Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (p. [119]-127).

Orientador: Ana Sílvia Alves Meira Tavares Moura / Banca: Heraldo César Gonçalves / Banca: Mônica Corrêa Ledur / Resumo: O objetivo deste trabalho foi identificar locos controladores de características quantitativas (QTLs) nos cromossomos 11 e 13 de galinhas (Gallus gallus) para características de desempenho e carcaça. A partir do cruzamento entre uma linhagem de corte e uma de postura, foi gerada a população experimental F2 na Embrapa Suínos e Aves. Foram avaliadas as seguintes informações fenotípicas: peso ao nascer, peso aos 35, 41 e 42 dias, ganho de peso, consumo de ração, eficiência e conversão alimentar dos 35 aos 41 dias e valores de hematócrito. As carcaças foram evisceradas e avaliados: o comprimento do intestino, peso dos pulmões, do fígado, do coração e da moela. Foram obtidos após quatro horas de resfriamento: peso da carcaça, gordura abdominal, peso de partes: peito, coxas, dorso, asas, cabeça e pés. Quatro e cinco marcadores microssatélites dos cromossomos 11 e 13, respectivamente, foram genotipados num total aproximado de 330 animais F2 em quatro famílias de irmãos-completos. Os mapas de ligação para ambos os cromossomos foram construídos e a análise de mapeamento de QTLs baseada no modelo genético de F2 foi realizada. No cromossomo 11 foram mapeados dois QTLs sugestivos: para peso de pés e de moela, ambos posicionados no intervalo entre ADL0123 e ADL0210. No cromossomo 13 foi mapeado um QTL sugestivo para peso de coração posicionado no intervalo entre MCW0110 e MCW0104. / Abstract: The objective of this study was to identify quantitative trait loci (QTLs) for performance and carcass traits in chicken (Gallus gallus) chromosomes 11 and 13. From the crossbreeding of a broiler and a layer line, an F2 experimental population was generated at the Embrapa Suínos e Aves. The following phenotypic data were recorded: body weights at birth, 35, 41 and 42 d; weight gain, feed consumption and feed conversion from 35 to 41 d; weights of carcass, carcass parts, organs and abdominal fat, hematocrit and length of intestine. Four and five microsatellite markers from chromosomes 11 and 13, respectively, were genotyped in approximately 330 F2 chickens from four full-sib families. The linkage maps for both chromosomes were constructed and the QTL mapping analyses were carried out based on an F2 genetic model. Two suggestive QTLs were mapped to chromosome 11: for feet and gizzard weights, both located in the interval between ADL0123 and ADL0210. On chromosome 13 one suggestive QTL for heart weight was detected in the interval between MCW0110 and MCW0104. / Mestre

This thesis describes the alignment of cloned DNA with the genetic map, and the identification of coding elements within the aligned DNA. The region of study was the dpy-5 unc-29 interval from chromosome I of the nematode Caenorhabditis elegans, with an emphasis on the region surrounding the gene dpy-14. The objectives of this thesis were: 1) to align the physical and genetic maps .of the region; 2) to identify and characterize the coding elements in the vicinity of dpy-14; and 3) to cross-hybridize the identified C. elegans coding elements to mammalian DNA in an attempt to identify evolutionarily conserved genes. Six polymorphisms from the dpy-5 unc-29 interval were mapped with respect to the free duplication sDp2. The polymorphisms hP5, sPl, and hP9 were found to.be inside the region spanned by sDp2 while the polymorphisms hP4, hP6, and hP7 were found to be outside this interval. In addition, these six polymorphisms were mapped with respect to visible markers from the dpy-5 unc-29 interval. These analyses demonstrated the genetic order to be dpy-5, hP5, unc-37, (dpy-14, sPl), hP9, unc-13, hP7, (hP4, hP6), unc-29. Lambda phage containing the hP5, sPl, and hP6 sites identified and anchored cosmid contigs to the genetic map. The interval from the left of hP5 to the right of unc-13 is contained in a single contig of approximately 1400 Kb. The amount of DNA in Kb across the hP5 and unc-13 interval was compared to the genetic distance in map units. The DNA per map unit value was found to vary in this interval with the greatest value found between hP9 and unc-13. Seven cosmids representing 173 Kb of N2 genomic DNA near the gene dpy-14 were isolated. Using cross-species hybridization to C. briggsae DNA ten conserved regions were identified within these seven cosmids. The ten conserved fragments were used to identify seven cDNAs, six of which also identified RNAs on Northern blots. The relative abundance of the isolated cDNAs varied 250 fold with the most abundant having a level similar to that found for actin. The first comprehensive survey of mammalian homologies in a contiguous set of ten coding regions found three coding elements to be. conserved. One was demonstrated to be the small nuclear RNA gene U1. Another shared sequence similarities with the gene S-adenosyl-L-homocysteine hydrolase. No detectable homologies were identified with the third. A formaldehyde-induced mutation that failed to complement the genes unc-37, unc-87, dpy-14, let-83 and let-86 was isolated. This mutation appeared to be the result of a DNA rearrangement which had one breakpoint within the cosmid C14A12. Using the conserved elements identified in this thesis together with the rearrangements and mapped genes from the region, a detailed physical and genetic map in the vicinity of dpy-14 was constructed. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Passiflora (Passifloraceae) é um grande gênero de espécies vegetais encontradas, principalmente, na flora tropical. Algumas passifloras são cultivadas como plantas ornamentais, frutíferas ou exploradas pelas suas propriedades medicinais. A principal espécie comercial brasileira, o maracujá-azedo (Passiflora edulis f. flavicarpa, 2n = 18), ocupa 95% da área plantada. Os frutos são consumidos in natura ou processados pela indústria de suco. Estudos genéticos e cromossômicos têm sido gerados para esta espécie. Entretanto, devido ao pequeno tamanho e a similaridade morfológica dos seus cromossomos, as medições do cariótipo convencional do maracujá-azedo têm levado a resultados inconsistentes, sendo necessário o desenvolvimento de marcadores cromossomos-específicos. Estes marcadores são produzidos a partir da identificação de sequências de cópia-única em clones de bibliotecas de BACs (Bacterial Artificial Chromosome), que são utilizadas como sondas em ensaios de FISH (Hibridização in situ Fluorescente). Neste trabalho, foi construída uma biblioteca genômica de maracujá-azedo em BACs contendo 82.944 clones, com tamanho médio dos insertos de 108 kb, e provendo uma cobertura de seis vezes o genoma. A biblioteca apresentou baixa contaminação com cpDNA e mtDNA (~0,04% e 0%, respectivamente), e foi possível o isolamento de oito clones contendo genes putativos de P. edulis f. flavicarpa. Estes clones foram marcados e utilizados como sondas em ensaios de FISH. Destas sondas, quatro apresentaram sinal único de hibridização, e foram mapeadas nos cromossomos 1 (gene ERS), 3 (gene ACCO) e 4 (genes G3PD e CYCD1). As demais sondas (genes LOX, NDID e MIPS) apresentaram sinais de hibridização subteloméricos ou pericentroméricos, indicando a presença de DNA repetitivo nos clones; a sonda contendo o gene EMB não revelou sinal fluorescente. Com base em análises de FISH, definiu-se, no presente trabalho, um novo cariótipo para o maracujá-azedo, com marcadores específicos para os cromossomos 1, 3 e 4, localizando, também, sítios de DNAr 45S nos cromossomos 7 e 8, e um sítio de DNAr 5S no cromossomo 5. A exploração da biblioteca de BAC, bem como o mapa físico aqui estabelecido, representa importantes avanços para guiar pesquisas futuras sobre o gênero Passiflora. / Passiflora (Passifloraceae) is a large genus of plant species essentially found in the tropical flora. Some passiflora are grown as ornamentals, cultivated for their edible fruits, or exploited due to their medicinal properties. The main Brazilian commercial species, the yellow passion fruit (Passiflora edulis f. flavicarpa, 2n = 18) occupies 95% of all planted orchards. The fruits are eaten fresh or used for industrial juice production. Genetic and chromosomal studies have been carried out on the species. However, due to the small size and morphological similarity of their chromosomes, the conventional measures of the karyotype have produced some inconsistent results, being imperative the development of chromosome-specific markers. These markers are produced by identifying BAC (Bacterial Artificial Chromosome) library clones that harbor single copy sequences, which are used as probes in FISH (Fluorescent in situ Hybridization) assays. In the present work, a yellow passion fruit genomic BAC library of 82.944 clones was constructed, with average insert sizes of 108 kb, and covering six times the genome equivalent. The library has shown a low level of cpDNA and mtDNA contamination (~0.04% and 0%, respectively), and it was possible the isolation of eight clones harboring putative genes of P. edulis f. flavicarpa. These clones were labeled and used as probes in FISH assays. Of these probes, four have shown single hybridization signals, and they were mapped on chromosome 1 (ERS gene), 3 (ACCO gene), and 4 (G3PD and CYCD1 genes). The other probes (LOX, NDID and MIPS gene) revealed subtelomeric or pericentromeric signals, suggesting the presence of repetitive DNA sequences in the clones; the probe harboring the EMB gene did not reveal any hybridization signal. Based on FISH analyses, a new karyotype for the passion fruit was established in the present work, with specific markers in chromosomes 1, 3 and 4; we also mapped 45S rDNA sites in chromosomes 7 and 8, and one 5S rDNA site in chromosome 5. The exploitation of the BAC library, as well as the physical map here established, represents novel and essential advances to guide future researches on the Passilfora genus.

