The human pathogenic Cryptococcus neoformans species complex are agents of a common AIDS-defining disease, which causes about 181,000 deaths each year. There are several specific features distinguishing this species from other fungi, including the presence of a polysaccharide capsule and melanin pigment production, both of which contribute to its virulence. A large number of studies about this pathogen used two model strains JEC20 and JEC21. In these studies, these two strains are assumed to be “isogenic”, differ only at the mating type region. Consequently, their phenotypic differences, including virulence, have been attributed to this region. Here, we applied second-generation sequencing and bioinformatics tools to identify sequence polymorphisms between the two genomes. Beside the Mating Type locus, two other regions were found to contain high frequencies of SNPs. To further understand the effects of these loci on the phenotypic differences, four phenotyping assays (mating ability, melanin pigment production, capsule formation, and high temperature growth ability) were conducted on the recombinant progeny obtained from the cross between JEC20 and JEC21. In addition, genomic sequences of these progeny were obtained to identify the complete distributions of other SNPs among the strains. Finally, we identified several novel SNPs contributing to virulence-related traits in this species, which suggest that caution should be placed in attributing phenotypic differences to specific genomic regions in “isogenic” strains derived from classical breeding experiments. / Thesis / Master of Science (MSc) / Cryptococcosis is a globally distributed infection that is prevalent among immune-compromised individuals, such as HIV/AIDS patients. This disease can be attributed to a group of opportunistic fungal pathogens – Cryptococcus neoformans species complex. During the past century, significant resources have been put in an effort to understand its ecology, evolution, life cycle, pathogenesis and virulence factors, and molecular and cellular processes. Most of the laboratory-based studies have relied on two model strains assumed to differ only at the mating type locus. My thesis investigated this assumption and found there are several additional significant genetic differences between these two strains and that such differences contribute to the observed phenotypic differences between them. My results highlight the complexity of genotype-phenotype relationships and the continued evolution of strains even in lab environments in C. neoformans.

Thesis / Master of Science (MS)

Present study was planned to compare the phenotypic variation of performances traits among commercial (CC) and heritage varieties of turkeys. Information about heritage turkey varieties continues to be limited. In addition, the emerging turkey genome sequence provides a unique opportunity to understand the DNA sequence variation and its associations with performance traits. The turClock, turPer3, turCry1 and turCry2 genes were screened and evaluated for its association with their performance traits. As expected, CC turkeys were superior to heritage birds in performance for most of the traits evaluated. However, heritage turkeys showed better reproductive performances compared to CC turkeys. A total of 41 SNPs were identified from the genes that screened. The haplogroups in the turClock gene were significantly associated with body weight (BW) at 309 d of age, feed conversion ratio (FCR) for 34 - 68 d and 69 - 159 d, egg production and average egg weight (P " 0.05). The haplogroups developed from turPeriod-3 gene were significantly associated with BW at 231 d of age, average daily gain (ADG) for 160 - 231 d, FCR for 69 - 159 d and 160 - 231 d, egg production traits, semen quality traits and plasma melatonin concentration (P " 0.05). In the turCry1 gene, haplogroups were significantly associated with ADG for 35 - 68 d, FCR for 160 - 231 d and 34 - 231 d, egg production and ejaculate volume (P " 0.05). The haplogroups identified from turCry2 gene were significantly associated with BW at 34, 68 and 231 d of age, ADG for 160 - 231 d, FCR for 34 - 68 d, average egg weight (P " 0.05). These findings reveal that phenotypic variation observed in growth and reproductive parameters among turkeys could be used for selecting birds for future breeding programs. DNA sequence variations at the nucleotide and haplotype levels are associated with some of growth, reproductive parameters and plasma melatonin of turkeys. Thus DNA sequence variations that identified of the circadian genes may have some regulatory role in the molecular mechanism of the circadian clock which may affect the circadian rhythm of the animal. / Ph. D.

Yes / MicroRNAs are short, non-coding RNAs that target messenger RNAs for degradation. miR-145 is a vascular-enriched microRNA that is important for smooth muscle cell (SMC) differentiation. Under healthy circumstances, SMC exist in a contractile, differentiated phenotype promoted by miR-145. In cases of disease or injury, SMC can undergo reversible dedifferentiation into a synthetic phenotype, accompanied by inhibition of miR-145 expression. Vascular disorders such as atherosclerosis and neointimal hyperplasia are characterised by aberrant phenotypic switching in SMC. This review will summarise the physiological roles of miR-145 in vascular SMC, including the molecular regulation of differentiation, proliferation and migration. Furthermore, it will discuss the different ways in which miR-145 can be dysregulated and the downstream impact this has on the progression of vascular pathologies. Finally, it will discuss whether miR-145 may be suitable for use as a biomarker of vascular disease.

