Plasmodium malariae é um parasita protozoário que causa malária em humanos e é geneticamente indistinguível de P. brasilianum, um parasita que infecta macacos do Novo Mundo nas Américas do Sul e Central. P. malariae possui uma ampla distribuição mundial em regiões tropicais e subtropicais, porém de forma pontual, sendo encontrado na América do Sul, Ásia e África. Entretanto, pouco se sabe sobre a genética destes parasitas e a similaridade entre eles pode devido ao pequeno número de sequências genômicas disponíveis para essas espécies de Plasmodium. Recentemente, seis marcadores microssatélites e a sequencia completa do gene que codifica a proteína de superfície do merozoíta 1 (MSP1) foram descritos para estes parasitas. Neste estudo, o polimorfismo genético de 24 isolados brasileiros de P. malariae e P. brasilianum obtidos de diferentes hospedeiros foi analisado através da utilização desses marcadores microssatélites e da região correspondente ao oitavo bloco da MSP1. Os dados epidemiológicos moleculares foram explorados em relação à origem geográfica e hospedeiros. Para todos os marcadores estudados, as amostras de símios apresentaram um polimorfismo mais elevado que as amostras humanas. Na análise de microssatélites, as amostras humanas foram polimórficas em apenas dois alelos, enquanto as amostras de símios foram polimórficas em cinco alelos. O alelo Pm42- 331 foi monomórfico em todas as amostras analisadas. Na análise do bloco 8 da MSP1, as amostras símias foram altamente polimórficas e as amostras humanas apresentaram quatro tipos alélicos, sendo que dois tipos alélicos (A5 e A7) foram encontrados em alta frequência (90%). Em ambas as análises, a amostra de mosquito foi mais similar a amostras simianas. Nossos dados também mostram que há uma possível ausência de dimorfismo alélico na MSP1 de P. malariae e P. brasilianum, ao contrário de outras espécies de Plasmodium. Pela primeira vez, amostras de humanos, símios e mosquito foram analisadas em conjunto e utilizadas para o primeiro estudo de polimorfismos genéticos de isolados de P. malariae e P. brasilianum do Brasil. / Plasmodium malariae is a protozoan parasite that causes malaria in humans and is genetically indistinguishable from P. brasilianum, a parasite infecting New World monkeys in Central and South America. P. malariae has a wide and patchy global distribution in tropical and subtropical regions, being found in South America, Asia, and Africa. However, little is known regarding the genetics of these parasites and the similarity between them could be because until now there are only a very few genomic sequences available from these Plasmodium species. Recently, six microsatellite markers and the complete sequence of the merozoite surface protein 1 (MSP1) gene have been described for these parasites. In this study, the genetic polymorphism of 24 P. malariae and P. brasilianum isolates obtained from different hosts was analyzed using these microsatellite markers and the corresponding region on the block 8 of MSP1. The molecular epidemiological data were explored in relation to geographical origin and hosts. For all markers studied, the simian samples showed a higher polymorphism than human samples. In microsatellite analysis, the human samples were polymorphic only in two alleles, while simian samples were polymorphic in five alleles. The allele Pm42-331 was monomorphic in all samples analyzed. In the analysis of Block 8 of MSP1, the simian samples were highly polymorphic and human samples showed four allele types with two allelic types (A5 and A7) frequently found (90%). In both analyzes, the mosquito sample was more similar to simian samples. Our data also show that there is a likely absence of allelic dimorphism of MSP1 from P. malariae and P. brasilianum, as opposed to other Plasmodium species. For the first time, samples of humans, monkeys and mosquito were analyzed together and used for the first study of genetic polymorphisms in P. malariae and P. brasilianum isolates from Brazil.

