Дисертації з теми "Genes families"

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.

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FACEPE
Primary brain calcification (PBC), also known as idiopathic brain calcification or Fahr's disease, is a rare neurological condition that is characterized by calcium phosphate deposits in the basal ganglia and adjacent areas, movement disorders, headache and neuropsychiatric symptoms. It presents autosomic dominant inheritance and it is associated with two inorganic phosphate transporter coding genes: SLC20A2 and XPR1. Two other genes related to the blood-brain barrier maintenance and integrity are also linked to PBC, the platelet-derived growth factor-β and its receptor (PDGFB and PDGFRB), although their roles in the formation mechanism of the calcifications is not clear yet. For this study, besides the four genes above mentioned, other members of the platelet-derived grown factor family (PDGFA, PDGFRA, PDGFC and PDGFD) have also been selected as candidate genes, for which new primer pairs were designed. All genes above were screened for new variants by Sanger sequencing in fifteen Brazilian unrelated patients with brain calcifications. Sequence in silico analysis was performed using CLC Main Workbench 6.9 software and online tools available in NCBI and GOLDENPATH platforms, resulting in the identification of the first de novo SLC20A2 mutation in a patient diagnosed with PBC (NM_006749.4:c.1158C>G; NP_006740.1:p.Y386*). SLC20A2 is to-date the main gene associated with PBC, with affecting-variants observed in ~50% cases. In order to find SLC20A2 deletions and/or duplications not detected by sequencing, all Brazilian probands were screened by QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) and a duplication of the terminal exon was found in a patient with brain calcifications and hyperparatiroidism. Simultaneously, twenty-four French unrelated patients with PBC were also analyzed by QMPSF and partial SLC20A2 deletions were detected in four patients: two with deletion of the exon 2, where the start codon is located; one with deletion of the exon 4; and one with deletion of exons 4 and 5. These results reinforce SLC20A2 role as the main gene associated to PBC, as well as demonstrate that copy number variation analyses, even when revealing only partial deletions or duplications of a gene, are complementary to sequencing and work side by side in the search of genetic variations involved in this disease.
Introdução: A calcificação cerebral primária (CCP), também conhecida como calcificação idiopática dos núcleos da base ou doença de Fahr, é uma condição neurológica caracterizada por depósitos de fosfato de cálcio dos núcleos da base e região de entorno, parkinsonismo e sintomas neuropsiquiátricos. Apresenta herança autossômica dominante e é associada a dois genes codificantes de transportadores de fosfato inorgânico: o SLC20A2 e o XPR1. Dois outros genes relacionados à manutenção e à integridade da barreira hemato-encefálica, o fator de crescimento plaquetário B e seu receptor (PDGFB e PDGFRB), também foram associados à CCP, embora seus papeis no mecanismo de formação das calcificações ainda não estejam claros. Materiais e Métodos: Além dos quatro genes acima, foram selecionados como candidatos outros genes da família dos fatores de crescimento plaquetário (PDGFA, PDGFRA, PDGFC e PDGFD) e das protocaderinas (PCDH12), para os quais foram confeccionados pares de primers utilizados no seu sequenciamento e para análise de variação de número de cópia. Resultados e Discussão: Quinze famílias brasileiras com CCP foram triadas para novas variantes nos genes candidatos por sequenciamento. A análise in silico do sequenciamento foi feita através do software CLC Combined Workbench versão 6.9 e das ferramentas disponíveis nas plataformas online do NCBI e do GOLDENPATH. A partir dessa análise, foi identificada em um probando a primeira mutação de novo do SLC20A2, o principal gene associado a CCP (NM_006749.4:c.1158C>G; NP_006740.1:p.Y386*). A fim de encontrar deleções e/ou duplicações do SLC20A2 não detectadas por sequenciamento, todos os probandos brasileiros com calcificações cerebrais foram triados através da técnica de QMPSF (do inglês, Quantitative Multiplex PCR of Short fluorescent Fragments). Foi encontrada uma duplicação do exon terminal do mesmo gene em um paciente brasileiro com calcificações cerebrais e hiperparatireoidismo. Simultaneamente, foram identificadas deleções parciais no mesmo gene em quatro famílias francesas com CCP. Conclusões: Esses resultados reafirmam o SLC20A2 como o principal gene associado a CCP, bem como demonstram que análises de variação de número de cópia (CNV), ainda que parciais, são complementares ao sequenciamento na busca por variantes genéticas relacionadas a esta doença.

Thesis (MScMed)--Stellenbosch University, 2008.
ENGLISH ABSTRACT: In hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder, hypertrophy is variable within and between families carrying the same causal mutation, suggesting a role for modifier genes. Associations between left ventricular hypertrophy and left ventricular pressure overload suggested that sequence variants in genes involved in the Renin-Angiotensin Aldosterone System (RAAS) may act as hypertrophy modifiers in HCM, but some of these studies may have been confounded by, amongst other things, lack of adjustment for hypertrophy covariates. To investigate this hypothesis, twenty one polymorphic loci spread across six genes (ACE1, AGT, AGTR1, CYP11B2, CMA and ACE2) of the RAAS were genotyped in 353 subjects from 22 South African HCM-families, in which founder mutations segregate. Genotypes were compared to 17 echocardiographically-derived hypertrophic indices of left ventricular wall thickness at 16 segments covering three longitudinal levels. Family-based association was performed by quantitative transmission disequilibrium testing (QTDT), and mixed effects models to analyse the X-linked gene ACE2, with concurrent adjustment for hypertrophy covariates (age, sex, systolic blood pressure (BP), diastolic BP, body surface area, heart rate and mutation status). Strong evidence of linkage in the absence of association was detected between polymorphisms at ACE1 and posterior and anterior wall thickness (PW and AW, respectively) at the papillary muscle level (pap) and apex level (apx). In single-locus analysis, statistically significant associations were generated between the CYP11B2 rs3097 polymorphism and PW at the mitral valve level (mit) and both PWpap and inferior wall thickness (IW)pap. Statistically significant associations were generated at three AGTR1 polymorphisms, namely, between rs2640539 and AWmit, rs 3772627 and anterior interventricular septum thickness at pap and rs5182 and both IWpap and AWapx. Furthermore, mixed effects model detected statistically significant association between the ACE2 rs879922 polymorphism and both posterior interventricular septum thickness and lateral wall thickness at mit in females only. These data indicate a role for RAAS gene variants, independent of hypertrophy covariates, in modifying the phenotypic expression of hypertrophy in HCM-affected individuals.
AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HCM), ‘n autosomale dominante afwyking, toon hoogs variërende hipertrofie binne en tussen families wat dieselfde siekte-veroorsakende mutasie het, hierdie dui op die moontlike betrokkenheid van geassosieerde modifiserende gene. Assosiasies tussen linker ventrikulêre hipertrofie en linker ventrikulêre druk-oorlading stel voor dat volgorde variasies in gene betrokke in die Renin-Angiotensin Aldosteroon Sisteem (RAAS) mag optree as hipertrofie modifiseerders in HCM. Sommige van hierdie soort studies is egter beperk omdat hulle nie gekompenseer het vir kovariante van hipertrofie nie. Om hierdie hipotese te ondersoek, is die genotipe bepaal by een-en-twintig polimorfiese lokusse, verspreid regoor ses RAAS gene (ACE1, AGT, AGTR1, CYP11B2, CMA and ACE2), in 353 kandidate vanuit 22 Suid-Afrikaanse HCM-families in wie stigter mutasies segregeer. Genotipes was vergelyk met 17 eggokardiografies afgeleide hipertrofiese indekse van linker ventrikulêre wanddikte by 16 segmente wat oor drie longitudinale vlakke strek. Familie-gebaseerde assosiasies was bestudeer deur kwantitatiewe transmissie disequilibrium toetsing (QTDT) en gemengde effek modelle om die X-gekoppelde geen ACE2 te analiseer, met gelyktydige kompensasie vir hipertrofie kovariate (ouderdom, geslag, sistoliese bloed druk (BP), diastoliese BP, liggaamsoppervlak area, hartritme en mutasie-status). Sterk indikasies van koppeling in die afwesigheid van assosiasie is waargeneem tussen ACE1 lokusse en posterior wanddikte (PW) asook anterior wanddikte (AW) by die papillêre spier vlak (pap) en die apeks vlak (apx). In enkel-lokus analises is statisties-betekenisvolle assosiasies gevind tussen die CYP11B2 rs3097 polimorfisme en PW by die mitraalklep vlak (mit) en beide die PWpap en inferior wanddikte (IW)pap. Statisties-betekenisvolle assosiasies was verder gevind by drie AGTR1 polimorfismes, naamlik, tussen rs2640539 polimorfisme en AWmit, rs3772627 en die anterior interventrikulêre septumdikte (aIVS) by die pap en rs5182 by beide die IWpap en AWapx. Gemengde-effek modelle het verder assosiasies aangetoon tussen die ACE2 rs879922 polimorfisme en die posterior interventrikulêre septumdikte en die laterale wanddikte by die mit, slegs in vrouens. Hierdie data dui op ‘n kovariaat-onafhanklike rol vir RAAS genetiese variante in die modifisering van die fenotipiese uitdrukking van hipertrofie in HCM-geaffekteerde individue.

Extended Spectrum β-Lactamases (ESBLs) are produced by the Enterobacteriaceae bacterial family, mainly by E. coli and K. pneumoniae. As these species are some of the main causes of urinary tract infections and sepsis, ESBL-production is of major concern. Occurrence of ESBLs also gives rise to concern as it is increasing epidemically. This because the genes coding for ESBLs (i.e. bla-genes) are located on plasmids replicating and spreading the replicated copies independently. Plasmids replicate by replicons. Plasmids with the same replicon variant are grouped into the same plasmid family. The aim of this study was to detect plasmid families carrying bla-genes in E. coli and K. pneumoniae from clinical (n = 6) and environmental water (n = 22) isolates. Plasmid family prevalence was examined. Association between plasmid families and bla-genes was also examined. Plasmid families were detected by a PBRT kit (PCR Based Replicon Typing), a multiplex PCR kit that detected 30 replicons, whereof 27 replicons representing the 27 plasmid families in Enterobacteriaceae, and three novel replicons. The IncF plasmid family was the most prevalent for both species in both clinical and environmental isolates. IncF seemed to be prevalent for all examined ESBLs, but it was difficult to associate one bla-gene with one plasmid family as most isolates carried several bla-genes and several plasmid families.
Extended Spectrum β-Lactamases (ESBLs) produceras av bakteriefamiljen Enterobacteriaceae, främst av E. coli och K. pneumoniae. Eftersom dessa arter är bland de vanligaste orsakerna till urinvägsinfektioner och sepsis är ESBL-produktion ett allvarligt problem. ESBL är också oroande eftersom det sprids epidemiskt. Detta möjliggörs av att generna som kodar för ESBLs (s.k. bla-gener) ligger på plasmider, som replikerar och sprider de replikerade plasmidkopiorna självständigt. Plasmider replikeras som s.k. replikon. Plasmider med samma replikonvariant tillhör samma plasmidfamilj. Syftet med detta arbete var att detektera plasmidfamiljer som bär bla-gener i E. coli och K. pneumoniae isolerade från kliniska prov (n = 6) och miljöprov (n = 22) från Helge Å. Plasmidfamiljernas prevalens undersöktes, liksom sambandet mellan plasmidfamiljer och bla-gener. Plasmidfamiljerna detekterades med ett PBRT-kit (PCR Based Replicon Typing), ett multiplext PCR-kit som detekterade 30 replikon varav 27 replikon som representerar de 27 plasmidfamiljer som finns i Enterobacteriaceae och tre nya replikon. Plasmidfamiljen IncF var vanligast förekommande i båda arter i både kliniska isolat och miljöisolat. IncF verkade förekomma för alla undersökta typer av ESBL, men det var generellt svårt att förknippa en bla-gen med en plasmidfamilj, eftersom de flesta isolaten bar flera bla-gener och flera plasmidfamiljer.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
No presente trabalho à feita a descriÃÃo de trÃs genes distintos que codificam lectinas ou proteÃnas relacionadas à lectina de Vatairea macrocarpa. As sequÃncias foram obtidas pela amplificaÃÃo de DNA genÃmico e cDNA de folhas utilizando primers semi-degenerados construÃdos a partir da informaÃÃo da sequÃncia de aminoÃcidos da lectina VML depositada no GenBank. O resultado do sequenciamento revelou a presenÃa de trÃs contigs. O contig1 corresponde à lectina VML desde que se assuma que a lectina depositada VML contenha heterogeneidades ou ambiguidades decorrentes na degradaÃÃo de Edman. A traduÃÃo dos contigs 2 e 3 mostram identidade de sequÃncia de 77% quando comparadas com VML. As sequÃncias, apesar de apresentar regiÃes conservadas, mostram diferenÃas de aminoÃcidos nos sÃtios de N-glicosilaÃÃo, sÃtios de ligaÃÃo a carboidrato e metais alÃm da presenÃa de resÃduos de cisteÃna sugerindo que tais proteÃnas podem ter outras atividades biolÃgicas. A anÃlise da sequÃncia obtida pelo 3â RACE se mostrou complementar ao contig3. Sendo assim, a sequÃncia hÃbrida contig3/contigA possui 2 resÃduos de cisteÃna alÃm de revelar diferenÃas de aminoÃcidos na regiÃo C-terminal quando alinhada com outras lectinas de leguminosas. AnÃlises filogenÃticas revelaram que os contigs observados formam um grupo monofiletico e tem alta similaridade com as lectinas de Sohora japonica e Robinia pseudoacacia, alÃm da proteÃna relacionada à lectina de Cladrastis lutea.
In this paper is made a description of three distinct genes that encode Vatairea macrocarpa lectin and related proteins. The sequences were obtained by amplification of genomic DNA and cDNA of leaves using semi - degenerate primers constructed from the information of the amino acid sequence of VML lectin deposited in GenBank. The result of sequencing rev eals the presence of three different genes, called contig 1, 2 and 3 . The VML lectin corresponds to contig1 long as one assumes that the lectin contains heterogeneities deposited VML or ambiguities arising in the Edman degradation . The translation of cont igs 2 and 3 show sequence identity of 77% compared to VML. Sequences, despite having conserved regions show differences in amino acid N - glycosylation sites, carbohydrate binding sites and metals and the presence of cysteine residues suggests that these pro teins may have other biological activities . The analysis of the sequence obtained by 3 'RACE proved complementary to contig3. Thus, the sequence contig3/contigA hybrid has two cysteine residues in addition to revealing differences in amino acid C - terminal region when aligned with other legume lectins. Phylogenetic analysis revealed that the observed contigs form a monophyletic group and has high similarity with lectins from Robinia pseudoacacia Sohora japonica and, in addition to the lectin - related protein Cladrastis lutea .

ALVES FILHO, João Garcia. Clonagem, sequenciamento e caracterização parcial dos genes relacionados à lectina de Vatairea macrocarpa. 2008. 81 f. Dissertação (Mestrado)-Universidade Federal do Ceará, Fortaleza-CE, 2008.
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In this paper is made a description of three distinct genes that encode Vatairea macrocarpa lectin and related proteins. The sequences were obtained by amplification of genomic DNA and cDNA of leaves using semi - degenerate primers constructed from the information of the amino acid sequence of VML lectin deposited in GenBank. The result of sequencing rev eals the presence of three different genes, called contig 1, 2 and 3 . The VML lectin corresponds to contig1 long as one assumes that the lectin contains heterogeneities deposited VML or ambiguities arising in the Edman degradation . The translation of cont igs 2 and 3 show sequence identity of 77% compared to VML. Sequences, despite having conserved regions show differences in amino acid N - glycosylation sites, carbohydrate binding sites and metals and the presence of cysteine residues suggests that these pro teins may have other biological activities . The analysis of the sequence obtained by 3 'RACE proved complementary to contig3. Thus, the sequence contig3/contigA hybrid has two cysteine residues in addition to revealing differences in amino acid C - terminal region when aligned with other legume lectins. Phylogenetic analysis revealed that the observed contigs form a monophyletic group and has high similarity with lectins from Robinia pseudoacacia Sohora japonica and, in addition to the lectin - related protein Cladrastis lutea.
No presente trabalho é feita a descrição de três genes distintos que codificam lectinas ou proteínas relacionadas à lectina de Vatairea macrocarpa. As sequências foram obtidas pela amplificação de DNA genômico e cDNA de folhas utilizando primers semi-degenerados construídos a partir da informação da sequência de aminoácidos da lectina VML depositada no GenBank. O resultado do sequenciamento revelou a presença de três contigs. O contig1 corresponde à lectina VML desde que se assuma que a lectina depositada VML contenha heterogeneidades ou ambiguidades decorrentes na degradação de Edman. A tradução dos contigs 2 e 3 mostram identidade de sequência de 77% quando comparadas com VML. As sequências, apesar de apresentar regiões conservadas, mostram diferenças de aminoácidos nos sítios de N-glicosilação, sítios de ligação a carboidrato e metais além da presença de resíduos de cisteína sugerindo que tais proteínas podem ter outras atividades biológicas. A análise da sequência obtida pelo 3’ RACE se mostrou complementar ao contig3. Sendo assim, a sequência híbrida contig3/contigA possui 2 resíduos de cisteína além de revelar diferenças de aminoácidos na região C-terminal quando alinhada com outras lectinas de leguminosas. Análises filogenéticas revelaram que os contigs observados formam um grupo monofiletico e tem alta similaridade com as lectinas de Sohora japonica e Robinia pseudoacacia, além da proteína relacionada à lectina de Cladrastis lutea.

Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia - UL
Grapevine (Vitis vinifera L.) is one of the most economically important fruit crops in the world. Abiotic stresses are likely to affect the plant development, yield and ultimately the quality of economic products. The cell wall (CW) is a dynamic structure that determines plant form, growth and response to environmental conditions. To investigate V. vinifera CW adaptative response to restrictions in major nutrients, essential to plant growth and development, N, P and S were excluded from V. vinifera callus and shoots growing substrates. Water stress on berry skin CW was studied in non-irrigated versus irrigated plants of two varieties. Specific CW components, namelly cellulose, were impacted in callus, shoots and berries subjected to abiotic stress. To overcome this, V. vinifera CWs suffered compositional changes and reorganization of deposition of several CW components, in particular the degree and pattern of pectin methyl-esterification, arabinan and XyG, which together promote stiffening of the CW. Moreover, polysaccharides of callus grown under mineral deficiency, mainly N, were more tightly bound in the CW. Under mineral stress the expression of genes from CW candidate enzyme families was affected for example downregulation of GH9C, XTHs with predicted hydrolytic activity and PMEs were observed. In shoots probed with CW-epitope monoclonal antibodies an increase in pectins with a low degree of methyl-esterification able to dimerise association through calcium ions was observed. CWs grown under N deficiency were enriched in 1,5-arabinan rhamnogalacturonan-I (RG-I) side chains. The impact on CW was dependent on the mineral stress, nitrogen leading to more pronounced responses, supporting the primary role of this major nutrient in plant development and metabolism. Water shortage affected berry skin by stiffening the CW, probably due to alterations in PGs, XTH and PME expression and pectin methyl-esterification in a variety dependent way

