M.Sc.(Med.), Faculty of Health Sciences, University of the Witwatersrand, 2008 / Introduction It is widely recognised that stroke is a multi-factorial disorder in which platelets play a crucial role in thrombus formation resulting in ischaemic stroke. Platelet adhesion and aggregation are initiated by the interaction of various platelet glycoproteins (GP’s) such as GPIbα, which binds to von Willebrand Factor and GPIIb/IIIa a fibrinogen receptor. Recent studies have shown that the GP’s are polymorphic and the polymorphisms described within GPIbα such as Kozak- 5T/C, the variable number of tandem repeats (VNTR) and the Human Platelet antigen 2 (HPA2), have been implicated in the development of stroke, while the PIA polymorphism of GPIIb/IIIa was found to contribute to “aspirin resistance”. Therefore, these polymorphisms may be potentially important for early detection and early intervention and thus setting the need to provide for a high volume genotype testing at health care centres. One of the most used techniques to determine platelet function is platelet aggregometry. However, the major disadvantages of platelet aggregation is that it is influenced by a number of environmental factors and its access is limited to tertiary health centres. Platelet aggregation measures the functional expression of platelets, which is known to deteriorate over time. It is for this reason that new methods at molecular level such as polymerase chain reaction (PCR) are needed to explore the role of genotypic expressions, which are not influenced by environmental factors. Currently, conventional PCR is used to detect platelet polymorphisms in the research settings and has limitations as a routine diagnostic test. Furthermore, it is time consuming and is prone to contamination. With the recent advances in real-time PCR it is possible to genotype large sample batches rapidly without compromising on the quality, accuracy and precision of results. This study aims to adapt conventional PCR methodology onto a real-time platform for detecting platelet polymorphisms that have been implicated in both stroke and aspirin resistance. Materials and methods A total of 60 caucasian patients classified as having ischaemic stroke by virtue of MRI and Doppler analysis from the Stroke Clinic at the Johannesburg Hospital were enrolled for this study. Healthy caucasian individuals (38), age and gender matched were enrolled as controls. DNA samples were extracted from all the subjects and the prevalence of the Kozak –5T/C, HPA-2, VNTR and GPIIIa PIA polymorphisms were determined first by using conventional PCR and then the real-time LightCycler TM PCR method. Results The frequency of the unfavourable alleles ( the PIA2 allele for the GPIIIa PIA polymorphism, the T allele for the Kozak –5T/C polymorphism, the B allele for the HPA-2 polymorphism and the C allele for the VNTR polymorphism) of the different GP’s were higher in the stroke patients when compared to the control subjects but did not reach statistical significance. There was complete statistical agreement between the results obtained for the conventional PCR as compared to the results obtained for real-time PCR except for the VNTR polymorphism, due to the difficulty in designing and the unavailability of probes for the real-time PCR assay. However, it is important to note that adapting the real-time PCR as a new methodology would greatly benefit both the patients and the clinicians by providing early detection and the possibility of early therapeutic intervention. Conclusion Therefore in conclusion, it is possible to perform not only conventional PCR for platelet polymorphism but also real-time PCR on a large scale without compromising on the quality, accuracy and precision on platelet polymorphisms that play a significant role in stroke and aspirin resistance. However, a larger population based study needs to be performed to confirm the findings.