As doenças cardiovasculares lideram as causas de morte em vários países, inclusive no Brasil, sendo a insuficiência cardíaca (IC) uma das enfermidades mais frequentes. Associações entre ancestralidade genética e susceptibilidade ao desenvolvimento da IC já foram relatadas em diferentes populações. O presente estudo teve como objetivo estimar as ancestralidades global e local de 492 pacientes com IC, identificar regiões e ancestralidades genômicas associadas à IC, seus fatores de risco (diabetes e hipertensão) e à mortalidade causada pela IC, por meio do mapeamento por miscigenação (AM, admixture mapping). Utilizando 182.090 Polimorfismos de Nucleotídeo Único (SNPs, Single Nucleotide Polymorphisms) comuns à nossa população e a três populações ancestrais (europeia, africana e ameríndia) foram realizadas as análises das ancestralidades global e local. Todos os pacientes apresentaram maior proporção de ancestralidade global europeia, média de 0,618±0,218. As análises de AM da IC e da diabetes não mostraram nenhuma região associada, nas três ancestralidades avaliadas. No estudo de AM da hipertensão houve associação estatisticamente significativa com a ancestralidade local africana no cromossomo 2 (chr2p25.3) (p=4,65x10-5). Nessa região estão mapeados 7 RNAs não codificantes, 2 RNAs longos de região intergênica não codificadores de proteína, 44,93% do gene Syntrophin Gamma 2 (SNTG2) e o gene que codifica uma proteína de transmembrana (TMEM18). A análise de AM da mortalidade por IC foi realizada com os mesmos 492 pacientes com IC, usando o modelo caso-controle, sendo o grupo de casos (n=248) composto por pacientes em óbito no final de 4 anos de avaliação, e o grupo de controles (n=244), de indivíduos que ainda estavam vivos no final desse mesmo período. Foi observada associação estatisticamente significativa com a ancestralidade local europeia no cromossomo 6 (chr6p22.3) (p=6,805x10-5). Nessa região estão mapeados 30,74% do gene Ataxina 1 (ATXN1) e o gene Guanosina Monofosfato Redutase (GMPR). O mapeamento fino nessa região, com 7.916 SNPs, apresentou dois marcadores genéticos, rs1042391 e rs2142672, com resultados significativos (p=1,140x10-4, p=3,921×10-4, respectivamente). O alelo em homozigose TT do rs1042391 foi associado a um aumento de 21% ao risco de morte por IC (HR=1.21, p=0,0013), enquanto que o alelo em homozigose CC do rs2142672 foi associado a um aumento de 51% ao risco de morte por IC (HR=1.51, p=0,0004). Estes são achados iniciais e, como tal, devem ser considerados como hipóteses geradoras. Apesar disso, ficou demonstrado a utilidade do estudo de AM para identificar regiões com diferentes ancestralidades genômicas que podem contribuir para o risco de doenças complexas em populações geneticamente miscigenadas. Esses dados podem auxiliar na compreensão da etiologia genética da hipertensão e da mortalidade em pacientes com IC, relacionados à ancestralidade, podendo servir como ponto de partida para estudos funcionais na tentativa de aprofundar os conhecimentos dos processos biológicos que levam à hipertensão e à IC. Estudos futuros são necessários para replicar as associações encontradas, detectar variáveis causais que conduzem essas associações e explorar aplicações clínicas para as regiões de genes consistentemente associadas a mortalidade em pacientes com IC e à hipertensão / Cardiovascular diseases lead the causes of death in several countries, including Brazil, with the heart failure (HF) being one of the most frequent diseases. Associations between genetic ancestry and susceptibility to the development HF have already been reported in different populations. The present study aimed to estimate the global and local ancestry of 492 HF patients, and identify genomic regions and ancestry associated with HF, HF risk factors (diabetes and hypertension) and HF mortality, through admixture mapping (AM). Using 182,090 Single Nucleotide Polymorphisms (SNPs) common to our population and to three ancestral populations (European, African and Amerindian) the analyses of global and local ancestry were performed. All patients showed a higher proportion of European global ancestry, mean of 0.618±0.218. The AM analysis of HF and diabetes did not show