Дисертації з теми "Genotype VII"

African swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in the southern African sub-region, where outbreaks regularly occur. Thereis anecdotal evidence suggesting that trans-boundary movement of infected animals may have played a role in precipitating widespread outbreaks in the past, however, since the1970s outbreaks have generally been more localised, particularly in those countries where control of animal movement is strictly regulated. The origin and relatedness of regional ASF outbreaks was investigated here by means of a two-step genetic characterisation approach whereby p72 gene sequencing was used to delineate genotypes, prior to intragenotypic resolution of viral relationships by central variable region (CVR) characterisation of the 9RL ORF. In this manner, regional virus heterogeneity and epidemiological links between outbreaks could be assessed for the first time through phylogenetic analysis of the C-terminal end of the p72 gene of viruses recovered from domestic pig outbreaks in southern Africa between 1973 and 1999. The phylogeny revealed the presence of 14 distinct p72 genotypes of which 6 (genotypes XVII–XXII) were considered novel. Eight of these were country-specific with the remaining six having a trans-boundary distribution. CVR products were heterogeneous in size ranging from 377 bp to 533 bp across the 14 southern African genotypes. Within-genotype CVR comparisons revealed the presence of a genotype XIX virus with an extended field presence in South Africa (1985–1996) and permitted discrimination between three genotype VII viruses that were identical across the p72 gene.

Em estudos de divergência genética por métodos multivariados, a distância euclidiana é a medida de distância mais amplamente utilizada e essa distância é a mais recomendada quando as unidades de cálculos são escores de componentes principais, como é o caso da análise AMMI (additive main effects and multiplicative interaction analysis). Tal análise permite a obtenção de estimativas mais precisas das respostas genotípicas e possibilita a análise da divergência genética por métodos aglomerativos. A análise dos modelos AMMI combina, num único modelo, componentes aditivos para os efeitos principais (genótipos e ambientes) e componentes multiplicativos para os efeitos da interação genótipos × ambientes. Os melhoristas de plantas compreendem que a interação genótipos × ambientes é de suma importância para a obtenção de variedades superiores e as estimativas de dissimilaridade atendem aos objetivos do melhorista, por quantificarem e informarem sobre o grau de semelhança ou de diferença entre pares de indivíduos. Entretanto, quando o número de indivíduos é grande, torna-se inviável o reconhecimento de grupos homogêneos pelo exame visual das estimativas de distância. Portanto, é importante proceder à análise de agrupamentos, obter dendrogramas por meio de métodos hierárquicos e posteriormente, analisar os grupos formados. A fim de determinar e classificar os grupos formados na clusterização hierárquica foram utilizados comandos específicos do programa computacional R que desenha no dendrograma os retângulos de cada grupo e os numera. Desta forma, o objetivo deste trabalho foi analisar a divergência genética via modelo AMMI, utilizando-se de técnicas multivariadas e reamostragem \"bootstrap\".
In studies of genetic diversity using multivariate approaches, the Euclidean distance is the most common measure used. This method is recommended when data are scores of principal components, such as in AMMI analysis (additive main effects and multiplicative interaction analysis). The AMMI method allows obtaining more precise estimates for genotypic results and also permits the use of genetic diversity analysis by using agglomerative approaches. Furthermore, this method combines additive components for the main effects (genotypes and environments) and multiplicative components for genotypes x environment interaction effects in a unique model. Plant breeders understand the importance of genotype and environment interaction to obtain superior varieties and the dissimilarity estimation meets breeders\" objectives since it quantifies and determines the similarity or the divergence between pairs of individuals. However, when the number of individuals is large it is unfeasible to recognize the group homogeneity by using a visual analysis of the distances estimation. Therefore, is important to use cluster analysis to obtain dendograms based on hierarchical methods and then analyze the groups obtained. In order to determine and classify the obtained groups from hierarchical cluster analysis specifics commands in the R software were used which shows in the dendrogram rectangles and numbers for each group. In this way, the objective of this work was to analyze the genetic divergence through AMMI model, by using multivariate approaches and \"bootstrap\" resampling.

Avaliaram-se 36 linhagens diaplóides de trigo, obtidas via cultura de anteras in vitro oriundas de plantas híbridas, em geração F1, divididas em dois ensaios com dezoito linhagens e dois cultivares controles (IAC-24 e IAC-289), nos anos de 1999 e 2000. Cada ensaio foi instalado em dois locais do Estado de São Paulo: Ensaio I - Estações Experimentais de Agronomia de Capão Bonito (solo ácido, sem aplicação de calcário e em condição de sequeiro) e Tatuí (solo ácido, com aplicação de calcário e em condição de irrigação por aspersão) e Ensaio II - Estações Experimentais de Agronomia de Tatuí e Monte Alegre do Sul (ambos em solo ácido com aplicação de calcário e condição de irrigação por aspersão). Em cada ensaio, avaliaram-se os seguintes parâmetros: acamamento, altura da planta, ciclos da emergência ao florescimento e da emergência à maturação, produção de grãos, resistência às moléstias, comprimento da espiga e componentes de produção. Todos os genótipos foram, também, avaliados quanto à tolerância à toxicidade de alumínio, em solução nutritiva, em condição de laboratório. No Ensaio I, destacaram-se, pela produção de grãos, as linhagens 4 (2.309 kg ha -1 ) e 5 (2.319 kg ha -1 ), provenientes do cruzamento PF70402/ALD'S'//PAT72160/ALD'S'/3/PEW'S'/4/OPATA/5/IAC-60; 9 (2.150 kg ha -1 ), provinda do cruzamento MLR'S'/BUC'S'//BUC'S'/3/IAC-24, 11 (2.102 kg ha -1 ) e 12 (2.056 kg ha -1 ), oriundas do cruzamento TEPOCA/IAC-24. A 13 (JUN/GEN//IAC-24) apresentou as plantas mais baixas (53 cm). As linhagens 2, 4 e 18, originárias dos cruzamentos JUN/GEN//IAC-24,PF70402/ALD'S'//PAT72160/ALD'S'/3/PEW'S'/4/OPATA/5/IAC-60 e TEPOCA/IAC-24, revelaram, ao mesmo tempo, moderada resistência aos agentes causais da ferrugem-da-folha e da mancha-da-folha. Todos os genótipos, com exceção do cultivar IAC-289 e da linhagem 13 (JUN/GEN//IAC-24), foram considerados tolerantes a 10 mg L -1 de Al 3+ , quando avaliados em solução nutritiva. No Ensaio II, a linhagem 8 (ANA/IAC-24) e o cultivar IAC-289 apresentaram elevadas produções de grão (3.311 e 3.341 kg ha -1 respectivamente). A linhagem 13 exibiu o porte mais baixo (61 cm) entre os genótipos estudados e a 3 (ANA/IAC-24//IAC-24), mostruo, ao mesmo tempo, resistência ao agente causal da ferrugem-da-folha, moderada resistência ao agente da mancha-da-folha e imunidade ao agente causal do oídio. As linhagens 8 (ANA/IAC-24) e 14 (PF70402/ALD”S”//PAT72160/ ALD”S”/3/PEW”S”/4/OPATA/5/ IAC-60) mostraram elevada tolerância à toxicidade de alumínio, associada a alta produção de grãos.
Thirty six dihaploid wheat lines, originated via anther culture from F1 hybrid plants were evaluated in two trials with eighteen lines plus two control cultivars (IAC-24 and IAC-289), in 1999 and 2000. Each trial was carried out in two locations of the State of São Paulo: trial I - Capão Bonito Agronomy Experiment Station (acid soil without lime application and upland condition) and Tatuí Agronomy Experiment Station (acid soil with lime application and sprinkler irrigation condition) and trial II - Monte Alegre do Sul and Tatuí Agronomy Experiment Station (both with acid soil with lime application and sprinkler irrigation condition). In each the genotypes were evaluated for lodging, plant height, cycle from emergence to flowering and from emergence to maturation, grain yield, resistance to disease, head length and yield components. All genotypes were also evaluated for aluminum toxicity tolerance, in nutrient solution, under laboratory condition. Considering trail I, the lines 4 (2.309 kg ha -1 ) and 5 (2.319 kg ha -1 ) originated from the cross PF70402/ALD'S'//PAT72160/ALD'S'/3/PEW'S'/4/OPATA/5/IAC-60, 9 (2.150 kg ha -1 ) from the cross MLR'S'/BUC'S'//BUC'S'/3/IAC-24, and the lines 11 (2.102 kg ha -1 ) e 12 (2.056 kg ha -1 ) from the cross TEPOCA/IAC-24, presented high grain yield. The line 13 (JUN/GEN//IAC-24) showed the shortest plants (53 cm). The lines 2, 4 and 18 originated from crosses JUN/GEN//IAC-24,PF70402/ALD'S'//PAT72160/ALD'S'/3/PEW'S'/4/OPATA/5/IAC-60 and TEPOCA/IAC-24, showed at the same time moderate resistance to the causal agents of leaf rust and leaf spot. All genotypes, with exception of the cultivar IAC-289 and the line 13 (JUN/GEN//IAC-24) , were considered tolerant to 10 mg L -1 Al 3+ , when evaluated in nutrient solutions. Considering trial II, the line 8 (ANA/IAC-24) and the cultivar IAC-289 presented high grain yield (3.311 e 3.341 kg ha -1 respectively). The line 3 (ANA/IAC-24//IAC-24) exhibited at the same time resistance to the causal agent of leaf rust, moderate resistance to the causal agent of leaf spot and immunity to the causal agent of powdery mildew. The lines 8 (ANA/IAC-24) and 14 (PF70402/ALD“S”//PAT72160/ALD“S”/3/PEW“S”/4/OPATA/5/IAC-60), also showed high tolerance to aluminum toxicity being associated to high grain yield, and so could be used in breeding programs with the objective to get cultivars for acid soils.

