Hepatitis B Virus (HBV) is a DNA virus and belongs to the genus Orthohepadnavirus of the Hepadnaviridae family which represents one of two animal viruses with a DNA genome which replicates by reverse transcription of a viral RNA intermediate. Nucleotide variation led to further sub-classification into 8 genotypes (A to H). The reverse transcription step within its life cycle is prone to the introduction of errors and recombination when dually infected. This leads to a viral quasispecies which forms during the course of infection with many minor population variants; such variants can however only be detected by means of ultra-deep sequencing. A recent study in the Department of Medical Virology (UP) by Mayaphi et al. identified a number of the specimens that partitioned away from the typical subgenotype A1 clades with high bootstrap values and longer branch lengths. Thus, the main objective of the current study was to characterize the full genome of all variants for the outliers observed in the aforementioned study, inclusive of potential recombination, dual infection and minor populations. Twenty samples were selected from a previous cohort for purposes of the present study. The viral DNA was extracted and amplified by PCR according to the methods described by Günther et al. with modified primer sets. Nineteen of the samples were successfully amplified and 15 of these were sequenced. Specimens were sequenced by NGS on the Illumina MiSeq™ sequencer and sequence data used to reconstruct the viral quasispecies of each specimen. Further analyses of the reconstructed variants included molecular characterization as well as phylogenetic analysis and screening for recombination and drug resistance mutations. Full genome coverage was obtained for twelve of the fifteen samples and full genome variants reconstructed, generating nearly 40 full genomes. Phylogenetic analysis showed that the majority of the samples are of genotype A, more specifically of subgenotype A1, differing by less than 4% from known sequences. The phylogenetic analysis revealed a similar clade of outliers, where four samples clustered together with significant bootstrap support (75%) and a fifth sample partitioned separate from, yet close to, this clade, away from the typical African A1 clade. This clade was assigned to genogroup III. Three samples were of the Asian A1 clade (genogroup I) with remaining specimens grouping within genotype D and E. The variants showed low diversity within each specimen with some differing at but a few positions across the genome while even the most diverse quasispecies differed by less than a percentage (32 positions). Several unique and atypical positional variations were observed amongst study samples of which some were present in but one of the variants for that sample. Twenty-six lead to shared amino acid changes. Some observed changes, such as A1762T/G1764A and G1896A, could explain the serological patterns such as HBeAg negativity while others, such as C2002T, were previously implicated in disease progression and severity. Sample N199 presented a longer branch length and revealed short regions within the genome that display evidence of recombination between HBV/A1 and HBV/A2. The results illustrate the utility of NGS technology in characterizing viral variants. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Medical Virology / MSc / Unrestricted

Thesis (M. Sc. (Agronomy)) -- University of Limpopo, 2021 / Cowpea (Vigna unguiculata (L.) Walp) is an important annual leguminous crop grown in arid and semi-arid areas in Sub Saharan Africa. Most of the cowpea production in South Africa is mainly used for domestic consumption and, as seed for planting and little gets to be used in food processing, thus, there is a need to expand the utilization of cowpea through food processing. This study aimed to evaluate cowpea genotypes for phenotypic and canning quality traits. Field experiments were conducted at two locations in Limpopo Province, namely Syferkuil Agricultural Experimental Farm and Ga-Molepo village during the 2017/2018 growing season. The field experiment was comprised of 100 cowpea genotypes laid out in an Alpha Lattice Design replicated twice. Cowpea canning analysis was done using tomato puree following ARC-GC in-house method at the Agricultural Research Council-Grain Crops in Potchefstroom, North West Province of South Africa. Collected data on yield was analysed using Genstat 18th edition and XLSTAT 2021.1.1.1081 software for canning quality data. The results revealed significant differences among the cowpea genotypes based on the number of seeds per pod and 100 seed weight for Syferkuil. Significant differences were also observed among the studied genotypes for 100 seed weight at Ga-Molepo. The highest yield recorded across locations was for genotypes RV 555 (875.4 kg/ha), RV 207 (756,3 kg/ha), RV 439 (694.6 kg/ha) and RV 554 (682.3 kg/ha) respectively. The number of pods per plant recorded a high positive association with pod number per plant and number of seeds per pod. Grain yield of RV 558, RV 556, RV 207, RV 439 and RV 553 was high at Syferkuil and at Ga-Molepo RV 353, RV 194, IT99K-494-6, RV 341 and RV 202 recorded the highest yield. The Principal Component Analysis (PCA) revealed the three most important PCs contributing to a total variation of 76.71%. PC1, PC2, and PC3 contributed 51.01, 13.97 and 11.73%, respectively. For canning ability, out of 79 cowpea canned genotypes, only 11 genotypes were spoiled and had a bad odour. About 68 genotypes were suitable for canning including genotypes that had an excellent appearance without cracks or loose skins and even colour. Furthermore, there was vast variability among the genotypes based on yield and yield components as well as canning quality traits. Genotypes with high grain yield and had canning ability are recommended xi for canning. Data produced from this study will add useful information to the database of the characteristics of these cowpea genotypes.