Les paraplégies spastiques héréditaires (PSH) font partie d’un groupe plus large de pathologies neurodégénératives associant une spasticité. J’ai exploré la variabilité clinique et moléculaire de ces pathologies à l’aide d’une cohorte de familles soudanaises. Nous avons recruté 41 familles soudanaises [337 individus/106 atteints de PSH]. J’ai extrait l’ADN génomique et constitué une banque. Le criblage de gènes candidats a été réalisé dans 4 familles en fonction du phénotype des patients. La technologie de séquençage de nouvelle génération (SNG) appliquée à 74 gènes de PSH a ensuite été appliquée aux 37 cas restants. Enfin, le séquençage de l’exome a permis de rechercher les gènes en cause dans les cas négatifs. Dans certains cas, des études fonctionnelles ont été utilisées afin de valider l’effet biologique des mutations. J’ai pu identifier la cause génétique dans 17 familles. Dans 12 familles, la mutation concernait un gène de PSH connu. Dans 3 familles, un nouveau gène a été identifié. 5 gènes candidats restent à départager dans 2 familles. Il est à noter que parfois, de multiple mutations ou maladies génétiques ségrégaient dans nos familles, dans la même branche ou dans des branches séparées. La complexité de ces familles fortement consanguines a rendu l’analyse des données du SNG difficile. Une autre particularité a été l’hétérogénéité clinique associée à des mutations du même gène entre patients de la même famille ou en comparaison avec la littérature. Ce travail est la première étude à grande échelle de patients soudanais avec PSH et rapporte de nouveaux gènes en cause, prérequis pour mieux comprendre dans le futur les mécanismes sous-jacents
Hereditary spastic paraplegias (HSP), a heterogeneous group of spastic neurodegenerative disorders which impose diagnostic challenges. I explored the clinical varieties and genetic pathways of spastic neurodegeneration in a familial Sudanese cohort. We recruited 41 Sudanese families [337 individuals/106 HSP patients]. I have established a genomic DNA bank and when necessary, skin biopsies and fibroblasts were also obtained. A phenotype-based candidate gene approach was followed in 4 families. A targeted next generation sequencing (NGS) for 74 HSP-related genes was the main screening strategy in all-remaining 37 families. Whole exome sequencing (WES) was done in search for novel mutations in new genes in families with negative screening results. Occasionally, functional studies were conducted when feasible and relevant. I identified the genetic cause in 17/41 families. In 12 families, the mutated genes were known HSP genes. In 3 families, novel genes were identified mutated. 5 candidate genes segregated with disease in 2 other families with more experiments needed to conclude. Analysis of the NGS screening panel and of WES data imposed certain challenges as multiple genetic disorders were sometimes found running in parallel in the same/different branches of highly inbred families. We could expand the phenotypic heterogeneity of these disorders due to clinical differences observed between Sudanese patients and patients of other origins even when caused by mutations by the same gene/variant. This is the first genetic screening in a large set of HSP families in Sudan. It describes new causative genes, paving the way for further deciphering of the underlying mechanisms

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Iron is an essential element for plant development, involved in metabolic processes, such respiration and photosynthesis. However, data regarding the genotype by environment interaction are lacking. Comparative analysis with lower plant groups and crop plants can increase the understanding about these processes. The use of bryophytes as model plants rise as a promising strategy since they present simpler patterns of development. The present work aimed to identify the occurrence patterns of molecular markers in model plant species, as well as to infer about the phylogenetical relationships of gene families related with iron homoestasis in plants, allowing the development of tranfer strategies of genomic data across model and orphan species. Using bioinformatics tools, a survey analysis was performed to detect repetitive elements in EST banks of eleven plant species. To validate the SSR markers found, 100 primer pairs were developed on the microsatelite sequences obtained for Physcomitrella patens Brid. and tested against genomic DNA of Polytrichum juniperinum Hedw. Phylogenetic and divergence time analysis was performed for the gene families Iron Regulated Transporter (IRT), Ferric Redectase Oxidase (FRO), Nicotinamide synthase (NAS), Yellow Stripe-Like (YSL) and Natural Resistance-Associated Macrophage Protein (NRAMP), related to the iron homoestasis, with help of the Bayesian inference and using the rice, Arabidopsis and P. patens genes for the Blast search in distinct land plants species. Also, primers for transposable elements recognizably related to Ysl genes were developed and applied jointly with the SSR primers by the IRAP/REMAP technic searching to find microsatellite markers associated to copies of this gene family. A total of 13,133 SSR markers were discovered in non- redundant EST databases made for all eleven species chosen for this study. The dimer motifs are more frequent in lower plant species, such as green algae and mosses, and the trimer motifs are more frequent for the majority of higher plant groups, such as monocots and dicots. Thirty percent of EST-SSE were successfully transferred with a relative polimorphism information across Physcomitrella patens Brid. and P. juniperinum, being promising for mapping and comparative genome analyses in plants. A total of 243 iron uptake gene sequences for 30 plant species were found using rice and Arabidopsis thaliana (L.) Heynh. homologues as queries. The evolutionary fingerprinting analyses suggested a positive selective pressure on iron uptake genes for most of the plant homologues analyzed, enabling an optimization and maintenance of gene function. The divergence time analysis indicates IRT as the most ancient gene family and FRO as the most recent. NRAMP and YSL genes appear as a close branch in the evolution of iron uptake gene families. No recent duplication in grasses were found based in the bayesian inference, and paralogue copies were only observed for dicot species. The Nramp cis-acting homology search indicated an ancestral duplication hypothesis for this gene family in grasses. Using IRAP/REMAP techniques, it was observed that YSL homologues in Physcomitrella are surrounded by copia-like retrotransposons as occurs in the maize ZmYSL1 copy. Also Polytrchum juniperinum Hedw. in vitro cultures were estabilished using spores as explants. Protonemal and gametophyte development were obtained using a growth regulator free culture medium.
O ferro é um elemento essencial para o crescimento e desenvolvimento das plantas, envolvido em processos metabólicos essenciais, como fotossíntese e respiração. Porém, são poucos os dados relacionando a interação entre diferentes genótipos e ambientes. Análises comparativas entre plantas inferiores e plantas cultivadas podem possibilitar o melhor entendimento destes processos. O uso de briófitas como modelo para estudos de processos biológicos em plantas surge como uma estratégia promissora devido ao padrão relativamente simples de desenvolvimento destas plantas. O presente trabalho objetivou identificar padrões de ocorrência de marcadores moleculares em plantas modelo, bem como inferir acerca da filogenia das famílias gênicas envolvidas na homoestase do ferro em plantas, possibilitando a criação de estratégias de transferência de informação genômica entre espécies modelo e espécies órfãs. Utilizando ferramentas de bioinformática foram realizadas análises exploratórias para detectar as ocorrências de elementos repetitivos em bancos de ESTs de onze espécies de plantas. Para a validação destes marcadores moleculares foram desenvolvidos 100 conjuntos de iniciadores a partir das sequências contendo microssatélites obtidas para Physcomitrella patens Brid. e testadas contra o DNA genômico de Polytrichum juniperinum Hedw. Foram realizadas análises filogenéticas e de divergência das famílias gênicas Iron Regulated Transporter (IRT), Ferric Redectase Oxidase (FRO), Nicotinamide synthase (NAS), Yellow Stripe-Like (YSL) e Natural Resistance-Associated Macrophage Protein (NRAMP), envolvidas na homoestase de ferro por meio de inferência bayesiana, utilizando genes de arroz, Arabidopsis e Physcomitrella patens Brid. na busca de homólogos em diferentes espécies de plantas terrestres, com o auxílio da ferramenta Blast (NCBI). Também foram desenvolvidos iniciadores para elementos transponíveis reconhecidamente associados a genes Ysl de milho e utilizados conjuntamente com os iniciadores EST-SSR por meio da técnica IRAP/REMAP buscando encontrar marcadores microssatélites associados a cópias desta familia gênica. Como resultados foram identificados 13.133 marcadores microssatélites em bancos de dados não redundantes de regiões expressas (EST) de onze espécies de plantas. Os motivos dinucleotídeos foram mais frequentes em espécies basais, enquanto os motivos trinucleotídeos foram mais frequentes em espécies derivadas. Em 30% dos conjuntos de iniciadores EST SSR testados contra o DNA de P. juniperinum, foi obtido bandas polimórficas promissoras para estudos de mapeamento comparativo e de diversidade genética. Foram encontrados 243 homólogos de genes relacionados as famílias gênicas envolvidas com a homoestase de ferro em trinta espécies de plantas. A análise de fingerprinting realizada sugere que a maioria destes genes estão submetidos a seleção positiva, indicando acúmulo de mutações adaptativas, essencial para a manutenção e otimização da resposta gênica. A análise de tempo de divergência indica que os genes IRT são mais basais e os genes FRO os mais recentes entre as familias gênicas estudadas. As famílias NRAMP e YSL são evolutivamente próximas. A análise bayesiana das sequências e de regiões promotoras dos genes NRAMP não indica duplicações recentes em gramíneas, sendo as duplicações provenientes de divergência ancestral a origem do grupo. Parálogos foram identificados somente em dicotiledôneas. Por meio da transferência de marcadores IRAP/REMAP é observado que genes YSL de P. patens estão cercados por retroelementos do tipo cópia, a exemplo do que ocorre com o gene ZmYSL1 em milho. Também foi estabelecido o cultivo, em condições axênicas, de Polytrichum juniperinum Hedw. utilizando esporos como explantes, onde foi observado que protonemas são obtidos utilizando meio de cultura livre de fitorreguladores, regenerando gametófitos em cultivo in vitro.

L'étude des familles de gènes et de leur histoire évolutive apporte des indices précieux pour l'annotation fonctionnelle. En effet, le transfert d’annotations fonctionnelles entre gènes dépend de leur histoire évolutive, que cela soit l’histoire des duplications ou la simple divergence de séquence. Afin d'aider la compréhension des familles de gènes, la reconstruction de l'histoire évolutive doit être associée à des annotations et indices fonctionnels liés aux gènes de la famille. Cette reconstruction de l'histoire évolutive des familles passe par l'intégration de plusieurs données hétérogènes. Les indices évolutifs peuvent provenir de plusieurs sources et faire appel à différents outils, il est alors important de bien identifier quelles sont les informations nécessaires à ces études, mais aussi de rendre possible leur intégration dans une analyse commune adapté aux organismes étudiés. Après avoir étudié les spécificités des génomes de plantes et les modes d'évolution spécifiques des familles de gènes nous avons apporté une réponse à cette problématique en développant un système intégratif dédié à l'analyse des familles de gènes, et particulièrement des familles de gènes impliquées dans la réponse aux stress environnementaux. Ce système contient un ensemble d'outils sélectionnés incluant une méthode originale d'intégration des données de synténie, et propose une visualisation synthétique des informations nécessaire à l'étude des familles de gènes. Ce système a été appliqué à l'analyse de familles d'intérêt impliquées dans la résistance aux stress abiotiques, qui nous permet de discuter de l'apport de ce système à l'étude des familles de gènes
The study of gene families and their evolutive history brings precious evidences for the functional annotation of families. The functional transfer depends on one hand on the relationship between genes, and on another hand on the sequence divergence. In order to facilitate the comprehension of gene families, the inference of their evolutive history must be correlated to functional evidences and annotations. This inference is possible through the integration of several heterogeneous data. Evolutive evidences can come from several different data sources and need several tools . It is therefore important to clearly identify both these sources and tools, but also to implement their integration in a common analysis specific to the studied organisms. After studying plant genomes specificities, and their specific mode of evolution, we responded to this problematic through the development of an integrative system containing expertingly chosen data, and implemented tools dedicated to the analysis of gene families. The system also propose a synthetic visualisation analytic tool and an original method to integrate syntenic data for gene family analysis. This system has then been used to study gene family of interest, implied in abiotic stress resistance in plants, that allows us to discuss the intake of the system for gene family analysis

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

MicroRNAs (miRNAs) are a class of small non-coding RNA, that guide RNA silencing of a complementary target mRNA. MiRNAs have been shown to act as post-transcriptional regulators, directing several essential processes in the plant. Despite the importance of miRNAs, the functions of many remain poorly characterized. During my research, two uncharacterised MIR gene families, one conserved and one non-conserved, were investigated. The hypothesis that highly conserved miRNAs regulate architectural and developmental processes while newly evolving miRNAs regulate temporal responses to environmental stresses is examined. The non-conserved MICRORNA163 (MIR163) has recently evolved by gene duplication events in the genus Arabidopsis. It was shown that miR163 regulates the expression of the S-ADENOSYL-METHYLTRANSFERASE (SAMT) family. Hormone treatment, fungal infection, wounding and herbivory each resulted in changes in miR163 and SAMT gene expression, indicating that this miRNA/target association is involved in stress adaptation responses. The highly conserved MIR394 family regulates the F-box protein gene LEAF CURLING RESPONSIVENESS (LCR) and disruption in the miR394/LCR association leads to developmental alterations in leaf polarity and shoot apical meristem organisation. Proteomic analyses identified MAJOR LATEX PROTEINS (MLPs) as probable targets of LCR F-box regulation, suggesting that the biological role of miR394 is to ensure de-repressed expression of MLPs in the shoot apical meristem, and that this is required for the stem cell homeostasis during normal development.

Over the last few decades, phylogenetics has emerged as a very promising field, facilitating a comparative framework to explain the genetic relationships among all the living organisms on earth. These genetic relationships are typically represented by a bifurcating phylogenetic tree — the tree of life. Reconstructing a phylogenetic tree is one of the central tasks in evolutionary biology. The different evolutionary processes, such as gene duplications, gene losses, speciation, and lateral gene transfer events, make the phylogeny reconstruction task more difficult. However, with the rapid developments in sequencing technologies and availability of genome-scale sequencing data, give us the opportunity to understand these evolutionary processes in a more informed manner, and ultimately, enable us to reconstruct genes and species phylogenies more accurately. This thesis is an attempt to provide computational methods for phylogenetic inference and give tools to conduct genome-scale comparative evolutionary studies, such as detecting homologous sequences and inferring gene families. In the first project, we present FastPhylo as a software package containing fast tools for reconstructing distance-based phylogenies. It implements the previously published efficient algorithms for estimating a distance matrix from the input sequences and reconstructing an un-rooted Neighbour Joining tree from a given distance matrix. Results on simulated datasets reveal that FastPhylo can handles hundred of thousands of sequences in a minimum time and memory efficient manner. The easy to use, well-defined interfaces, and the modular structure of FastPhylo allows it to be used in very large Bioinformatic pipelines. In the second project, we present a synteny-aware gene homology method, called GenFamClust (GFC) that uses gene content and gene order conservation to detect homology. Results on simulated and biological datasets suggest that local synteny information combined with the sequence similarity improves the detection of homologs. In the third project, we introduce a novel phylogeny-based clustering method, PhyloGenClust, which partitions a very large gene family into smaller subfamilies. ROC (receiver operating characteristics) analysis on synthetic datasets show that PhyloGenClust identify subfamilies more accurately. PhyloGenClust can be used as a middle tier clustering method between raw clustering methods, such as sequence similarity methods, and more sophisticated Bayesian-based phylogeny methods. Finally, we introduce a novel probabilistic Bayesian method based on the DLTRS model, to sample reconciliations of a gene tree inside a species tree. The method uses MCMC framework to integrate LGTs, gene duplications, gene losses and sequence evolution under a relaxed molecular clock for substitution rates. The proposed sampling method estimates the posterior distribution of gene trees and provides the temporal information of LGT events over the lineages of a species tree. Analysis on simulated datasets reveal that our method performs well in identifying the true temporal estimates of LGT events. We applied our method to the genome-wide gene families for mollicutes and cyanobacteria, which gave an interesting insight into the potential LGTs highways.

QC 20161010

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
As formas familiares de carcinomas não-medulares da tiróide (FNMTC) representam 5% das neoplasias da tiróide. Foram já mapeados 8 loci de susceptibilidade para o FNMTC, no entanto, até à data, apenas o gene DICER1 foi identificado. O envolvimento de diferentes loci sugere a existência de heterogeneidade genética para o FNMTC, contudo, a sua base molecular, é essencialmente desconhecida. Os factores de transcrição NKX2-1, FOXE1, PAX8 e HHEX estão envolvidos na morfogénese e diferenciação da tiróide. Estudos recentes levaram à identificação de mutações germinais no gene NKX2-1 em famílas com FNMTC. No entanto, continua por esclarecer o papel destes factores de transcrição na etiologia do FNMTC. A nova tecnologia de sequenciação global do exoma (WES) tem facilitado a identificação de genes de susceptibilidade para diferentes doenças hereditárias. Este projecto teve como objectivos o estudo do papel do gene FOXE1 em FNMTC e a identificação de novos genes de susceptibilidade para esta doença, utilizando a WES. Desenvolveram-se estudos funcionais para a variante p.A248G do gene FOXE1, identificada numa família com FNMTC, usando como modelos células de tiróide normal (PCCL3) e uma linha celular de carcinoma papilar da tiróide (TPC-1). Nestes ensaios, observou-se que a variante p.A248G promovia a proliferação e migração celular, sugerindo que esta variante poderá contribuir para a tumorigénese na tiróide. Por WES, identificou-se uma nova variante (p.T22I) no gene C8orf48. Esta variante segregava com a doença na família. O gene C8orf48 interage com proteínas da via de sinalização WNT. Em estudos preliminares de expressão génica, identificaram-se alguns genes-alvo desta via (CCND1 e MYC) que apresentavam sobre-expressão no tumor da tiróide relativamente à tiróide normal no probando. Estudos funcionais poderão esclarecer o papel desta variante na tumorigénese. Neste trabalho, foram identificadas variantes genéticas, potencialmente patogénicas nos genes FOXE1 e C8orf48, que constituem a primeira evidência do seu envolvimento em FNMTC.

Mutations in the melanocortin 2 receptor (MC2R) and its accessory protein (MRAP), in the ACTH signalling pathway, and the antioxidant genes nicotinamide nucleotide transhydrogenase (NNT) and thioredoxin reductase 2 (TXNRD2) have been associated with familial glucocorticoid deficiency (FGD). Using a tandem affinity purification and mass spectrometry approach to identify interacting partners of MC2R and MRAP failed to identify putative candidate genes for further FGD cases. However in a male patient a homozygous mutation in another antioxidant gene, glutathione peroxidase 1 (GPX1), was identified. In vitro studies showed H295R cells with knockdown of GPX1 had 50% less basal GPX activity and were less viable than wild-type when exposed to oxidative stress. Adrenals from Gpx1-/- mice showed no gross morphological changes and corticosterone levels were not significantly different from their wild-type counterparts (in contrast to the Nnt mutants). Sequencing of >100 FGD patients did not reveal any other GPX1 mutations. This equivocal data lead to the hypothesis that there could be a second gene defect present in this proband contributing to his disease. Whole exome sequencing revealed a homozygous loss-of-function mutation in peroxiredoxin 3 PRDX3 (p.Q67X) in this patient, that was also present in his unaffected brother. In vitro studies revealed both single and double knockdown of the two genes in H295R cells reduced cell viability, but redox homeostasis and cortisol production were unaffected. GPXs and PRDXs work simultaneously to reduce H2O2, preventing cellular damage. My data suggest that loss of PRDX3 alone is insufficient to cause adrenal failure and further that mutation in GPX1, either alone or in combination with PRDX3 mutation, may tip the redox balance to cause FGD.

A Neoplasia Endócrina Múltipla tipo 1 (NEM1, OMIM 131100) é uma doença essencialmente caracterizada por sua complexidade clínica. A NEM1 afeta tanto tecidos endócrinos quanto tecidos não-endócrinos; apresenta tanto tumores malignos quanto tumores benignos; e apresenta extensa variabilidade clínica inter e intra-familiar quanto aos tipos de tumores e quanto à ordem de desenvolvimento e detecção clínica desses tumores. Em sua forma familiar, a NEM1 é transmitida por um padrão de herança autossômico dominante com elevada penetrância e é identificada pela presença de um parente de primeiro grau apresentando ao menos um tumor NEM1-relacionado. A realização do diagnóstico de NEM1 pode ser: a) clínico, pelo reconhecimento em único paciente de tumores em pelo menos duas das três glândulas endócrinas-alvo principais (paratireóides, hipófise e pâncreas endócrino) e/ou b) genético, pela identificação de mutação germinativa no gene responsável pela doença (MEN1). A grande maioria dos casos NEM1 (90%) apresentam mutações inativadoras no gene MEN1. Não há correlações descritas até o momento entre o genótipo e o fenótipo. Não há também regiões preferenciais (hot-spots) para as mutações no gene MEN1. Além disto, há perda de heterozigose nos tumores NEM1, corroborando com a hipótese que o MEN1 seja um gene supressor de tumor. No presente trabalho, objetivamos a) identificar mutações germinativas no gene MEN1 em pacientes índices com NEM1 típica; b) rastrear parentes dos pacientes que se apresentavam sob-risco para NEM1; c) adicionalmente, estimamos preliminarmente alguns dos possíveis impactos deste do rastreamento gênico familiar no seguimento clínico desses pacientes no Hospital das Clínicas, SP. Para identificação das mutações nos casos-índices com NEM1, foi realizado seqüenciamento automático de todas as regiões codificadoras (éxons 2-10) e fronteiras éxon/íntron do gene MEN1. Para o rastreamento gênico dos familiares, foi realizado seqüenciamento direcionado ao éxon mutado no casosíndices. Quatorze (14) famílias brasileiras com NEM1 e 141 familiares sob risco foram estudados clinica e geneticamente. Doze (12) diferentes mutações MEN1 causadoras de doença foram aqui identificadas, sendo que sete (7) dentre estas mutações não haviam sido previamente descritas: 308delC, 375del21, 1243del1, I147F, L413R, L414P e W471C. As famílias com as mutações recorrentes, 360del4 e L413R, não eram relacionadas. Pela análise evolutiva, viu-se que as quatro mutações novas de ponto aqui relatadas estavam localizadas em resíduos altamente conservados, enquanto que as três novas mutações do tipo deleção ocorriam em regiões repetitivas ricas em GCs. Estas mutações são preditas codificarem proteínas truncadas, o que leva a inativação da ação anti-tumorigênica da proteína menin, e portanto, à doença NEM1. Cento e quarenta e um (141) parentes de pacientes sob-risco de apresentarem NEM1 participaram desse rastreamento. Ao todo, 53 indivíduos foram documentados serem portadores de mutação germinativa no MEN1. Os casos geneticamente diagnosticados foram convidados a aderirem ao rastreamento clínico para NEM1. De modo preliminar, estimamos também os eventuais impactos do rastreamento gênico familiar na conduta clínica da NEM1. Assim, os casos afetados foram subdivididos em 3 grupos e analisados separadamente: casos-índices (grupo I), familiares diagnosticados clinicamente (grupo II) e genicamente (grupo III). A idade média ao diagnóstico no grupo III (27±14,0 anos) foi significativamente menor que a dos grupos II (39.5±15.7; p = 0.03) e III (42.4±15.0; p = 0.01). A maioria dos pacientes dos grupos I e II apresentou 2 ou 3 tumores, enquanto que 81,8% dos casos do grupo III apresentavam 1 ou nenhum tumor relacionado à NEM1. Além disto, 45,4% dos casos no grupo III eram assintomáticos, não sendo observados nenhuma metástase ou óbito. Contrariamente, nos grupos I e II havia ocorrência de metástases provindas de tumores NEM1-relacionados e quatro mortes ligadas a tumores NEM1-relacionados foram relatadas. Os 102 familiares que não herdaram mutação MEN1 foram excluídos do rastreamento clínico. Um caso de fenocópia NEM1 foi também localizado. Em conclusão, relatamos no presente trabalho, a) a identificação de sete (7) novas mutações causadoras de doença no gene MEN1, todas elas localizadas ou em regiões evolutivamente conservadas ou em áreas repetitivas em GCs. b) foi aqui relatado o primeiro rastreamento genético sistemático de famílias com NEM1 na América do Sul, no qual 141 parentes de pacientes com NEM1 foram genotipados. Ao todo, 53 pacientes foram caracterizados como portadores de mutações germinativas no gene MEN1. c) estimamos preliminarmente os eventuais impactos do rastreamento gênico familiar na conduta clínica da NEM1. Os dados desse trabalho suportam a necessidade de se implementação de um sistemático programa de rastreamento na NEM1 em nosso País.
Multiple endocrine neoplasia type 1 (MEN1; OMIM 131100) is a high-penetrance tumor syndrome mainly characterized by the triad: parathyroid (95-100%), pituitary (30%) and enteropancreatic tumors (50%). MEN1 is clinically diagnosed by the occurrence of MEN1-related tumors in at least two of these endocrine glands in a same patient. The familial form of the disease has an autosomal dominant pattern of inheritance and it is identified when a first-degree relative presents at least one MEN1-related tumor. High prevalence of inactivating mutations in the MEN1 has been reported leading to MEN1 syndrome. No mutation hot-spots or genotypephenotype correlations have been observed. In addition, loss of heterozygosity has been found, indicating that MEN1-tumorigenesis is in accordance with Knudson´s classical hypothesis for tumor suppressor genes. This study aimed to characterize clinical features and identify MEN1 germline mutations in Brazilian families with MEN1. Fourteen Brazilian families with MEN1 and 141 at-risk relatives were clinically and genetically studied. Twelve (12) different MEN1 disease-causing mutations were identified, seven of them were previously unreported: 308delC, 375del21, 1243del1, I147F, L413R, L414P and W471C. Families with the recurrent mutations 360del4 and L413R were shown to be unrelated. The four novel missense mutations were found to be located at highly conserved residues by evolutionary analysis, whereas the three novel deletion/frameshift mutations occurred at GC-rich repetitive regions. Familial genetic screening was performed by direct sequencing. Taken together, 53 subjects were found to carry MEN1 germline mutation. To gain preliminary insights on the possible impacts of such familial genetic screening on clinical management of these MEN1 cases, they were separated in three groups: MEN1 index-cases (group I), MEN1 clinically diagnosed at-risk relatives (group II) and genetically diagnosed at-risk family members (group III). The age at the diagnosis in group III (27.0±14.0 y-old) was significantly lower than in groups I (39.5±15.7; p = 0.03) and II (42.4±15.0; p = 0.01). Patients in groups I-II mostly presented two or three MEN1 tumors, while 81.8% of cases in group III presented one or no one MEN1-related tumor. Further, in group III 45.4% of cases were asymptomatic and no metastasis or death were verified. Conversely, in groups I and II, metastases from MEN1-related tumors were frequent and four deaths due to MEN1-related tumors were reported. Moreover, one hundred and two (102) no mutation carriers were excluded of MEN1 surveillance, including one MEN1 phenocopy. In conclusion, it is reported the first systematic genetic screening of MEN1 families in South America and seven novel MEN1 disease-causing mutations were identified. Also, this study underscores the need for implementing a systematic MEN1 screening program in Brazil. At our Institution, we have begun to establish such program.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Universidade Estadual Paulista (UNESP)
Os carcinomas de células escamosas de cabeça e pescoço (CCECP) constituem 90% de todos os cânceres de cabeça e pescoço e estão associados a altas taxas de mortalidade, devido ao seu alto potencial infiltrativo. Este é o sexto tumor mais incidente na população Brasileira. Pouca atenção tem sido dada a fatores hereditários envolvidos em sua etiologia, mas estudos iniciais revelaram que os carcinomas orais tendem a se agregar em famílias. Evidências adicionais são fornecidas pela ocorrência de múltiplos tumores primários em pacientes com CCECP, pela associação com história familial, aparecimento precoce da doença e por membros afetados sem hábitos tabagista e etilista. Em estudos prévios do grupo foi descrita uma associação entre a perda de 22q e história familial de câncer em pacientes com CCECP, após análise citogenética, análise de LOH e PCR quantitativa em tempo real. O gene PHF21B (PHD finger protein 21B), mapeado em 22q13.31, codifica uma proteína reguladora transcricional e foi selecionado como um gene candidato associado a predisposição familial em CCECP. Os resultados da PCR quantitativa em tempo real mostraram associação estatisticamente significativa com relação à perda de seqüências do gene e história familial de câncer em parentes de 1º grau (P<0,0001). Estes dados sugerem que o PHF21B é um gene supressor tumoral candidato envolvido com história familial em carcinomas de cabeça e pescoço. Para investigar a inativação de um gene supressor tumoral (Knudson, 1971), foram selecionados 13 pacientes com CCECP que mostravam história familial positiva de câncer e outros 14 casos sem história familial. Foram considerados como critério para história familial positiva: parentes de primeiro e segundo graus com câncer; não-fumantes e não-consumidores de álcool; idade igual ou inferior a 56 anos quando do diagnóstico...
Head and neck squamous cell carcinomas (HNSCC) represents 90% of all cancers and are associated with high mortality, due to high infiltrative potential. This tumor is the sixth most incident in the Brazilian population. Little attention have been given to hereditary factors involved in their etiology, but early studies showed that oral carcinomas tend to aggregate themselves in families. Additional evidences are provided for primary multiple tumors occurrence in HNSCC patients, to family history association, early disease development and no tobacco and alcohol consumptions. Previously we described an association between 22q loss and cancer family history in HNSCC patients by cytogenetics, LOH and real time PCR analysis. The PHF21B gene (PHD finger protein 21B), mapped at 22q13.31, codifies a transcriptional regulatory protein and was selected as a putative gene associated with HNSCC familial predisposition. The real time PCR results showed statistical significant association related to gene loss and first degree family history (P<0,0001). This data suggests that PHF21B is a tumor suppressor gene candidate involved in HNSCC family history. To investigate the tumor suppressor gene inactivation (Knudson, 1971), were selected 13 HNSCC patients that had positive cancer family history and other 14 without HNSCC family history. It was considered for positive family history: first and second relatives with cancer; no tobacco and alcohol users; age 56 or less at diagnosis. Investigation analysis was performed by direct sequencing at exons: 7, 8 and 9. These exons were selected for its relation to PHD zinc-finger domain, responsible for PHF21B protein function. No mutations or significative variations were found in these exons. The SNPs (single nucleotide polymorphisms) analysis in gene extension showed a reduced SNPs number in exonic regions, however 16 SNPs were found at 3´UTR region... (Complete abstract click electronic access below)

Orientador: Silvia Regina Rogatto
Banca: Benedito Mauro Rossi
Banca: Ivan de Godoy Maia
Resumo: Os carcinomas de células escamosas de cabeça e pescoço (CCECP) constituem 90% de todos os cânceres de cabeça e pescoço e estão associados a altas taxas de mortalidade, devido ao seu alto potencial infiltrativo. Este é o sexto tumor mais incidente na população Brasileira. Pouca atenção tem sido dada a fatores hereditários envolvidos em sua etiologia, mas estudos iniciais revelaram que os carcinomas orais tendem a se agregar em famílias. Evidências adicionais são fornecidas pela ocorrência de múltiplos tumores primários em pacientes com CCECP, pela associação com história familial, aparecimento precoce da doença e por membros afetados sem hábitos tabagista e etilista. Em estudos prévios do grupo foi descrita uma associação entre a perda de 22q e história familial de câncer em pacientes com CCECP, após análise citogenética, análise de LOH e PCR quantitativa em tempo real. O gene PHF21B (PHD finger protein 21B), mapeado em 22q13.31, codifica uma proteína reguladora transcricional e foi selecionado como um gene candidato associado a predisposição familial em CCECP. Os resultados da PCR quantitativa em tempo real mostraram associação estatisticamente significativa com relação à perda de seqüências do gene e história familial de câncer em parentes de 1º grau (P<0,0001). Estes dados sugerem que o PHF21B é um gene supressor tumoral candidato envolvido com história familial em carcinomas de cabeça e pescoço. Para investigar a inativação de um gene supressor tumoral (Knudson, 1971), foram selecionados 13 pacientes com CCECP que mostravam história familial positiva de câncer e outros 14 casos sem história familial. Foram considerados como critério para história familial positiva: parentes de primeiro e segundo graus com câncer; não-fumantes e não-consumidores de álcool; idade igual ou inferior a 56 anos quando do diagnóstico... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Head and neck squamous cell carcinomas (HNSCC) represents 90% of all cancers and are associated with high mortality, due to high infiltrative potential. This tumor is the sixth most incident in the Brazilian population. Little attention have been given to hereditary factors involved in their etiology, but early studies showed that oral carcinomas tend to aggregate themselves in families. Additional evidences are provided for primary multiple tumors occurrence in HNSCC patients, to family history association, early disease development and no tobacco and alcohol consumptions. Previously we described an association between 22q loss and cancer family history in HNSCC patients by cytogenetics, LOH and real time PCR analysis. The PHF21B gene (PHD finger protein 21B), mapped at 22q13.31, codifies a transcriptional regulatory protein and was selected as a putative gene associated with HNSCC familial predisposition. The real time PCR results showed statistical significant association related to gene loss and first degree family history (P<0,0001). This data suggests that PHF21B is a tumor suppressor gene candidate involved in HNSCC family history. To investigate the tumor suppressor gene inactivation (Knudson, 1971), were selected 13 HNSCC patients that had positive cancer family history and other 14 without HNSCC family history. It was considered for positive family history: first and second relatives with cancer; no tobacco and alcohol users; age 56 or less at diagnosis. Investigation analysis was performed by direct sequencing at exons: 7, 8 and 9. These exons were selected for its relation to PHD zinc-finger domain, responsible for PHF21B protein function. No mutations or significative variations were found in these exons. The SNPs (single nucleotide polymorphisms) analysis in gene extension showed a reduced SNPs number in exonic regions, however 16 SNPs were found at 3'UTR region... (Complete abstract click electronic access below)
Mestre

Orientador: Carmen Silvia Bertuzzo
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Mutações germinativas no gene APC (Polipose adenomatosa coli) são responsáveis pela ocorrência de polipose adenomatosa familiar (PAF). Mutações somáticas levam à transformação maligna de adenomas. O objetivo desse trabalho foi identificar mutações germinativas no gene APC. No presente estudo, 20 pacientes com PAF foram estudados. A determinação das mutações germinativas no APC foi realizada por meio de sequenciamento, e as mutações foram comparadas com marcadores clínicos (sexo, idade no momento do diagnóstico, tabagismo, estádio TNM, classificação Coller-Astler e o grau de diferenciação do adenocarcinoma). Os dados foram comparados por meio do programa SPSS , com o teste de Fisher e teste de ?2 , considerando ? = 0,05. De acordo com os principais resultados da nossa amostra, 16 alelos com mutações deletérias (80 % dos pacientes) foram identificados, enquanto 7 (35%) pacientes não tinham mutações deletérias. Houve um predomínio de mutações nonsense (45% dos pacientes) e de mutações frameshift (20% dos pacientes). Não houve significância estatística entre as mutações germinativas identificadas e as variáveis clínicas consideradas em nosso estudo. Apenas a fase TNM foi associada com a presença de mutações deletérias. Os portadores com mutações deletérias tinha uma OR , 0,086 ( IC = 0,001-0,984 ); TNM I + II em comparação com III + IV , quando comparado com os pacientes sem mutações deletérias identificados. Neste estudo, demonstramos a heterogeneidade molecular de mutações germinativas no APC em portadores de PAF e a dificuldade para realizar diagnóstico molecular em uma população brasileira
Abstract: Adenomatous polyposis coli (APC) germline mutations are responsible for the occurrence of familial adenomatous polyposis (FAP). Somatic mutations lead to malignant transformation of adenomas. In this context, considering the significance of APC germline mutations in FAP, we aimed to identify APC germline mutations. In the present study, 20 FAP patients were enrolled. The determination of APC germline mutations was performed using sequencing, and the mutations were compared with clinical markers (gender, age at diagnosis, smoking habits, TNM stage, Astler-Coller stage, degree of differentiation of adenocarcinoma). The data were compared using the SPSS program, with the Fisher's exact test and ?2 test, considering ?=0.05. According to the main results in our sample, 16 alleles with deleterious mutations (80% of the patients) were identified while 7 (35%) patients had no deleterious mutations. There was a predominance of nonsense (45% of the patients) and frameshift (20% of the patients) mutations. There was no statistical significance between the APC germline mutations identified and the clinical variables considered in our study. Only TNM stage was associated with the presence of deleterious mutations. Patients with deleterious mutations had an OR, 0.086 (IC=0.001-0.984); TNM stage I + II in comparison with III + IV, when compared with the patients with no deleterious mutations identified. In this context, as a conclusion, we demonstrated the molecular heterogeneity of APC germline mutations in FAP and the difficulty to perform molecular diagnostics in a Brazilian population, considering the admixed population analyzed
Doutorado
Clinica Medica
Doutora em Ciências

Migraine is a painful temporarily incapacitating disorder that affects an estimated 12% of the general population including 18% of adult women and 6% of adult men. The disorder involves two main subtypes termed migraine with or without aura (MA and MO respectively). Migraine can present with a variety of symptoms that vary between individuals and between episodes experienced by a single individual. This disorder causes significant social and economic burden and alarmingly is often poorly treated. A direct cause of this is a lack of understanding of the underlying pathology of migraine. Migraine is believed to be a neurogenic disorder that involves temporary disruption of pathways that receive and respond to sensory signals. While numerous environmental triggers may have been identified the exact mechanisms that cause the disruption are still largely unknown. However, familial aggregation of migraine suggests significant genetic contributors.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Orientador: Marcelo Carnier Dornelas
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Na planta modelo Arabidopsis thaliana o desenvolvimento do fruto foi amplamente estudado e uma série de genes foram relacionados com a ontogênese e o amadurecimento. A identidade dos tecidos dos frutos secos de Arabidopsis é determinada principalmente pelos genes FRUITIFULL (FUL) e SHATTERPROOF (SHP), pertencentes à família multigênica MADS-box. Homólogos a estes genes foram encontrados em frutos carnosos, no entanto a expressão destes homólogos e o papel biológico dos mesmos durante o desenvolvimento dos frutos carnosos são desconhecidos. No banco de dados de genes expressos em Citrus (CitEST), sequências homólogas a FUL e SHP foram encontradas. Uma vez que os frutos de Citrus são carnosos e não apresentam as mesmas estruturas dos frutos de Arabidopsis, decidiu-se caracterizar detalhadamente o desenvolvimento dos frutos em Citrus sinensis e Citrus reticulata e estudar as possíveis funções dos homólogos de FUL e SHP durante o desenvolvimento dos frutos em C. sinensis. Técnicas de microscopia óptica e eletrônica de varredura foram utilizadas para caracterização do desenvolvimento dos frutos. Resultados de RT-PCR mostraram a expressão diferencial dos homólogos de Citrus de FUL e SHP em frutos maduros. Os resultados de hibridização in situ mostraram que o homólogo de FUL expressou-se diferencialmente durante o desenvolvimento do fruto, principalmente nas células secretoras das cavidades oleíferas e das vesículas de suco, nos tecidos do epicarpo, mesocarpo e endocarpo. O homólogo de SHP também se expressou no epicarpo, mesocarpo, endocarpo e nas vesículas de suco. Os resultados obtidos poderão auxiliar num melhor entendimento do desenvolvimento do fruto em Citrus.
Abstract: Fruit development is extensively studied in the model plant Arabidopsis thaliana and a number of genes is being related to the control of fruit ontogenesis and ripening. The FRUITFULL (FUL) and SHATTERPROOF (SHP) genes, which belong to the MADS-box multigenic family are, in large part, responsible for determining the identity of the Arabidopsis dry fruit tissues. Homologs to these genes have been described for species harbouring fleshy fruits, but the expression patterns and biological functions for these genes during fruit development are unknown. Sequences showing similarity to FUL and SHP were found at the database of the CitEST Project (expressed sequence tags in Citrus). Since Citrus fruits are fleshy and do not have the same structures of the Arabidopsis fruits, we decided to characterize in detail the development of fruits in Citrus sinensis and Citrus reticulata and to study the possible functions of FUL and SHP homologues during the development of C. sinensis fruits. These sequences were characterized by bioinformatic's tools and phylogenetic analysis. The use of light and scanning electron microscopy (SEM) techniques allowed the morpho-anatomical characterization of Citrus fruit development. The RT-PCR results showed a preferential expression of FUL and SHP in developing fruits. The in situ hybridization results showed that the C. sinensis homolog of the FUL gene is differentially expressed in the secretory cells of the oil cavity as well as in the juice vesicles, epicarp, mesocarp and endocarp tissues. The homolog of the SHP gene also expressed in epicarp, mesocarp, endocarp and juice vesicles. All the results taken together might contribute to a better understanding of the processes involved in Citrus fruit development.
Mestrado
Mestre em Biologia Vegetal

Colorectal cancer in the commonest internal malignancy in western society today. At least a third of the incidence is likely to be due entirely or in part to inherited genetic factors. Over the last 15 years several genes have been described in which germline mutation leading to increased colorectal cancer risk may occur. The commonest are Hereditary Non-Polyposis Colorectal Cancer (HNPCC), which accounts for around 1-4% of colorectal cancer diagnosis without polyposis and is caused by mutations in mismatch repair genes and Familial Adenomatous Polyposis caused by mutations in the AFC gene. In this thesis two related themes are addressed. Firstly I examine clinical, pathologic and molecular genetic information in kindreds recruited from family cancer clinics in order to investigate several relevant clinical problems relating to decisions regarding genetic testing and clustering of non-HNPCC families. Secondly, the group of individuals and families with multiple colorectal polyps without known genetic cause are investigated in several ways. A candidate gene analysis is undertaken looking for germline changes, an analysis of adenomas from such individuals for informative somatic changes is performed and I describe a new inherited syndrome of colorectal cancer and polyposis, MYH associated polyposis as well as the pathway of tumourigenesis in affected individuals.

Les glycophorines a et b (gpa et gpb), sont les deux sialoglycoproteines majeures de la membrane du globule rouge humain. Nous avons etudie, chez des individus normaux et variants, la structure des genes gpa et gpb, ainsi que celle d'un troisieme gene que nous avons mis en evidence, le gene gpe. Les trois genes sont tres similaires. Seul le gene gpa possede une region 3 differentes des deux autres. Cette brusque divergence est due a une recombinaison ayant eu lieu entre des sequences alu au cours de l'evolution. Les trois promoteurs ne presentent que quelques differences ponctuelles sur 300 paires de bases et les points d'initiation de la transcription sont identiques, bien que les quantites d'arnm detectes pour les trois genes soient tres differents. L'etude chez les variants des deletions dues a des mecanismes de recombinaisons illegitimes, nous a permis de proposer que les genes sont dans l'ordre gpa-gpb-gpe sur le chromosome 4

Familial cutaneous mastocytosis is an exceptional condition of unknown etiology. In this study we report the largest series of patients with familial cutaneous mastocytosis without other manifestations (18 affected subjects from seven unrelated families), and we investigate the role of germ-line KIT mutations in the pathogenesis of the disease. The mean age at onset was 5.4 years (range from birth to 22 years), and the clinical behavior was variable over a mean follow up period of 15.1 years (range 2-36): improvement in seven, stability in eight and worsening in the remaining three patients. The pattern of inheritance was compatible with an autosomal dominant trait with incomplete penetrance; a female preponderance (14 females vs 4 males, ratio 3.5:1) was noted; among the six women who have been pregnant at least once, three experienced important clinical changes during pregnancy. No germ-line mutation was found in the exons 10, 11, and 17 of the KIT proto-oncogene, which are the most commonly mutated exons in sporadic mastocytosis. However, in the majority of affected subjects we found the Met541Leu polymorphic variant of the KIT gene, which seems to confer a growth advantage to mast cells in vitro. This observation further suggests that the Met541Leu may be a predisposing factor of cutaneous mastocytosis, although it seems to be neither necessary nor sufficient for the development of the disease.

Familial cutaneous mastocytosis is an exceptional condition of unknown etiology. In this study we report the largest series of patients with familial cutaneous mastocytosis without other manifestations (18 affected subjects from seven unrelated families), and we investigate the role of germ-line KIT mutations in the pathogenesis of the disease. The mean age at onset was 5.4 years (range from birth to 22 years), and the clinical behavior was variable over a mean follow up period of 15.1 years (range 2-36): improvement in seven, stability in eight and worsening in the remaining three patients. The pattern of inheritance was compatible with an autosomal dominant trait with incomplete penetrance; a female preponderance (14 females vs 4 males, ratio 3.5:1) was noted; among the six women who have been pregnant at least once, three experienced important clinical changes during pregnancy. No germ-line mutation was found in the exons 10, 11, and 17 of the KIT proto-oncogene, which are the most commonly mutated exons in sporadic mastocytosis. However, in the majority of affected subjects we found the Met541Leu polymorphic variant of the KIT gene, which seems to confer a growth advantage to mast cells in vitro. This observation further suggests that the Met541Leu may be a predisposing factor of cutaneous mastocytosis, although it seems to be neither necessary nor sufficient for the development of the disease.

Potato cyst nematodes (PCN) are economically relevant plant parasites that infect potato crops. The genomes of three PCN species are available and genome data have been generated for several populations of PCN, to address questions related to the molecular basis of plant parasitism. In this thesis, I employ approaches of comparative genomics to highlight differences and similarities between PCNs and other nematode species. I present two new software solutions to address challenges associated with the field of comparative genomics: BlobTools, a taxonomic interrogation toolkit for quality control of genome assemblies, and KinFin, a solution for the analysis of protein orthology data. I apply both software solutions to genomic datasets of nematodes, platyhelminths, and tardigrades. Based on KinFin analysis of plant parasitic nematodes, I identify protein families in PCNs likely to be involved in host-parasitic interaction, termed effectors, and discuss their functions. I highlight examples of horizontal gene transfer from bacteria to plant parasitic nematodes. Through genomic data of European and South American populations of PCNs, I address variation in populations, infer phylogenetic relationships, and try to estimate the effect of selection on effector genes identified through KinFin. Furthermore, I estimate the rate of variation across the reference genomes of two PCNs.

La hipomagnesèmia familiar amb hipercalciúria i nefrocalcinosi (HFHNC) és una tubulopatia minoritària autosòmica recessiva causada per mutacions als gens CLDN16 o CLDN19, que codifiquen per la claudina-16 i -19, respectivament, que s’expressen a la porció gruixuda de la nansa ascendent de Henle, i estan implicades en el transport iònic paracel·lular. Aquesta malaltia es caracteritza per la pèrdua urinària massiva de calci i magnesi, nefrocalcinosi bilateral i progressió inexorable de la malaltia renal crònica, que desencadena en fallida renal. Addicionalment, la majoria de pacients amb mutacions a CLDN19 també desenvolupen alteracions oculars, ja que aquest s’expressa a les cèl·lules epitelials de la retina. Característicament, existeix una gran variabilitat fenotípica entre els pacients, inclús entre aquells que comparteixen la mutació c.59G>A; p.G20D a CLDN19 (mutació Hispànica), i entre membres d’una mateixa família. Aquest fenomen suggereix que possiblement existeixen altres processos moleculars que determinen la progressió clínica dels pacients. Sota aquesta hipòtesi, aquest treball s’ha focalitzat en l’estudi dels factors genètics i epigenètics que podrien modular la progressió de la malaltia renal. Per això, s’han obtingut les dades clíniques i mostres d’orina i sang de 30 pacients afectes d’HFHNC i 6 individus control d’arreu d’Espanya, essent la majoria dels pacients diagnosticats d’HFHNC en aquest territori. L’anàlisi de les dades clíniques va permetre avaluar les diferències en l’evolució de la malaltia renal en funció del sexe i el genotip, classificar els pacients segons la pèrdua anual de funció renal (progressió renal ràpida, moderada i lenta), identificar biomarcadors clínics de pronòstic, i evidenciar l’alta variabilitat fenotípica intrafamiliar. L’estudi de variants gèniques en homozigosi associades als fenotips més extrems (progressió ràpida i lenta) s’ha realitzat amb les dades obtingudes de la seqüenciació massiva de l’exoma dels 30 pacients de la cohort. Seguint aquesta estratègia, s’han identificat un total de 45 variants gèniques. D’entre aquestes, per la funció fisiològica dels gens on es localitzen, en destaquen la rs11207827 (al gen PATJ) i la rs1050171 (al gen EGFR). Per determinar els factors epigenètics implicats en la fisiopatologia de la HFHNC i en la seva progressió, s’han utilitzat els urinary exosome-like vesicles (uEVs) com a font no invasiva d’informació dels processos cel·lulars renals. Mitjançant microarrays, s’ha analitzat el perfil d’expressió dels miRNAs continguts als uEVs dels 20 pacients que mantenien els ronyons natius funcionals identificant-se 24 miRNAs diferencialment expressats en el conjunt de pacients respecte el grup control, i 43 en el subconjunt dels pacients homozigots per la mutació p.G20D a CLDN19. La comparació de pacients d’ambdós sexes, va mostrar únicament la infraexpressió del miR 1915 5p en aquells de sexe masculí. Per últim, s’han identificat 4 miRNAs diferencialment expressats en els pacients d’HFHNC amb progressió renal moderada, en comparació amb el de progressió lenta, i 8 en el subgrup de pacients homozigots per la mutació p.G20D a CLDN19. La biologia de sistemes i l’ús de xarxes neuronals d’intel·ligència artificial han permès associar els resultats obtinguts amb processos biològics crucials en la fisiopatologia de la HFHNC, com la fibrosi renal i el transport de calci i magnesi, entre d’altres. Aquest treball ha permès incrementar el coneixement de la fisiopatologia de la HFHNC i determinar factors clínics, genètics i epigenètics implicats en la progressió de la malaltia renal i identificar nous possibles biomarcadors pronòstics de la HFHNC, i dianes terapèutiques que podrien permetre modular la severitat de la malaltia i, en el millor dels casos, curar-la.
La hipomagnesemia familiar con hipercalciuria y nefrocalcinosis (HFHNC) es una tubulopatía minoritaria autosómica recesiva causada por mutaciones en los genes CLDN16 o CLDN19, que codifican para la claudina-16 y -19, respectivamente, expresadas en la porción gruesa del asa ascendente de Henle, e implicadas en el transporte iónico paracelular. Esta enfermedad se caracteriza por la pérdida urinaria de calcio y magnesio, nefrocalcinosis bilateral y progresión inexorable de la enfermedad renal crónica, que desencadena en fallo renal. Adicionalmente, la mayoría de pacientes con mutaciones en CLDN19 también desarrollan alteraciones oculares, ya que este se expresa en las células epiteliales de la retina. Existe una gran variabilidad fenotípica entre pacientes, incluso entre aquellos que comparten la mutación c.59G>A; p.G20D en CLDN19 (mutación Hispánica), y entre miembros de una misma familia. Este fenómeno sugiere que, más allá de la mutación causante de la enfermedad, posiblemente existen otros procesos moleculares que determinan la progresión clínica de los pacientes. Bajo esta hipótesis, este trabajo se ha focalizado en el estudio de los factores genéticos y epigenéticos que podrían modular la progresión de la enfermedad renal. Para ello, se han obtenido los datos clínicos, orina y sangre de 30 pacientes afectos de HFHNC y 6 individuos control de diferentes lugares de España, siendo estos la mayoría de pacientes diagnosticados de HFHNC en este territorio. El análisis de los datos clínicos permitió evaluar las diferencias en la evolución de la enfermedad renal en función del sexo y del genotipo, clasificar los pacientes según la pérdida anual de función renal (progresión renal rápida, moderada y lenta), identificar biomarcadores clínicos de pronóstico, y evidenciar la alta variabilidad fenotípica intrafamiliar. El estudio para la identificación de variantes génicas en homocigosis asociadas a los fenotipos más extremos (progresión renal rápida y lenta) se realizó con los datos obtenidos de la secuenciación masiva del exoma de los 30 pacientes de la cohorte. Con esta estrategia, se identificaron 45 variantes génicas. De entre estas, por la función fisiológica de los genes donde se localizan, destacan la rs11207827 (en el gen PATJ) y la rs1050171 (en el gen EGFR). Para determinar los factores epigenéticos implicados en la fisiopatología de la HFHNC y en su progresión, se utilizaron los urinary exosome-like vesicles (uEVs) como fuente no invasiva de información de procesos celulares renales. Mediante microarrays, se analizó el perfil de expresión de los miRNAs contenidos en los uEVs de los 20 pacientes que mantenían los riñones nativos funcionales, identificándose 24 miRNAs diferencialmente expresados en el total de pacientes respecto al grupo control, y 43 en el subconjunto de pacientes homocigotos para la mutación p.G20D en CLDN19. La comparación de pacientes de ambos sexos mostro únicamente la infraexpresión del miR-1915-5p en aquellos de sexo masculino. Finalmente, se identificaron 4 miRNAs diferencialmente expresados en los pacientes con progresión renal moderada, en comparación con el de progresión lenta, y 8 en el subgrupo de pacientes homocigotos para la mutación p.G20D en CLDN19. La biología de sistemas y el uso de redes neuronales de inteligencia artificial han permitido asociar los resultados obtenidos con procesos biológicos cruciales en la fisiopatología de la HFHNC, como la fibrosis renal y el transporte de calcio y magnesio, entre otros. Este trabajo ha permitido incrementar el conocimiento de la fisiopatología de la HFHNC y determinar factores clínicos, genéticos y epigenéticos implicados en la disfunción renal. Desde el punto de vista traslacional, se han identificado nuevos posibles biomarcadores pronósticos de la HFHNC, y dianas terapéuticas que podrían permitir modular la severidad de la enfermedad y, en el mejor de los casos, curarla.
Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubulopathy caused by mutations in either CLDN16 or CLDN19 genes, which encode for claudin-16 and -19, respectively, expressed in the thick ascending loop of Henle, and involved in paracellular ion transport. This disease is characterized by urinary loss of calcium and magnesium, bilateral nephrocalcinosis, and inexorable progression of chronic renal disease leading to renal failure. Besides, most patients with CLDN19 mutations also develop ocular anomalies, as it is expressed in the retinal epithelial cells. There is great phenotypic variability among patients, even in those who share the c.59G>A; p.G20D mutation in CLDN19 (Hispanic mutation), and also among members of the same family. This phenomenon suggests that, beyond the mutation causing the disease, other molecular processes could determine the clinical progression of patients. Under this hypothesis, this work has focused on the study of genetic and epigenetic factors that could modulate renal disease progression. For this purpose, clinical data, urine and blood from 30 patients affected by FHHNC and 6 control individuals were collected from around Spain, being them the majority of patients diagnosed of FHHNC in this territory. The analysis of the clinical data allowed evaluating the differences in the evolution of the renal disease according to sex and genotype, classifying patients according to the annual decline of renal function (fast, moderate and slow renal progression), identifying clinical biomarkers of prognosis, and evidencing the high intrafamilial phenotypic variability. The study for the identification of gene variants in homozygosis associated with both extreme phenotypes (fast and slow renal progression) was carried out with data obtained from exome sequencing of the 30 patients included in the cohort. With this strategy, 45 gene variants were identified. Among these, due to the physiological function of the genes where they are located, the rs11207827 (in the PATJ gene) and the rs1050171 (in the EGFR gene) were highlighted. To determine the epigenetic factors involved in FHHNC physiopathology and disease progression, the urinary exosome-like vesicles (uEVs) were used as a non-invasive source of information on renal cellular processes. Microarray technique was used to analyze the expression pattern of miRNAs contained in uEVs of the 20 patients who maintained functional native kidneys. Twenty-four miRNAs were identified differentially expressed in all patients when compared with the control group, and 43 in the subset of patients homozygous for the p.G20D mutation in CLDN19. The comparison of patients of both sexes showed only the under-expression of the miR-1915-5p in males. Finally, 4 miRNAs were differentially expressed in patients with moderate to slow progression of renal disease, and 8 within the subgroup of patients homozygous for the p.G20D mutation in CLDN19. Systems biology and the use of artificial neuronal networks have allowed to associate the results obtained with crucial biological processes in FHHNC physiopathology such as renal fibrosis and calcium and magnesium transport, among others. This work, through two complementary strategies, has allowed increasing the knowledge of the physiopathology of FHHNC and, for the first time, determining clinical, genetic and epigenetic factors involved in renal disease progression. From a translational perspective, this thesis has identified new possible prognostic biomarkers of FHHNC as well as novel therapeutic targets that could allow modulating the severity of the disease and, in the best case, to cure it.

Haemonchus contortus is a blood-feeding Strongylid parasite that is economically significant worldwide. Due to the increasing problem of anthelmintic resistance, alternative approaches are urgently required for parasitic nematode control. H. contortus cathepsin B gut cysteine proteases have received attention as potential vaccine candidates because of their proposed role in blood feeding. The increasing amount of H. contortus genome information has now enabled detailed identification and annotation of cathepsin B protease gene families. In this study H. contortus BAC 18f22 was annotated and found to encode eight tandemly arranged cysteine proteases related to the previously identified AC family, but with six novel genes identified. Annotation of supercontig and scaffold sequence identified many more members of the HmCP and GCP-7 cathepsin B families. In total this work has shown that the H. contortus genome encodes at least 41 cathepsin B protease genes, more than in other nematodes, to date. In contrast, Hc-cpr-6 is present as a single copy gene that is highly conserved in a number of species, suggesting an important conserved function. Further work examined regulation of gut gene expression in H. contortus, in particular the H. contortus ELT-2 GATA transcription factor (TF), as it has been shown to be the major TF in C. elegans controlling gut gene expression. A high throughput assay was developed and used to screen an integrated C. elegans worm strain expressing GFP in the gut and hypodermis (Ce-cpl-1::gfp) against 594 chemical compounds. Compounds were identified that specifically cause a decrease in gut GFP expression, affect larval development and show a degree of lethality. Further work on two of the compounds identified an embryonic effect, with a significant decrease in number of progeny. To conclude, this thesis identified a number of novel cathepsin B genes as well as two compounds potentially interfering with TF activity and gut gene expression which may be of use as novel anthelmintics.