Lactobacilos são um grupo de bactérias relacionadas com cárie dental. Há falta de estudos sobre a biologia populacional dos lactobacilos na cárie dental. O objetivo do estudo foi avaliar o efeito das modificações ambientais na composição, diversidade genética dos lactobacilos e Identificar a filogenia dos Lactobacillus paracasei isolados do biofilme. Lactobacilos foram isolados em um modelo de formação de biofilme in situ antes e depois de 28 dias de exposição à solução de sacarose 20%. As colônias foram randomicamente selecionadas do meio Rogosa Ágar e subcultivadas (n=222, 31 antes e 191 após período de exposição à sacarose). Os isolados foram identificados usando o seqüenciamento parcial dos genes pheS ou rpoA. As espécies de lactobacilos predominantes encontradas foram L. paracasei, L. fermentum e L. rhamnosus. A diferença na composição e na diversidade genética de lactobacilos associada às modificações ambientais no biofilme foi analisada através de PCR utilizando palíndromes repetitivos extragênicos (REP-PCR). Após a fase com sacarose, um maior número de lactobacilos pode ser encontrado no biofilme (p=0,001). A prevalência de L.fermentum foi similar na fase sem sacarose (6/11 indivíduos colonizados) e na fase com sacarose (8/11 indivíduos colonizados) (p=0,721). A prevalência de L. rhamnosus e L. paracasei aumentou no biofilme de 2/11 para 8/11 indivíduos (p= 0,028) e de 2/11 para 7/11 indivíduos colonizados (p=0,012) após exposição a sacarose, respectivamente. A prevalência de L. gasseri foi baixa e em ambas as fases (p=1,00). As espécies de Lactobacilos apresentaram maior diversidade na fase com sacarose (2 a 3 espécies por indivíduo) do que na fase sem sacarose (0 a 2 espécies por indivíduo) (p=0,045). Na maioria dos casos, diferentes genótipos estavam presentes na fase sem sacarose em comparação à fase com sacarose (p=0,01). Aqueles lactobacilos identificados como L. paracasei foram também submetidos à tipificação através do seqüenciamento de múltiplos loci (MLST). No MLST, foi obtida a sequência parcial de 7 genes de referência: fusA, ileS, lepA, leuS, pyrG, recA, e recG. Sete indivíduos apresentavam L. paracasei (n=75) e 14 seqüências de tipificação (ST) foram encontradas. Verificou-se que indivíduos não relacionados podem apresentar uma mesma ST e que múltiplas STs estão presentes por indivíduo. Três indivíduos apresentavam STs previamente isoladas de produtos alimentícios lácteos. Resultados conflitantes na comparação entre REP-PCR e MLST foram observados na genotipagem de L. paracasei. Diferentes números de padrões foram obtidos para os L. paracasei de acordo com o método molecular utilizado (14 com MLST versus 25 com REP-PCR). Para algumas cepas, REP-PCR foi mais discriminatório do que MLST. Em outros casos, cepas pertencentes a diferentes STs foram agrupadas pelo REP-PCR, mostrando que o MLST foi mais discriminatório do que o REP-PCR. Em poucos casos, MLST e REP-PCR apresentaram o mesmo poder discriminatório. Em geral, REP-PCR mostrou um maior poder discriminatório em comparação ao MLST, mas pouca concordância foi apresentada entre os dois métodos. Assim, MLST pode contribuir na identificação da diversidade genética obtida pelo REP-PCR em alguns casos. Os dados obtidos permitiram concluir que após exposição a sacarose observa-se (a) aumento no número de lactobacilos e de superfícies colonizadas; e (b) aumento na diversidade genética e de espécies. Além disso, observou-se que (c) alguns lactobacilos orais podem apresentar origem exógena, e que (d) a combinação dos métodos MLST e REP-PCR aumenta o poder discriminatório quanto à diversidade genética de L. paracasei. / Lactobacilli are a group of bactéria related to dental caries. There is a lack of studies on the population biology of this organisms in dental caries. The aim of the study was to evaluate the effect of the environmental changes in the composition, genetic diversity and identify the filogeny of Lactobacillus paracasei isolated from biofilm. Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a 28-day period of exposure to 20% sucrose solution. The lactobacillus colonies were randomly selected from Rogosa Agar medium and subcultured (n=222, 31 prior to and 191 following a sucrose exposure period). The isolates were identified using pheS or rpoA gene sequence analysis. The predominant lactobacilli were L. paracasei, L. fermentum and L. rhamnosus. The difference in composition and genetic diversity of lactobacilli related to environmental changes in biofilm was analysed by repetitive extragenic palindromic PCR (REP-PCR). After the sucrose phase, a higher number of lactobacilli could be found in dental biofilm (p=0.001). The prevalence of L. fermentum was similar in the non-sucrose (6/11 subjects) compared to the sucrose phase (8/11 