When provided with spatial niches by growth in static nutrient medium, Pseudomonas fluorescens diversifies through adaptive radiation into several well-defined phenotype classes. One of these classes, named wrinkly spreader (WS) for its morphology on agar medium, forms a biofilm at the air-liquid interface through mutations in one of several loci including the genes wspF and awsX. These genes code for negative regulators of di-guanylate cyclases (DGCs). These DGCs catalyse synthesis of cyclic-di-GMP, a second messenger, overproduction of which effects physiological changes leading to overproduction of a cellulose polymer and the WS phenotype. Intriguingly, a diverse range of wspF mutations leads to diversity both in colony morphology and strain fitness.In this study, I investigate genetic and fitness diversity in wrinkly spreaders with the aim of identifying the causal factors that link genetic diversity and physiological factors with diversity in fitness. I approach the subject from several directions, examining the historical context of genetic diversity in wspF and awsX, distribution of control over output in the Wsp pathway and overall fitness effects of different causal factors. I investigate the genetic basis of wrinkly spreader evolution through generation of a large number of novel WS strains and exploration of the distribution of mutations in the wspF and awsX genes. In combination with this I calculate estimates of the past rates of mutation in these genes, derived from a phylogenetic investigation of a group of orthologues. I examine the response of the Wsp pathway to change in WspF function through a novel computational analysis that is capable of revealing valuable information on control in a biological system based purely on model structure. In addition I show how this analysis can be refined through specification of broad estimates of system parameters, thereby avoiding issues related to over-reliance on specific parameter values. Finally, I investigate the fitness implications of these factors, as well as a variety of others, through assays of fitness in a group of WS strains combined with machine learning analyses of predictive relationships between protein and mutation characteristics and experimentally measured strain fitness, and consider the implications of this analysis in the context of intermediate physiological effects.I find that mutations in the WspF protein that lead to the WS phenotype tend to be located in regions of historically strong conservation, the first time that any such pattern to WS mutations has been identified. Mutations in AwsX, on the other hand, do not fit such a pattern. Computational analysis of the Wsp pathway shows that, regardless of model parameters, pathway output is always more sensitive to changes in methylesterase activity by WspF than to changes in phosphorylation of WspF, which may explain the greater frequency of mutations fixed in vivo seen in the methylesterase domain. Despite these patterns, none of a wide range of mutation and sequence-based biochemical characteristics, including local rates of past evolution and size and position of mutations, exhibited any predictive power over WS fitness. Overall, the findings in this study point towards an essential role for complex pleiotropic effects in strongly modulating the fitness effect of different mutations in wspF.

The diptericidal $\textit{Bacillus thuringiensis}$ (Bt) ssp. $\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\textit{am1}$ and $\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\delta$-endotoxins (Cry19A from Bt ssp. $\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\textit{240A}$ from Bti and an insertion sequence associated with the $\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\alpha$/$\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\alpha$/$\beta$-type SASP from $\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\textit{Bacilli}$.

Philosophiae Doctor - PhD / Apple scab, powdery mildew and woolly apple aphid are a major concern for apple breeders and producers. Control of these diseases is a significant economic and marketing priority for the South African apple industry. Application of chemicals and orchard management practices are the main methods for controlling these diseases. These diseases require an average of 15 chemical sprays per season, which leads to increased production costs for the farmer. The increased cost of chemical based control programs and demand from consumers for ‘organic apples’ grown with very little to no chemical sprays makes it important to breed for commercial apple cultivars with endogenous disease resistance genes (R-genes). The use of genetic tools (apple genetic linkage maps and the apple genome sequence) to track and introgress endogenous R-genes in breeding and to confer durable disease resistance in commercial apple cultivars will lead to a more cost effective means of disease control for apple producers. Historically, most breeding programmes rely on recurrent conventional breeding systems. This involves the crossing of apple selections showing resistance to a given disease with a susceptible elite variety. This is followed by phenotyping the progeny to identify trees exhibiting segregating field resistance. Several crosses and backcrossing are required to produce resistant varieties and to fix the resistance trait using this breeding strategy. This breeding technique is time consuming, especially in perennial tree species such as apples, which have a long juvenile period. Molecular markers have enabled the building of genetic maps, which has allowed for tracking of the inheritance of genes contributing towards the observed resistances. This has given breeders the opportunity to start the implementation of marker-assisted-breeding (MAB) and marker-assistedselection (MAS). MAB and MAS greatly reduce the time required to select for favourable genotypes, given that MAB facilitates efficient selection for inherited traits at the seedling stage. With the publication of the apple genome sequence, the identification of the genes involved in disease resistances has been made possible and this will allow researchers to venture into cisgenics for apples, which will further reduce the time required for the introgression of desirable genes into commercial cultivars. The main thrust of this research was to generate dense genetic linkage maps for three mapping populations segregating for apple scab, woolly apple aphid and powdery mildew resistance. The three mapping populations are ‘Mildew Resistant’ x ‘Golden Delicious’, ‘Russian Seedling’ x ‘Golden Delicious’ and Malus platycarpa x ‘Mildew Resistant’ and are Malus full-sib outbreed mapping populations. The generation of the genetic maps was for use in the subsequent identification candidate disease resistance QTLs/genes that can be implemented in apple cisgenics. Integrated genetic maps using SSRs, DArTs and SNP marker data were generated for all the three crosses. The integrated map of ‘Mildew Resistance’ x ‘Golden Delicious’ consists of 1, 563 markers with a total map length of 1, 298.8 cM. The ‘Russian Seedling’ x ‘Golden Delicious’ genetic map is composed of 979 markers with a total map length of 1, 729.9 cM. The Malus platycarpa x ‘Mildew Resistant’ integrated map has 616 markers and a total map length of 1,324.3 cM. Due to the fragmentation of some of the linkage groups in the ‘Russian Seedling’ x ‘Golden Delicious’ and in the Malus platycarpa x ‘Mildew Resistant’ genetic maps, a phylogenetic analysis was performed to evaluate the genetic distances between the parents of the crosses in order to understand the cause of the fragmentation of these two integrated genetic maps. QTLs were detected through the statistical correlation of the phenotypic and map data using restricted Multiple QTL Mapping (rMQM) from MapQTL® 6.0. The genome-wide LOD score minimum QTL detection thresholds were determined using 10 000 permutations for each population. The minimum QTL detection threshold for accepting a putative QTL was then determined to be 4.5 for ‘Mildew Resistant’ x ‘Golden Delicious’ and 4.6 for both the ‘Malus platycarpa’ x ‘Mildew Resistant’ and ‘Russian Seedling’ x ‘Golden Delicious’ mapping populations. A total of 17 putative QTLs were detected for the ‘Mildew Resistant’ x ‘Golden Delicious’ population, 10 putative QTLs for the Malus platycarpa x ‘Mildew Resistant’ population and nine putative QTLs for the ‘Russian Seedling’ x ‘Golden Delicious’ population were detected for the three diseases under study. The two putative QTLs for apple scab resistance detected on LG 02 of the ‘Russian Seedling’ x ‘Golden Delicious’ map coincided with the loci previously identified as encoding two apple scab resistance genes Vh2 and Vh4 on ‘Russian apple’. The QTL for apple scab resistance identified on the proximal QTL of LG 02 co-localized with SNP marker R_8936738_Lg2 on the loci where Vh4 was previously identified. The distal QTL on LG 02 shown to encode the Vh2 R-gene was linked with the SNP marker R_32981524_Lg2. With ‘Russian apple’ being known to carry a natural pyramid of R-genes for apple scab on LG 02, therefore, the ‘Russian Seedling’ used in this study was screened by a set of 14 SSR markers to determine if it was related to ‘Russian apple. The 14 SSRs produced identical alleles to those amplified by ‘Russian apple’, which means “Russian Seedling’ and ‘Russian apple’ are closely related or identical. The LG 02 pseudo-chromosome sequence was extracted from the NCBI database housing the apple genome sequence and was then used to mine for the putative R-genes within the two QTL regions. The region corresponding to the Vh2 loci, which was roughly a 600 kb region, had two clusters of ABC (PDR) disease resistance related genes. These were predicted using a full Pfam domain search and were only detected on the negative strand. The 60 kb region corresponding to the Vh4 loci comprised a cluster of LRR domains that were also detected on the negative strand using a full Pfam domain search. This 60 kb region was further analysed using Phytozome and Genome Database for Rosaceae (GDR) leading to two candidate disease resistance genes being identified. Ten consensus gene sequences were present within the 60 kb region, with only two transcripts MDP0000657246 and MDP0000128458 identified as being disease resistance related genes. The MDP0000657246 was identified on the contig MDC000294 of the Malus x domestica reference genome as being a Leucine Rich Repeat protein kinase family, which is one of the most abundant disease resistance family mainly involved in the gene-for-gene resistance mechanism. The MDP0000128458 locus was identified on contig MDC015161 as being a Ser/Thr phosphatase 7. The Ser/Thr phosphatase genes have been associated with the regulation of MAP kinase cascades that have been shown to induce the hypersensitive response (HR) in tobacco. Therefore these two genes are likely to be the loci associated with the hypersensitive response associated with the infection of apples with race 4 of apple scab, carrying the Vh4 apple scab resistance gene. Recurrent putative QTLs were detected that still need to be validated in order to be used for MAB. The ‘Russian Seedling’ x ‘Golden Delicious’ cross produced a single powdery mildew resistance QTL located on LG08 and conferring a 1:1 resistance to susceptible phenotypic segregation ratio. These results indicate that the source of the resistance thus was a single dominant resistance gene. The ‘Mildew Resistant’ x ‘Golden Delicious’ mapping population also showed two stable QTLs one for powdery mildew on LG 03, which co-segregated with SNP GD_LG03snp00866 and in addition SNP R_13071892_Lg10 was also identified to be co-segregating with the QTL for apple scab resistance on LG10. However, none of these recurrent QTLs co-localized with known genes or QTLs. For the phylogenetic analysis, re-sequenced data using the Illumina® sequencing technologies and the apple SNP chip data for ‘Russian Seedling’, ‘Mildew Resistant’, Malus platycarpa, a Chinese accession of Malus sieversii and ‘Anna’ where used to infer relatedness of the five genotypes. The Chinese accession of Malus sieversii was included in the analysis since ‘Russian Seedling’ was thought to be relatively close genetically. Whilst ‘Anna’ is known to be a low chilling cultivar of Malus x domestica (Borkh) and therefore would add in the phylogenetic placement of ‘Mildew Resistant’ and Malus platycarpa. These were sequenced to coverage of approximately 60X for ‘Russian Seedling’ and 6X for the other four genotypes. The sequence data was aligned to the reference Malus x domestica cv Golden Delicious mitochondrial genome sequence. Phylogenetic analysis was then performed using both the data from the apple SNP-chip and the aligned mitochondrial genomes. The results from both sets of data supported the putative evolutionary distances between the five genotypes. ‘Russian Seedling’ and M. sieversii were closely related, while both were genetically divergent from the closely related ‘Anna’ and ‘Golden Delicious’ commercial cultivars. This analysis however indicated that ‘Mildew Resistant’ was relatively closely related to ‘Golden Delicious’ and hence the low number of markers showing segregation distortions for the ‘Mildew Resistant’ x ‘Golden Delicious’ population in the 17 LGs of the integrated map. However, the other two mapping population exhibited a high number of markers with segregation distortions. Markers which are closely associated with disease resistance to apple scab powdery mildew and woolly apple aphid resistance will play a major role in the identification of the genes responsible for the resistances being observed. The identification of the two candidate genes for the Vh4 gene associated with apple scab resistance will be the platform from which a cisgenic programme can be implemented in the South African apple breeding program.