In the semi-arid west, moisture is often the limiting factor in plant growth. High crop production is dependent upon rainfall and supplemental moisture supplied through irrigation. Plants for these areas, which are most efficient in use of water, other things being equal, would be more desirable.

Alleles, genotypes and haplotypes (combinations of alleles) have been widely used in gene-disease association studies. More recently, association studies using diplotypes (haplotype pairs on homologous chromosomes) have become increasingly common. This article reviews the rationale of the four types of association analyses and discusses the situations in which diplotype-based analyses are more powerful than the other types of association analyses. Haplotype-based association analyses are more powerful than allele-based association analyses, and diplotype-based association analyses are more powerful than genotype-based analyses. In circumstances where there are no interaction effects between markers and where the criteria for Hardy-Weinberg Equilibrium (HWE) are met, the larger sample size and smaller degrees of freedom of allele-based and haplotype-based association analyses make them more powerful than genotype-based and diplotype-based association analyses, respectively. However, under certain circumstances diplotype-based analyses are more powerful than haplotype-based analysis.

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Les dystrophies héréditaires vitelliformes de transmission autosomique dominante représentent la 2ème cause de maculopathie après la maladie de Stargardt, maladie récessive monogénique (ABCA4). BEST1 et PRPH2 sont les deux gènes connus associés aux dépôts vitellins. L'étude d'une large cohorte de 88 patients ayant une dystrophie vitelliforme juvénile ou de l'adulte avec un criblage systématique des deux gènes BEST1 et PRPH2 nous a permis d'établir des recommandations en fonction des trois critères : l'âge, l'histoire familiale et le rapport d'Arden. Nous avons ensuite recherché de larges réarrangements (délétions, insertions) dans les familles négatives par MLPA. Deux cas de délétion exonique ont été retrouvés (délétion de l'exon 4 du gène BEST1, délétion de l'exon 2 du gène PRPH2). L'étude de l'exome d'une grande famille (3 générations, 10 sujets atteints) n'ayant pas de mutations exoniques ou de réarrangements, a permis de démontrer l'implication du gène IMPG1 qui code pour une glycoprotéine de la matrice interphotoréceptrice. La même mutation faux-sens hétérozygote a été retrouvée dans deux autres familles. Nous avons ensuite testé son paralogue IMPG2 qui code également une protéine de la matrice interphotoréceptrice. Une seule famille avec une forme modérée de dystrophie vitelliforme a une mutation faux-sens hétérozygote dans ce second gène. IMPG1 et IMPG2, deux gènes de la matrice interphotoréceptrice sont désormais à ajouter à la liste des gènes des dystrophies vitelliformes après BEST1 le gène majeur et PRPH2. / Vitelliform dystrophies represent the second cause of inherited macular dystrophies after Stargardt disease (monogenic disease linked to ABCA4). To date, BEST1 and PRPH2 are the only known genes involved in vitellin deposits. Considering a large cohort of 88 unrelated patients with juvenile or adult form of vitelliform dystrophy and after a systematic screening of both genes, we propose a rational for BEST1 and PRPH2 analysis according to age of onset, positive family history and Arden ratio. The second step was to consider large deletions or insertions in these genes in patients negative for BEST1 and PRPH2. Exonic deletions are rare: one exon 4 deletion of BEST1 and one exon 2 deletion of PRPH2. Whole exome sequencing in a large family (3 generations, 10 affected patients) revealed a hetezogygous missense variation in IMPG1 an interphotoreceptor matrix gene. IMPG1 was the causal gene in two additionnal families. In the same way, its paralog IMPG2 have been tested : only one family with an heterozygous missense mutation was found. IMPG1 and IMPG2 are two new genes involved in vitelliform dystrophies after BEST1 the main gene and PRPH2.