Natal dispersal outcomes are an interplay between environmental conditions and individual phenotypes. Peripheral, isolated populations may experience altered environmental conditions and natal dispersal patterns that differ from populations in contiguous landscapes. We document nonphilopatric, sex-biased natal dispersal in an endangered small mammal, the Mt. Graham red squirrel (Tamiasciurus hudsonicus grahamensis), restricted to a single mountain. Other North American red squirrel populations are shown to have sex-unbiased, philopatric natal dispersal. We ask what environmental and intrinsic factors may be driving this atypical natal dispersal pattern. We test for the influence of proximate factors and ultimate drivers of natal dispersal: habitat fragmentation, local population density, individual behavior traits, inbreeding avoidance, competition for mates, and competition for resources, allowing us to better understand altered natal dispersal patterns at the periphery of a species' range. A juvenile squirrel's body condition and its mother's mass in spring (a reflection of her intrinsic quality and territory quality) contribute to individual behavioral tendencies for movement and exploration. Resources, behavior, and body condition have the strongest influence on natal dispersal distance, but affect males and females differently. Male natal dispersal distance is positively influenced by its mother's spring body mass and individual tendency for movement; female natal dispersal distance is negatively influenced by its mother's spring body mass and positively influenced by individual tendency for movement. An apparent feedback between environmental variables and subsequent juvenile behavioral state contributes to an altered natal dispersal pattern in a peripheral population, highlighting the importance of studying ecological processes at the both range center and periphery of species' distributions.

Le maintien de l'intégrité de l'épithélium des voies aériennes est assuré par un mécanisme de réparation épithéliale pouvant être dérégulé dans la bronchopneumopathie chronique obstructive (BPCO). La BPCO, définie par une obstruction irréversible des débits aériens et émaillée d'épisodes d'exacerbation, présente différents phénotypes, pour lesquels l'évaluation de la sévérité tient compte de la fonction respiratoire et de données cliniques. L'objectif de cette thèse était d'étudier du point de vue phénotypique des patients BPCO lors des exacerbations virales et au cours du processus de réparation épithéliale. Nous avons étudié prospectivement 51 patients BPCO suivis mensuellement et lors des exacerbations. Virus et bactéries étaient recherchés dans l'expectoration induite par PCR et culture. Les résultats analysés en fonction des données cliniques et fonctionnelles respiratoires ont montré qu'une coinfection, présente dans 25% des exacerbations, n'était pas associée au phénotype des patients ni à la sévérité ou à la récidive de l'exacerbation. Nous avons ensuite étudié la réparation de cellules épithéliales bronchiques primaires dans un modèle de réparation de lésion. L'analyse des paramètres de fermeture de lésion (vitesse de fermeture, sécrétions, prolifération cellulaire) en fonction des données cliniques, fonctionnelles et histologiques a montré notamment que la vitesse de fermeture était associée à la sévérité de l'obstruction bronchique. Ces résultats suggèrent le rôle d'anomalies de réparation épithéliale dans la physiopathologie de la BPCO et soulignent la complexité du phénotypage des patients atteints de BPCO. / The integrity of airway epithelium is provided by a complex mechanism of epithelial repair that can be dysegulated in chronic obstructive pulmonary disease (COPD). COPD is a heterogeneous condition defined by an irreversible obstructive airflow limitation with frequent acute episodes of exacerbations. COPD patients can present different phenotypes, for which severity evaluation must take into account the severity of lung function and clinical data. The aim of this thesis was to study the COPD patients in a phenotypic point of view during viral exacerbations and during the process of epithelial repair.We prospectively studied 51 COPD patients monitored monthly and during exacerbations. Induce sputum were analyzed for viruses and bacteria detection by PCR and culture. These results analyzed according to the clinical and functional respiratory data showed that co-infection was present in 25 % of exacerbations and was not associated with the phenotype of patients or the severity or recurrence of exacerbation.We then studied primary bronchial epithelial cells repair in a model of wound closure. Associations between wound closure parameters (speed of wound closure, secretions, cell proliferation) and clinical, histological and functional data were analyzed. We showed that the speed of wound closure was associated with the severity of bronchial obstruction. These results suggest the role of abnormal epithelial repair in the pathogenesis of COPD and highlight the complexity of phenotypic characterization of COPD patients.