subjects) (p=0.721). Prevalence of L. rhamnosus and L. paracasei increased in biofilm from 2/11 to 8/11 subjects (p= 0.028) and from 2/11 to 7/11 subjects (p=0.012) after sucrose exposure, respectively. L. gasseri prevalence was low in both phases (p=1.00). Lactobacilli exhibited greater species diversity (2 or 3 species per subject) in the sucrose phase, than those isolated from the non-sucrose phase (0-2 species per subject) (p=0.045). In most of the cases different genotypes were present in the non-sucrose phase in comparison to the sucrose phase. Those lactobacilli identified as L. paracasei were subjected to multilocus sequencing typing (MLST). In MLST, partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG was obtained. Seven subjects harboured L. paracasei (n=75) and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. There were mixed results among REP-PCR patterns compared with the MLST in the L. paracasei genotyping. Different numbers of patterns were obtained for L. paracasei according to the molecular technique used (14 MLST versus 25 REP-PCR patterns). For some strains, REP-PCR was more discriminatory than MLST. In other cases, strains belonging to different ST were grouped together by REP-PCR, showing that MLST was more discriminatory than REP-PCR. In few cases MLST and REP-PCR presented the same discriminatory power. REP-PCR showed a greater discriminatory power in comparison to MLST, but little agreement was observed between this two methods. Therefore, MLST could enhance the genetic diversity obtained by REP-PCR. The present data support that after sucrose exposure there is (a) an increase in the number of lactobacilli and colonized surfaces; and (b) increase in lactobacilli species and genetic diversity. Also it was found that (c) some oral lactobacilli may be of exogenous origin; and (d) the combination of MLST and REP-PCR increased the discriminatory power in genotyping L. paracasei.

Rhipicephalus microplus belongs to the Ixodidae, the largest family of ticks, which are of great economic importance due to their negative socio-economic impact on agriculture (BROUQUI 2011; PORTO NETOA et al. 2011; SONENSHINE 1991). Acaricides have been the first choice in tick control for cattle farmers, but R. microplus rapidly acquires resistance to these chemicals. Replication slippage and recombination drive genetic diversity in tick populations (BAFFI et al. 2007; GUERRERO et al. 2007; LI et al. 2007); generating point mutations and frame shifts within the genes targeted by acaricides, resulting in resistance (BAFFI et al. 2007; HERNANDEZ et al. 2002; HERNANDEZ et al. 2000; JONSSON et al. 2010; MORGAN et al. 2009). In addition, resistance can quickly accumulate in a population due to the pangamy mating structure of ticks (CHEVILLON et al. 2007b; CUTULLÉ et al. 2010) and their ability to produce multiple generations within one season (BUDELI et al. 2009; LI et al. 2007). Vaccines have become increasingly important to control ticks, as acaricide resistance can be acquired by field tick populations within two years (RODRIGUEZ-VIVAS et al. 2011). Although Bm86 has been successful against multiple-acaricide resistant ticks, recent reports indicate that the Bm86 vaccine has become ineffective, possibly due to resistance (PARIZI et al. 2009). Also, Bm86 vaccines display great variability in terms of their efficacy against ticks isolated across Argentina (GARCIA-GARCIA et al. 2000; PARIZI et al. 2009). This is hypothesised to be due to the genetic variability between R. micoplus populations. The majority of phylogenetic studies on ticks have been based on slow evolving sequences, such as 18S or 28S rRNA, which provide genus-level resolution. The COI, D3, ANT and ITS2 genes have the potential to resolve intra-specific and interspecies variation, and may assist with the identification of cryptic speciation within R. microplus of South Africa (ANSTEAD et al. 2011; BARKER 1998; CAREW et al. 2009; MURRELL et al. 2000; SONGA et al. 2011). Phylogeography is a multidisciplinary field that utilises phylogenetic (molecular evidence of speciation) and population genetic principles (coalescence theory), in combination with additional data (such as geography and population history), to determine the genetic relationships of populations within a species (AVISE 2009) and was one of the main aspects of this study. The phylogenetic and population genetic structure of R. microplus will provide valuable information to geneticists, farmers and acaricide/vaccine suppliers about the different R. microplus tick populations of South Africa. The information will facilitate more efficient and targeted tick control whether acaricide or vaccine based as opposed to the inefficient approaches generally adopted to tick control. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Genetics / MSc / Unrestricted