Knowledge of physiological and morfological mechanisms which are related to drought tolerance is essential in breeding high-yielding and tolerant plants, which would not suffer unfavourable environmental conditions. Drought stress can lead to oxidative damage which causes serious disorders in physiological and biochemical processes in plant cells. Antioxidants can reduce those negative effects. Morphological (dry mass of shoot and roots, height of shoot, number of leaves), physiological (photosynthetic efficiency, pigment content, relative water content-RWC) and antioxidative (catalase-CAT, ascorbate peroxidase-APX, proline) parameters were evaluated in this study using two genotypes of Vicia faba L. differing in drought susceptibility. Drought resistant (Merkur) and sensitive (Piešťanský) genotypes were exposed to 10 days of drought. Activity of CAT and APX of stressed plants rather decreased. Proline content oscillated and no evident trend or significant differences were observed in relation to drought stress. Decrease in RWC was expressed more at susceptible plants, electron-transport chain activity and pigment content were not affected much by drought. Drought susceptible genotype expressed more serious negative effects of drought in morphological parameters, however this genotype was a bit bigger than...

Organismos do gênero Acanthamoeba são muito pouco investigados no Brasil, não se conhecendo a diversidade genética e o potencial patogênico das espécies prevalentes no país. Este estudo examinou a presença de Acanthamoeba em 118 amostras de solo e água de diversas regiões do Brasil e, como resultado, 38 novas culturas axenicas foram estabelecidas. A partir do sequenciamento da subunidade menor do gene ribossômico foi possível genotipar, avaliar o polimorfismo genético das amostras brasileiras e também inferir relações filogenéticas entre isolados clínicos e de ambiente do Brasil e entre esses e as demais espécies/isolados de Acanthamoeba. Para alguns grupos de isolados brasileiros, o potencial patogênico, inferido por indicadores indiretos de virulência (termo- e osmotolerância, secreção de peptidases e efeito citopático), foi também investigado. / Organisms of the genus Acanthamoeba are poorly investigated in Brazil, not knowing the genetic diversity and pathogenic potential of the species prevalent in the country. This study examined the presence of Acanthamoeba in 118 soil and water samples from different regions of Brazil and as a result, 38 new axenic cultures were established. From the sequencing of small subunit ribosomal gene was possible to genotype, to assess the genetic polymorphism of Brazilian samples and also to infer phylogenetic relationships among clinical and environmental isolates from Brazil and between these and other species/isolates of Acanthamoeba. For some groups of Brazilian isolates, pathogenic potential, inferred by indirect indicators of virulence (thermo-and osmotolerance, secretion of peptidases and CPE), was also investigated.

O sistema nervoso (SN) autonômico é fundamental na regulação cardiovascular. A análise da variabilidade da freqüência cardíaca (VFC), no domínio do tempo e da freqüência, expressa a modulação autonômica cardíaca, pois reflete a atividade do SN simpático (receptores adrenérgicos) e do SN parassimpático (receptores muscarínicos) sobre Nó Sinoatrial. As variantes genéticas funcionais (polimorfismos) do receptor adrenérgico beta2 vêm sendo associadas a diferentes estados funcionais do receptor e a diversos fenótipos cardiovasculares. Investigamos em 218 de jovens normotensos (entre 18 a 30 anos) a associação dos polimorfismos do receptor adrenérgico beta2 tipo Gln27Glu (Gln/Gln, Gln/Glu e Glu/Glu) e tipo Arg16Gly (Arg/Arg, Arg/Gly e Gly/Gly) com o perfil antropométrico e com os fenótipos cardiovasculares: o balanço autonômico para o coração (análise da VFC), a noradrenalina sérica, e variáveis hemodinâmicas, que foram registradas durante o repouso (5min) e o tilt test (teste de estresse postural, 5 min). Resultados: polimorfismo beta2 tipo Gln27Glu - comparados aos portadores do genótipo Gln/Gln, os indivíduos com genótipos Glu/Glu e Gln/Glu apresentaram uma menor relação cintura/quadril (p=0,008) e uma maior atividade simpática em resposta ao tilt teste, ou seja, maior aumento do componente LF (p=0,027) e maior relação LF/HF (p=0,014); polimorfismos beta2 tipo Arg16Gly - portadores do genótipo Arg/Arg apresentaram maior queda do índice alfa durante o tilt test, comparados aos outros genótipos; associação de polimorfismos (haplótipos) - portadores do haplótipo Gln27Gln/Arg16Gly apresentaram maior incremento da FC quando comparados aos portadores dos haplótipos Gln27Gln/Gly16Gly (p=0,06). Conclusão: foi possível detectar que os polimorfismos do receptor adrenérgico beta2 tipo Gln27Glu e tipo Arg16Gly têm impacto sobre o balanço autonômico cardíaco, respectivamente, aumentando a atividade simpática para o coração e diminuindo a atividade baroreflexa durante manobra de estresse postural, em indivíduos jovens normotensos / The autonomic nervous system (NS) has a pivotal role in cardiovascular control. Time domain and spectral analyzes of heart rate variability (HRV) indicates cardiac autonomic modulation, since it reflects the sympathetic (adrenergic receptors) and parasympathetic (muscarinic receptors) nerve activity over the Sinoatrial Node. Polymorphisms of the adrenergic receptor beta2 have been associated to different functional state of receptor and cardiovascular phenotypes. We analyzed in 218 young normotensive subjects (18-30 years old) the association of polymorphisms of the adrenergic receptor type Gln27Glu (Gln/Gln, Gln/Glu e Glu/Glu) and type Arg16Gly (Arg/Arg, Arg/Gly e Gly/Gly) with anthropometric data and cardiovascular phenotypes: cardiac autonomic balance (HRV), norephinefrine levels, and hemodynamic parameters, which were registered during rest (5min) and tilt test (5 min). Results: beta2 polymorphism type Gln27Glu - compared to subjects with genotype Gln/Gln, subjects with genotype Glu/Glu e Gln/Glu showed a lower WHR (p=0,008) and a higher increase in sympathetic activity during tilt teste, i.e., a higher increase in LF component (p=0,027) and LF/HF relation (p=0,014); beta2 polymorphism type Arg16Gly - subjects with genotype Arg/Arg demonstrated a higher decrease in alpha index during tilt test, compared to other genotypes; polymorphism association (haplotype) - subjects with Gln27Gln/Arg16Gly had a higher increase in hear rate compared to subjects with haplotype Gln27Gln/Gly16Gly (p=0,06). Conclusion: it was possible to detect in young normotensive subjects that polymorphisms of the adrenergic receptor type Gln27Glu and type Arg16Gly have an impact over cardiac autonomic balance, respectively, increasing the cardiac sympathetic activity and decreasing the baroreflex sensibility during tilt test