Memoria para optar al título profesional de Ingeniero Agrónomo
La clorosis férrica es uno de los principales problemas nutricionales que afectan a viñedos injertados sobre portainjertos híbridos de Vitis americana establecidos en zonas calcáreas, las cuales se caracterizan por la presencia de elevados contenidos de carbonato de calcio y bicarbonatos en los suelos. El presente trabajo evalúa, a nivel fisiológico y bioquímico, las respuestas del portainjerto de vid 140 Ruggeri a la presencia de bicarbonato, en condiciones de déficit de Fe en la solución nutritiva. Plantas del genotipo 140 Ruggeri fueron crecidas en condiciones hidropónicas, con dos niveles de Fe y bicarbonato (-Fe, +Fe; 0, 10 µM de Fe; -BIC, +BIC; 0 y 5mM de KHCO 3 respectivamente). Determinaciones de crecimiento y peso de la planta, índice SPAD y clorofila, fotosíntesis y conductancia estomática, pH de la solución, actividad enzimática de raíces (PEPC, MDH, CS, NADP + −IDH) y concentración de proteínas, concentración de ácidos orgánicos en raíces y líquido xilemático fueron realizadas. Los resultados obtenidos indican que el genotipo de vid 140 Ruggeri cuando es sometido a un déficit directo de Fe, responde activamente disminuyendo el crecimiento del brote y la biomasa de la parte aérea, e incrementando la concentración de ácidos orgánicos y sus enzimas de síntesis en raíces. Además, el efecto del déficit de Fe directo se manifiesta mediante una reducción en el contenido de clorofila, la fotosíntesis neta y la conductancia estomática de las hojas. Por su parte, la presencia de bicarbonato en la solución nutritiva induce una reducción en el crecimiento del tallo y las feminelas, y un incremento en la biomasa radical. Adicionalmente, la presencia de bicarbonato incrementa la concentración de clorofila foliar, y la concentración de ácido tartárico en el líquido xilemático. Los resultados confirman la probabilidad de que la presencia de bicarbonato en la solución nutritiva contribuye a mitigar el efecto del estrés en las plantas sometidas a déficit de Fe. Lo anterior se fundamenta en que, ante un déficit de Fe, las plantas cultivadas en presencia de bicarbonato muestran una menor actividad de la PEPC y concentración de ácido tartárico en raíces, asimilándose al control. Estos resultados permiten concluir que el genotipo tolerante al déficit de Fe 140 Ruggeri responde con mecanismos correspondientes a una planta de Estrategia I en condiciones limitantes de Fe. Además frente presencia de bicarbonato, presenta respuestas similares a la especie calcícola Parietaria diffusa como la reducción del crecimiento, mayor biomasa radical, y aumento de la concentración de ácidos orgánicos, sin embargo más investigaciones son necesarias para determinar con precisión el rol del bicarbonato en el metabolismo del portainjerto 140 Ruggeri.
Iron (Fe) chlorosis is one of the main nutritional disorders in vineyards grafted on Vitis americana hybrids rootstocks established in calcareous zones which are characterized by high levels of calcium carbonates and bicarbonates in the soil. The present work determine, at physiological and biochemical level, the responses of the grapevine rootstock 140 Ruggeri to the presence of bicarbonate under Fe deficiency conditions. The 140 Ruggeri plants were grown in hydroponic conditions, and were submitted to two levels of Fe and bicarbonate (-Fe, +Fe; 0, 10 µM de Fe; -BIC, +BIC; 0 y 5mM de KHCO 3, respectively). Determinations regarding the plants biomass and growth, chlorophyll content and SPAD value, photosynthesis and stomatal conductance, nutrient solution pH, enzyme activity (PEPC, MDH, CS, NADP + −IDH) and protein concentration in roots and organic acids content in roots and xylem sap were done during the experiment. The results obtained indicate that the grapevine genotype 140 Ruggeri, when it was submitted to a direct Fe depletion in the nutrient solution, reacted reducing the length and biomass of the shoots, and increasing the organic acids concentration and the activity their related enzymes in roots. Moreover, Fe deficiency reduced the chlorophyll content, the net photosynthesis, and the stomatal conductance of the leaf. In addition, the presence of bicarbonate in the nutrient solution induced a reduction in the main and lateral shoots growth, and increased the root biomass. Moreover, the presence of bicarbonate increased the leaf chlorophyll content, and the tartaric acid concentration in the xylem sap. Data obtained confirm the probability that the presence of bicarbonate in the nutrient solution contributes to mitigate the stress in plants submitted to Fe deficiency. This is based on that, under Fe deficiency conditions, the plants grown under the presence of bicarbonate show a PEPC activity and tartaric acid concentration in roots similar to control plants. Such results, allow to conclude that the Fe deficiency tolerant genotype 140 Ruggeri reacts to Fe deficiency activating response mechanisms corresponding to a Strategy I plants. In addition, under the presence of bicarbonate, the genotype shows similar responses to Parietaria diffusa species, such as the growth reduction, the higher root biomass, and the root organic acids increase. However, more investigations are necessary to determine the role of bicarbonate in the 140 Ruggeri rootstock metabolism.

Linha de pesquisa: Melhoramento animal.
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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)
Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG)
Objetivou-se com este estudo, caracterizar a intera??o gen?tipo x ambiente em codornas de corte, alimentadas com dietas contendo diferentes rela??es de triptofano:lisina, via normas de rea??o. Para realizar o experimento foram utilizadas duas linhagens de codornas de corte, LF1 e LF2. A ra??o fornecida no per?odo de crescimento, nascimento aos 21 dias de idade, continha uma rela??o de 0,20% de triptofano:lisina, e no per?odo final do crescimento, 22 a 35 dias, as codornas foram separadas em 5 tratamentos com as rela??es: 0,15; 0,20; 0,25; 0,30; 0,35% de triptofano:lisina. As aves foram pesadas aos 28 e 35 dias e abatidas aos 35 dias de idade. As caracter?sticas avaliadas foram os pesos pr?-abate, peso 28, peso 35, peso e rendimentos da carca?a, peito, coxa+sobrecoxa e as caracter?sticas de qualidade da carne. As an?lises foram realizadas utilizando modelos de regress?o aleat?ria considerando efeito fixo de sexo e o efeito aleat?rio gen?tico aditivo direto como fun??es das rela??es triptofano:lisina da dieta, admitindo-se homogeneidade de vari?ncia residual. Os resultados para desempenho, caracter?sticas de carca?a e caracter?sticas de qualidade da carne indicam que o coeficiente de regress?o do intercepto (b0), foi maior que o coeficiente de regress?o aleat?rio (b1). A herdabilidade e a vari?ncia gen?tica aditiva foram influenciadas pelas diferentes rela??es triptofano:lisina da dieta. Houve intera??o gen?tipo x ambiente para as caracter?sticas rendimento de carca?a, rendimento de peito e rendimento de coxa+sobrecoxa. As caracter?sticas de qualidade da carne CRA, b*, c* e H* (LF1) e CRA, PPC, L* e H* (LF2) apresentaram intera??o gen?tipo x ambiente. A intera??o gen?tipo x ambiente observada para as caracteristicas de rendimento e qualidade da carne indicam que a sele??o seja feita na rela??o triptofano:lisina em que as codornas ser?o criadas.
Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Zootecnia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016.
We animed of this experiment was to characterize the genotype x environment interaction in the production of meat quails, fed diets containing different tryptophan:lysine ratios, via reaction standards. Two lines of cutting quails, LF1 and LF2, were used to perform the experiment. The feed provided in the growing period, from birth to 21 days, contained a 0.20% tryptophan:lysine ratio and in the final growth period, from 22 to 35 days of age, quails were separated into 5 treatments containing the Ratios of 0.15; 0.20; 0.25; 0.30; 0.35% tryptophan: lysine. The birds were weighed at 28 and 35 days of age and slaughtered at 35 days of age. The evaluated characteristics were the pre-slaughter weights, weight 28, weight 35, weight and yields of the carcass, breast, thigh + sobrecoxa and the quality traitsof the meat. The analyzes were performed using random regression models considering fixed sex effect and direct additive genetic random effect as functions of the tryptophan:lysine ratios of the diet, assuming homogeneity of residual variance. The results for performance, carcass traitsand meat quality traitsindicate that the regression coefficient of the intercept (b0) was higher than the random regression coefficient (b1). Heritability and additive genetic variance were influenced by the different tryptophan:lysine ratios of the diet. There was no genotype x environment interaction for the traitsof carcass yield, yield of breast and yield of thigh + overcoat. The quality traits of the meat CRA, b *, c * and H * (LF1) and CRA, PPC, L * and H * (LF2) presented genotype x environment interaction. It is possible to conclude that for yield traitsthe selection can be made in the lowest tryptophan: diet lysine ratios. For meat quality traitsthe sensitivity of the values indicates that the selection must be made in the tryptophan:lysine ratio in which the quails will be created.

La cryptococcose neuroméningée (CNM) est la seconde infection opportuniste chez les patients infectés par le VIH. Il s’agit de la 4ème cause de décès dus aux maladies infectieuses en Afrique avec une mortalité annuelle de 600.000 cas. Les levures responsables appartiennent au complexe d’espèces Cryptococcus neoformans / C. gattii. Notre étude décrit, l’épidémiologie et la résistance aux antifongiques de souches environnementales et cliniques de cryptocoques en Côte d’Ivoire. Les isolats sont issus d’une file active de 1750 PVVIH et de 667 prélèvements réalisés dans l’environnement de vie des patients. Nous démontrons une grande diversité génotypique au sein de notre cohorte, la présence de plusieurs espèces de cryptocoques dans un seul prélèvement chez un même patient ainsi que dans des prélèvements issus de suivi de patients, ce qui n’avait jamais été démontré en Afrique de l’Ouest. Nous avons constaté que la récurrence de la CNM est due à des infections multiples par des souches différentes au cours du temps. Nos résultats décrivent également pour la première fois, l’isolement de cryptocoques à partir de fientes de pigeons à Abidjan. Et nous constatons que les génotypes des isolats environnementaux et cliniques sont très différents, ce qui exclut les fientes de pigeons comme source de contamination des patients dans notre échantillon. Enfin, la majorité des isolats est sensible aux antifongiques de référence mais un patient peut être contaminé par des isolats de sensibilité différente
Cryptococcal meningitis (CM) is the second opportunistic infection in HIV infected patients. It is the fourth cause of death due to infectious diseases in Africa with an annual mortality of 600,000. The yeasts responsible belong to the C. neoformans / Cryptococcus gattii species complex. Our study describes epidemiology and resistance to antifungal of environmental and clinical strains of Cryptococcus in Ivory Coast. The isolates are from an active list of 1,750 patients VIH positive and 667 samples taken in the living environment of patients. We demonstrate a high genotypic diversity within our cohort and the presence of several species of Cryptococcus in one sample from the same patient as well as in samples from patients follow up, which had never been shown in West Africa. We found that the recurrent cryptococcosis is caused by multiple infections by different strains over time. Our results describe also, for the first time, the isolation of Cryptococcus from pigeon droppings from Abidjan. And we notice that, as the genotypes of environmental and clinical isolates are very different, that excludes contamination of patients by pigeon droppings. Finally, most of the isolates were susceptible to reference antifungal but a patient might be contaminated by isolates with different susceptibility

O objetivo deste trabalho é propor uma nova metodologia de interpretação da estabilidade dos métodos de agrupamento, para dados de vegetação, utilizando a metodologia AMMI e a reamostragem (bootstrap), para ganhar confiabilidade nos agrupamentos formados. Os dados utilizados são provenientes do departamento de genética da Escola Superior de Agricultura \"Luiz de Queiroz\", e visam à produtividade de soja. Primeiramente aplica-se a metodologia AMMI e então, é estimada a matriz de distâncias euclidianas - com base nos dados originais e obtidos via reamostragem (bootstrap) - para a aplicação dos métodos de agrupamento (vizinho mais próximo, vizinho mais distante, ligação média, centroide, mediana e Ward). Para a verificação da validade dos agrupamentos formados utiliza-se o coeficiente de correlação cofenética, e pelo teste de Mantel, é apresentada a distribuição empírica dos coeficientes de correlação cofenética. Os agrupamentos obtidos pelos diferentes métodos são, em sua maioria, semelhantes indicando que, em princípio, qualquer um desses métodos seria adequado para a representação. O método que apresenta resultados discrepantes em relação aos outros (tanto para os dados originais, quanto pelos dados obtidos via bootstrap) - na representação gráfica em dendrograma - é método de Ward. Este estudo é promissor na análise da validade de agrupamentos formados em dados de vegetação.
The objective of this work is to propose a new interpretation methodology of clustering methods for vegetation data stability, using the AMMI and bootstrap methodology, to gain reliability in the clusters formed. The database used is from the Departament of Genetics of Luiz de Queiroz College of Agriculture, aiming soybean yield. Firstly AMMI is applied, then the Euclidian distance matrix is estimated - based on the original data and on the acquired by the bootstrap method - for the application of clustering methods (nearest neighbor, furthest neighbor, average linkage, centroid , median and Ward). In order to assess the validity of clusters formed the cophenetic correlation coefficient is used, and the Mantel test, in order to show the empirical distribution of the cophenetic correlation coefficients. The clusters obtained by different methods are, in most cases, quite similar, indicating that in principle, any of these methods would be suitable for the representation. The method that presents discrepant results (for both the original and bootstrap method obtained data) - on the dendrogram graphical representation, compared to the others - is the Ward\'s. This study is promising in the analysis of validity of clusters formed in vegetation data.