Aim. The human organic cation transporter subtype 2 (OCT2) is highly expressed in the renal tubular epithelium and can play an important role in the renal clearance of many drugs and drug-drug interactions occurring in the kidney. The purposes of this study are: (1) to investigate the genetic polymorphisms of OCT2 in Chinese population; (2) to evaluate the potential genotype-phenotype relationship involving OCT2 polymorphism in human subjects; and (3) to identify the SNPs in proximal promoter/enhancer region and assess their functional significance. / Conclusion. The present study demonstrated the existence of genetic polymorphisms of OCT2 gene in the Chinese population and for the first time showed that a ncSNP 808G>T is associated with a reduced renal transport function and can significantly impact the magnitude of drug interactions. Our study also for the first time found that a promoter polymorphism (-1283 T>C) is associated with an altered promoter activity in vitro, but no such relationship was observed with this SNP in the in vivo metformin study. Thus, it was the ncSNP 808G>T but not the -1283T>C in promoter that was associated with variations in the metformin renal clearance. (Abstract shortened by UMI.) / Method. One hundred and twelve Hong Kong Chinese subjects were recruited and their DNA samples were obtained. / Results. A total of 13 SNPs were identified in the coding and surrounding non-coding regions of OCT2, with minor allele frequencies (MAF) ranged from 4.5% to 24.7%. From these SNP data sets, 28 haplotypes were inferred with 4 being the common ones (frequencies ranged from 5.4% to 50.4%). Only one non-synonymous coding region SNP (ncSNP), 808G>T in the exon 4, was observed among all the identified SNPs. Significant differences were observed in the renal clearance of metformin in subjects with different mutation status for this variant. The mean renal tubular clearance (CLt) values of metformin were 8.54 +/- 1.86, 7.72 +/- 0.64, and 6 36 +/- 0.98 ml/kg/min for subjects with GG (n = 6), GT (n = 5) and TT (n = 4) genotypes respectively (P = .043. 1-way ANOVA). After a 6-day cimetidine treatment, a mean decrease of 50.7%, 34.6% and 18.9% in metformin CLt was observed in the GG, GT and TT genotype groups (P =.013, P =.002 and P = .065 respectively compared to metformin alone). The decrease of CLt was significantly lower in the TT genotype group than that in the GG group (P = .027). Five SNPs were identified and 5 haplotypes inferred (frequencies ranged from 2.7% to 38.4%) in promoter/enhancer area. One haplotype, characterized by the presence of -1283 T>C, was associated with a significantly lower luciferase activity in vitro (26.7% decrease in comparison to wild-type, P = .016), but not with metformin CLt in Chinese Subjects. / Wang, Zhijun. / "Aug 2007." / Adviser: Moses Chow. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0966. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 148-168). / 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. / Abstracts in English and Chinese. / School code: 1307.

The distribution of epidermal hairs (trichomes) is an important taxonomic character in the genus Antirrhinum. Most species in subsection Antirrhinum produce trichomes from lower internodes and leaves, then have bald stems and leaf blades after the third node and resume trichomes production again in the inflorescence (the "bald" phenotype). All species in subsection Kickxiella produce trichomes throughout development (the "hairy" phenotype). Populations of some species are polymorphic for trichome distribution-both bald and hairy individuals were observed in A. australe, A. graniticum, A. latifolium and A. meonanthum. Antirrhinum species also varied in trichome morphology. Five types were recognized according to length and the presence or absence of a secretory gland. Some types were present in all species and had similar distributions-for example short glandular trichomes were found on the adaxial midribs of all leaves in all species, and the lower leaves and internodes of all species shared longer glandular and long eglandular trichomes. However, the trichomes on leaf blades and stems at higher vegetative nodes of hairy species and in the inflorescences differed in morphology between species, suggesting that they are regulated differently from trichomes at more basal positions. Other species in the tribe Antirrhineae showed similar variation in trichome morphology and distribution to Antirrhinum, suggesting that the control of trichome development might be conserved within the tribe. To understand the genetic basis for variation in trichome distribution, a near-isogenic line (NIL) was generated by introducing regions of the genome of A. charidemi (hairy, subsection Kickxiella) into the genetic background of