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Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The olive industry has emerged as an important industry in Australia with increasing demand for both olive oil and table olives. To meet the domestic demand for olive products, it is necessary to increase production. Studies have shown that only 1-2% of olive flowers mature into fruits (Martin, 1990). Insufficient pollination due to self and cross incompatibility is a major factor affecting fruit set. The various methods used for studies on compatibility relationships have often shown conflicting results, with the same cultivar being found to be self-compatible in some studies, and self-incompatible in others (Sibbett et at., 1992; Caruso et al., 1993). Also, most of these studies have been conducted in the northern hemisphere where the environmental conditions and combination of cultivars growing nearby are expected to be different from Australia. It is therefore necessary to carry out studies on compatibility relationships under natural conditions in the Australian environment. The use of molecular markers has been found to be an effective and reliable method for paternity analysis studies. Using polymorphic and codominant markers, fingerprints of embryos may be compared to markers present in the mother plant, and therefore, the paternal contribution of alleles may be identified. By comparing these alleles with the genotype of all the potential pollen donors the pollinating genotype can be identified. The aim of this project was to identify the most compatible pollen donors for five olive cultivars (Barnea, Corregiola, Koroneiki, Kalamata, and Mission) and to observe the effect of morphological characters (bloom time, percentage pollen vitality, and percentage of complete flowers) and weather conditions (temperature, rainfall, and wind direction) on pollination. The study was conducted in a mixed olive orchard in Gumeracha, South Australia over the 2002-2003 and 2003-2004 growing seasons. Prior to the study, the genotypes of the trees were compared with the standards in the database (Guerin et al., 2002) and it was found that most of the trees matched with the standard cultivars. However, the trees considered to be Manaki by the grower did not match with the standard Manaki and were therefore referred to as atypical Manaki. Also, some Pendolino, Corregiola, and Kalamata trees did not match with the standard and were also referred to as atypical. The maximum and minimum temperatures, rainfall, and wind direction were recorded for the bloom period of both the years. The range of maximum temperature minimum temperatures during the bloom period was similar in both years. There was more rainfall in the bloom period during the first year than during the second year. Wind direction data during the bloom period showed that the wind direction was similar in both years. The winds were mainly easterly or westerly in the mornings and mainly westerly in the afternoon. However, there were winds of lower intensities blowing in the other directions as well, thus ensuring adequate wind movement for pollen dissemination. Dates of the start of bloom, full bloom and end of bloom for each cultivar were recorded for both years. It was observed that most of the cultivars overlapped in their bloom time, although some such as Kalamata flowered late in both years. Bloom time dates for replicate trees of a cultivar were similar, but there were differences in the dates between cultivars. The percentage of complete flowers was recorded for all cultivars in both years and it was observed that King Kalamata had the lowest value (42.5%) in the first year and Koroneiki had the lowest value (29%) in the second year. Leccino, atypical Manaki, and Corregiola had high percentages of complete flowers in both years. Percentage pollen vitality observations ranged from 23.5% in King Kalamata to 72.3% in Koroneiki in the first year. In the following year, UC13A6 had the lowest percentage pollen vitality (19.7%) and Leccino had the highest value (65.5%). The flowers sampled from Verdale and atypical Manaki did not contain pollen in both the years. Paternity analysis showed that: Barnea embryos were mainly fertilised by Pendolino and Mission; Corregiola embryos were mainly fertilised by Mission, Kalamata, and atypical Manaki; Koroneiki embryos were mainly fertilised by Mission; Kalamata embryos were mainly fertilised by Koroneiki; and Mission embryos were mainly fertilised by Koroneiki. There were also unidentified pollen donors pollinating a significant proportion of embryos. No apparent effect of direction of canopy and distance of pollen donors was observed and it was concluded that wind movement was not a limitation for movement of pollen in the orchard. Temperature and rainfall did not have any apparent effect on the overall bloom period. Pollen vitality, time of flowering, number of trees in the orchard, and tree age may have affected the effectiveness of some cultivars as pollen donors. The results highlighted the importance of cross-pollination for fruit set. Only two instances of self-pollination were observed suggesting that cross-pollination is more effective than selfing. The results also suggest that there is genetically controlled compatibility relationship operating among the cultivars and this determined which pollen type lead to successful fruit formation. However little is known about the mechanism of incompatibility operating in olives. There were differences in the effectiveness of some pollen donors over the two years which suggests that having more than one compatible pollen donor in the orchard is important. The results obtained in this study may be used as a basis for studying the mechanism of incompatibility in olives. The compatible pollen donors identified can be used to make recommendations to olive growers regarding the combinations of olive cultivars that will maximise yield and hence boost the production of olives in Australia. The method can also be extended to other cultivars to identify compatible pollen donors and also to compare the effect of different environmental conditions on pollination. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1148163 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2004

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Artículo sustentado el 30 de Enero 2014 para la obtención del título profesional Médico Cirujano en la Escuela de Medicina, Facultad de Ciencias de la Salud. Universidad Peruana de Ciencias Aplicadas - UPC. / Artículo publicado el 27 de Abril de 2014 en la Revista Transactions of the Royal Society of Tropical Medicine & Hygiene (Oxford University Press). / Background: Gastroenteritis by rotavirus is responsible for approximately 810 annual deaths/year in children under 5 years in Peru and emerging rotavirus genotypes have led to concerns regarding cross-protection by the vaccines available. Moreover, there are no reports on the molecular-epidemiology of rotavirus diarrhea in Peru Methodology: A total of 131 stool samples were obtained from children under 5 years old hospitalized from January 2010 to December 2012 in the Hospital Regional de Cajamarca, Peru. ELISA and RT-PCR techniques were performed for rotavirus detection. G and P typing of rotavirus-positive samples were obtained by semi-nested multiplex RT-PCR and sequencing was performed to confirm the PCR results. Results: Of the 117 samples available, 18.80% (22/117) tested positive for rotavirus by ELISA and 35.90% (42/117) by RT-PCR. Among the G-genotype identified, G9 in 35.71% (15/42) and G12 in 33.33% (14/42) were the most prevalent. With the most common combination being G12/P6 in 23.81% (10/42). Conclusions: A high prevalence of the G12/P6 genotype was detected. It is know that this genotype is not covered by the current vaccines available. More in depth studies are needed to know the current rotavirus genotypes presents in Peru.