any associated regions, in the three ancestries evaluated. In AM of hypertension there was a statistically significant association with African local ancestry on chromosome 2 (chr2p25.3) (p=4,65x10-5). In this region are mapped 7 non-coding RNAs, 2 long intergenic non-protein coding RNAs, 44.93% of the Syntrophin Gamma 2 gene (SNTG2) and the gene encoding a transmembrane protein (TMEM18). The AM analysis of HF mortality was performed with the same 492 HF patients, using case-control model, case group (n=248) was composed of patients who died at the end of 4 years of evaluation, and control group (n=244) included individuals who were still alive at the end of that period. A statistically significant association with European local ancestry on chromosome 6 (chr6p22.3) (p=6.805x10-5) was observed. In this region are mapped 30.74% of the gene Ataxin 1 (ATXN1) and the Guanosine Monophosphate Redutase gene (GMPR). The fine mapping in this region, with 7,916 SNPs, presented two genetic markers, rs1042391 and rs2142672, with significant results (p=1,140x10-4, p=3,921×10-4, respectively). The TT homozygous allele of rs1042391 was associated with 21% increase risk of HF death (HR=1.21, p=0,0013), while the CC homozygous allele of rs2142672 was associated with 51% increase risk of HF death (HR=1.51, p=0.0004). These are initial findings and, as such, should be considered as generating hypotheses. Despite this, it was demonstrated the utility of the AM study to identify regions with different genomic ancestry that may contribute to the risk of complex diseases in genetically mixed populations. These data may contribute to understand the genetic etiology of hypertension and mortality in HF patients, related to ancestry, and may serve as a starting point for functional studies in an attempt to deepen the knowledge of the biological processes that lead to hypertension and HF. Future studies are needed to replicate the associations found, to detect causal variables that lead these associations, and to explore clinical applications for gene regions consistently associated with death in patients with HF and hypertension

Coronaviruses are a diverse group of large, single-stranded RNA virus that cause respiratory and enteric diseases in mammalian and avian species. Phylogenetic analysis shows that SARS-CoV is an unique branch of coronavirus showing no close relationship to other groups of coronaviruses. The genome size of SARS-CoV is about 30 kilobase and the genome, like other coronaviruses, is composed of replicase (rep), spike (S), envelope (E), membrane (M) and nucleocapsid (N) and 8 additional unknown open reading frames (ORFs) (ORF 3a, ORF 3b, ORF 6, ORF 7a, ORF 7b, ORF8a, ORF 8b and ORF 9b). The 3a gene, the largest unknown ORF, encodes a viral protein which is predicted to be a transmembrane protein. In this study, we showed that the 3a protein was expressed in SARS patients' lung and intestinal tissues, and it is localized to the endoplasmic reticulum (ER) in 3a-transfected monkey kidney Vero E6 cells. Results from experiments including chromatin condensation, DNA fragmentation and antibody microarray suggest that the 3a protein may trigger apoptosis through a caspase-8-dependent pathway and possibly a PKR-mediated FADD-caspase-8 pathway. Our data show that over-expression of the SARS-CoV protein can induce apoptosis in vitro. / Severe acute respiratory syndrome (SARS), an atypical form of pneumonia, is first recognized in Guangdong Province, China in November 2002 and later spread to Hong Kong in mid February 2003. It is believed that the etiological agent of SARS is a previously unknown coronavirus - SARS-CoV. Over 8,400 cases and 789 deaths were reported to World Health Organization (WHO) from over 28 countries around the world including Hong Kong. Up to now, there are still no efficient antiviral drugs to treat the disease, and the detailed pathology of SARS-CoV infection and the host response to the viral infection are still unknown. During the epidemic, we have done complete genome sequencing for five SARS-CoV isolates and we postulate that at least two SARS-CoV strains with distinct etiological origins exist in the environment during the epidemic. / Law Tit-wan Patrick. / "Aug 2005." / Adviser: Stephen K. W. Tsui. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 156-172). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