Camarea St.-Hil. (Malpighiaceae) é um gênero endêmico da América do Sul, constituído por nove espécies. O objetivo do trabalho foi a reconstrução da filogenia do gênero, por meio de evidências químicas e moleculares. Foram avaliados nove terminais, sete dos quais são espécies correntemente reconhecidas, um é uma espécie que entrou em sinonímia (C. triphylla = C. axillaris) e outro é um suposto híbrido. Como grupos externos, foram utilizadas as espécies Peixotoa reticulata e Janusia guaranitica. Foram analisados os n-alcanos das ceras epicuticulares e os flavonóides de todas as espécies. Os n-alcanos principais foram C29, C31 e C33, todos da série normal. Como flavonóides característicos de Camarea, foram identificados glicosídeos de apigenina, luteolina, crisoeriol, campferol e quercetina. A análise de agrupamentos baseada na distribuição de alcanos, usando UPGMA e distâncias euclideanas, resultou em dois grupos principais, um com C29 como homólogo principal, constituído por C. hirsuta, C. affinis x C. hirsuta, C. affinis e C. ericoides. O outro grupo caracteriza-se por homólogos principais C31 ou C33, e é formado por C. elongata, C. humifusa, C. sericea, C. axillaris e C. triphylla (= C. axillaris). Esses dois principais agrupamentos contêm grupos internos menores. Uma análise de UPGMA usando coeficiente de DICE e baseada na distribuição de agliconas de flavonóides forneceu um dendrograma com alguns agrupamentos coerentes com as afinidades reveladas pela distribuição de alcanos, como as associações: 1) C. hirsuta, C. affinis e C. affinis x hirsuta; 2) C. elongata e C. axillaris; 3) C. sericea e C. humifusa. Uma inferência filogenética molecular foi obtida com seqüências de duas regiões do cloroplasto (trnL-F e rps16) e uma nuclear (ITS). Dentre as análises com um só marcador, resultados mais consistentes foram conseguidos com ITS, que forneceu 49 caracteres filogeneticamente informativos, enquanto trnL-F e rps16 forneceram 10 e 18 caracteres informativos, respectivamente. A análise de consenso estrito de quatro árvores mais parcimoniosas de uma análise combinando-se as três seqüências resultou em um cladograma em que Camarea é um grupo monofilético, com \"bootstrap\" (BS) 100, várias politomias e clados com baixa consistência. Foi realizada análise por AFLP utilizando quatro combinações de iniciadores seletivos, obtendo-se 217 fragmentos polimórficos. Uma análise combinando as evidências moleculares resultantes de seqüências de DNA e marcadores AFLP forneceu uma única árvore mais parcimoniosa com uma politomia agrupando C. affinis, C. ericoides, C. hirsuta e C. affinis x C. hirsuta, mas boa resolução e elevada sustentação em outros clados. A combinação de todas as evidências moleculares e químicas (estas compreendendo três caracteres derivados da análise de alcanos e cinco de flavonóides) resultou numa única árvore mais parcimoniosa completamente resolvida. Os resultados apóiam a fusão de C. triphylla em sinonímia com C. axillaris e indicam forte associação entre: 1) C. humifusa e C. sericea (BS 94); 2) C. affinis, C. affinis x hirsuta, C. hirsuta e C. ericoides (BS 83); 3) C. axillaris e C. elongata (BS 100). C. affinis, C. hirsuta e C. affinis x C. hirsuta compartilham a presença crisoeriol. C. affinis e C. affinis x C. hirsuta, compartilham também luteolina e formam um clado com BS 70. O presente trabalho demonstra a utilidade de caracteres químicos para melhorar a resolução de filogenias e elevar a sustentação de clados. / Camarea St.-Hil. (Malpighiaceae) is a genus with eight species endemic in South America. The purpose of the present work was the attainment of a phylogenetic inference of the genus by means of chemical and molecular evidences. Nine accessions were analyzed: seven correspond to currently recognized species; one is a species sunk into synonymy (C. triphylla = C. axillaris); and a last one is a hypothesized hybrid. Peixotoa reticulata and Janusia guaranitica were used as out-groups. n-Alkanes from epicuticular waxes and flavonoids were analyzed from all species. The main alkanes of all distributions were either C29 or C31 or C33, all from the normal series. The characteristic flavonoids of Camarea were shown to be apigenin, luteolin, chrysoeriol, kaempferol and quercetin. A cluster analysis based on the alkane distribution using UPGMA and Euclidean distances provided two main clusters. One cluster is characterized by C29 as main homologue and is formed by