At least since the Neolithic, humans have largely lived in networks of small, traditional communities. Often socially isolated, these groups evolved distinct languages and cultures over microgeographic scales of just tens of kilometers. Population genetic theory tells us that genetic drift should act quickly in such isolated groups, thus raising the question: do networks of small human communitiesmaintain levels of genetic diversity over microgeographic scales? This question can no longer be asked in most parts of the world, which have been heavily impacted by historical events that make traditional society structures the exception. However, such studies remain possible in parts of Island Southeast Asia and Oceania, where traditional ways of life are still practiced. We captured genome-wide genetic data, together with linguistic records, for a case-study system-eight villages distributed across Sumba, a small, remote island in eastern Indonesia. More than 4,000 years after these communities were established during the Neolithic period, most speak different languages and can be distinguished genetically. Yet their nuclear diversity is not reduced, instead being comparable to other, evenmuch larger, regional groups. Modeling reveals a separation of time scales: while languages and culture can evolve quickly, creating social barriers, sporadic migration averaged over many generations is sufficient to keep villages linked genetically. This loosely-connected network structure, once the global norm and still extant on Sumba today, provides a living proxy to explore fine-scale genome dynamics in the sort of small traditional communities within which the most recent episodes of human evolution occurred.

Maize is the staple food for the majority of Tanzanians. However, maize production in the Southern highlands of Tanzania (SHT) is highly reduced by gray leaf spot disease (GLS) caused by the fungus Cercospora zea maydis. GLS reduces grain yield, kernel and silage quality. The most common GLS control methods in Tanzania include amongst others; fungicides, crop rotation, field sanitation, host resistance. These methods except host resistance are, however, either expensive or less effective or unsafe to the environment. Furthermore, conventional breeding strategies are not very effective for traits, which are lowly inherited such as GLS resistance. Lastly, to date there are few GLS resistant commercial hybrids in SHT. Thus, this study aimed to produce more commercial GLS resistant hybrids, increase farmers’ hybrid choices of growing genetically different GLS insensitive hybrids, which will also provide a constant supply of GLS resistant maize cultivars in case of GLS resistance breakdown due to new GLS pathotypes. This research combined conventional breeding with molecular technologies to increase the efficacy of selecting GLS resistant hybrids and assist breeders in predicting best inbred combinations for commercial hybrid production. Studies conducted to meet the main aims were on; the prediction of best line combiners and heterosis in Tanzanian maize breeding lines through the use of amplified fragment length polymorphism, (AFLP), an association of AFLPs and the performance of phenotypic traits in maize, evaluation of maize hybrids for gray leaf spot resistance in multienvironments and finally a preliminary study on gray leaf spot PCR-based marker development with the long term objective of implementing cleaved amplified polymorphic markers (CAPS) in a marker assisted selection (MAS) strategy in the SHT maize breeding programme. Results from the study revealed that pairwise GD (genetic distance) of the lines varied from a GD of 0.13 to 0.5. High coancentry coefficients were exhibited by these lines. Joint data analyses showed that there were tighter associations between line GD and F1 traits or MPH in the intergroup than in the intragroup crosses. Combined analyses revealed that hybrids 48, 90 and 45 recorded higher stable yields and consistently low GLS scores in multienvironments. Fifteen CAPS marker bands were identified that are putatively linked to the GLS resistant genes. In summary, it was noted that strong selection during inbreeding programs should be avoided as it reduces germplasm variability. Local landraces/varieties can be improved by introgressing desirable genes into them. AFLP marker system could be effectively used for inbred genetic diversity studies in Tanzania. Intergroup crosses with high GD-MPH should be the main target for commercial hybrid production but field testing of them is inevitable to confirm their yielding potentials. Intergroups and intragroup crosses with low GD-MPH should be discarded to avoid field costs. Better