by Peter, Wei Gong Chang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 103-124). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Human genome project --- p.5 / Chapter 1.3 --- Human adult heart cDNA library --- p.7 / Chapter 1.4 --- Human fetal heart cDNA library --- p.8 / Chapter 1.5 --- Sequencing of a human heart cDNA clone --- p.9 / Chapter 1.6 --- Knowledge of the role of selenium --- p.13 / Chapter 1.7 --- Mouse 56kDa selenium binding protein and acetaminophen-binding protein --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Plating out the cDNA library --- p.20 / Chapter 2.1.1 --- "Mediums, buffers and solutions" --- p.20 / Chapter 2.1.2 --- Bacteriophage clones preparation --- p.21 / Chapter 2.2 --- cDNA clone amplification by PCR --- p.23 / Chapter 2.3 --- Cycle sequencing of PCR products --- p.25 / Chapter 2.3.1 --- "Media, buffers and solutions" --- p.25 / Chapter 2.3.2 --- Preparation of sequencing reaction --- p.25 / Chapter 2.4 --- Gel electrophoresis using an automated A.L.F sequencer --- p.27 / Chapter 2.5 --- DNA sequence analysis --- p.29 / Chapter 2.6 --- Preparation of competent E. coli for transformation --- p.30 / Chapter 2.7 --- Transformation of plasmid into competent E. coll --- p.31 / Chapter 2.8 --- Mini-preparation of plasmid DNA --- p.32 / Chapter 2.9 --- Large scale plasmid DNA preparation by QIAGEN --- p.34 / Chapter 2.10 --- Cloning the human 56 kDa selenium binding protein (hSP56) into the pAED4 vector --- p.36 / Chapter 2.10.1 --- Bacterial strains and vector --- p.36 / Chapter 2.10.2 --- "Media, buffers and solutions" --- p.38 / Chapter 2.10.3 --- Primers design and PCR --- p.42 / Chapter 2.10.4 --- Purification of PCR products by Geneclean --- p.43 / Chapter 2.10.5 --- Restriction digestion of purified PCR product and pAED4 --- p.44 / Chapter 2.10.6 --- Ligation and transformation of hSP56 --- p.45 / Chapter 2.10.7 --- Screening and purification ofpAED4hSP56. --- p.47 / Chapter 2.11 --- Expression of hsp56 --- p.50 / Chapter 2.11.1 --- Induction of hSP56 expression --- p.50 / Chapter 2.11.2 --- SDS-PAGE and protein detection --- p.51 / Chapter 2.12 --- Northern hybriddization of hSP56 --- p.53 / Chapter 2.12.1 --- Animals & human tissue --- p.53 / Chapter 2.12.2 --- "Mediums, buffers and solutions" --- p.53 / Chapter 2.12.3 --- Preparation of total RNA --- p.56 / Chapter 2.12.4 --- Formaldehyde agarose gel electrophoresis --- p.57 / Chapter 2.12.5 --- Preparation of radioactive probe --- p.58 / Chapter 2.12.6 --- RNA transfer and Northern hybridization --- p.59 / Chapter 2.13 --- Chromosomal mapping of the hSP56 gene --- p.62 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- The sequencing results of 553 cDNA clones --- p.63 / Chapter 3.2 --- Catalogues of genes expressed --- p.65 / Chapter 3.3 --- Sequence analysis of hSP56 --- p.71 / Chapter 3.4 --- Northern hybridization of hSP56 --- p.84 / Chapter 3.5 --- Cloning of hSP56 into pAED4 --- p.87 / Chapter 3.6 --- Expression of the hSP56 in E. coli --- p.89 / Chapter 3.7 --- Chromosomal mapping of the hSP56 gene --- p.92 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- General discussion --- p.94 / Chapter 4.2 --- The possible roles of hSP56 and mSP56 --- p.101 / Chapter 4.3 --- Future prospects --- p.102 / REFERENCES --- p.103 / APPENDIX 1 --- p.125 / APPENDIX.2 --- p.127

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum com uma prevalência aproximada de 3% em pacientes com mais de 64 anos. A doença é esporádica, mas o parkinsonismo primário (PP) familiar, decorrente de defeitos genéticos específicos, tem sido encontrado em cerca de 10% dos casos diagnosticados como DP. Os objetivos deste trabalho são analisar o DNA de pacientes com PP acompanhados no ambulatório do Grupo de Estudo de Distúrbios do Movimento da Clínica Neurológica do Hospital das Clínicas da FMUSP que apresentam início precoce (< 40 anos) ou história familiar positiva com o intuito de rastrear mutações responsáveis pela doença e descrever as características clínicas desse grupo de pacientes e dos familiares acometidos. Entre Janeiro de 2004 a Janeiro de 2006 foram selecionados 53 probandos com PP, sendo que 29 eram esporádicos, 16 com história familiar sugestiva de herança autossômica dominante (AD) e 8 com história familiar sugestiva de herança de autossômica recessiva (AR). No total, 100 amostras de DNA foram coletadas, 70 de pacientes ou familiares com PP, 1 com parkinsonismo secundário ao uso de neuroléptico e o restante de familiares sem PP. Dos casos afetados, 45 eram do sexo masculino e 25 feminino, a idade média de início dos sintomas foi de 38,3 anos (10-72) e a média de idade no momento da investigação foi de 49,8 anos (22-72). Todos apresentaram instalação assimétrica do quadro, curso lento e progressivo e boa resposta ao tratamento com levodopa ou agonista dopaminérgico. Pacientes com padrão de herança AD foram testados para a mutação Gli2019Ser que é o defeito mais comum do gene LRRK2 (PARK8) sendo encontradas duas famílias afetadas. A análise mutacional dos genes PARK6 e PARK7 está em andamento. Todos os casos esporádicos e com padrão de transmissão AR foram testados para mutações do gene PARK2 e foram encontradas as seguintes mutações homozigóticas em 4 famílias: 255delA, deleção de exon 3-4, deleção do exon 2-3 e uma nova mutação IVS1+1G/T. Num paciente com parkinsonismo juvenil (idade de início dos sintomas <21 anos) foi encontrada uma nova mutação homozigótica no gene ATP13A2 (PARK9) no exon 15 que determina a substituição Gli504Arg na proteína codificada. Em grande parte dos casos estudados os achados genéticos e clínicos são similares aos descritos na literatura. Entretanto, encontramos novas mutações do gene PARK2 e PARK9 e no paciente com a mutação ATP13A2 os achados clínicos diferem em alguns aspectos da descrição clássica. / Parkinson disease (PD) is the second most common neurodegenerative disorder affecting approximately 3% of the population over age 64. Most cases of PD manifest in sporadic form, but familial primary parkinsonism (PP) due to specific genetical abnormalities has been found in about 10% of cases diagnosed as PD. The aims of this study were to analyze the DNA of PP patients seen at the Group for the Study of Movement Disorders of the Neurology Department of Hospital das Clinicas of the University of São Paulo who presented early onset of the disease (< 40 years of age) or positive family history, with the purpose of screening possible candidate mutations for the disease, and to describe the clinical features of this group of patients and affected members of their families. Between January 2004 and January 2006, 53 probands were selected of whom, 29 were sporadic cases, 16 had probable autosomical dominant (AD) pattern of inheritance, and 8 autosomical recessive (AR). In total 100 samples of DNA were collected, 70 from PP patients or affected relatives, one case with neuroleptic-induced parkinsonism, and the rest from not affected members. Forty five affected individuals were men and 25 women, the median age of the symptoms onset was 38.3 years (10-72), and the median age at the moment of the examination was 49.8 years (22-72). All patients had asymmetric installation of the disease, slow progression of the PP, and good response to levodopa or dopaminergic agonist therapy. Patients with AD inheritance were screened for Gly2019Ser mutation, which is the most common defect in PD due to LRRK2 gene, and two families carried this mutation. The screening of PARK6 and PARK7 is ongoing. All sporadic and AR inheritance cases were tested for mutation of (PARK2) and the following mutation were found in 4 families in homozygous state: 255delA, exon 3-4 deletion, exon 2-3 deletion, and a novel mutation IVS1+1G/T. In a juvenile parkinsonism proband (age of onset < 21 years) a novel missense homozygous mutation in ATP13A2 (PARK9) gene was found in exon 15 which resulted the Gly504Arg change in the encoded protein. In general the genetical and clinical findings of this series of patients are similar to those reported in the literature, although novel mutation in PARK2 and PARK9 were obtained. Some clinical features of the patient with ATP13A2 mutation differed from the classical descriptions.