Um total de 8.961 amostras fecais coletadas de pacientes com diarréia aguda, no período de 1996 a 2006, no Estado de São Paulo foi testado para rotavírus por EIE. Destas, 20,0% foram positivas e posteriormente realizadas a caracterização dos rotavírus em genótipos G e P por nested RT-PCR. O genótipo G1 de rotavírus foi o mais freqüente, detectado em 35,2% das amostras, seguidos dos tipos G9, G2, G3, G4, infecção mista e G12. A associação mais freqüente foi a P[8]G1 e P[8]G9. Foi realizada a sequencia nucleotídica do gene 9 (VP7) de 38 rotavirus genótipo G1. Duas cepas foram analisadas dos anos de 1997, 1998, 2001 e 2002, três cepas dos anos 1996, 1999 e 2003, quatro cepas em 2000, sete cepas em 2004 e 2005, e cinco em 2006. Os 38 rotavírus G1 foram classificados em duas linhagens distintas, linhagem G1-I e linhagem G1-II. A linhagem G1-I foi detectada durante seis anos, 1996-1997, 2001-2002 e 2004-2006, e a linhagem G1-II foi detectada durante os anos de 1998-2001, e 2003-2005. Análises preliminares mostraram que Rotarix ® foi eficiente contra estas linhagens G1. / Rotavirus (RV) infections are recognized as a major cause of severe gastroenteritis in children worldwide. In March 2006, a monovalente P[8]G1, human RV vaccine (Rotarix® GlaxoSmithKline Biologicals) was introduced in Brazil into the routine childhood immunization schedule. Therefore, the study of genetic diversity among rotavirus strains before and after the introduction of this vaccine may be important for the development of vaccination strategies. A total of 8,961 fecal samples collected from patients with acute diarrhea, during the 11-year period surveillance in São Paulo State (1996 to 2006) were tested for rotavirus by ELISA. One thousand seven hundred eighty- four (1,784, 20.0%) were positive, and the characterization of the G and P genotypes was performed on 1,300 rotavirus samples by nested RT-PCR. The G1 type was the most prevalent rotavirus strain (35.2%). The second most prevalente was the G9 type (31.2%), followed by G2 (4.0%), G3 (3.5%), G4 (2.2%), mix infection (1.8%) and G12 (0.5%). The more frequent association was P[8]G1 and P[8]G9. We performed a sequence analysis of 38 P[8]G1 rotavirus strains, selected from a total of 341 P[8]G1.Two strains from 1997, 1998, 2001, and 2002 were analyzed; three strains from 1996, 1999, and 2003; four strains from 2000; seven strains from 2004, and 2005; and five strains from 2006. All 38 rotavirus G1 sequence in this study were found to be classified into two distinct lineages, lineage I with 44.7% (17/38) and lineage II with 55.3% (21/38). The G1I lineages were detected during six rotavirus seasons 1996-1997, 2001- 2002, and 2004-2006 whereas and lineage G1- II was detected during 1998-2001, and 2003-2005. Preliminary analyses 4 demonstrated that Rotarix® has been efficacious against these G1 lineages.