F1 hybrid performance predictions can be achieved by integrating inbred GD and F1 phenotypic data. Hybrids with low GLS/high GLS resistance could be used to produce other breeding populations. Hybrids 45, 48 and 90 can be commercially preleased. Lastly a study to characterize the GLS fungus in the SHT is imperative since information on virulence of isolates is needed for long term breeding strategies against the fungus. Finally, the SHT maize germplasm has potential GLS resistant inbred lines which could be used in the deployment of genes to susceptible lines and in the development of commercial GLS resistant hybrids/open pollinated varieties/doubled haploid hybrids. / Thesis (PhD)--University of Pretoria, 2008. / Genetics / unrestricted

Toxigenic Aspergillus species produce mycotoxins that are carcinogenic, hepatotoxic and teratogenic immunosuppressing agents in both human and animals. Kenya frequently experiences outbreaks of aflatoxicosis with the worst occurring in 2004, which resulted in 125 deaths. This study sought to find possible reasons for frequent aflatoxicosis outbreaks in Kenya by isolating Aspergillus flavus strains from maize kernels sampled from different climatic regions of Kenya. Using diagonal transect random sampling, maize kernels were collected from Makueni, Homa Bay, Nandi, and Kisumu regions. The genetic diversity and variation among the isolates was examined by characterising the strains according to morphology, phenotype, vegetative compatible groups and molecular systematics. Selected atoxigenic and aflatoxigenic A. flavus isolates were also further analysed for aflatoxin production potential using quantitative real-time PCR and various bioanalytical techniques. The influence of the maize lines grown in Kisumu, Homa Bay, Nandi and Makueni region on A. flavus infection and aflatoxin production was also examined and served as the basis for an in vitro biocontrol assay. Out of 37 isolates identified, nitrate non-utilizing auxotroph’s complementation test revealed 20 vegetative compatibility groups. These groups were further designated using the prefix ʻʻKVCGʼʼ, where ʻʻKʼʼ represented Kenya and consequently assigned numbers 1 to 20 based on our findings. KVCG14 and KVCG15 had highest distribution frequency (n = 13; 10.8 %). The distribution of the L, S and S/L- morphotypes across the regions were 57 % (n = 21); 7 % (n = 3) and 36 % (n = 13) respectively. The phylogenetic analysis exhibited high diversity of A. flavus isolates from Makueni. ITS1 and ITS2 markers did not reveal significant information within intraspecies speciation of A. flavus. Furthermore, a unique isolate (KSM015) was identified that had characteristics of S-morphotype, but produced both aflatoxins B and G. Coconut agar medium (CAM) assay, TLC, HPLC and LCMS/MS analyses confirmed the presence or absence of aflatoxins in selected toxigenic and atoxigenic isolates. qPCR analysis revealed aflP, aflS, aflR and aflO transcripts as the most upregulated genes across the tested isolates whereas false detection of aflD gene transcript was observed in both induced and uninduced A. flavus isolates. Diversity Index (H) analyses ranged from 0.11 (Nandi samples) to 0.32 (Kisumu samples). Heterokaryon compatibility ranged from 33 % (for the Makueni samples, n = 3) to 67 % (Nandi samples, n = 6). The KDV1 maize line was more sensitive to A. flavus infection in comparison to GAF4. We also tested the biocontrol of atoxigenic isolates to inhibit toxin production by aflatoxigenic strains on infected maize kernels. It was shown that the atoxigenic strain (KSMO12) could inhibit the aflatoxigenic strain (KSM014) depending on the atoxigenic concentration during infection. To our knowledge, this is the first reported study for A. flavus genetic diversity, variation and distribution in Nandi, Homa Bay and Kisumu regions in comparison to and could assist researchers in the selection of biocontrol strategies to mitigate aflatoxin contamination, especially in Makueni and neighbouring regions.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18122号 / 理博第4000号 / 新制||理||1577(附属図書館) / 30980 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 伊谷 原一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(情報学) / 甲第18401号 / 情博第516号 / 新制||情||91(附属図書館) / 31259 / 京都大学大学院情報学研究科社会情報学専攻 / (主査)教授 守屋 和幸, 教授 松田 哲也, 教授 荒井 修亮 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DGAM

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13020号 / 論農博第2830号 / 新制||農||1042(附属図書館) / 学位論文||H28||N4966(農学部図書室) / 32948 / (主査)教授 祝前 博明, 教授 今井 裕, 教授 廣岡 博之 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20956号 / 理博第4408号 / 新制||理||1633(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)准教授 渡辺 勝敏, 教授 曽田 貞滋, 教授 中川 尚史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM