, non-invasive and electronic method of DNA sensing that harnesses natural unwinding properties of DNA helicase by attaching it to Carbon Nanotubes. Methods: a. A literature review on methods of attaching proteins to carbon nanotubes was conducted b. A design of the biosensor was developed based on previously reported attachment methods for other proteins c. A part of the sensor was developed by attaching DNA helicase to carbon nanotubes d. The result was tested for preservation of helicase functionality and carbon nanotube electronic structure integrity Results: a. Helicase was successfully attached to carbon nanotubes b. Helicase was found to retain its NTP hydrolysis function, DNA binding and DNA unwinding ability upon attachment c. Carbon nanotube electronic structure and function was not compromised upon attachment Conclusions: Non-specific attachment of helicase to carbon nanotubes preserves enzyme structure and function, allowing rapid DNA unwinding at an in vitro rate comparable to DNA helicase.

L'analyse chromosomique sur puce ADN (ACPA) tend à devenir le principal examen diagnostique dans la déficience intellectuelle (DI). Parmi les techniques d'ACPA, les puces SNP ont l'intérêt de pouvoir détecter les pertes d'hétérozygotie, et par conséquent d’identifier les isodisomies uniparentales (iUPD) et les zones d'identité liées à la consanguinité. Nous avons étudié une cohorte de 1 187 patients atteints de DI, dans un cadre diagnostique, sur puces SNP. Nous avons réalisé, par cette étude, 145 diagnostics (12%) dont 2 iUPD et 6 délétions n'incluant qu'un seul gène. De plus, nous avons détecté 639 CNV rares non décrits chez des sujets contrôles et incluant des séquences codantes, ce qui nous a permis d'identifier 11 gènes candidats dans la DI : CAMTA1, SP3, CNTNAP4, NUDT12, STXBP6, DOCK8, DOCK10, SMARCA2, NYAP2, ATAD3A et ATAD3B. Nous avons tenté de valider l'implication de ces gènes par séquençage, mais n'avons trouvé de seconde mutation pour aucun d'entre eux. Toutefois, des réarrangements de CAMTA1 ont été retrouvés dans 2 autres familles avec un phénotype homogène (DI et ataxie congénitale) ce qui nous a permis d’affirmer qu'il s'agit d'un gène de DI. Par ailleurs, l'homozygosity mapping, réalisé avec puces SNP, a identifié, par séquençage whole exome, une mutation non-sens homozygote du gène BUD13 dans une famille de DI syndromique. Enfin, de façon fortuite, nous avons caractérisé en ACPA une translocation familiale entraînant une disruption d'un gène d'ataxie spino-cérébelleuse, ATXN10, ce qui a permis de mieux comprendre la physiopathologie de cette maladie. Au total, notre étude démontre l'intérêt des puces SNP dans la DI, d'une part en diagnostic et d'autre part pour l'identification de nouveaux gènes responsables de DI. / Chromosomal Microarray Analysis (CMA) has become the main diagnostic test in the field of intellectual disability (ID). Among CMA techniques, SNP arrays have the advantage of identifying losses of heterozygosity in addition to Copy Number Variants (CNVs). Therefore they can detect uniparental isodisomies (iUPD) and regions of identity by descent. We screened a cohort of 1,187 patients with ID, in diagnostic setting, by CMA using SNP arrays. Causal abnormalities, including 2 iUPDs and 6 deletions comprising only one gene, were detected in 145 patients (12%). Moreover we found 639 rare CNVs, absent from control individuals, which included coding sequences. Our results allowed us to identify 11 genes possibly involved in or contributing to ID: CAMTA1, SP3, CNTNAP4, NUDT12, STXBP6, DOCK8, DOCK10, SMARCA2, NYAP2, ATAD3A and ATAD3B. We then screened additional patients with similar phenotypes in order to find a second mutation, but no second mutation was identified in any of them. Besides, CAMTA1 rearrangements were also found among two other families with homogeneous phenotypes (ID and congenital ataxia), which confirms that this gene is involved in ID. Furthermore, thanks to homozygosity mapping made possible by SNP arrays combined with exome sequencing, we identified a homozygous nonsense mutation in the BUD13 gene, in a family with syndromic ID. Finally we incidentally characterized a familial translocation resulting in the disruption of ATXN10, a gene responsible for spinocerebellar ataxia. This translocation has helped to better understand the pathophysiology of the disease. Overall, our study shows the importance of SNP arrays for the molecular diagnosis of ID and as a tool to identify genes responsible for ID.

Ultra-high throughput DNA sequencing technologies have rapidly changed the face of genomic research projects. Technologies such as mRNA-Seq have the potential to rapidly profile the expressed gene-catalog of non-model organisms, albeit with significant bioinformatics related costs and support required. This study developed automated data analysis workflows focused on the quality evaluation of mRNA-Seq reads, de novo transcriptome assembly, transcriptome annotation and digital gene expression profiling making use of data analysis tools available in the public domain and novel tools developed for this purpose. The developed workflows were made available in a private instance of the Galaxy workflow management system. The developed workflows were used to perform the de novo assembly of a gene-catalog of a Eucalyptus plantation tree. The fast growing and good wood properties of Eucalyptus tree species and their hybrids make them excellent renewable resources of fiber for pulp and paper, and woody biomass for bioenergy production. We produced an expressed gene-catalog of 18 894 de novo assembled contigs from Illumina deep mRNA-Seq of six sampled plant tissues. Using a novel coverage-assisted re-assembly approach, we were able to assemble near full-length biologically relevant transcripts. The assembly was evaluated in terms of contig quality and contiguity, and functional annotations were assigned. Digital expression profiling (FPKM values) of each contig across the tissues were calculated, which was used to identify of tissue-specific sets of expressed genes. Polymorphism analysis of 13 806 high-confidence contigs revealed a combined exon and untranslated region SNP density of 0.534 SNPs/100 bp, which provides a good opportunity for designing high-density SNP assays in the expressed regions of the Eucalyptus genome. The assembled and annotated gene catalog was made available for public use in a user-friendly, web-based interface as the Eucspresso database (http://eucspresso.bi.up.ac.za). The developed database acts as a prelude to a more comprehensive mRNA-Seq whole-transcriptome repository, the Eucalyptus Genome Intergrative Explorer (EucGenIE), a resource that will focus on identifying transcriptional networks active during woody biomass development. Results from the study proved that current bioinformatics software tools and approaches can be used to successfully assemble and characterize a large proportion of the transcriptome of a complex eukaryotic organism. This approach can be used to characterise the gene catalog of a wide range of non-model organisms using only data derived from uHTS experiments. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted

abstract: Next-generation sequencing is a powerful tool for detecting genetic variation. How-ever, it is also error-prone, with error rates that are much larger than mutation rates. This can make mutation detection difficult; and while increasing sequencing depth can often help, sequence-specific errors and other non-random biases cannot be de- tected by increased depth. The problem of accurate genotyping is exacerbated when there is not a reference genome or other auxiliary information available. I explore several methods for sensitively detecting mutations in non-model or- ganisms using an example Eucalyptus melliodora individual. I use the structure of the tree to find bounds on its somatic mutation rate and evaluate several algorithms for variant calling. I find that conventional methods are suitable if the genome of a close relative can be adapted to the study organism. However, with structured data, a likelihood framework that is aware of this structure is more accurate. I use the techniques developed here to evaluate a reference-free variant calling algorithm. I also use this data to evaluate a k-mer based base quality score recalibrator (KBBQ), a tool I developed to recalibrate base quality scores attached to sequencing data. Base quality scores can help detect errors in sequencing reads, but are often inaccurate. The most popular method for correcting this issue requires a known set of variant sites, which is unavailable in most cases. I simulate data and show that errors in this set of variant sites can cause calibration errors. I then show that KBBQ accurately recalibrates base quality scores while requiring no reference or other information and performs as well as other methods. Finally, I use the Eucalyptus data to investigate the impact of quality score calibra- tion on the quality of output variant calls and show that improved base quality score calibration increases the sensitivity and reduces the false positive rate of a variant calling algorithm. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020