C. hirsuta, C. affinis, C. affinis x hirsuta and C. ericoides. The other cluster has species with either C31 or C33 as main homologue and is formed by C. elongata, C. humifusa, C. sericea, C. axillaris and C. triphylla (= C. axillaris). These two main clusters contain smaller inner clusters. An analysis using UPGMA and DICE coefficients and based on the distribution of flavonoid aglycones provided a dendrogram with clusters congruent with affinities revealed by the alkane evidence, such as the groupings: 1) C. hirsuta, C. affinis and C. affinis x hirsuta; 2) C. elongata and C. axillaris; 3) C. sericea and C. humifusa. A phylogenetic molecular inference was obtained with sequences from two chloroplast (trnL-F and rps16) and one nuclear (ITS) DNA regions. Among the analyses based on a single marker, more consistent results were obtained with ITS, which provided 49 informative phylogenetic characters, while trnL-F and rps16 provided 10 and 18 informative characters, respectively. A strict consensus analysis based on four more parsimonious trees from an analysis combining sequences of the three DNA regions gave a cladogram showing Camarea as a monophyletic group with bootstrap support (BS) 100. The cladogram contains several polytomies and clades with low support. An AFLP analysis, using four combinations of selective primers, provided 217 polymorphic fragments. Data from sequencing and AFLP were combined in a phylogenetic analysis. A sole more parsimonious tree was obtained, with most clades completely resolved with and high support. A polytomy remained, grouping C. affinis, C. ericoides, C. hirsuta and C. affinis x hirsuta. The combination of all molecular and chemical evidences (the latter comprising three alkane and five flavonoid characters) was used for a phylogenetic analysis. A sole more parsimonious tree was obtained, completely resolved and with clades highly supported. The results support sinking C. triphylla into synonymy of C. axillaris and indicate strong kinship: 1) between C. humifusa and C. sericea (BS 94); 2) among C. affinis, C. affinis x C. hirsuta, C. hirsuta and C. ericoides (BS 83); 3) between C. axillaris and C. elongata (BS 100). C. affinis, C. hirsuta and C. affinis x C. hirsuta share the possession of chrysoeriol. C. affinis and C. affinis x C. hirsuta share the possession also of luteolin and form a clade with BS 70. The present work reveals the utility of chemical characters to improve resolution of phylogenies and increment clade support.

Dengue é uma doença infecciosa não contagiosa causada pelo vírus da dengue (gênero Flavivirus, família Flaviviridae), transmitida pela picada de artrópodes do gênero Aedes, principalmente Aedes aegypti, e sendo atualmente um importante problema de saúde pública em todo o mundo. Segundo a Organização Mundial de Saúde, cerca de 50 a 100 milhões de pessoas se infectam anualmente em mais de 100 países de todos os continentes. A dengue apresenta-se em três formas clínicas principais; doença febril indiferenciada, febre clássica do dengue (DF) e dengue hemorrágico com ou sem choque (DHF/DSS). Recentemente, viu-se um dramático aumento do número de casos de DHF/DSS nas Américas, e este aumento coincidiu com a introdução do dengue tipo 3, genótipo III. Neste trabalho, caracterizamos completamente o genoma de um vírus brasileiro de dengue tipo 3 (D3BR/RP1/2003) isolado de um paciente com dengue clássica. O genoma viral possui um tamanho de 10707 nucleotídeos e contém uma única região codificadora (posição 95 a 10264) flanqueada por duas regiões não codificadoras (RNC5, 1 a 94; RNC3, 10268 a 10707). A comparação do genoma viral com outros vírus isolados de pacientes com DF e DHF não mostrou diferenças significativas que sugerissem a presença de um fator genético associado à virulência. A analise filogenética baseada no genoma completo, mostra que a cepa D3BR/RP1/2003 pertence ao genótipo III e encontra-se proximamente relacionada a outro vírus isolado no Rio de Janeiro em 2002. Entretanto, quando essa análise filogenética é realizada com as regiões codificadoras das proteínas E e NS5 individualmente, a cepa D3BR/RP1/2003 encontra-se mais proximamente relacionada a um vírus isolado na Ilha de Martinica no Caribe. Este achado sugere que o isolado D3BR/RP1/2003 pode ter sido originado através de um evento de recombinação entre duas cepas virais distintas antes ou após sua introdução no Brasil. Dados sugestivos de recombinação foram observados também quando