Master of Science / Agronomy / Dorivar A. Ruiz Diaz Suarez / The effect of tillage on crop yield, early growth, and soil nutrient stratification can be influenced by fertilizer placement. In addition, deeper root systems can enhance the crop ability to uptake water and nutrients. A thorough understanding of how these factors interact can result in increased grain yields and profitability for the producer. Three studies were completed to describe and evaluate different aspects of crop root system and response to fertilizer placement and tillage. The objective of the first study was to characterize the root system of two genotypes of corn (Zea mays) and soybean (Glycine max (L.) Merr.) using image analysis in the greenhouse and in the field, as well as evaluate dry weight accumulation and nutrient uptake patterns by shoot and root plant parts for both crops. Two different genotypes of each crop were sampled during the growing season to access root characteristics such as biomass, length, surface area, average diameter and volume. Significant differences were found in corn where the P1151 AM hybrid had greater root length, surface area and volume than the P1105 AM hybrid. In soybean, the differences were found in nutrient uptake with overall greater nutrient uptake values for the poor drainage variety (PD) compared to the good drainage variety (GD). The objective of the second study was to evaluate the effect of fertilizer placement and tillage system on corn with different genotypes. Three fertilizer treatments were combined with two different corn genotypes selected based on contrasting root systems and two different tillage systems. The three fertilizer placements were sub-surface band, broadcast, and control. The two hybrids of corn used were a P1151 AM hybrid and P1105 AM hybrid. The two tillage systems were no-till (NT) and strip-till (ST). Corn hybrids showed different response in root biomass but did not show a consistent response in other characteristics evaluated. Broadcast and sub-surface band increased nutrient uptake and grain yields over the control but were not significantly different from each other. Tillage showed no difference in corn response. The objective of the third study was to evaluate the effect of fertilizer placement and tillage system on contrasting soybean genotypes. Three fertilizer treatments were combined with two different genotypes selected based on contrasting root systems and two different tillage operations. The three fertilizer placements were sub-surface band, broadcast, and control. The two varieties of soybean used were one recommended for poor drainage (PD) and one recommended for good drainage (GD). The two tillage operations were NT and ST. Soybean root biomass differences were observed by varieties. Sub-surface band treatment favored early soybean growth, biomass and P uptake at the V3 growth stage, but it did not turn into yield increase. Soybean grain yields did not respond to fertilization in this study. Yield was affected significantly by variety selection and response varies by site-year.