A. majus subsp. majus (bald, subsection Antirrhinum). One NIL segregated bald and hairy progeny, with the same trichome distributions as the parent species, in a ratio that suggested a single locus is responsible for the differences and baldness is dominant. The locus was named as Hairy and assumed to act as a suppressor of trichome formation. Progeny of the NIL were used in genome resequencing of bulked phenotype pools (Pool-seq) to map Hairy. No recombination between Hairy and a candidate gene (GRX1) from the Glutaredoxin gene family, was detected in the mapping population. In addition, RNA-seq revealed that GRX1 was expressed in bald parts of bald progeny, but not in the same parts of hairy progeny, and in situ hybridisation showed GRX1 RNA was restricted to epidermal cells, which form trichomes in the absence of Hairy activity. A virus-induced gene silencing (VIGS) method was also developed to test GRX1 function further. Reducing GRX1 activity allowed ectopic trichome formation in the bald NIL. Together, this evidence strongly supported Hairy being GRX1. To investigate evolution of Hairy and its relationship to variation in trichome distribution, the NIL was crossed to other Antirrhinum species. These allelism tests suggested that Hairy underlies variation in trichome distribution throughout the genus, with the exception of A. siculum, which has a bald phenotype but might lack activity of hairy and a gene needed for trichome formation. Hairy sequences were obtained from representative of 24 Antirrhinum species and two related species in the tribe Antirrhineae. The conserved trichome-suppressing function of the sequence from one of these species (Misopates orontium, bald phenotype) was confirmed by VIGS. Gene phylogenies combined with RNA expression analysis suggested that the ancestral Antirrhinum had a bald phenotype, that a single mutation could have given rise to the hairy alleles in the majority of Kickxiella species, that these alleles were also present in polymorphic populations in the other subsections, consistent with transfer from Kickxiella by hybridisation, and that multiple, independent mutations had been involved in parallel evolution of the hairy phenotype in a minority of Kickxiella species. Phylogenetic analysis of GRX proteins suggested that Hairy gained its trichome-repressing function relatively late in the evolutionary history of eudicots, after the Antirrhineae-Phrymoideae split, but before divergence of the lineages leading to Antirrhinum and Misopates. A yeast two-hybrid screen identified members of the TGA and HD-Zip IV transcription factors as potential substrates of the Hairy GRX.

Les paraplégies spastiques héréditaires (PSH) sont des maladies cliniquement et génétiquement hétérogènes, qui se manifestent par la présence de signes pyramidaux (spasticité, réflexes myotatiques vifs et diffusés) et d’un déficit moteur des membres inférieurs. On distingue des formes pures et complexes de PSH, ces dernières étant associées à la présence de signes additionnels tels que troubles cognitifs, neuropathie périphérique, signes cérébelleux, etc. Les mécanismes physiopathologiques des PSH sont également hétérogènes, incluant une anomalie du transport axonal (SPG3A, SPG4, SPG10, SPG20), du métabolisme mitochondrial (SPG7, SPG13), une anomalie de la formation de la myéline (SPG1) ou un dysfonctionnement du développement neuronal (SPG2). Elles peuvent se transmettre selon le mode autosomique dominant (AD), récessif (AR), ou récessif lié au chromosome X. Actuellement, 13 loci dont 9 gènes de PS-AD sont connus, mais seulement 5 gènes responsables de PS-AR ont été identifiés, alors que 14 loci sont connus. De par leur hétérogénéité clinique, génétique et physiopathologique, les PSH sont encore des maladies mal connues. Une meilleure connaissance du phénotype associé à chaque locus/gène permettrait aux cliniciens de mieux orienter les analyses moléculaires pour un diagnostic rapide. L’établissement de corrélations génotype-phénotypes et de la fréquence des gènes impliqués dans les PSH permettrait tant aux cliniciens qu’aux biologistes de cibler les gènes, les exons à analyser ou les mutations à rechercher en priorité. L’identification des mécanismes physiopathologiques des mutations est une première étape vers des études fonctionnelles et des traitements spécifiques. Nous avons montré que la forme de PS AD liée à des mutations dans le gène SPG3A était caractérisée par un début très précoce, avant l’âge de 10 114 ans. Cette forme en générale pure de PS peut se compliquer, notamment par une neuropathie périphérique ou un syndrome cérébelleux après une longue durée d’évolution de la maladie. Ces résultats permettent d’orienter les analyses moléculaires vers le gène SPG3A avant le gène SPG4, devant tout patient qui a débuté la