La variabilité phénotypique a tendance à augmenter lorsque l'environnement est variable dans le temps ou l'espace. Cette thèse traite plus précisément de la variabilité des traits d'histoire de vie dans le cadre de populations fragmentées. En région Méditerranéenne, le Pélodyte ponctué, Pelodytes punctatus, se reproduit à la fois au printemps et en automne, en réponse aux variations temporelles de l'environnement. J'ai cherché à comprendre l'origine et les conséquences évolutives de ces stratégies de reproduction. Les deux périodes de reproduction produisent efficacement des descendants (plus en automne) et les têtards de printemps semblent souffrir de la compétition avec les têtards d'automne. Ces derniers se métamorphosent plus tôt et à une plus grande taille. Ces différences de développement ne s'expliquent pas par des différences génétiques entre des populations saisonnières. Au contraire, elles sont liées à la plasticité phénotypique et répondent à des conditions environnementales drastiquement différentes. Il semble plus favorable de se reproduire en automne. Le maintien des deux stratégies correspond à du bet-hedging ou un simple opportunisme. Par ailleurs, la fragmentation, phénomène croissant lié à l'anthropisation des milieux, diminue la taille des populations et augmente l'hétérogénéité génétique au sein d'une population. Il n'y a cependant pas de consanguinité au sein des populations étudiées mais une forte structuration en familles qui induit des associations allèles/phénotypes.Ce travail éclaire les variations de traits d'histoire de vie du Pélodyte et indique une grande plasticité phénotypique face aux variations de l'environnement
Phenotypic variability tends to increase in temporally and spatially variable environments. This thesis deals with the variability of life-history traits in fragmented populations. In Mediterranean regions, Parsley frog, Pelodytes punctatus, breeds both in spring and in autumn, in response to temporal variations of its environment. I studied the origin and evolutionary consequences of its breeding strategies.Both breeding periods produce offspring (much more in autumn, though) and spring tadpoles suffer from intraspecific competition with older autumn tadpoles. Autumn laid juveniles are bigger and emerged sooner from the ponds. These developmental differences are not due to genetic differences between seasonal populations. They are explained by phenotypic plasticity in response to drastically different conditions. Even if it seems more favourable to breed in autumn, both strategies are maintained either by bet-hedging or pure opportunism.Besides, fragmentation, which increases with global changes, tends to reduce population effective size and increase genetic heterogeneity within populations. However, no inbreeding was found in the studied populations but a high family structure induced alleles/fitness correlations.Together, these results enlighten the variability of breeding strategies and larval traits in Parsley frog and indicate a high phenotypic plasticity in response to environmental variations

O presente estudo teve por objetivo estudar os efeitos da interação genótipo x ambiente sobre as características peso à desmama, peso ao sobreano e ganho de peso da desmama ao sobreano em bovinos da raça Nelore. Foram analisados 58.032 registros de peso à desmama ajustados para 205 dias (PD), 46.032 registros de peso ao sobreano ajustados para 550 dias (PS) e 45.844 registros de ganho de peso da desmama ao sobreano ajustados para 345 dias (GP), originários de três rebanhos distintos. Os dados foram submetidos a dois métodos de análises: no primeiro, processaram-se análises unicaracterísticas para os rebanhos individuais e para o conjunto formado pelos três rebanhos, e análises tri-características para os dados de cada rebanho, em que as mesmas características foram consideradas como variáveis distintas. Foi utilizado o programa GIBBS2F90, sob abordagem bayesiana. As estimativas dadas pelas médias dos coeficientes de herdabilidade para PD, PS e GP variaram de 0,09 a 0,24, 0,24 a 0,44 e 0,09 a 0,31, respectivamente. Nesta mesma ordem, as correlações genéticas das mesmas características nos diferentes ambientes variaram de 0,88 a 0,93, 0,85 a 0,98 e 0,75 a 0,97. As correlações entre as DEPs dos touros nos ambientes variaram de 0,97 a 0,99, 0,69 a 0,95 e 0,77 a 0,98 para PD, PS e GP, respectivamente. No segundo método, utilizou-se um modelo de regressão aleatória para descrever alterações nos valores genéticos dos animais em função do gradiente ambiental, formado pelos grupos contemporâneos. As análises foram feitas pelo programa INTERGEN, também sob enfoque bayesiano. As estimativas dos coeficientes de herdabilidade para PD, PS e GP variaram de 0,06 a 0,44, 0,19 a 0,63 e 0,20 a 0,40, respectivamente. As correlações genéticas entre intercepto e inclinação das normas de reação foram de 0,75, para PD, 0,76 para PS e 0,34 para GP. As correlações entre os valores genéticos dos touros nos ambientes variaram de -0,38 a 0,99, 0,79 a 1,00 e 0,68 a 0,99 para PD, PS e GP, respectivamente. Os resultados de ambos os métodos apontaram efeito da interação genótipo x ambiente sobre as características nos rebanhos incluídos neste estudo, especialmente sobre a classificação dos touros.
The objective of the present study was to evaluate the genotype by environment interaction effect on weaning weight, post-weaning weight and post-weaning weight gain in Nellore cattle. It were analyzed 58,032 records of weaning weight adjusted for 205 days (PD), 46,032 records of post-weaning weight adjusted for 550 days (PS) and 45,844 records of post-weaning weight gain adjusted for 345 days (GP), originated from three distinct herds. Those data were analyzed applying two different methods: in the first proceeding, the data set of the three herds separately and the data set composed by all herds in one was submitted to single-trait analysis, while a three-trait analysis considered the same trait as a distinct variables in different herds. The variance components were estimated by GIBBS2F90, under bayesian inference. The estimates given by the means of heritability coefficients for PD, PS and GP ranged from 0.09 to 0.24, 0.24 to 0.44 and 0.09 to 0.31, respectively. In the same sequence, the genetic correlation among the same traits in different environments varied from 0.88 to 0.93, 0.85 to 0.98 and 0.75 to 0.97. The correlation between sire\'s EPDs in the environments ranged from 0.97 to 0.99, 0.69 a 0.95 and 0.77 to 0.98 for PD, PS and GP, respectively. In the second method, a random regression model was performed in order to describe changes in breeding values as a function of the gradient environment, arranged by contemporary groups. The analyses were performed by INTERGEN, also under bayesian inference. The estimates of heritability coefficients for PD, PS and GP ranged 0.06 to 0.44, 0.19 to 0.63 and 0.20 to 0.40, respectively. The genetic correlation between level and slope of reaction norms were 0.75 for PD, 0.76 for PS and 0.34 for GP. The correlation between sire\'s breeding values in the environments ranged from - 0.38 to 0.99, 0.79 to 1.00 and 0.68 to 0.99 for PD, PS and GP, respectively. The results of both methods shown effect of genotype by environment interaction over the traits in herds included in this study, especially over the ranking of sires.