analisada a relação filogenética entre vírus isolados na Ásia. / Dengue is an infectious disease caused by dengue virus (genus Flavivirus, family Flaviviridae), transmitted by the bite of arthropods of the Aedes genus, mainly Aedes aegypti, and being currently an important problem of public health worldwide. According to the World Health Organization, about 50 the 100 million people are infected annually in more than 100 countries of all continents. Dengue can be presented in three main clinical forms; undifferentiated febrile illness, classic dengue fever (DF) and hemorrhagic dengue fever with or without shock (DHF/DSS). Recently, a dramatically increase of DHF/DSS cases in the Americas have been seen, and this increase coincided with the introduction of the dengue type 3, genotype III. In this work, we have completely characterized the genome of a Brazilian dengue type 3 (D3BR/RP1/2003 strain) isolated from a DF patient. The viral genome possesses a size of 10707 nucleotides and has a unique open reading frame (position 95 to 10264), flanked by two untranslated regions (UTR5\', 1 to 94; UTR3\', 10268 to 10707). The comparison of the viral genome with other viruses isolated from patients with DF and DHF did not show significant differences to suggest the presence of a genetic factor associate with virulence. Phylogenetic analysis based on the complete genome showed that D3BR/RP1/2003 strain belongs to genotype III and is close related to another virus isolated in Rio de Janeiro in 2002. However, when the phylogenetic analysis was based on the individual coding regions of E and NS5 proteins, D3BR/RP1/2003 strain was closely related to a virus isolated in the Island of Martinique in the Caribbean. This finding suggests that D3BR/RP1/2003 strain could have been originated through an event of recombination between different virus strains before or after its introduction to Brazil. Data supporting recombination events between viruses isolated in Asia have also been observed.

A fibropapilomatose é uma neoplasia caracterizada pela formação de múltiplos tumores que acomete, mais frequentemente, a espécie de tartaruga marinha Chelonia mydas. Estudos recentes apontam o Chelonid herpesvirus 5 (ChHV5) como o provável agente etiológico dessa doença, embora a associação com ambientes antropogenicamente alterados parecem contribuir para o desenvolvimento da doença. Nesse estudo, biópsias de tumores e secreções de tartarugas verdes capturadas no litoral do Estado de São Paulo, Brasil, foram submetidas a análises histológicas e moleculares visando detectar e caracterizar ChHV5. Em 45,5 % dos casos, os achados histopatológicos revelaram células epiteliais balonizantes com corpúsculos de inclusão intranucleares. ChHV5 foram detectados nas biópsias de pele e oculares dos animais e em secreções oculares e saliva por PCR. A análise das sequências parciais do gene da polimerase do ChHV5 detectadas revelou duas sequências gênicas distintas entre si. A análise filogenética indicou que as amostras brasileiras são similares às amostras de ChHV5 do grupo filogeográfico do Atlântico, compartilhando o mesmo clado que amostras provenientes do Golfo da Guiné e de Porto Rico, sugerindo um possível fluxo dos vírus entre essas três regiões. / Fibropapillomatosis is a neoplastic disease characterized by the formation of multiple tumors affecting different species of sea turtles and, most often, Chelonia mydas. Recent studies indicate that Chelonid herpesvirus 5 (ChHV5) is the etiological agent of this disease, though its association with anthropogenically altered environments also appears to contribute to disease expression and tumor formation. In this study, tumor biopsy and secretions from green turtles captured off the coast of São Paulo State, Brazil, were used in histological and molecular analyses to detect and characterize ChHV5. In 45.5 % of cases, the tumor histopathological findings revealed ballooning degeneration with intranuclear inclusion bodies. ChHV5 was detected using polymerase chain reaction on the animals skin, ocular tumor biopsies, and ocular and oral secretions. The analysis of the detected ChHV5 sequences revealed two distinct genetic sequences together. Phylogenetic analysis indicated that Brazilian samples were similar to ChHV5 samples described for the Atlantic phylogeographic group and are therefore part of the same clade as the Gulf of Guinea and Puerto Rico samples. This similarity suggests a possible flow of the virus between these three regions.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22282号 / 理博第4596号 / 新制||理||1659(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 朝倉 彰, 准教授 下村 通誉, 教授 曽田 貞滋 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

>Magister Scientiae - MSc / Molecular methods were used to investigate the microbial diversity and community structure of ammonia-oxidizing bacteria (AOB) associated with the roots of the Proteaceae plant family. The identification of ammonia oxidizing bacteria in this ecosystem is of particular interest since Proteaceae are adapted to acidic, low nutrient (e.g. nitrogen) soils. The ammonia monooxygenase operon was used as a molecular marker to identify ammonia-oxidizing bacteria associated with the proteoid roots of the three Proteaceae members and compared to non-plant associated soil. PCR amplification using primer sets targeting the ammonia monooxygenase gene (amoA subunits) were used to construct a clone library. Sequence diversity was determined by RFLP analysis of amoA to identify major groups of AOB of the ~-subclass of Proteobacteria in total community DNA, and DNA sequencing and phylogenetic analysis were also applied. DGGE analysis was performed to determine the community structure and distribution of ammonia-oxidizing bacteria in plant-associated and non-plant associated soils. The AOB genotypic diversity was similar in the plant-associated samples and non-plant associated soil. All AOB phylotypes belonged to Nitrosospira species and clustered with Nitrosospira cluster 3. The abundance of the amoA was quantified to be approximately 4.2 x 107 copies/g of dry soil, using a real-time PCR assay. These data suggest that the Nitrosospira species are the dominant phylotypes in that environment. This investigation provides new insights into the relationships between plants and ammonia-oxidizing bacteria in natural Fynbos ecosystems.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21687号 / 医博第4493号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 清水 章, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bovine viral diarrhea virus (BVDV) is an important viral pathogen affecting all ages of cattle, resulting in significant economic losses worldwide. BVDV infection is associated with a diverse array of symptoms including gastrointestinal disorder, respiratory distress, fetal malformation, stillbirth, abortions, and mucosal disease (MD). Transplacental infections of fetuses between 42 and 125 days of gestation can result in immune-tolerance and the surviving fetuses become persistently infected (PI). PI animals are major reservoir of BVDV and it becomes problematic to control the disease. The objectives of this dissertation were to: 1) develop a cost-effective testing scheme to detect BVDV PI animals from exposed herds, 2) characterize two virulent BVDV-2 Mississippi isolates associated with severe hemorrhagic diseases, and 3) perform phylogenetic analysis based on sequences of 5'UTR, E2, and NS5B regions. First, we developed a BVDV testing scheme by combining pooled real-time RT-PCR with antigen capture enzyme-linked immunosorbent assay (ACE) to screen cattle herds. From positive pools individual positives were identified using ACE. Data from a three year period indicated that 92.94% PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'UTR of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a. Second, two virulent BVDV isolates, M10-3432 and M10-5347, were successfully recovered from an adult beef breeding cow and feedlot calf respectively. When compared to the reference strain BVDV-2 125c, five and three unique amino acids in E2 regions were different from M10-5347 and M10-3432 respectively. Phylogenetic analysis of E2 region grouped both Mississippi isolates in BVDV-2a, a subtype containing high virulent strains. M10-3432 was clustered with high virulent strain 890 while M10-5347 was clustered with high virulent strain CD87. Third, we compared the phylogenetic analyses of BVDV based on the sequences of 5'UTR, E2, and NS5B at either nucleotides or amino acids level. Although slight differences were observed, the virulent BVDV isolates were consistently classified into BVDV-2a cluster regardless of region of sequences used. Furthermore, phylogenetic tree constructed using combined two or more regions had higher posterior probability and bootstrap value than phylogenetic trees constructed using a single region