Dissertations / Theses on the topic 'Genes'

ZNF804A was (at the time this work started) one of only a few robustly implicated schizophrenia susceptibility genes, due to replicated genome-wide significant evidence for association between a polymorphism in the gene and schizophrenia. Determining the function of the ZNF804A protein, which is currently unknown, may provide a way of elucidating the pathophysiology of this relatively common, complex disorder. Based on the hypothesis that the ZNF804A protein regulates gene expression or splicing, the aim of this thesis was to identify genes that exhibit altered expression or splicing in brain tissue from mice in which the orthologue Zfp804a carries a nonsense mutation. No robust evidence was obtained that showed the effects of the mutation on differential expression in individual genes. Although this finding does not support the hypothesis that ZNF804A acts directly to regulate gene expression, the results may reflect the possibility that effects on gene expression may be too subtle to be detected using the methods applied. Evidence was obtained to show the mutation affected the alternative splicing of a number of individual genes, which could suggest a role for ZNF804A in the direct or indirect regulation of alternative splicing. Through RNA sequencing, I identified a novel transcript in Zfp804a with an alternative exon upstream of the Refseq exon 1. I also showed that a proportion of the significant splicing differences identified in mutants were artefacts of strain differences in gene sequences that are likely to affect the efficiency of hybridisation on the exon array. Genes identified as differentially spliced between mutants and wildtypes were enriched in axon guidance and cell adhesion pathways, both thought to be important during development. The findings of this thesis suggest the novel hypothesis that ZNF804A effects risk for schizophrenia via aberrant splicing in the above pathways that are critical to normal brain development. Further studies with increased power are required to understand the effects on gene expression.

Entamoeba histolytica is the protozoan parasite that causes amoebic dysentery and amoebic liver abscesses in humans. For many years it was believed to be a primitive organism because it lacks many typical eukaryotic features including mitochondria. Recently, two genes that in other organisms encode proteins normally found in the mitochondrion have been isolated, giving evidence for the secondary loss of mitochondrial function in E. histolytica. These are the pyridine nucleotide transhydrogenase (PNT) and the mitochondrial chaperonin cpn60 genes. In this study we isolated and characterised a gene encoding a mitochondrial-type heat shock protein 70 from E. histolytica. cDNA and genomic library clones have been isolated and sequenced. Comparison with previously published sequences confirmed the assumption that E. histolytica comes from mitochondrion - bearing ancestors. Southern blot hybridisation revealed there are two copies of the gene in the genome. Northern blot analysis revealed two transcripts hybridising to the mt-hsp70 probe that differ in length and which are induced by heat shock. In addition, an apparently noncoding, polyadenylated RNA that is also induced by heat shock is encoded immediately upstream of the mitochondrial-type hsp70 gene. Expression analysis was also performed in four other Entamoeba species. Partial cpn60, PNT, and mt-hsp70 genes were isolated and the size of the mRNAs and their heat shock induction levels were investigated by hybridisation to these probes. The similarity of the mt-hsp70 amino terminus to those of hydrogenosomal proteins in conjunction with the phylogenetic analyses suggests it is also likely to be targeted to the mitochondrion-derived organelle of E. histolytica known as the mitosome.

Polymorphism in coding regions and regions controlling gene expression is the major determinant of adaptive differences in natural populations. Genes of the major histocompatibility complex (MHC) possess a high level of genetic variation, which is maintained by selection over long coalescence times. MHC genes encode antigen-presenting molecules in the adaptive immune system, which protects the host from infectious diseases. However, MHC molecules may also present self-peptides and for most autoimmune diseases there is a genetic factor associated with the MHC. MHC genes have been used to learn about the interplay of selection and historical population events. In domestic dogs and their progenitor, the wolf, I explored factors associated with domestication and breed formation and their influence not only on MHC coding regions but also on the haplotypic structure of the class II region. Polymorphism and strong selection was demonstrated in the proximal promoters of MHC genes in dogs and wolves. Hence, genetic variation associated with MHC gene expression may have at least equal importance for a well functioning immune system. Associations between promoter sequences and particular coding alleles suggested allele-specific expression patterns. SNP haplotypes of the MHC class II region revealed ancestral as well as convergent haplotypes, in which combinations of alleles are kept by selection. Interestingly, weaker allelic associations were found between different genes and between coding regions and promoters in dogs compared to wolves. Potentially, this could cause insufficient defense against infections and predispose dogs to autoimmune diseases. For example, I identified a site in the promoter region that showed a consistent difference between haplotypes conferring susceptibility and protection to diabetes in dogs, which should be investigated further. Furthermore, I investigated how selection and demographic changes associated with glacial and inter-glacial periods have affected MHC variation in European hedgehogs and extended the prevailing knowledge concerning their population history.

Migraine is a serious neurological disorder that affects the central nervous system causing painful attacks of headache. Attacks of head pain vary widely in intensity, frequency and duration lasting from anywhere between 4-72 hours and are often accompanied by further symptoms of nausea, vomiting, photo- and phonophobia. In 1988, a group of world leaders in the diagnosis of migraine formed the International Headache Society (IHS) and compiled and published a consensus set of diagnostic criteria known as International Classification of Headache Disorders, ICHD-I 1988. This was the first classification system and was subsequently updated in 2004, ICHD-II 2004 and more recently a 3rd Edition beta version has been released (ICHD-3rd Ed Beta Version) and is the gold standard for diagnosing headache disorders. Migraine displays two main subtypes termed migraine with or without aura (MA and MO respectively). The two forms are distinguished from each other based on the development of aura, a period of variable and diverse neurological symptoms that precede the headache phase.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Science, Environment, Engineering and Technology
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Les répertoires de gènes sont des objets extrêmement dynamiques : Des gènes sont dupliqués et perdus, transférés d’un génome à l’autre et des nouveaux gènes sont créés. L’étude de ces processus et de leur impact sur l’évolution des répertoires de gènes est fondamentale pour notre compréhension de l’énorme diversité de la vie sur terre. J’ai reconstruit les familles des gènes homologues chez les levures du clade Lachancea et je les ai classées en trois catégories selon leur présence chez les espèces en dehors du clade en: transmises verticalement (98.2 %), transmises horizontalement (0.15 %) et spécifiques aux Lachancea (1.63 %). Ensuite, j’ai reconstruit l’évolution de chaque famille de gènes le long de l’arbre phylogénétique des Lachancea en terme de gains et de pertes depuis l’origine du clade. Mes résultats suggèrent que les réarrangements chromosomiques balancés (translocations, inversions) peuvent interrompre, au niveau de leurs points de cassure, la séquence codante des gènes, et entraîner jusqu’à 14 % des pertes de gènes observées (rupture de gène). En outre, j’ai observé des corrélations entre le taux de divergence des séquences codant pour des protéines et les taux de duplication de gènes, de translocations et d’inversions, et de rupture de gène, suggérant l’existence d’une horloge génomique qui coordonnerait ces processus. Par la suite, je me suis focalisé sur l’émergence de nouveaux gènes de novo à partir de séquences non-codantes, dont l’impact global sur les génomes n’est pas encore connu. J’ai pour cela analysé les gènes taxonomiquement restreints aux levures des clades Lachancea et Saccharomyces sensu stricto et j’ai pu identifier un ensemble de 596 gènes ayant fort probablement émergé de novo. Le taux d’émergence de novo est constant chez les levures au sein du même clade mais varie d’un ordre de grandeur entre les 2 clades (2.8 gènes/ma chez les Saccharomyces et 0.27 gènes/ma chez les Lachancea). Ces nouveaux gènes sont distribués uniformément sur les chromosomes. Ils sont le plus souvent orientés de façon divergente par rapport à leur voisin en 5’, ce qui suggère que leur transcription pourrait être initiée au niveau de promoteurs divergents, favorisant ainsi la transition d’une séquence intergénique non transcrite à une séquence codante transcrite (puis traduite). Enfin, j’ai montré que dans certains cas, seul un petit nombre de mutations permettent la création d’un gène bien adapté à son environnement génomique, en comparaison avec des gènes plus «anciens». Cela signifie que sous certaines conditions la transition d’une séquence non-codante vers une séquence codante peut être relativement rapide. Globalement, mes résultats suggèrent que l’émergence de novo est un processus évolutif non négligeable, représentant une source importante de création de nouvelles protéines
Gene repertoires are highly dynamic : Genes are duplicated, lost, transferred from one genome toanother and new genes are formed. Studying these processes and how they shape gene repertoireevolution is fundamental to our understanding of how the enormous diversity of life on earth came to be. I reconstructed the homologous gene families of the yeasts of the Lachancea genus and categorized them based on their conservation in species outside the genus into vertically inherited (98.2%), horizontally transferred (0.15%) and taxonomically restricted (1.63%). Then, I inferred the evolution of each family along the genus’ phylogeny and identified the gene gain and loss events that occurred since the genus’ origin. I found that balanced chromosomal rearrangements may be responsible for up to 14% of gene losses by disrupting the coding sequence at their breakpoints and detected 3 cases with clear traces of the disruption at the sequence level. Additionally, I found that correlations exist between the rate of protein-coding sequence divergence and the rates of gene duplication, chromosomal inversions and translocations, and gene disruptions by balanced rearrangements, suggesting the existence of a genomic clock coordinating these processes. Next, I focused on the emergence of new genes de novo from non-coding sequences, a process whose overall impact remains a matter of debate. I thus analyzed taxonomically restricted genes in the two model yeast genera Lachancea and Saccharomyces sensu stricto and identified a robust set of 596 genes that have likely emerged de novo. I found that de novo emergence rates are constant among yeasts of the same genus but differ by an order of magnitude between the two genera with 2.8 genes/my in the Saccharomyces and 0.27 genes/my in the Lachancea. De novo genes are uniformly distributed on yeast genomes and are found divergently oriented relative to their 5’ neighbors suggesting that divergent transcription might play a role in their transition from non-transcribed intergenic sequences to transcribed (and translated) coding sequences. Moreover, through specific examples I was able to show that a few enabling mutations are sufficient for a young de novo gene to emerge already well-adapted relative to older genes, indicating that the transition from non-coding to coding can happen rapidly. Overall, my results support de novo emergence as a ubiquitous evolutionary process and a potent source of novel proteins

Les répertoires de gènes sont des objets extrêmement dynamiques : Des gènes sont dupliqués et perdus, transférés d’un génome à l’autre et des nouveaux gènes sont créés. L’étude de ces processus et de leur impact sur l’évolution des répertoires de gènes est fondamentale pour notre compréhension de l’énorme diversité de la vie sur terre. J’ai reconstruit les familles des gènes homologues chez les levures du clade Lachancea et je les ai classées en trois catégories selon leur présence chez les espèces en dehors du clade en: transmises verticalement (98.2 %), transmises horizontalement (0.15 %) et spécifiques aux Lachancea (1.63 %). Ensuite, j’ai reconstruit l’évolution de chaque famille de gènes le long de l’arbre phylogénétique des Lachancea en terme de gains et de pertes depuis l’origine du clade. Mes résultats suggèrent que les réarrangements chromosomiques balancés (translocations, inversions) peuvent interrompre, au niveau de leurs points de cassure, la séquence codante des gènes, et entraîner jusqu’à 14 % des pertes de gènes observées (rupture de gène). En outre, j’ai observé des corrélations entre le taux de divergence des séquences codant pour des protéines et les taux de duplication de gènes, de translocations et d’inversions, et de rupture de gène, suggérant l’existence d’une horloge génomique qui coordonnerait ces processus. Par la suite, je me suis focalisé sur l’émergence de nouveaux gènes de novo à partir de séquences non-codantes, dont l’impact global sur les génomes n’est pas encore connu. J’ai pour cela analysé les gènes taxonomiquement restreints aux levures des clades Lachancea et Saccharomyces sensu stricto et j’ai pu identifier un ensemble de 596 gènes ayant fort probablement émergé de novo. Le taux d’émergence de novo est constant chez les levures au sein du même clade mais varie d’un ordre de grandeur entre les 2 clades (2.8 gènes/ma chez les Saccharomyces et 0.27 gènes/ma chez les Lachancea). Ces nouveaux gènes sont distribués uniformément sur les chromosomes. Ils sont le plus souvent orientés de façon divergente par rapport à leur voisin en 5’, ce qui suggère que leur transcription pourrait être initiée au niveau de promoteurs divergents, favorisant ainsi la transition d’une séquence intergénique non transcrite à une séquence codante transcrite (puis traduite). Enfin, j’ai montré que dans certains cas, seul un petit nombre de mutations permettent la création d’un gène bien adapté à son environnement génomique, en comparaison avec des gènes plus «anciens». Cela signifie que sous certaines conditions la transition d’une séquence non-codante vers une séquence codante peut être relativement rapide. Globalement, mes résultats suggèrent que l’émergence de novo est un processus évolutif non négligeable, représentant une source importante de création de nouvelles protéines
Gene repertoires are highly dynamic : Genes are duplicated, lost, transferred from one genome toanother and new genes are formed. Studying these processes and how they shape gene repertoireevolution is fundamental to our understanding of how the enormous diversity of life on earth came to be. I reconstructed the homologous gene families of the yeasts of the Lachancea genus and categorized them based on their conservation in species outside the genus into vertically inherited (98.2%), horizontally transferred (0.15%) and taxonomically restricted (1.63%). Then, I inferred the evolution of each family along the genus’ phylogeny and identified the gene gain and loss events that occurred since the genus’ origin. I found that balanced chromosomal rearrangements may be responsible for up to 14% of gene losses by disrupting the coding sequence at their breakpoints and detected 3 cases with clear traces of the disruption at the sequence level. Additionally, I found that correlations exist between the rate of protein-coding sequence divergence and the rates of gene duplication, chromosomal inversions and translocations, and gene disruptions by balanced rearrangements, suggesting the existence of a genomic clock coordinating these processes. Next, I focused on the emergence of new genes de novo from non-coding sequences, a process whose overall impact remains a matter of debate. I thus analyzed taxonomically restricted genes in the two model yeast genera Lachancea and Saccharomyces sensu stricto and identified a robust set of 596 genes that have likely emerged de novo. I found that de novo emergence rates are constant among yeasts of the same genus but differ by an order of magnitude between the two genera with 2.8 genes/my in the Saccharomyces and 0.27 genes/my in the Lachancea. De novo genes are uniformly distributed on yeast genomes and are found divergently oriented relative to their 5’ neighbors suggesting that divergent transcription might play a role in their transition from non-transcribed intergenic sequences to transcribed (and translated) coding sequences. Moreover, through specific examples I was able to show that a few enabling mutations are sufficient for a young de novo gene to emerge already well-adapted relative to older genes, indicating that the transition from non-coding to coding can happen rapidly. Overall, my results support de novo emergence as a ubiquitous evolutionary process and a potent source of novel proteins

Siglecs and CEA are two families of cell surface proteins belonging to the immunoglobulin superfamily. They are thought to be involved in cell-cell interactions and have various other biological functions. We used the GENECONV program that applies statistical tests to detect gene conversion events in each family of five primate species. For the Siglec family, we found that gene conversions are frequent between CD33rSiglec genes, but are absent between their conserved Siglec genes. For the CEA family, half of gene conversion events detected are located in coding regions. A significant positive correlation was found between the length of the conversions and the similarity of the converted regions only in the Siglec gene family. Moreover, we found an increase in GC-content and similarity in converted regions compared to non-converted regions of the two families. Furthermore, in the two families, gene conversions occur more frequently in the extracellular domains of proteins, and rarely in their transmembrane and cytoplasmic regions. Finally, these two families appear to be evolving neutrally or under negative selection.

A doença de Parkinson é freqüente no mundo todo, atingindo indivíduos de todas as idades e etnias. Embora comum e muito estudada, seus mecanismos causais ainda não são plenamente conhecidos e ainda não há tratamento curativo. Atualmente, são conhecidos alguns fatores ambientais e genéticos associados ao desenvolvimento da doença de Parkinson. Dentre os fatores genéticos, foram identificados diversos genes que podem, ou determinar a ocorrência da doença de forma mendeliana (genes principais), ou apenas aumentar o risco de seu surgimento (genes de suscetibilidade). Embora os fatores genéticos, em conjunto, sejam responsáveis por uma minoria dos casos, permanece relevante esta investigação, para promover um aconselhamento genético adequado para os portadores de formas mendelianas, para adequar medidas de tratamento e reconhecer características clínicas e de prognóstico, oportunizando, inclusive, ampliar o entendimento dessa condição. O presente estudo analisou pacientes portadores de doença de Parkinson em acompanhamento no Hospital de Clínicas de Porto Alegre, que apresentavam baixa idade de início dos sintomas, história familiar positiva ou presença de manifestações atípicas da doença. Essas características foram utilizadas como critério de seleção dos pacientes por estarem associadas com maior probabilidade de detecção de causas genéticas. Os pacientes foram submetidos à avaliação clínica e à testagem molecular para diversos genes principais e de suscetibilidade. Foram, posteriormente, comparadas as características clínicas dos pacientes positivos com relação aos demais pacientes estudados. Os resultados são apresentados sob a forma de três artigos, que descrevem, respectivamente, os achados moleculares da investigação para os genes causais autossômicos recessivos (genes PARK2, PARK6, PARK7 e PARK8), autossômicos dominantes (genes SCA1, SCA2, SCA3, SCA6 e SCA7) e o gene de suscetibilidade (gene GBA).

A identidade na segmentação do corpo de diversos organismos, durante o desenvolvimento, é devida, em grande parte, à ação das proteínas homeóticas. Em especial, dois grupos de proteínas, Trithorax (trxG) e Polycomb (PcG) têm um papel fundamental na manutenção, respectivamente, da ativação e da repressão da transcrição gênica, associando-se à cromatina. A importância das PcG nos estimulou a buscar a caracterização das proteínas humanas ortólogas ao \"Enhancer of Polycomb\" (Epc) de Drosophila, até então não descritas no genoma humano. Para tanto, buscamos: - obter a sequência completa e mapear o cDNA do novo gene humano homólogo ao \"Enhancer of Polycomb\" de Drosophila; - analisar sua expressão em tecidos fetais, adultos e tumorais e fazer estudos buscando sua caracterização funcional. Encontramos, mapeamos e obtivemos a seqüência completa de dois genes humanos, ortólogos de Epc1 (10p11-22) e de Epc2 (2q21-23) de camundongo, publicando estes dados em 2001 (Camargo et al., 2001). Ambos os genes são bastante conservados entre várias espécies, sendo que o cDNA de hEPC2 humano, por exemplo, é 94% idêntico ao Epc2 de camundongo e possui 96% de identidade ao nível de proteína, sugerindo que a função do gene deve ter sido mantida durante a evolução. No entanto, as seqüências protéicas de hEPC1 e hEPC2 humanos possuem apenas 68% de identidade entre si. Portanto, é provável que após a duplicação dos parálogos, estes tenham divergido funcionalmente. A expressão de ambos os genes foi avaliada utilizando \"dot-blots\" contendo 76 mRNAs de amostras de tecidos fetais, adultos e tumorais, mostrando-se fraca e ubíqua. Análises in silico sugeriram a existência de 4 isoformas de splicing para hEPC2, as quais foram validadas por RT-PCR ou \"Northern blots\". Uma das isoformas (de 2.7 Kpb) se mostrou mais abundante em todas as linhagens tumorais estudadas através de análises de \"Northern blot\", principalmente nas linhagens de linfoma de Burkitt\'s Raji e na linhagem de leucemia pró-mielocítica HL-60. Esta isoforma é gerada através de um sítio alternativo de poli-adenilação, que reduz sua porção 3\'UTR, retirando 4 dos 5 \"elementos ricos em adenilatos e uridilatos\" (AREs), envolvidos com a degradação de mRNAs lábeis que codificam proteínas regulatórias. Estes resultados se encontram em um manuscrito recentemente submetido à publicação (anexo à tese). Interação entre hEPC2 e SMADs e sua modulação por TGF-β. Durante a montagem da seqüência completa de hEPC2, verificamos que duas ESTs patenteadas mostravam alta identidade com o gene. Estas seqüências foram descritas como sendo parte de uma nova proteína de interação com as proteínas da família SMAD, envolvidas com transdução de sinais desencadeados por TGF-β. Esta citocina por sua vez, regula a proliferação, diferenciação e morte celular. Partimos para a avaliação da possível interação entre hEPC2 e as SMADs, em colaboração com o grupo do Dr. Aristidis Moustakas, do Ludwig Institute for Cancer Research de Uppsala, Suécia. Os resultados de co-imunoprecipitação sugeriram que as SMADs 2, 3, 4, 7 e 8 interagem com hEPC2, sendo que a interação entre as SMAD2, SMAD3, SMAD4 e hEPC2 nas células tratadas com TGF-β1, mostraram uma redução na co-imunoprecipitação. Este resultado sugere que TGF-β1 modula negativamente a interação entre essas proteínas. Da mesma maneira, foi observada uma redução na interação de hEPC2 com SMAD8 após o tratamento com BMP-7. Esse resultado é ainda mais destacado para as SMADs 2 e 3. Estes dados foram observados para ambas as construções de hEPC2, o que sugere fortemente a veracidade da interação entre estas proteínas. A localização celular de hEPC2, e também sua co-localização com SMAD2 foram investigadas através de imunofluorescência indireta e confirmaram a predição do programa PSORTII, de que hEPC2 se localiza no núcleo. No entanto, não foi possível observar a co-localização entre hEPC2 e SMAD2. É possível que hEPC2 não se ligue diretamente ao DNA, necessitando se associar como parceiro de um fator de transcrição. Esta foi uma das hipóteses para a atuação de hEPC2, como um co-fator que se associe com uma das SMADs e se ligue a um elemento específico de ligação a SMAD (SBE). Para investigar essa hipótese um ensaio de gene repórter foi feito utilizando uma construção de um repórter contendo 12 repetições da seqüência CAGA (seqüência específica de ligação das SMADs 2,3 e 4) fusionado com o gene da luciferase. No entanto, este ensaio não demonstrou que a transcrição de SMAD2 é dependente de hEPC2 e o experimento deverá ser repetido. Para confirmar a interação entre hEPC2 e as SMADs, será feito um experimento de \"pull-down\". Para tal o cDNA de hEPC2 foi clonado no vetor pET-32A de expressão indutível em bactérias. A proteína recombinante já foi produzida, tendo sido induzida e posteriormente purificada em condições desnaturantes. Apesar de dezenas de genes PcG terem sido caracterizados em Drosophila, poucos destes genes foram estudados em mamíferos. Portanto, a descrição do gene hEPC2 e seus transcritos alternativos, contribui para o conhecimento de PcG humanos, indicando a associação de maior expressão de uma de suas isoformas em linhagens celulares tumorais. Em relação à interação de hEPC2 com as SMADs, é interessante observar que nenhuma outra proteína foi descrita por possuir a particularidade de interagir com as SMADs de diferentes categorias. Talvez este seja um dado importante, que indique o papel singular de hEPC2 na sinalização de TGF-β1.
The identity of body segmentation in several organisms during development is, to a large extent, due to the action of the homeotic proteins. In particular, two groups of proteins, the Trithorax (trxG) and Polycomb (PcG), have a major role in maintenance of respectively, transcription activation and repression, when associated to the chromatin. The importance of PcGs has motivated us to pursue the isolation and characterization of two new human proteins that are orthologs of the \"Enhancer of Polycomb\" (Epc) of Drosophila. To achieve this goal we undertook the task of the cloning and mapping of complete cDNA sequence of the novel genes hEPC1 and hEPC2, analyzing its expression in fetal, adult and tumoral tissues and functionally characterizing the hEPC2 protein. In 2001, we published the mapping and cloning of the complete cDNA sequences of both genes, as being orthologs of the mouse Epc1 (10p11-22) and Epc2 (2q21-23), together with the strategy used to obtain the full-length cDNAs (Camargo et al., 2001). Both genes are shown to be highly conserved among several species. Thus, the human hEPC2 cDNA is 94% identical to the mouse Epc2 and displays 96% identity at the protein level, suggesting maintenance of its function during the evolution. However, the protein sequences of the human hEPC1 and hEPC2 display only 68% identity. Therefore, it is likely that they have undergone a functional divergence after their duplication. The expression of both genes was evaluated using \"dot-blots\" containing 76 mRNAs samples from fetal, adult and tumoral tissues and is shown to be weak and ubiquitous. \"In silico\" analysis suggested the existence of 4 hEPC2 splicing isoforms that were validated by RT-PCR and/or Northern-blots. One of the isoforms (of 2.7 Kbp) is shown to be more abundant in all of the tumoral cell lines evaluated using Northern-blot analysis, mainly in the Burkit\'s Raji lymphoma and in the promyelocytic leukemia HL-60. This isoform results from the use of an alternative polyadenylation site that reduces the 3\'UTR, abolishing 4 of 5 \"adenylates and urilates rich elements\" (AREs), involved in the degradation of labile mRNAs that codify to regulatory proteins. These results have been recently submitted to publication (manuscript attached to this thesis). Interaction between the hEPC2/SMADs and its modulation by TGF-β. During the assembly of the hEPC2 full-length cDNA sequence, we found two patented ESTs that tagged a portion of the gene. These sequences were described as partial sequences of a \"new SMAD interacting protein\", involved in signal transduction of TGF-β, a cytokine that regulates cell proliferation, differentiation and death. To evaluate this putative interaction between hEPC2 and the SMADs proteins, we begun a collaboration with the TGF-β signalling group of the Dr. Aristidis Moustakas, from the Uppsala Ludwig Institute for Cancer Research, Sweden. The results of co-imunoprecipitation assays suggested that SMADs 2, 3, 4, 7 e 8 interact with hEPC2. Moreover, the interaction among SMAD2, SMAD3, SMAD4 and hEPC2 in cells treated with TGF-β1 showed decreased co-imunoprecipitation. This result suggests that TGF-β1 negatively modulates the interaction of these proteins. Likewise, we observed a reduction in hEPC2 interaction with SMAD8 upon BMP-7 treatment. This effect was even more dramatic for SMADs 2 and 3. These data were observed for both hEPC2 plasmid constructs, which strongly suggest the veracity of these proteins interaction. The cell localization of the hEPC2 protein, as well as its co-localization with the SMAD2, were investigated through indirect immunofluorescence assay, confirming the predicted localization of hEPC2 in the cell nucleus using the PSORTII program. However, we were not able to confirm the co-localization of hEPC2 and SMAD2. It is possible that hEPC2 does not bind directly to the DNA, requiring an association with a partner such as a transcription factor. This raises the hypothesis of hEPC2 having a role as a co-factor associated to one of the SMADs and binding to a \"SMAD binding element\" (SBE). To investigate this hypothesis, gene reporter assays were undertaken using a reporter construct containing 12 CAGA sequence repetitions (specific binding sequence of the SMADs 2, 3 and 4) fused to the luciferase gene. However, this assay could not demonstrate that the transcription of the SMAD is dependent on hEPC2. This experiment must be repeated. To confirm the interaction of hEPC2 and SMADs, a pull-down assay will be performed. To this end, the coding region of hEPC2 was cloned into the pET-32A bacterial inducible expression vector. The recombinant protein was already produced, having been induced and purified under denaturing conditions. Despite the dozens of PcG genes that were described in Drosophila, only a few of these genes have been characterized in mammals. Therefore, the description of the hEPC2 and its alternative transcripts is a contribution to better knowledge of the human PcGs. Regarding the hEPC2 and SMADs interaction, it\'s it is noteworthy that this is the first protein described to interact with SMADs of distinct categories. This may be an important indication of a unique role for hEPC2 in the TGF-β1 signaling pathway.

Thesis (M.S.)--West Virginia University, 2005.
Title from document title page. Document formatted into pages; contains viii, 99 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 82-99).

Cherax quadricarinatus (Redclaw), C. destructor (Yabby) and C. cainii (Marron) are a group of economically important freshwater crayfish and have been developed for aquaculture production in many countries. As crayfish are farmed in a wide range of culture conditions, optimisation of water quality parameters, are crucial for their maximum growth performance. Previous reports have shown that fluctuations in water quality can negatively impact on growth of crayfish. Therefore, this project aims to identify and characterize the major genes that enable freshwater crayfish to persist in different water chemistries and evaluate their patterns of expression under different water parameters. Overall, this project found a number of candidate genes in all three species and determined that water chemistry had a strong influence on the expression of candidate genes. This information is important in the optimization of water quality parameters in freshwater crayfish aquaculture production.

The $ beta$-tubulin gene family of the parasitic nematode, Brugia pahangi consists of three to five $ beta$-tubulin sequences. Two genomic clones containing $ beta$-tubulin sequences were isolated and characterized. The $ beta$1-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mRNA of 1.7 kb which codes for a protein of 448 amino acids. A partial nucleotide sequence of the second clone confirmed the isolation of a distinct $ beta$-tubulin sequence, $ beta$2-tubulin. The $ beta$1-tubulin transcript is found in microfilariae and adult worms, whereas the $ beta$2-tubulin transcript is predominant in male adult worms but absent from microfilariae. Results of this study also indicate that the maturation of the $ beta$1-tubulin message involves the acquisition of the conserved nematode 22-nucleotide splice leader sequence. Antipeptide IgGs raised against the divergent carboxy-terminal region of $ beta$1-tubulin recognize the same $ beta$-tubulin isoform pattern as a phylum cross-reactive monoclonal antibody. This result suggests that the $ beta$1-tubulin is highly represented in B. pahangi adults and microfilariae.

Telomere shortening and an increased telomerase activity are associated with poor prognosis and disease progression in many cancers. In Chronic Myeloid Leukaemia (CML) telomere shortening has a strong correlation with disease progression. Expression of hTERT, the catalytic component of telomerase, was evaluated in the CD34+ cells of CML patients. This revealed that expression of hTERT was significantly reduced in chronic phase CML and decreased with disease progression to accelerated phase and blast crisis. .It could therefore be concluded that reduced hTERT expression contributes to reduced telomere length in CML. Additionally, expression of c-Myc, which increases hTERT transcription, correlated with hTERT expression suggesting decreased hTERT is partly caused by reduced c-Myc. hTERT promoter methylation and mutation status were investigated and this revealed that the hTERT promoter was not methylated and mutation rates were low suggesting that these are not contributing to reduced hTERT expression. cDNA microarrays were used to analyse gene expression in neutrophils of patients with Essential Thrombocythaemia (ET) harbouring the JAK2 V617F mutation which, like the BCRlABL translocation in CML, results in an activated kinase. Neutrophils of ET patients exhibited a gene expression profile close to that of controls despite the presence of the mutation. Affymetrix microarrays were used to investigate the role of telomerase related genes in Myelodysplastic Syndromes (MDS). Genes decreased in patients with del(5q) include positive regulators of telomere length and genes with higher expression were associated with increased telomerase activity.

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Evidences indicate that by 2030 an increase in 40% of rice production is necessary so that it can meet the demand of the world population. The aim of this study was to quantify the expression of rice genes homologs to Arabidopsis genes previously related to yield (RUBISCO, AVP1, DA1 and TOR) by RT-qPCR analysis. The genotypes used in the study were the upland rice cultivars BRSMG Curinga and Primavera, and the old variety Douradão, which were evaluated in a yield experiment under two soil fertility levels, in a factorial scheme, in completely randomized design with two replications. Plant tissue samples were collected in vegetative and reproductive stages, which were used for total RNA isolation and subsequent cDNA syntheses. The cDNA was then subjected to RT-qPCR to evaluate the expression of four genes studied. Significant differences were observed in the expression of RUBISCO gene in leaves of upland rice plants in the vegetative stage, where there was a higher expression in high-level fertility in the soil, while in the reproductive stage, there was a higher expression in the low fertility treatment. DA1 that negatively regulates cell proliferation was less expressed in the vegetative stage in the treatment with high level of fertility, suggesting the suppression of this gene. AVP1 was more expressed in the reproductive stage, probably in order to increase the availability of P in a fundamental phase for the formation of the grain. TOR was the most expressed gene in this work, with a greater expression in adequate conditions of fertility, confirming its action under favorable conditions of cultivation. This study indicates that even two species that diverged over 120 million years have conserved productivity related metabolic routes. Thus, genes previously studied and validated in the model species Arabidopsis and that are of interest to economically important crops such as rice, may be the starting point for the development of cultivars with higher performance and better agronomic traits.
Evidências indicam que, até 2030, será necessário um aumento em 40% da produção de arroz para que ele possa atender a demanda da população mundial. O objetivo desse estudo foi quantificar a expressão de genes de arroz homólogos a genes de Arabidopsis anteriormente relacionados à produtividade (RUBISCO, AVP1, DA1 e TOR) por meio da análise de RT-qPCR. Os genótipos usados no estudo foram as cultivares de arroz de sequeiro BRSMG Curinga e Primavera, e a cultivar antiga Douradão, as quais foram avaliadas em um experimento de rendimento sob dois níveis de fertilidade do solo, em esquema fatorial, em delineamento experimental inteiramente casualizado, com duas repetições. Amostras de tecido foliar foram coletadas nas fases vegetativa e reprodutiva, que foram utilizadas para o isolamento de RNA total e posterior síntese de cDNA. O cDNA foi, em seguida, submetido à RT-qPCR para avaliar a expressão dos quatro genes estudados. Foram observadas diferenças significativas na expressão do gene RUBISCO (Ribulose bifosfato Carboxilase/Oxigenase) em folhas de plantas de arroz de terras altas no estádio vegetativo, em que houve uma maior expressão em solo com fertilidade recomendada, enquanto que na fase reprodutiva, houve uma maior expressão no tratamento de baixa fertilidade. DA1 (Receptor de Ubiquitina), que regula negativamente a proliferação celular foi menos expresso no estádio vegetativo, no tratamento com elevado nível de fertilidade, sugerindo a supressão deste gene. AVP1(Arabidopsis Vacuolar Pirofosfatase) foi mais expresso no estádio reprodutivo, provavelmente para aumentar a disponibilidade de P em uma fase fundamental para a formação do grão. TOR (Alvo de rapamicina) foi o gene com maior nível de expressão, principalmente no solo com maior nível de fertilidade, confirmando sua ação em condições favoráveis de cultivo. Esse estudo indica que mesmo duas espécies que divergiram mais de 120 milhões anos, como é o caso de Arabidopsis e arroz, têm conservadas rotas metabólicas relacionadas à produtividade. Assim, os genes previamente estudados e validados na espécie modelo Arabidopsis e que são de interesse para as culturas economicamente importantes, como o arroz, podem ser o ponto de partida para o desenvolvimento de cultivares com maior desempenho e melhores características agronômicas.

The goal of this project was to clone and express the antimicrobial peptide protegrin 1 (PG-1). Initially a yeast system was chosen but was discarded due to technical difficulties. Invitrogen's bacterial T7 expression system was chosen next to express the peptide. PG-1 expression was verified by anti-his immunoblot and then the peptide was purified by IMAC. Its activity was verified using a Bacillus subtillis radial diffusion assay.

A comparação de genomas, genes ou até sequências de nucleotídeos não condificantes é uma importante tarefa na qual a bioinformática pode ser aplicada, uma vez que ela auxiliar em diversas atividades, por exemplo, análises filogenéticas. Análise filogenética, por sua vez, busca analisar a relação evolutiva de cada espécie, considerando suas características genéticas. Esses processos e as técnicas que os implementam se baseiam em sequências de nucleotídeos sequenciadas e armazenadas em bancos de dados de genomas públicos. Com análise filogenética também é possível identificar possíveis origens de um gene. Essa tarefa é de grande importância, pois auxilia na identificação da origem de genes patogênicos, podendo auxiliar no combate e prevenção do surgimento de doenças. Um problema potencial dessas sequências é a possibilidade de haver erros nas anotações (marcações de sequências como genes). Esses erros são pouco explorados por pesquisadores atualmente. Outro tema pouco explorado é a análise filogenética de genes exclusivos, que são genes que se manifestam em apenas uma espécie, considerando um grupo de espécies próximas. A identificação de genes exclusivos de alguma espécie pode servir para a correta identificação de, por exemplo, a espécie que causa uma doença, de forma a permitir o uso do tratamento mais específico e adequado. A importância da descoberta de filogenias de genes exclusivos e a dificuldade de garantir a consistência nas anotações genéticas motivaram este trabalho, que teve como objetivo implementar ferramentas para interpretar dados de comparação genética, identificando potenciais erros em anotação de genes exclusivos e criando estratégias para identificar a origem desses genes. As origens de genes exclusivos exploradas neste trabalho envolvem a possibilidade dos genes exclusivos terem derivado de outras famílias de genes do próprio organismo, ou, os genes exclusivos se diferenciaram muito dos genes ancestrais. Essas hipóteses, juntamente com a hipótese da existência de erros de anotação, foram exploradas em experimentos utilizando as ferramentas desenvolvidas. Os experimentos visaram a analisar a aplicabilidade da estratégia desenvolvida. Foram utilizados genomas de bactérias do gênero Xanthomonas, que contém um grande grupo de bactérias que causam doenças em plantas. Os resultados obtidos demonstram que existe uma quantidade considerável de potenciais erros de anotação nos genomas considerados, provando a hipótese de que a inconsistência nas anotações genômicas possui grande influência para a dificuldade na identificação de filogenias (tanto de genes exclusivos como para não exclusivos). Os resultados também demonstraram que boa parte dos genes exclusivos possivelmente se originaram de outras famílias de genes do próprio genoma. Ou ainda, que esses genes sofreram modificações em relação aos genes ancestrais, mas ainda possuem certas semelhanças com sequências de nucleotídeos que não codificam genes em outras espécies mais distantes. Por fim, a estratégia desenvolvida se mostrou útil na análise filogenética das bactérias estudadas, sendo este um forte indício de que a mesma abordagem pode ser utilizada para problemas similares com outras espécies de seres vivos
Comparison of genomes, genes or even non-coding nucleotide sequences is an important task in which bioinformatics can be applied, since it allows the application of phylogenetic analyses. Phylogenetic analysis, in its turn, seeks to analyze the evolutionary relation of each species, considering its genetic characteristics. These processes and the techniques that implement them are based on nucleotide sequences sequenced and stores in databases of public genomes. With phylogenetic analysis it is also possible to identify possible origins of a gene. This task has a great importance, because it allows the identification of the origin of pathogenic genes, which may help to combat or prevent deseases. A potencial problem of these sequences is the possibility of having annotation errors (sequences marking as genes). These errors are little explored by researchers nowadays. Another unexplored topic is the phylogenetic analysis of exclusive genes, which are genes thaht manifest in only one species, considering a group of nearby species. The identification of exclusive genes of a species may serve to correctly identify, for example, a desease, in order to allow the use of a more especific and appropriate treatment. The importance of discovering phylogenies of exclusive genes and the difficulty of guaranteeing the consistency of genetic annotations motivated this work, whose objective was to implement tools to interpret data of genetic comparison, identifying annotation errors in exclusive genes and creating strategies to identify the origin of these genes.The origins of exclusive genes explored in this work involve the possibility of the exclusive genes have derived of other gene families of the organism itself, or, the exclusive genes differed a lot from the ancestral genes. Theses hypotheses, with the hypotesis of the existance of annotation errors, were explored in experiments using the developed tools. The experiments aimed to analyse the applicability of the developed strategy. Genomes of bacteria of the genus Xanthomonas were used, which contains a large group of bacteria that cause diseases in plants. The results show that there is a considerable amount of annotation errors on the genomes, proving the hypothesis that the inconsistency in genomic annotations has a great influence on the difficulty in identifying phylogenies (both exclusive and non-exclusive genes). The results also show that much of exclusive genes possibly originated from other gene families of the genome itself. Furthermore, these genes may have sufferedmodifications in relation to the ancestral genes, but still have certain similarities with nucleotide sequences that don\'t encode genes in other more distant species. Finally, the strategy developed proved useful on phylogenetic analysis of the studied bacteria, which is a strong indication that the same approach can be used for similar problems with other species of living beings

The desert ant Cataglyphis fortis lives in the Sahara desert where it is exposed to extreme temperatures up to 70° C. In other words, the organism is considered as a thermophile. Until now the genome remains unknown but the fact that C. fortis provides heat stable proteins makes it very interesting in the field of protein studies and maybe even therapeutical research later on. This thesis focuses on trying to find genes that are expressed in C. fortis. Different genes were chosen and capable primers designed. After fishing for the enzyme GAPDH a fragment was found and sequenced. The sequence showed 31% homology on amino acid level with protein disulfide isomerase (PDI) in Apis mellifera (honey bee) and Drosophila melanogaster (fruitfly). The received sequence can be used to design new primers that match exactly. Gene fishing can also be continued by using the other primers that were designed during this project.

We employed a large scale gene trap approach to identify genes involved in osmotic stress responses. This strategy allows the analysis of stress-induced Gus expression, so we realized a selective screening of the EXOTIC (EXOn Trapping Insert Consortium) collection, by using Mannitol as selective agent. Moreover this strategy allows the analysis of cell-specific Gus expression, so we searched the EXOTIC lines for guard cell-specific activation of the reporter gene. We identified 3 lines that showed GUS expression only after exposure to Mannitol. In one line we mapped the transposon insertion in the At3g03350 gene, coding for a putative dehydrogenase/reductase protein. At3g03350 is specifically activated by Mannitol in roots, but it is constitutively expressed in the endosperm. We observed an arrest of embryo development at the globular stage in homozygous mutant seeds and verified that it is due to knockout of the At3g03350 gene. Both mutant embryo and endosperm did not show morphological defects. This phenotype suggests that the enzyme is involved in early seed development, particularly in energy metabolism.Subsequently we employed the gene trap approach to identify genes expressed in guard cells. We identified five lines in which the reporter gene was exclusively or preferentially expressed in guard cells. In 3 lines we mapped the DsG insertion in intergenic regions while in 2 lines we mapped the DsG insertion in two annotated genes. CYP86A2, coding for a cytochrome P450 protein, is involved in cutin monomers biosynthesis in stomata and we hypothesize that it is involved in stomatal responses to pathogen attacks. AtPDR3 (pleiotropic drug resistance 3) is an ABC transporter and we provide evidence that it is involved in the modulation of guard cell responses to ABA.

Even today, many genes are without any known homolog. These "orphans" are found in all species, from Viruses to Prokaryotes and Eukaryotes. For a portion of these genes, we might simply not have enough data to find homologs yet. Some of them are imported from taxonomically distant organisms via lateral transfer; others have homologs, but mutated beyond the point of recognition. However, a sizeable fraction of orphan genes is unambiguously created via "de novo" mechanisms. The study of such novel genes can contribute to our understanding of the emergence of functional novelty and the adaptation of species to new ecological niches. In this work, we first survey the field of orphan studies, and illustrate some of the common issues. Next, we analyze some of the intrinsic properties of orphans proteins, including secondary structure elements and Intrinsic Structural Disorder; specifically, we observe that in young proteins the relationship between these properties and the G+C content of their coding sequence is stronger than in older proteins. We then tackle some of the methodological problems often found in orphan studies. We find that using evolutionarily close species, and sensitive, state-of-the art homology recognition methods is instrumental to the identification of a set of orphans enriched in de novo created ones. Finally, we compare how intrinsic disorder is distributed in bacteria versus eukaryota. Eukaryotic proteins are longer and more disordered; the difference is to be attributed primarily to eukaryotic-specific domains and linker regions. In these sections of the proteins, a higher frequency of the disorder-promoting amino acid Serine can be observed in Eukaryotes.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Manuscript.

Introduction Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). Human Major Histocompatibility complex (HLA) class I and II genes are associated with MS pathogenesis. Natural killer cell receptors (KIRs) bind to different MHC class I molecules activating NK cells to produce immune-regulatory cytokines. Sphingosine 1-phosphate receptor 1 (S1PR1) polymorphism and increasing IL-17 levels in blood of MS patients associate with non­responding to fingolimod treatment. Aim: To investigate HLA class I and II alleles, MOG (V142L) variant, KIR receptor genes, S1PR1 gene polymorphisms and a possible biomarker, serum IL-17, association with MS in Iranian patients. Material and methods: 489 patients and 350 controls from Iran were recruited. PCR, Direct Sanger sequencing, ELISA, and PAGE, Real time PCR were applied for genotyping. Statistical analysis such as Chi squared test, independent sample t-test, Principal component analysis (PCA) and Logistic regression analysis, was performed to identify the associations of traits with MS. Bonferroni correction and Power calculation of study was calculated. Results Iranian Multiple Sclerosis (IRAMS) database containing 247 parameters of patients was generated. Association between HLA-DRBl*1501 (P=3.70xl0-5, OR=2.10 (95% CI: 1.50-3.00)) and HLA-A02 (P= 0.014, OR= 0.671 (95% CI: 0.489-0.922)) with MS was observed. Significant association (P<0.05) between HLA class I, II with motor symptoms, bladder dysfunction, optic symptoms, and neuropsychological signs was shown. There was significant association between HLA-C6 allele and MS (P= 0.027 and OR= 1.445 (1.040-2.007), but not between KIRs gene variants and MS. Respond to fingolimod was not associated with either S1PR1 polymorphism or IL-17 levels. Discussion This study has shown clear association between genetic variants and MS related traits. Novel variants in S1PR1 have been identified.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
O esgotamento das reservas de energia nÃo-renovÃvel, como petrÃleo, carvÃo e gÃs natural, tÃm estimulado a busca por fontes alternativas geradoras de energia. Neste contexto, o pinhÃo manso (Jatropha curcas L.) tem recebido especial atenÃÃo por parte dos pesquisadores, devido à presenÃa de grande quantidade de Ãleo em suas sementes, que pode ser convertida em biodiesel. O objetivo deste trabalho foi investigar o comportamento de genes relacionados ao metabolismo de lipÃdios durante os processos de desenvolvimento e germinaÃÃo da semente de pinhÃo manso. Para quantificar com exatidÃo os nÃveis de expressÃo gÃnica por RT-qPCR foi feita uma seleÃÃo entre nove candidatos a genes de referÃncia. Nossos resultados mostraram que na anÃlise de sementes em desenvolvimento, os genes GAPDH, UCP, ACT11, PP2A2 e CICLOF foram os mais estÃveis. Para as sementes em germinaÃÃo, os genes considerados mais estÃveis foram EF1-α, PP2A2, GAPDH, PUB3 e ACT11. Para validar nossos resultados com genes de referÃncia, foi utilizado o padrÃo de expressÃo do gene que codifica a proteÃna oleosina no qual foi observado que o mesmo foi similar aos observados em artigos cientÃficos pesquisados, indicando que os genes de referÃncia estavam apropriados para normalizaÃÃo dos dados de RT-qPCR. ApÃs obtenÃÃo desses dados, efetuou-se um estudo da expressÃo por RT-qPCR de 20 genes envolvidos com o metabolismo de lipÃdios. Nossos resultados revelaram que os genes oleosina, β-cetoacil-ACP Sintase I e II, tioesterase A e triacilglicerol lipase I, bem como outros genes envolvidos na biossÃntese de lipÃdios, alcanÃaram altos nÃveis de expressÃo no desenvolvimento da semente. Os genes acil-CoA sintetase, tiolase e triacilglicerol lipase II, relacionados com a degradaÃÃo de lipÃdios apresentaram altos nÃveis de transcritos na germinaÃÃo da semente. Os dados obtidos neste trabalho contribuem para o entendimento das vias metabÃlicas estudadas, fornecendo subsÃdios para a produÃÃo, via engenharia genÃtica, de variedades melhoradas do pinhÃo manso.
The depletion of non-renewable energy such as oil, coal and natural gas, has stimulated the search for alternative sources of energy generation. In this context, physic nut (Jatropha curcas L.) has received special attention from researchers due to the presence of large amounts of oil in its seeds that can be converted into biodiesel. The objective of this study was to investigate the behavior of genes related to lipid metabolism during the development and germination of physic nut seeds. To accurately quantify the levels of gene expression by RT-qPCR, a selection of nine candidates for reference genes was performed. Our results showed that the GAPDH, UCP, ACT11, PP2A2 and CICLOF were the most stable genes during the development of the seeds. In germinating seeds, EF1-α, PP2A2, GAPDH, PUB3 and ACT11 were considered the most stable genes. To validate our findings with reference genes, we used the expression profile of the gene encoding the oleosin protein in which that was similar to those observed in the literature evaluated, indicating that they were suitable reference genes for data normalization by RT-qPCR. After obtaining these data, we performed a gene expression study by RT-qPCR of 20 genes involved in lipid metabolism. Our results revealed that the oleosin, β-ketoacyl-ACP Synthase I and II, thioesterase A and triacylglycerol lipase I genes, as well as other genes involved in lipid biosynthesis, achieved high expression levels in developing seeds. Acyl-CoA synthetase, thiolase and triacylglycerol lipase II, genes related to the degradation of lipids, showed high transcript levels in germinating seeds. The data obtained in this study contribute to the understanding of the metabolic pathways studied, providing subsidies for production of improved varieties of physic nut via genetic engineering.

HOX11 is a developmental regulator that plays a crucial role in the normal development of the spleen and is also aberrantly activated by the t(10;14)(q24;q11) and variant t(7;10)(q35;q24) translocations in a subset of T-cell acute lymphoblastic leukaemias (TALLs). The recent finding that HOX11 is deregulated in up to 40% of childhood TALLs when abnormalities not detected by cytogenetics are included, suggests that the over-expression of HOX11 and subsequent deregulation of downstream target genes are critical events in the progression of this tumour type. To date, three candidate HOX11 target genes have been reported, two of which are Aldehyde Dehydrogenase 1a1 (ALDH1A1) and Four and a Half LIM domain Protein 1 (FHL1). This investigation focused on two aspects of HOX11 function, namely its roles as a transcriptional regulator and as a nuclear oncoprotein capable of inducing neoplastic transformation. More specifically, we sought to further understand the role of HOX11 in tumorigenesis by 1) Confirming target gene status of ALDH1A1 and FHL1 by assessing whether their proximal promoter regions are transcriptionally regulated by HOX11, 2) Investigating the regulatory elements/transcriptional complexes involved in the response of ALDH1A1 to HOX11 in both a T-cell and an erythroid cell line in order to gain an insight into the mechanism(s) responsible for mediating a HOX11 activity and 3) Assessing the ability of ALDH1A1 and FHL1 to perturb normal patterns of haematopoiesis, on the basis that the transforming capabilities of HOX11 are thought to derive from its ability to affect haematopoietic cell differentiation. To confirm ALDH1A1 and FHL1 as target genes, they were both characterised in terms of the ability of their proximal promoters to be transcriptionally regulated by HOX11 using luciferase reporter assays. Significant repression of the proximal promoters of ALDH1A1 and FHL1 by HOX11 was observed in PER-117 T-cells which provided further evidence for their status as target genes. In the case of ALDH1A1, a CCAAT box (-74/-70bp) was identified as the primary cis-regulatory element involved in ALDH1A1 transcription and repression by HOX11 appeared to occur, either directly or indirectly, via interactions at the CCAAT box. Electromobility shift assays (EMSAs) revealed the disruption of a specific complex at this site by HOX11, which also altered the formation of complexes at a non-canonical TATA box (a GATA box at -34/-29bp). Significantly, HOX11 was shown to have the potential to interact with TFIIB, a member of the basal transcriptional complex. This, together with the presence of a TFIIB responsive element immediately 5' of the GATA box, suggested that HOX11 may repress transcription by interfering with members of a preinitiation complex on the ALDH1A1 promoter. The transcriptional repression by HOX11 demonstrated in T-cells was dependent on DNA binding helix 3 of the homeodomain, suggesting that repression may require DNA binding. Alternatively, this region may be required for stable protein-protein interactions. In support of this, the in vitro association of HOX11 with TFIIB was disrupted upon deletion of helix 3, and the HOX11 H3 mutant switched from a transcriptional repressor to a potent activator of transcription. Together, this data supports a model whereby HOX11 represses transcription by interfering with activation complexes at the CCAAT box and at the GATA box possibly via protein-protein interactions involving the homeodomain helix 3, whereas deletion of the region disables repressor-specific interactions, resulting in potent activation by HOX11. Luciferase reporter gene assays investigating the response of nested deletions of the ALDH1A1 promoter to HOX11 in the HEL900 erythroleukaemic cell line, also identified the CCAAT box (-74/-70bp) as the primary cis-regulatory element involved in ALDH1A1 transcription. However, in stark contrast to the its effect in T-cells, HOX11 was shown to activate transcription in the HEL cell line, both from the empty pGL3Basic luciferase reporter vector and from the ALDH1A1 promoter, in a manner independent of the homeodomain DNA binding helix 3. HOX11 thus appears to be a dichotomous regulator, capable of both transcriptional activation and repression depending on the circumstances. The mechanisms underlying these two functions are also appear to be distinct, with repression but not activation requiring the presence of homeodomain helix 3. ALDH1A1 encodes an enzyme involved in the irreversible conversion of retinaldehyde to the biologically active metabolite, retinoic acid (RA) and appears to be physiologically regulated by Hox11 in the developing spleen. Since RA is a potent modulator of cellular differentiation, proliferation and apoptosis, the dysregulation of RA synthesis is likely to have severe consequences for the cell and may constitute a mechanism whereby overexpression of HOX11 predisposes T-cells to malignant transformation. FHL1 also appears to have potential relevance to tumorigenesis, given that it encodes protein isoforms with suspected roles in transcriptional regulation. As a starting point to investigate a possible link between these HOX11 target genes and leukaemogenesis, the effect of overexpressing ALDH1A1 and FHL1 on murine haematopoiesis was assessed following reconstitution of lethally irradiated mice with retrovirally-transduced primary murine bone marrow cells. The enforced expression of ALDH1A1 in bone marrow was associated with a marked increase in myelopoiesis and a decrease in B and T-lymphopoiesis. By contrast, overexpression of FHL1 was not associated with perturbations in myelopoiesis or lymphopoiesis, although a slight increase in erythropoiesis was observed in the bone marrow. While further work is required to clarify the possible oncogenic roles of both of these HOX11 target genes, these findings have served to identify ALDH1A1 in particular, as a gene which could potentially be involved in HOX11-mediated tumorigenesis.

Trabalho Complementar apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciado em Ciências da Nutrição
Obesity is a global epidemy and in children the number of individuals with obesity increased 2,28 times in the last three decades. Obesity comes with a massive amount of complications such as type 2 diabetes, dyslipidemia and insulin resistance. Obesity is a multifactorial disease and there many risk factors interacting in its development, genetic, behavioral and environmental factors. This paper aims to understand the genetic factors behind this disease and the interactions between the genes and diet that prompts childhood obesity. For the purpose of this paper the literature was searched in English language in the PubMed Central® search engine, totaling 110 papers. During the last decade, research has tried to understand the gene(s) responsible for the predisposition for childhood obesity, so many studies were conducted such as genome-wide association studies (GWAS). Childhood obesity appear to result from the presence of many risk gene variants, and their response to obesogenic environments. There is evidence that genes like FTO, MC4R, POMC, LEP and LEP receptor have an influence in weight gain and in the development of related complications since early ages. Diet is one of the most important environmental factors believed to contribute to obesity development. Currently, there are two approaches trying to understand interactions between genes and diet, nutrigenetics and nutrigenomics. Both are based in the premise that nutrients/diet components can influence the gene expression process and affect different metabolic pathways that finally will origin the individual’s phenotype. Most of the studies accomplished are related to fatty acids metabolism, so to further understand other metabolic pathways more studies are needed. FTO, APOA2 and NPC1 genes are some of the genes that already have some evidence supporting their interaction with dietary fat intake in weight gain, including in children for whom less evidence exists. Genome-wide association studies have increased the knowledge in this area, but they have some limitations, which means that more studies and with different approaches are needed to further understand the relation between genes and environmental factors in obesity. A more personalized diet (under prior knowledge of obesity-related polymorphisms) is currently under discussion in the scientific arena.
A obesidade é ume epidemia global; o número de crianças com obesidade aumentou cerca 2.28 vezes nas últimas três décadas. A obesidade tem associadas inúmeras complicações como diabetes tipo 2, dislipidemia e resistência à insulina. A obesidade é uma doença multifatorial que possui vários fatores de risco que interagem no seu desenvolvimento, como genéticos, comportamentais e ambientais. Este artigo ambiciona perceber os fatores genéticos inerentes a esta doença bem como as interações entre os genes e a alimentação que potenciam a obesidade infantil. Para a realização deste artigo procedeu-se a uma revisão bibliográfica da literatura em língua Inglesa no motor de busca PubMed Central®, totalizando 110 artigos. Durante a última década, a investigação tem tentado perceber quais os genes responsáveis por uma maior predisposição à obesidade infantil. Nesse sentido vários estudos foram conduzidos, como por exemplo os genome-wide association studies (GWAS). A obesidade infantil parece resultar da presença de genes de risco, bem como da sua resposta face a ambientes obesogénicos. Existe já evidência da influência de genes como o FTO, MC4R, POMC, LEP e recetor de LEP no aumento de peso e no desenvolvimento de complicações associadas desde idades precoces. A alimentação é um dos fatores ambientais mais importantes para o desenvolvimento da obesidade. Atualmente, existem duas abordagens para perceber as interações entre os genes e a alimentação, a nutrigenética e a nutrigenómica. Ambas se baseiam na premissa de que os nutrientes/componentes da alimentação podem influenciar a expressão génica e afetar diferentes vias metabólicas, que finalmente irão originar o fenótipo do indivíduo. A maioria dos estudos conduzidos até hoje estão relacionados com o metabolismo dos ácidos gordos, pelo que para melhor compreender outras vias metabólicas são necessários mais estudos. A interação com a ingestão de gordura no aumento de peso já está evidenciada para genes como o FTO, APOA2 e NPC1, incluindo estudos realizados em crianças para as quais a evidência é mais escassa. Os genome-wide association studies permitiram o aumento de conhecimento nesta área, mas apresentam algumas limitações, o que significa que são necessários mais estudos e com diferentes abordagens para melhor entender a relação entre os genes e os fatores ambientais no desenvolvimento da obesidade. Uma dieta mais personalizada (com prévio conhecimento de polimorfismos relacionados com a obesidade) está atualmente sob discussão nos fóruns científicos.
N/A

Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. Following this initial work, we investigated the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, as well as the regulation of Hox genes expression. We find that H1 TKO ESCs are more resistant to spontaneous differentiation, impaired in embryoid body differentiation, and largely blocked in neural differentiation. We present evidence that H1 contributes to efficient repression of the expression of pluripotency factors, Oct4 and Nanog, and participates in establishment and maintenance of DNA methylation and histone modification necessary for silencing pluripotency genes during stem cell differentiation and embryogenesis. In addition, we find reduced expression of a distinct set of Hox genes in embryos and ESCs, respectively. Furthermore, by characterizing H1c−/−; H1d−/−; and H1e−/− single-H1 null ESCs established in this study, we showed that individual H1 subtypes regulated specific Hox genes in ESCs. Finally, we demonstrate that the levels of H3K4me3 were significantly diminished at the affected Hox genes in H1 TKO- and single-H1 KO- ESCs, whereas H3K27me3 occupancy is modestly increased at specific Hox genes. Our results suggest that marked reduction of H1 levels and decondensation of bulk chromatin affect the expression of pluripotency genes and Hox genes in embryos and ESCs, which may be in part mediated through establishment and maintenance of epigenetic marks.

Tandemly arrayed genes (TAG) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. We identified all the TAGs in the genomes of human, chimp, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG gene orientations and intergenic distances, and TAG gene functions. TAGs account for about 14-17% of all the genomic genes and nearly one third of all the duplicated genes in the four genomes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests while others are enriched with TAG deserts. The majority of TAGs are of size two for all genomes, similar to the previous findings in C. elegans, A. thaliana, and O. sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes with parallel orientation in the genome. The analysis of GO function indicate that genes with receptor or binding activities are significantly over-represented by TAGs. Simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. It is noteworthy to mention that gene family sizes are significantly correlated with the extent of tandem duplication, suggesting that tandem duplication is a preferred form of duplication, especially in large families. There has not been any systematic study of TAG genes' expression patterns in the genome. Taking advantage of recent large-scale microarray data, we were able to study expression divergence of some of the TAGs of size two in human and mouse for which the expression data is available and examine the effect of sequence divergence, gene orientation, and physical proximity on the divergence of gene expression patterns. Our results show that there is a weak negative correlation between sequence divergence and expression similarity between the two members of a TAG, and also a weak negative correlation between physical proximity of two genes and their expression similarity. No significant relationship was detected between gene orientation and expression similarity. Moreover, we compared the expression breadth of upstream and downstream duplicate copies and found that downstream duplicate does not show significantly narrower expression breadth. We also compared TAG gene pairs with their neighboring non-TAG pairs for both physical proximity and expression similarity. Our results show that TAG gene pairs do not show any distinct differences in the two aspects from their neighboring gene pairs, suggesting that sufficient divergence has occurred to these duplicated genes during evolution and their original similarity conferred by duplication has decayed to a level that is comparable to their surrounding regions.
Master of Science

Orientadores: IÍscia Teresinha Lopes Cendes, Cláudia Vianna Maurer-Morelli
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: As epilepsias formam um grupo de doenças neurológicas crônicas caracterizadas por crises epilépticas, as quais podem ser definidas como um distúrbio intermitente do sistema nervoso causado por descarga elétrica anormal, súbita e sincronizada dos neurônios cerebrais. A epilepsia de lobo temporal (ELT) é a mais frequente, representando aproximadamente 50% dos casos em adultos e tem como manifestação típica, a crise parcial complexa. Além disso, é frequentemente refratária ao tratamento medicamentoso. Os principais sintomas gerados pela ELT são predominantemente pelo acometimento das estruturas mediais do lobo temporal, sendo a ELT mesial (ELTM), a forma mais comum de ELT. Atualmente é ainda controversa a participação de fatores genéticos contribuindo na etiologia das epilepsias, principalmente da ELTM, que não teve até hoje nenhum gene inequivocamente associado a sua predisposição. O objetivo deste trabalho foi investigar o papel de dois genes candidatos: o PTPRM e o IL1B na predisposição à ELTM. Para tanto utilizamos as seguintes modalidades de estudo em pacientes com ELTM (i) estudo de associação genética através da genotipagem de polimorfismos de nucleotídeo único (SNPs) localizados nos referidos genes candidatos (esta etapa do estudo foi realizada durante o mestrado); (ii) quantificação dos transcritos de ambos os genes, por PCR em tempo real em tecido das estruturas mediais do lobo temporal (principalmente hipocampo) que foi obtido através da realização de cirurgia para tratamento das crises refratárias. (iii) Para o PTPRM, foi também realizada a localização do transcrito pela técnica de hibridação "in situ" em tecido hipocampal de pacientes e de controle. Além disso, como existem evidências do envolvimento do PTPRM em etapas importantes do desenvolvimento cerebral, e pouco se conhece da função específica desse gene no cérebro realizamos (iv) a quantificação do transcrito de PTPRM durante o desenvolvimento em cérebro de camundongos. (v) Finalmente, com o objetivo específico de avaliar se o aumento de expressão de IL1B no tecido hipocampal se refletia também na circulação realizamos a quantificação do transcrito e proteica do IL1B no plasma de pacientes com ELTM. Nossos resultados revelaram associação genética entre SNPs localizados em ambos os genes investigados e o fenótipo estudado. No entanto, em nenhum dos estudos uma variante funcional pode ser identificada. A quantificação dos transcritos em tecido hipocampal dos pacientes com ELTM indicou que ambos os genes PTPRM e IL1B estão hiper-regulados em pacientes quando comparados ao tecido controle. Não identificamos variação significativa na expressão do transcrito de PTPRM no cérebro de camundongos nas diferentes etapas de desenvolvimento. Não identificamos variação significativa na quantificação do transcrito e proteica de IL1B no plasma dos pacientes com ELTM quando comparados aos controles. Em conclusão, nossos resultados dos estudos de associação indicam um papel de PTPRM e de IL1B na predisposição à ELTM, porém não fomos capazes de encontrar uma variante funcional associada ao fenótipo. Corroborando o papel de ambos os genes nosso estudo de expressão gênica no tecido acometido indicou um aumento de expressão de ambos os genes. No entanto, o aumento de expressão de IL-1beta no tecido hipocampal não se traduziu pelo aumento no plasma dos pacientes. Finalmente, nosso estudo do perfil de expressão de PTPRM durante o desenvolvimento cerebral não aponta para um papel desse gene em etapas específicas do desenvolvimento
Abstract: The epilepsies are a group of chronic neurological disorders characterized by seizures, which can be defined as an intermittent disorder caused by an abnormal and sudden electrical discharge of neurons in the brain. Temporal lobe epilepsy (TLE) is the most frequent form, representing approximately 50% of cases in adults, and it is often refractory to drug treatment. The main symptoms in TLE are generated by the involvement of the medial temporal lobe structures, characterizing mesial TLE (MTLE). The contribution of genetic factor to MTLE it is still controversial and to date, no gene has been unequivocally associated with the predisposition to MTLE. Therefore, the aim of this study was to investigate the role of two candidate genes: PTPRM and IL1B in the predisposition to MTLE. To achieve this we use the following type study modalities in patients with MTLE (i) genetic association study by genotyping of single nucleotide polymorphisms (SNPs) located in these two candidate genes; (ii) quantification of the transcripts of both genes by real-time PCR in hippocampal tissue obtained from epilepsy surgery for the treatment of refractory seizures. (iii) For PTPRM we also performed in situ hybridization experiments in order to localize the transcript in hippocampal tissue from patients and controls. Furthermore, since there is evidence that PTPRM could be involved in key stages of brain development and little is known about the specific role of this gene in the brain, we performed (iv) quantification of its transcript during development in mouse brain. (v) Finally, with the specific objective of assessing whether the increase of IL1B expression in hippocampal tissue was also seen outside the central nervous system we quantified IL1B transcript and protein in plasma of patients with MTLE. Our results revealed genetic association between SNPs located in both genes and the phenotype. The quantification of transcripts in hippocampal tissue of patients with MTLE indicated that both genes are hyper-regulated when compared to control tissue. We did not find any significant variation in transcript expression of PTPRM in mouse brain during developed. In addition, no difference in transcript expression and protein levels of IL1B was observed in plasma of patients with MTLE. In conclusion, our results indicate an involvement of PTPRM and IL1B in the predisposition to MTLE; however, we are unable to find a functional variant associated with the phenotype. Corroborating the role of both genes in MTLE gene expression in affected tissue (hippocampus) indicated an up-regulation of both genes. However, the increase in IL1B expression in hippocampal tissue was not reflected by an increase of transcript or protein in plasma of patients with MTLE. Finally, our expression profile of PTPRM during brain development does not point to a role for this gene in specific stages of development
Doutorado
Fisiopatologia Médica
Doutora em Ciências

In the present study, DNA sequences associated with an apomixis gene inPennisetum ciliare were isolated from a distantly related grass species, Tripsacum dactyloides. Primers were developed for two bioinformatics-identified candidate genes (Pca2l and Pca24) for apomixis in Pennisetum ciliare. Homologous gene sequences were successfully isolated from both diploid (2n=36) and tetraploid (4n=72) Tripsacum using the primers and polymerase chain reaction (PCR) amplification. Bioinformatics analysis of the purified, cloned and sequenced PCR products revealed that the isolated homolog of the Pca2l gene varies significantly between the diploid and the tetraploid Tripsacum. Comparative genome analyses against Oryza, Zea, Arabidopsis, Pennisetum, Tripsacum and the National Center for Biotechnology Information (NCBI) nucleotide collection (nr) have shown that the PCR-generated sequences are reproductive specific. Analysis of the Trip2lT3c sequence was shown to be a Rab2 homolog with an e-value of 9e-23. Further proteomics analyses of the putative gene products have revealed that the Pca2l and the Tripsacum sequences may be partially conserved, with the Trip2lT3c sequence more highly conserved than the Trip21D3a.
Department of Biology

Bird migration is an adaptive strategy evolved to exploit the seasonal changes of resources by moving to different areas at different times of the year, as these become suitable. Migratory birds reach the breeding grounds when the food supplies enable them to breed and leave towards more suitable areas before the ecological conditions deteriorate; to this end, they must anticipate the changes in ecological conditions by weeks or even months to prepare for migration. Tis is allowed by the by the existence of an endogenous control of the annual schedule, that is kept in phase with seasons by means of external cues, such as photoperiod. The occurrence of a tight endogenous control of migration hint that migration is under a strong genetic influence and the broad among-species, -populations and -individuals differences in migratory traits are partly triggered by variation at genes controlling migratory behaviour. Identifying which genes underline the phenotypic variation observed in natural populations, and to which extent, is of broad evolutionary and conservation interest. It has been suggested that variation in migratory traits could be the outcome of polymorphism at relatively few regions or loci with additive effects on a variety other genes. Hence, in recent years, several studies have focused on the polymorphism of phenological candidate genes, that may explain the variability of behavioural traits. Most of the phenological candidate genes studied so far are involved in the ‘core circadian oscillator’ (CCO), an auto-regulated negative feedback loop that modulates the photoperiodic response and sets the circadian and circannual rhythms. The Clock and Adcyap1 genes are among the best studied candidate genes involved in the photoperiodic response and in the circannual rhythmicity. Both the genes show a polymorphism in short tandem repeat sequences that may affect the gene function or the post-transcriptional processes and that have been linked to variation in the timing of seasonal events and other behavioural traits in several vertebrate species. However, no study has investigated the effect of Clock and Adcyap1 genotype on migration phenology and on migration distance directly in wild birds. Hence, the aim of this thesis is to investigate whether polymorphism at candidate genes affects the timing of migration and the migration distance in avian species, with particular reference to Clock and Adcyap1 genes. I found Clock allele size to predict the timing the timing of migration in different long-distance migratory bird species. The direction of the association was coherent with previous studies and across species (longer allele being associated with a delayed phenology), the only exception being the willow warbler (Phylloscopus trochilus). Moreover, Adcyap1 allele size was associated with the migration distance in a Nearctic-Neotropical migratory bird. In such species the genotype-phenotype association was stronger among the northern, long-distance migratory populations than among the southern ones. Finally, I found that Clock allele size increased with breeding latitude across species and that Clock gene diversity was depleted among the species migrating over longer distances, that show delayed and more concentrated migration periods. To conclude, the findings from this thesis deepen our understanding of the role of polymorphism at candidate gene in shaping avian behaviour, by highlighting that Clock and Adcyap1 polymorphisms are involved in the control of migration.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Pentatomomorpha é composta por cerca de 14.000 espécies, distribuídas em seis superfamílias, entre as quais estão inclusas Pentatomoidea e Coreoidea. Pentatomoidea possui 7.000 espécies distribuídas em 15 famílias, das quais Pentatomidae é a maior, com 4.500 espécies e 760 gêneros, estando representados no Estado de São Paulo 163 espécies. A superfamília Coreoidea é composta por cinco famílias, contudo apenas Coreidae, Rophalidae e Alydidae estão presentes no Neotrópico. A família Coreidae possui 1.884 espécies, divididas em quatro subfamílias, das quais Coreinae tem oito tribos com representantes no Brasil. Embora as famílias Pentatomidae e Coreidae apresentem um significante papel como pragas de culturas agrícolas, são escassas análises cladísticas envolvendo esses táxons, o que resulta na ausência de uma única hipótese de classificação, tornando pesquisas com abordagens de sistemática filogenética necessárias para essas famílias. Portanto, o presente estudo buscou caracterizar as relações filogenéticas das famílias Pentatomidae e suas subfamílias por meio dos genes 16S, ND5 (mitocondriais), 18S e 28S (nucleares) e da família Coreidae e sua respectiva subfamília Coreinae por meio dos genes 18S e 28S. Objetivou-se também, confirmar a classificação atual das tribos e subfamílias das espécies de Pentatomidae e Coreidae, que é embasada em caracteres morfológicos, assim como avaliar a variabilidade genética das sequências correspondentes aos genes mitocondriais e nucleares analisados. A partir dos resultados foi observado que os genes nucleares são conservados ao passo que os mitocondriais são altamente variáveis e com tendência as bases AT. Este estudo evidenciou que os genes nucleares 18S e 28S não são ideais para resoluções filogenéticas em nível de famílias, subfamílias e tribos de Coreidae e Pentatomidae, visto que os suportes dos ramos não permitem que haja confiabilidade nas análises. Além disso, alguns grupos que são considerados monofiléticos, com base em caracteres morfológicos, como as subfamílias de Pentatomidae, não tiveram sua monofilia resgatada. Contudo ao considerar as topologias obtidas com os genes nucleares aliados aos mitocondriais, para a família Pentatomidae, obteve-se alto suporte nos ramos, e, portanto, grande confiabilidade nas análises, resgatando a monofilia da maioria das tribos estudadas. Dessa forma, estudos utilizando diferentes marcadores moleculares, em especial, genes mitocondriais, são necessários, pois podem auxiliar no esclarecimento e suporte da classificação atual das relações intrafamiliares de Pentatomidae e Coreidae que permanecem pendentes.
Pentatomomorpha consists about 14.000 species distributed in six superfamilies, to which Pentatomoidea and Coreoidea are included. Pentatomidae has 7000 species in 15 families, of which Pentatomidae is the largest, with 4500 species and 760 genera, although in the state of São Paulo only 163 species are present. The Coreoidae superfamily consists of five families, however only Coreidae, Rophalidae and Alydidae are present in the Neotropics. The Coreidae family has 1884 species, divided into four subfamilies, of which Coreinae has eight tribes with representatives in Brazil. Although Pentatomidae and Coreidae families have a significant role as pests of agricultural crops, the cladistic analysis involving these taxa are scarce, which results in the absence of a single hypothesis classification, making research on phylogenetic systematic approaches necessary for these families. Therefore, this study aimed to characterize the phylogenetic relationships of Pentatomidae families and subfamilies through the 16S, ND5, 18S and 28S genes and Coreidae family and their respective Coreinae subfamily through the 18S and 28S genes. We aimed also to confirm the current classification of tribes and subfamilies of Pentatomidae and Coreidae, which is grounded on morphological characters, as well as to evaluate the genetic variability of the sequences corresponding to the mitochondrial and nuclear genes analyzed. From the results it was observed that the nuclear genes are conserved while the mitochondrial are highly variable and tend to AT base. Our study showed that nuclear genes 18S and 28S are not ideal for phylogenetic resolution at the level of families, subfamilies and tribes of Coreidae and Pentatomidae, because the supports of the branches do not allow that there is confiability in the analysis. Furthermore, groups considered monophyletic, based on morphological characteristics, such as Pentatomidae subfamilies, have not had their monophyly rescued. But when we consider the topologies obtained with nuclear genes combined with mitochondrial genes for the Pentatomidae family, we had high support in the branches, and thus high confiability in the analysis, rescuing the monophyly of most of the studied tribes. Thus, studies using different molecular markers, in particular mitochondrial genes are needed, because they can help in understanding and support of the current classification of intra-family relations Pentatomidae and Coreidae that remain pending.