Em experimentos de genótipos ambiente são comuns à presença de valores ausentes, devido à quantidade insuficiente de genótipos para aplicação dificultando, por exemplo, o processo de recomendação de genótipos mais produtivos, pois para a aplicação da maioria das técnicas estatísticas multivariadas exigem uma matriz de dados completa. Desta forma, aplicam-se métodos que estimam os valores ausentes a partir dos dados disponíveis conhecidos como imputação de dados (simples e múltiplas), levando em consideração o padrão e o mecanismo de dados ausentes. O objetivo deste trabalho é avaliar a eficiência da imputação múltipla livre da distribuição (IMLD) (BERGAMO et al., 2008; BERGAMO, 2007) comparando-a com o método de imputação múltipla com Monte Carlo via cadeia de Markov (IMMCMC), na imputação de unidades ausentes presentes em experimentos de interação genótipo (25) ambiente (7). Estes dados são provenientes de um experimento aleatorizado em blocos com a cultura de Eucaluptus grandis (LAVORANTI, 2003), os quais foram feitas retiradas de porcentagens aleatoriamente (10%, 20%, 30%) e posteriormente imputadas pelos métodos considerados. Os resultados obtidos por cada método mostraram que, a eficiência relativa em ambas as porcentagens manteve-se acima de 90%, sendo menor para o ambiente (4) quando imputado com a IMLD. Para a medida geral de exatidão, a medida que ocorreu acréscimo de dados em falta, foi maior ao imputar os valores ausentes com a IMMCMC, já para o método IMLD estes valores variaram sendo menor a 20% de retirada aleatória. Dentre os resultados encontrados, é de suma importância considerar o fato de que o método IMMCMC considera a suposição de normalidade, já o método IMLD leva vantagem sobre este ponto, pois não considera restrição alguma sobre a distribuição dos dados nem sobre os mecanismos e padrões de ausência.
In trials of genotypes by environment, the presence of absent values is common, due to the quantity of insufficiency of genotype application, making difficult for example, the process of recommendation of more productive genotypes, because for the application of the majority of the multivariate statistical techniques, a complete data matrix is required. Thus, methods that estimate the absent values from available data, known as imputation of data (simple and multiple) are applied, taking into consideration standards and mechanisms of absent data. The goal of this study is to evaluate the efficiency of multiple imputations free of distributions (IMLD) (BERGAMO et al., 2008; BERGAMO, 2007), compared with the Monte Carlo via Markov chain method of multiple imputation (IMMCMC), in the absent units present in trials of genotype interaction (25)environment (7). This data is provisional of random tests in blocks with Eucaluptus grandis cultures (LAVORANTI, 2003), of which random percentages of withdrawals (10%, 20%, 30%) were performed, with posterior imputation of the considered methods. The results obtained for each method show that, the relative efficiency in both percentages were maintained above 90%, being less for environmental (4) when imputed with an IMLD. The general measure of exactness, the measures where higher absent data occurred, was larger when absent values with an IMMCMC was imputed, as for the IMLD method, the varied absent values were lower at 20% for random withdrawals. Among results found, it is of sum importance to take into consideration the fact that the IMMCMC method considers it to be an assumption of normality, as for the IMLD method, it does not consider any restriction on the distribution of data, not on mechanisms and absent standards, which is an advantage on imputations.

Les mutations de résistance génotypiques constituent un problème majeur pour l’optimisation du traitement antirétroviral chez les patients infectés par le VIH-1 naïfs au traitement ou prétraités. Cependant, l’analyse de l’impact des mutations sur la réponse au traitement est compliquée par i) le nombre élevé de mutations, ii) la colinéarité possible entre ces mutations, iii) le faible nombre de patients inclus dans les études et iv) la définition du critère de jugement. Les objectifs de cette thèse sont 1) de donner une vue d’ensemble et de discuter, en collaboration avec le réseau européen NEAT (European AIDS treatment network), les critères de jugement utilisés dans les essais cliniques récents et ceux utilisés lors de l’analyse des mutations de résistance, 2) d’évaluer l’impact des mutations génotypiques sur la réponse au traitement chez les patients naïfs dans le cadre d’une grande collaboration Européenne (EuroCoord-CHAIN) et 3) de comparer des méthodes adaptées pour les données à haute-dimension dans le but de construire un score génotypique pour la prédiction de la réponse virologique chez les patients prétraités. Les critères de jugement composites sont les plus utilisés dans les essais cliniques récents mais un critère purement virologique devrait être utilisé pour l’analyse de l’impact des mutations génotypiques. Les mutations de résistance transmises impactent sur la réponse à la première ligne de traitement si le traitement antirétroviral n’est pas adapté au génotype du virus du patient. L’analyse en composantes principales et l’analyse partial least square avaient une bonne capacité à prédire la réponse virologique mais n'étaient guère meilleures que le score génotypique. Nous allons continuer à travailler sur la comparaison de ces méthodes utilisant des critères de jugement différents dans le cadre de notre collaboration avec le Forum for collaborative HIV research
Genotypic resistance mutations are a major concern for antiretroviral treatment optimisation in HIV-1 infected treatment naïve and treatment experienced patients. However, the analysis of the impact of genotypic mutations on treatment outcome is hampered by methodological issues such as the i) high number of possible mutations, ii) the potential collinearity between mutations, iii) the low number of patients included in those studies and iv) the definition of a virological endpoint. The objective of this thesis are 1) to give an overview and to discuss endpoints used in recent clinical trials in collaboration with European AIDS treatment network (NEAT) and those used in the context of drug resistance analysis, 2) to investigate the impact of genotypic resistance mutations on treatment outcome in treatment naïve patients in a huge European collaboration EuroCoord-CHAIN and 3) to compare methods adapted for high-dimensional data in order to construct a genotypic score to predict treatment outcome in treatment experienced patients. We saw that most of the endpoints used in recent clinical trials are composite endpoints but pure virological outcomes should be used for the evaluation of drug resistance mutations. Transmitted drug resistance mutations impact on virological outcome of initial antiretroviral therapy if the treatment of the patient is not adapted to the viral genotype the patient is harbouring. Principal component analysis and partial least square showed a good performance but had only a slightly better predictive capacity for a virologal outcome compared to the genotypic score. We continue working on the comparison of these and other methods using different endpoints in the context of a collaboration with the Forum for collaborative HIV research

Pour se développer, survivre ou se reproduire, les organismes ont besoin d’énergie, généralement acquise par l’alimentation. Or cette ressource alimentaire est présente en quantité fluctuante et limitée dans l’environnement, obligeant les êtres vivants à faire des compromis et à répartir leur énergie entre différentes fonctions. Ces compromis évolutifs sont visibles à l’échelle d’une population sous la forme d’une relation négative entre traits appelée trade-off. Les trade-offs ont longtemps été considérés comme étant uniquement la résultante d’une allocation différentielle des ressources et comme étant immuables. Ainsi, allouer davantage d’énergie à un trait, la survie par exemple, réduit nécessairement la part pouvant être redistribuée aux autres traits tel que la fécondité ou la croissance. Or l’allocation différentielle des ressources est un processus régulé par un mécanisme endocrinien, lui-même codé génétiquement et donc capable d’évoluer. L’objectif de cette thèse a été de comprendre, de manière théorique, comment l’évolution du mécanisme endocrinien impacte la forme des trade-offs et comment la forme des trade-offs elle-même évolue. Pour ce faire, j’ai développé des modèles évolutifs où l’allocation des ressources est régie par un système endocrinien. Ce système peut évoluer, sous l’effet de mutations impactant l’expression et la conformation des hormones et des récepteurs qui composent ce système endocrinien. J’ai ainsi pu montrer que les relations négatives entre traits peuvent évoluer et que leur forme dépend fortement d’un paramètre rarement considéré : le coût du stockage. Dans un second temps, j’ai étudié l’impact de la variabilité temporelle dans l’abondance d’une ressource alimentaire sur l’allocation différentielle et les mécanismes endocriniens sous–jacents. Mon projet de thèse comprend un troisième volet, complémentaire à la partie théorique, qui s’attache à tester certaines prédictions empiriquement. J’ai mené une expérience de sélection artificielle dans laquelle je contrôlais la topologie d’un paysage de fitness, permettant de sélectionner des combinaisons de trait n’appartenant pas à la relation phénotypique habituellement observée. Cette expérience, conduite chez Drosophila melanogaster durant 10 générations, a montré que l’évolution peut effectivement se produire dans ce contexte, remettant partiellement en cause notre compréhension des mécanismes sous-tendant l’expression des traits phénotypiques
In order to grow, survive or reproduce, all organisms need energy, usually acquired through diet. However, this food resource is present both in fluctuating and limited quantities in the environment, forcing living beings to compromise and thus to divide their energy between their different functions. These evolutionary compromises, visible at the scale of a population in the form of a negative relationship between traits, are called trade-off. Trade-offs have long been considered as the result of a differential resource allocation and as immutable. Therefore, allocating more energy to a trait such as survival necessarily reduces the amount that can be redistributed to other traits, such as fecundity or growth. It is noteworthy that the differential allocation of resources is a process regulated by an endocrine mechanism, itself genetically coded and thereby able to evolve. The aim of my PhD thesis was to understand, theoretically, (i) how the evolution of the endocrine mechanism impacts the shape of trade-offs and (ii) how the shape of trade-offs itself evolves.To do so, I first developed evolutionary models where the allocation of resources is governed by an endocrine system. This system can evolve under the effect of mutations that impact both the expression and the conformation of hormones and receptors constituting this endocrine system. Thanks to this model, I show that the negative relationships between traits can evolve and that their shape strongly depends on a parameter rarely considered: the cost of storage. In a second step, I studied the impact of temporal variability in food abundance on the endocrine mechanisms responsible for the differential allocation of resources.Lastly, my thesis project includes a component complementary to the theoretical part, which attempts to empirically test certain of the expressed predictions. I conducted an artificial selection experiment in which I controlled the topology of a fitness landscape, thus allowing to select combinations of traits not belonging to the phenotypic relationship usually observed. This experiment, implemented in Drosophila melanogaster for 10 generations, has shown that evolution can indeed occur in this context, thereby partially challenging our understanding of the mechanisms underlying the expression of phenotypic traits

A incidência de doença respiratória pelo vírus sincicial respiratório humano (VSRH) avaliada em uma coorte de recém-nascidos, entre dezembro/2002 a setembro/2005, foi de 9,84/1000 criança-mês.Um total de 316 amostras de lavado de nasofaringe, foram processadas por três diferentes técnicas (isolamento viral, imunofluorescência direta e PCR) para detecção de vírus respiratório sincicial humano (VSRH). Destas, 36 (11,4%) foram positivas para o VSRH. A PCR foi à técnica mais sensível, sendo positiva em 35 (11,1%) das amostras, seguido da imunofluorescência direta (25/316, 7,9%) e isolamento viral (20/315, 6,3%) (p < 0,001). Os dados do presente estudo sugerem que o conceito de isolamento viral como "padrão ouro" no diagnóstico do VSRH seja revisto.
The incidence of respiratory illnesses caused by the human respiratory syncytial virus (HRSV) in a cohort of neonates between December 2002 and September 2005 was 9.84/1000 children/month. A total of 316 samples of nasopharyngeal lavage were processed using three different techniques (viral isolation, direct immunofluorescence and PCR) to detect the human respiratory syncytial virus (HRSV). Of these, 36 (11.4%) were positive for HRSV. PCR was the most sensitive technique. It was positive in 35 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). The findings of this study suggest that the view that viral isolation is the "gold standard" for diagnosis of HRSV should be reconsidered.

Les deux virus d’ARN VIH et VHC attirent beaucoup d’attention de la santé publique parce qu’ils partagent des mêmes facteurs de risque de transmission par le sang et par des contacts sexuels. En plus, les maladies associées aux infections par le VIH et par le VHC sont des causes principales de mortalité et de morbidité globalement. Néanmoins, grâce à l’introduction des thérapies d’antirétroviraux pour le traitement de l’infection par le VIH et les antiviraux à action directe (AADs) pour le traitement de l’infection par le VHC, les patients infectés par ces virus constatent une amélioration significative de leur qualité de vie. Cependant, le taux de réplication élevé et l'absence de mécanisme de correction d'erreur de ces virus génèrent une population virale diversifiée appelée des quasi-espèces. Sous la pression sélective de traitement, les quasi-espèces virales sélectionnent des variants de résistance au médicament correspondant et rendent la thérapie inefficace, en particulier dans les cas où une surveillance appropriée du traitement n'est pas assurée. Afin de réserver un large choix de traitement antirétroviral tout au long de la vie chez les patients infectés par le VIH et, parallèlement, de réduire le coût de traitement des infections par le VIH et le VHC, il est fondamental de mener des recherches ciblées sur la détection, la surveillance et la transmission des mutations de résistance aux antiviraux. Dans cette thèse, nous avons utilisé les technologies de séquençage haut-débit (UDS) ou de séquençage de nouvelle génération (NGS) pour chercher des variants de résistance minoritaires (MiRVs) qui sont classiquement considérés comme représentant moins de 15%-25% de la population virale et ne sont pas trouvés par la technique de séquençage Sanger. La présence des MiRVs à baseline est responsable possiblement de l'échec de traitement et leur présence à l'échec peut limiter les options des traitements ultérieurs. Dans cette thèse, nous avons évalué la prévalence et l’impact clinique des MiRVs sur le gène de l’intégrase chez les patients infectés par le VIH qui ont été en échec d’un régime thérapeutique contenant un inhibiteur de l’intégrase. Nous avons également évalué l’impact des MiRVs chez les patients infectés par le VHC de génotype 3 et de génotype 4 et ayant échoué aux AADs. De plus, nous avons utilisé la technique UDS pour identifier et caractériser les chaines de transmission du VHC parmi une population clé d'hommes ayant des rapports sexuels avec des hommes, co-infectés par le VIH ou ayant un risque élevé d'acquisition du VIH. Nous avons également observé plusieurs cas d’infections mixtes de génotype du VHC dans cette population, probablement à cause du risque élevé de multiples expositions au VHC. Les questions sur l’avantage de l'UDS dans la recherche en virologie et l'applicabilité de cette technique en clinique ont été posées et vérifiées au travers de multiples types de projets dans cette thèse. L’UDS n’a pas été établi de manière concluante comme étant plus intéressant et bénéfique que la technique de séquençage de Sanger pour prévenir l’échec au traitement chez les patients infectés par le VIH ou le VHC et pour identifier les chaînes de transmission à grande échelle si le coût et le temps de l’expérience pour l’analyse des données sont pris en compte. Cependant, le développement dynamique des nouvelles technologies de l’UDS et les efforts sans cesse afin d’optimiser des procédures d'analyse montrent un rôle prometteur de l’UDS. Et l'applicabilité de l'UDS en pratique clinique devra encore être élucidée dans différents types de projets de recherche
The two RNA viruses HIV and HCV are getting a lot of public health concerns because both of them have overlapping risk factors for transmission through direct blood and sexual contacts. Furthermore, HIV and HCV infections are the leading cause of mortality and morbidity globally due to related diseases. However, with the introduction of antiretroviral therapy (ART) for the treatment of HIV infection and direct-acting antivirals (DAAs) for the treatment of HCV infection, patients infected by these viruses are witnessing significant improvement in their quality of life. However, the high replication rate and the lack of error correction mechanism of these viruses result in a diverse viral population referred to as quasispecies. Under drug- selective pressure, the viral quasispecies select resistance variants against corresponding drug and render the therapy ineffective especially in cases an appropriate treatment monitoring is not ensured.To reserve a wide range of possibilities for a life-long ART in HIV-infected patients and in parallel to reduce cost for treatment of both HIV and HCV infection, research focusing on detection, surveillance and transmission of resistance mutations is fundamental to prevent treatment failure on antivirals. In this PhD, we employed the ultra-deep sequencing (UDS) or next-generation sequencing (NGS) technologies to look for minority resistant variants (MiRVs) which are conventionally considered to represent less than 15%-25% of viral population and undetectable by Sanger sequencing. The presence of MiRVs at baseline is possibly responsible for the treatment failure and their presence at failure may limit options for subsequent therapies. In this PhD, we evaluated the prevalence and clinical impact of MiRVs on integrase gene in HIV-infected patients failing an integrase inhibitor containing regimen. We also evaluated the impact of MiRVs in HCV genotype 3 and genotype 4-infected patients failing DAAs. Furthermore, we used the UDS technique to identify and characterize the HCV transmission networks among a key population of men having sex with men either co-infected with HIV or at high risk of HIV acquisition. We also discovered several cases of mixed HCV genotype infections in this population probably for their high risk of multiple HCV exposures. The advantages of UDS in virology research and the applicability of this technique in clinic have been questioned and verified throughout multiple types of projects in this PhD. UDS has not been conclusively established to be more interesting and beneficial than Sanger sequencing in prevention of treatment failure in patients infected by HIV or HCV and in identifying the viral transmission networks at large scale if taking into account the experiment cost and time for data analysis. However, the dynamic development of UDS technologies and the continuing attempts in optimizing analysis procedures display a promising role of UDS. And the applicability of UDS in clinical practice still needs to be elucidated in different kinds of research projects

碩士
國立屏東科技大學
動物疫苗科技研究所
103
Newcastle disease virus (NDV), also known as avian paramyxovirus serotype 1, is a member of the family Paramyxoviridae, belongs to OIE List A, and causes a highly contagious respiratory, neurological, or enteric disease in chickens. Historically, NDV isolates have been classified into 10 genotypes (genotype I to X), based on the nucleotide sequences of fusion protein. The virulent live vaccine strains LaSota, B1 and the virulent inactivated vaccine strain Sato presently in worldwide use. In contrast, the LaSota, B1 and Sato vaccine strains were isolated some 60 years ago and belong to genotype II and III. Currently, genotype VII strains are the predominant virulent strains circulating and have recently been isolated in broiler, layer, and breeder farms worldwide. Although the different genotypes of NDV all belong to one serotype, it is still difficult to confer cross-protection under stressful environmental conditions. In this work, we have attenuated a field genotype VII strain by 39 serial passages through BHK-21 cells. The hemagglutination test (HA) reached 256, the MDT of NDV strains is higher than 159 hours, and the intracerebral pathogenicity indexes (ICPI) had decreased to 0. The attenuated strain, KGM-01, is then inactivated and mixed with oil adjuvants for vaccine evaluation. Safety, protective efficacy, stability of the vaccine was evaluated in eggs. Hemagglutination inhibition test showed that the inactivated vaccine elicited high antibody titer two weeks after immunization. In challenge tests, little virus shedding was detected using real-time PCR when challenged with the Sato strain and field type VII strains, and all chickens remained healthy. Thus, KGM-01 is a low virulent, type VII genotype strain with high antigenicity suitable for inactivated NDV vaccine development.

Since it was discovered in 1926 in England and Indonesia, Newcastle Disease Virus (NDV), especially from Genotype VII (GVII), has caused death in chickens that have been vaccinated using the NDV vaccine Genotype II (GII), known as a heterologous vaccine. Vaccines from the GVII strain, also known as homologous vaccines, have prevented NDV outbreaks. Determination of the differences between the two vaccines was done using a transcriptomic method to determine the response of chickens at the genetic level; with serology approach and viral loading counting; and differences between the two strains genetically using whole-genome sequences. Thirty, three-week-old specific-pathogen-free (SPF) chickens were divided into three groups. The first group was a negative control, the second group was vaccinated with GIIvacc, and the third group was vaccinated with GVIIvacc. Treatment groups were immunized with vaccines on day 14 and day 28. Sera were obtained from all groups on day 28 for the serology tests. On day 42, the spleen was collected for transcriptomic. Meanwhile, the whole genome sequence samples were obtained from the NDV outbreak in 2015 in Indonesia, Genotype II strain, and challenge strain to determine vaccine effectiveness Spleen transcriptomic showed that GVIIvacc down-regulates the neuroinflammation pathway but increases the communication activity among neurons as part of the synaptogenesis pathway. Thus, it is speculated that suppressing the neuroinflammation pathway is associated with protecting the nervous system in chickens from excess leukocytes and cytokine activity. Meanwhile, GIIvacc only prevents apoptosis by suppressing PERK/ ATF4/CHOP as part of the unfolded protein response (UPR) pathway. Thus, the use of GVIIvacc should be considered in countries where GVII strain causes NDV outbreaks. The transcriptomic result aligned with serological and challenged virus test that homologous vaccine (GVIIvacc) gave better protection by reducing the viral shedding and had higher protective antibodies than a heterologous vaccine (GIIvacc). In particular, the Hemagglutination Inhibition (HI) test showed that antibody titers were higher when tested with homologous antigen. However, the cleavage site of the Fusion (F) protein from GII and GVII were used as alternative antigens in an ELISA, did not perform well to obtain the relevant antibody titer. After being challenged with GVII, viral shedding from vaccinated chickens with GVIIvacc was significantly reduced compared to chickens vaccinated with GIIvacc. Both chicken groups showed no clinical signs. The whole-genome sequence and phylogenetic tree results showed that GVII is still the dominant NDV strain that causes NDV outbreaks in Indonesia. In addition, ITA strains for testing the vaccine's efficacy belong to GVI. Hence, using GVI as a heterologous strain from the field as a challenge strain for effective vaccine testing should be considered in veterinary laboratories, especially in Indonesia. All the results from my research study suggested that to combat NDV, the vaccine, antigen for antibody titer, and antigen as challenge strain for effectivity vaccine need to be homologous or coming from the NDV genotype, which causes an outbreak in the field.
Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 2021

Since it was reported in Indonesia in 1926, Newcastle disease (ND) is an endemic disease in Indonesia resulting in devastating economic losses in poultry. Despite heavy vaccination programs, NDV infection still occurs among commercial chicken farms, including vaccinated birds in Southeast Asia, especially in Indonesia. Therefore, the first objective was to detect and perform full genome sequencing of highly virulent circulating NDVs in vaccinated chicken flocks to identify the most adequate NDV strains as vaccine candidates. Our full genome sequencing analysis on two selected isolates from vaccinated birds in Indonesia has shown that both of them belong to highly virulent NDV-GVII.2 strains, while only 0.4% of the vaccine strains used in this country are genotype VII. Moreover, sequencing analysis of the existing genotype VII vaccine revealed that it has significant differences from GI vaccine strain, which is not sufficiently representative of genotype VII viruses. This finding illustrates that virulent NDV-GVII.2 strains are mainly responsible for the high morbidity and mortality of recent ND outbreaks in poultry in Indonesia. Virulent NDV-GVII strains were previously characterised as immunopathological phenotypes triggering severe tissue damage probably through apoptosis and necrosis of lymphoid tissues. However, the underlying molecular mechanism of pathogenesis of ND remains to be fully understood. Hence, the second objective was to identify and characterise the molecular mechanism of lymphotropic behaviour of NDV-GVII by focusing on detecting key biomarkers and cellular immune response signalling pathways that contribute to severe lymphocyte destruction in infected birds using RNA sequencing, bioinformatics tools and PPI network analysis. Transcriptomic profiling indicates that virulent NDV-GVII significantly downregulates immunologically regulated genes and innate immune regulating pathways including fMLP signalling in neutrophils, Fcy receptor-mediated phagocytosis in macrophages and monocytes, and leukocyte extravasation signalling and NF-kB activation by viruses. These transcriptional changes may lead to widespread immunosuppression and enhanced replication of the virus. As a result, the host’s immune response may be diminished, delayed, incomplete or display overly strong induction that can cause severe tissue damage. This finding also implies that virulent NDV-GVII strains appear to possess the capability to inhibit the induction of immune responses by targeting lymphocytes and destroying these cells, which may be one indispensable factor of the pathogenesis of NDV-GVII. Moreover, PPI network analysis revealed that the top three significantly enriched gene modules were phagocytosis, immune response-related terms, and glutamate receptor signalling pathway. We identified novel genes EGF, LPAR5, AGT, AGTR1, RAC2, CD4, CD3D, IL7R, NPY, GRM3, and GRAP2 as potential biomarkers. This study provides valuable information to help understand novel immune evading mechanisms of highly pathogenic NDV-GVII from a host-pathogen interaction point of view. Interactions between the virus and the host determine the success of the viral infection. Hence, the third objective was to identify any particular genes of the virus that may have an important role in virulence and pathogenicity by profiling the transcription of virus in infected birds. This study revealed the transcriptional gradient of these highly pathogenic NDV-GVII genes: NP:P:M:F:HN:L, in which there were a slight attenuations at the NP:P and HN:L gene boundaries. Our result also provides a fully comprehensive qRT-PCR protocol for measuring viral transcript abundance that may be more convenient for laboratories where accessing HTS is not feasible.
Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 2022

Congenital factor VII deficiency (FVIID) is the most frequent of the rare autosomal recessive bleeding disorders, comprising a wide molecular spectrum that correlates with plasma FVII levels and sometimes a bleeding phenotype. Establishing clinical significance for a variant is essential for an efficient diagnosis resulting in better patient care. For this, the variants must be submitted to the same methodological analysis of variant classification according to the ACMG recommendations. In a cohort of 550 patients from central Portugal, diagnosed with FVII deficiency over a period of 20 years, between February 2001 and June 2021, at the Laboratory of Molecular Hematology - SHC/CHUC, data of the functional and molecular studies were analyzed. After evaluating the genotype-phenotype of the variants identified in the F7 gene, these were then reclassified according to the international guidelines of the ACMG. For some cases it was necessary to perform molecular analyzes using PCR, MLPA, and direct Sanger sequencing. 404 unrelated patients (probands) had FVII levels<60% when compared with healthy individuals. Individuals with FVII levels<1% showed variants in homozygosity and lethal cases have been described. We found some unusual cases such as a double heterozygous patient for partial deletion of F7 and F10. A total of 32 rare F7 variants were found in a group of 185 individuals with polymorphisms and pathogenic variants, the majority being missense. Of these 32 variants, eleven were considered new, i.e., not previously described. This study allowed prenatal diagnosis to be available when requested. All variants were reclassified according to the ACMG international guideline, where most are deemed pathogenic or likely pathogenic. It was concluded that all the information found in this study is of relevant importance to understand the genotype/phenotype factors that contribute to the development of the knowledge of FVII deficiency in the Portuguese population. This combination of procedures ensured a more efficient diagnosis: analyzing the severity of FVII deficiency with greater precision for a more effective therapeutic approach.
A deficiência congénita de fator VII (FVIID) é o mais frequente dos distúrbios hemorrágicos autossómicos recessivos, compreendendo um amplo espectro molecular que se correlaciona com os níveis plasmáticos de FVII e, por vezes, um fenótipo de hemorragia. Estabelecer o significado clínico para uma variante é essencial para um diagnóstico eficiente resultando num melhor atendimento ao doente. Para isso, as variantes devem ser submetidas à mesma análise metodológica de classificação de variantes conforme as recomendações da American College of Medical Genetics and Genomics (ACMG). Num grupo de 550 pacientes do centro de Portugal, diagnosticados com a deficiência de FVII ao longo de um período de 20 anos, entre fevereiro de 2001 e junho de 2021, no Laboratório de Hematologia Molecular - SHC / CHUC, foram analisados dados dos estudos funcionais e moleculares. Após avaliado o genótipo fenótipo das variantes identificadas no gene F7, em seguida as mesmas foram reclassificadas de acordo com as guidelines internacionais ACMG. Para alguns casos em que se verificou ser necessário, as análises moleculares foram efetuadas por meio de PCR, MLPA e sequenciação direta de Sanger. Nesta grupo, 404 doentes não relacionados (propósitos) tiveram níveis de FVII<60% quando comparado com os indivíduos saudáveis. Os indivíduos com níveis FVII:c<1% apresentaram variantes em homozigotia e foram descritos alguns casos letais. Foram também encontrados alguns casos raros como o de um doente duplo heterozigótico para deleção parcial do F7 e F10. Um total de 32 variantes raras no F7 foram encontradas num grupo de 185 indivíduos com polimorfismos e variantes patogénicas, sendo a maioria missense. Destas 32 variantes, onze foram consideradas novas, i.e., não descritas previamente. Este estudo permitiu disponibilizar o diagnóstico pré-natal quando solicitado. Todas as variantes foram reclassificadas de acordo com as guidelines internacionais ACMG, onde a maioria foi considerada patogénica ou provável patogénica. Concluímos que todas as informações encontradas nesse estudo foram de importância relevante para compreender o genótipo/fenótipo destes doentes e, assim, elucidar e desenvolver o conhecimento sobre a deficiência do FVII da população em Portugal. Este conjunto de procedimentos possibilitou um diagnóstico mais eficiente: analisar a gravidade da deficiência do FVII com maior precisão para uma abordagem terapêutica mais eficaz.
Mestrado em Bioquímica

North Atlantic and South Atlantic swordfish (Xiphias gladius L.) are currently managed as two stocks separated at 5°N. While previous studies of genetic population structure using both mitochondrial and nuclear DNA confirm two genetically distinct stocks, sampling coverage has not been uniform or representative of all areas and estimates of admixture in areas of contact have not been provided. In this study, we examined: 1) the applicability of high-resolution melting analysis (HRMA) in population genetic studies of non-model organisms, 2) the use of nuclear markers in Atlantic swordfish and the methodology whereby nuclear gene variation can be quickly screened, identified, and genotyped using short-amplicon (SA) HRMA and unlabeled probe (UP) HRMA, and 3) the use of HRMA to characterize nuclear markers to study the genetic population structure of Atlantic swordfish using representative samples of the entire basin to provide an estimation of population admixture by means of Bayesian individual assignment. High resolution melting analysis (HRMA) is shown to be a highly sensitive, rapid, closed-tube genotyping method amenable to high throughput and, though until recently primarily confined to clinical studies, suitable for population studies in non-model species. Ten nuclear markers were genotyped primarily by SA- and UP-HRMA in North Atlantic (n=419), South Atlantic (n=296), and Mediterranean (n=59) swordfish. Comparisons of pairwise FST, AMOVA, PCoA, and Bayesian individual assignments were congruent with previous finding of three discrete populations with comparatively low levels of estimated gene flow for a marine organism (FST = 0.039-0.126). Population admixture was identified and estimated in the Northeast Atlantic and appeared to be asymmetrical, with swordfish from the South Atlantic found among North Atlantic localities but no North Atlantic migrants identified in the South Atlantic. The Mediterranean boundary currently at the Strait of Gibraltar is found to extend west into Atlantic waters to approximately 8°W. Similarly, the boundary between North and South Atlantic swordfish should be revised to a line that extends north from 0°N 45°W to 25°N 45°W and from that position, as a nearly horizontal line, eastwards to the African coast. Finally, I show that Bayesian individual assignment using the developed marker set can be used for mixed stock allocation in the Northeast Atlantic.