Passiflora (Passifloraceae) é um grande gênero de espécies vegetais encontradas, principalmente, na flora tropical. Algumas passifloras são cultivadas como plantas ornamentais, frutíferas ou exploradas pelas suas propriedades medicinais. A principal espécie comercial brasileira, o maracujá-azedo (Passiflora edulis f. flavicarpa, 2n = 18), ocupa 95% da área plantada. Os frutos são consumidos in natura ou processados pela indústria de suco. Estudos genéticos e cromossômicos têm sido gerados para esta espécie. Entretanto, devido ao pequeno tamanho e a similaridade morfológica dos seus cromossomos, as medições do cariótipo convencional do maracujá-azedo têm levado a resultados inconsistentes, sendo necessário o desenvolvimento de marcadores cromossomos-específicos. Estes marcadores são produzidos a partir da identificação de sequências de cópia-única em clones de bibliotecas de BACs (Bacterial Artificial Chromosome), que são utilizadas como sondas em ensaios de FISH (Hibridização in situ Fluorescente). Neste trabalho, foi construída uma biblioteca genômica de maracujá-azedo em BACs contendo 82.944 clones, com tamanho médio dos insertos de 108 kb, e provendo uma cobertura de seis vezes o genoma. A biblioteca apresentou baixa contaminação com cpDNA e mtDNA (~0,04% e 0%, respectivamente), e foi possível o isolamento de oito clones contendo genes putativos de P. edulis f. flavicarpa. Estes clones foram marcados e utilizados como sondas em ensaios de FISH. Destas sondas, quatro apresentaram sinal único de hibridização, e foram mapeadas nos cromossomos 1 (gene ERS), 3 (gene ACCO) e 4 (genes G3PD e CYCD1). As demais sondas (genes LOX, NDID e MIPS) apresentaram sinais de hibridização subteloméricos ou pericentroméricos, indicando a presença de DNA repetitivo nos clones; a sonda contendo o gene EMB não revelou sinal fluorescente. Com base em análises de FISH, definiu-se, no presente trabalho, um novo cariótipo para o maracujá-azedo, com marcadores específicos para os cromossomos 1, 3 e 4, localizando, também, sítios de DNAr 45S nos cromossomos 7 e 8, e um sítio de DNAr 5S no cromossomo 5. A exploração da biblioteca de BAC, bem como o mapa físico aqui estabelecido, representa importantes avanços para guiar pesquisas futuras sobre o gênero Passiflora. / Passiflora (Passifloraceae) is a large genus of plant species essentially found in the tropical flora. Some passiflora are grown as ornamentals, cultivated for their edible fruits, or exploited due to their medicinal properties. The main Brazilian commercial species, the yellow passion fruit (Passiflora edulis f. flavicarpa, 2n = 18) occupies 95% of all planted orchards. The fruits are eaten fresh or used for industrial juice production. Genetic and chromosomal studies have been carried out on the species. However, due to the small size and morphological similarity of their chromosomes, the conventional measures of the karyotype have produced some inconsistent results, being imperative the development of chromosome-specific markers. These markers are produced by identifying BAC (Bacterial Artificial Chromosome) library clones that harbor single copy sequences, which are used as probes in FISH (Fluorescent in situ Hybridization) assays. In the present work, a yellow passion fruit genomic BAC library of 82.944 clones was constructed, with average insert sizes of 108 kb, and covering six times the genome equivalent. The library has shown a low level of cpDNA and mtDNA contamination (~0.04% and 0%, respectively), and it was possible the isolation of eight clones harboring putative genes of P. edulis f. flavicarpa. These clones were labeled and used as probes in FISH assays. Of these probes, four have shown single hybridization signals, and they were mapped on chromosome 1 (ERS gene), 3 (ACCO gene), and 4 (G3PD and CYCD1 genes). The other probes (LOX, NDID and MIPS gene) revealed subtelomeric or pericentromeric signals, suggesting the presence of repetitive DNA sequences in the clones; the probe harboring the EMB gene did not reveal any hybridization signal. Based on FISH analyses, a new karyotype for the passion fruit was established in the present work, with specific markers in chromosomes 1, 3 and 4; we also mapped 45S rDNA sites in chromosomes 7 and 8, and one 5S rDNA site in chromosome 5. The exploitation of the BAC library, as well as the physical map here established, represents novel and essential advances to guide future researches on the Passilfora genus.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.