maladie précocement, quelque soit le tableau clinique. Nous avons montré que les mutations dans le gène SPG3A, dont les mutations récurrentes p.R239C et p.R495W dans les exons 7 et 12 peuvent apparaître de-novo, justifiant l’analyse de ce gène chez des patients isolés. Nous avons étendu le phénotype des PS AD liées à des mutations dans le gène SPG4, qui doit être analysé chez les patients présentant une PS associée à un retard mental sans malformation cérébrale. De plus, nous avons montré que les délétions de ce gène ne sont pas rares, atteignant une fréquence de 20% chez les patients présentant une PS-AD sans mutation retrouvée par DHPLC. Ceci entraîne un changement des stratégies d’analyses moléculaires utilisées chez les patients atteints de PS, avec l’instauration systématique d’un MLPA chez chaque patient. Nous avons précisé le tableau clinique des paraplégies spastiques AR liées aux loci SPG26 et 27. Nous avons réduit l’intervalle génomique de ces loci. L’identification d’autres familles liées à ces loci permettra de réduire encore plus leurs intervalles génomiques, voire d’identifier les gènes responsables de ces maladies.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer de esôfago é uma malignidade altamente freqüente e letal. Uma característica específica das áreas de alta incidência de câncer de esôfago é a grande proporção de duplas mutações no gene TP53, sendo, ao menos uma delas, uma transição G para A em sítios CpG. Essas transições resultam de malpareamentos GT causados pela desaminação espontânea da 5-metilcitosina em ilhotas CpG. A enzima de reparo de DNA Timina-DNA Glicosilase (TDG) é responsável pelo primeiro passo na remoção da timina de malpareamentos GT em CpG. A alta proporção de mutações em sítios CpG em câncer de esôfago das áreas de alta incidência sugere que a via de reparo de DNA iniciada pela TDG pode estar prejudicada. A presença de duplas mutações, sendo ao menos uma delas em CpG, levantou a hipótese de que a primeira mutação no TP53 reduz a atividade da via de reparo iniciada pela TDG, que acarretaria a segunda mutação em sítios CpG. Dessa forma, o objetivo desse trabalho foi analisar o efeito da p53 sobre a expressão e atividade da TDG. Os resultados obtidos mostram que a expressão de TDG é regulada transcricionalmente pela p53 numa gama de linhagens celulares e é induzida pelo dano ao DNA, de forma p53-dependente. Além disto, os resultados apontam um possível papel da proteína p53 ativa na migração nuclear e atividade da TDG. Estes resultados ainda nos levam à conclusão de que o silenciamento de TDG aumenta a sensibilidade à morte celular induzida por MMS quando a p53 é encontrada na forma selvagem, mas não quando esta proteína é mutada, e de que o status mutacional de TP53 parece afetar a expressão de TDG em CEE primários. Juntos esses resultados sugerem que a p53 regula o reparo de DNA mediado pela TDG e que a inativação de p53 em células tumorais pode contribuir para a aquisição de um mutator phenotype. / Esophageal squamous cell carcinoma (ESCC) is a highly frequent and fatal malignancy in the world. A peculiar characteristic of the high incidence areas of esophageal cancer is the large proportion of double mutations in TP53 gene, being, at least one of them, a G to A transition at CpG sites. These transitions result from GT mismatches caused by the spontaneous deamination of 5-methylcytosine at CpG sites. The DNA repair enzyme Thymine-DNA Glycosylase (TDG) is responsible for the first step in the removal of the thymidine from the GT mismatches at CpG sites. The high proportion of mutations at CpG sites in esophageal tumors in the high incidence areas suggests that the DNA repair pathway initiated by TDG might be impaired. The large number of double mutations, with one being at a CpG site, raised the possibility that the first mutation in TP53 reduces the activity of the TDG base excision repair pathway, increasing the chance of a second mutation event at a CpG site. In this way, the aim of this work was to analyze the effect of p53 on the expression and activity of TDG. The results achieved show that TDG expression is regulated by p53 in a variety of cells lines at the trancriptional level and induced by DNAdamage in a p53-dependent manner. Furthermore, these results point out a possible role of active p53 in the nuclear migration and activity of TDG. The results further support the notion that TDG silencing increases the sensitivity to cell death induced by Methylmethane sulphonate when p53 is found in a wild-type, but not in a mutant form, and that TP53 mutation seems to affect TDG expression in primary ESCC. Together, these results suggest that p53 regulates TDG-mediated repair and that p53 inactivation in cancer cells may contribute to a mutator phenotype through loss of TDG function.