AIM To evaluate magnitude/direction of changes in peripheral lipid profiles in patients undergoing direct acting therapy for hepatitis C by genotype. METHODS Mono-infected patients with hepatitis C were treated with guideline-based DAAs at a university-based liver clinic. Patient characteristics and laboratory values were collected before and after the treatment period. Baseline demographics included age, ethnicity, hypertension, diabetes, hyperlipidemia, treatment regimen, and fibrosis stage. Total cholesterol (TCHOL), high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TG), and liver function tests were measured prior to treatment and ETR. Changes in lipid and liver function were evaluated by subgroups with respect to genotype. Mean differences were calculated for each lipid profile and liver function component (direction/magnitude). The mean differences in lipid profiles were then compared between genotypes for differences in direction/magnitude. Lipid profile and liver function changes were evaluated with Levene's test and student's t test. Mean differences in lipid profiles were compared between genotypes using ANOVA, post hoc analysis via the Bonferroni correction or Dunnett T3. RESULTS Three hundred and seventy five patients enrolled with 321 (85.6%) achieving sustained-viral response at 12 wk. 72.3% were genotype 1 (GT1), 18.1% genotype 2 (GT2), 9.7% genotype 3 (GT3). Baseline demographics were similar. Significant change in lipid profiles were seen with GT1 and GT3 (Delta GT1, p and Delta GT3, p), with TCHOL increasing (+ 5.3, P = 0.005 and + 16.1, P < 0.001), HDL increasing (+ 12.5, P < 0.001 and + 7.9, P = 0.038), LDL increasing (+ 7.4, P = 0.058 and + 12.5, P < 0.001), and TG decreasing (-5.9, P = 0.044 and -9.80 P = 0.067). Among genotypes (Delta GT1 v.Delta GT2 v.Delta GT3, ANOVA), significant mean differences were seen with TCHOL (+ 5.3 v. + 0.1 v. + 16.1, P = 0.017) and HDL (+ 12.3 v. + 2 v. + 7.9, P = 0.040). Post-hoc, GT3 was associated with a greater increase in TCHOL than GT1 and GT2 (P = 0.028 and P = 0.019). CONCLUSION Successful DAA therapy results in increases in TCHOL, LDL, and HDL and decrease in TG, particularly in GT1/ GT3. Changes are most pronounced in GT3.

Les carbapénèmes sont des antibiotiques de la famille des β-lactamines utilisées en dernier recours à cause de leur stabilité vis-à-vis de la plupart des mécanismes de résistance. Cependant, on assiste chez les entérobactéries à l’émergence de carbapénèmases capables d’inactiver ces molécules. Les objectives de ce travail étaient d’explorer l’émergence de ces mécanismes de résistance dans des entérobactéries isolées au Nord Liban entre 2008 et 2012, d’analyser leur support génétique, ainsi que le fond génétique et la pathogénicité des souches porteuses. Nous avons observé une augmentation d’un facteur quatre de la prévalence des entérobactéries de sensibilité diminuée ou résistantes aux carbapénèmes dans les prélèvements cliniques hospitaliers entre 2008 et 2012. Un portage intestinal a été également observé chez 1,5% des individus dans une population d’enfants issus de la communauté. Le phénotype de résistance observé était lié à la production de la carbapénèmase OXA-48. Bien que sept espèces productrices ont été identifiées, la plupart des isolats étaient des souches non-redondantes appartenant aux espèces K. pneumoniae et surtout E. coli. Le vecteur de la diffusion de OXA-48 dans ces bactéries était trois plasmides du groupe d’incompatibilité IncL/M de 49 kb, 63 kb et 84 kb. Cependant, 67% des souches E. coli portaient le gène codant OXA-48 (blaOXA-48) sur le chromosome. La caractérisation des supports génétiques par des approches de séquençage à haut débit a montré qu’ils étaient apparentés et le produit de remaniements génétiques impliquant le transposon Tn21-like, la séquence d'insertion IS1R ou un nouveau transposon composite appelé Tn6237. L’insertion chromosomique de blaOXA-48 résultait de la transposition de Tn6237 qui est capable de transférer un fragment plasmidique de 20 kb dans différents sites du chromosome de E. coli. Les souches K. pneumoniae produisant OXA-48 n’appartenaient pas aux génotypes capsulaires hautement virulents K1 et K2, mais portaient des facteurs identifiées pour favoriser la virulence ou la colonisation de l’hôte. OXA-48 a été observée dans tous les phylogroupes de E. coli, y compris les phylogroupes B2 et D connus pour contenir les souches pathogènes extra-intestinales. Une souche se distinguait par une accumulation sans précédent de huit îlots de pathogénicité. Cette souche induisait une létalité inhabituellement élevée dans un modèle murin de sepsis. En conclusion, l’acquisition du gène blaOXA-48 est donc liée à la diffusion de plasmides apparentés, qui sont marqués par une plasticité conduisant à une localisation chromosomique du gène pouvant favoriser sa persistance. Elle conduit à une diffusion multi-clonale de souches K. pneumoniae et surtout E. coli potentiellement hautement virulentes. Cette association entre de l’espèce E. coli et la carbapénèmase OXA-48 est inquiétante, car E. coli constitue à la fois un réservoir dont la taille peut être considérable et un pathogène responsable d’infections fréquentes pouvant parfois mettre en jeu le pronostic vital. / In the β-lactam family, carbapenems are the most effective antimicrobial agents used as a last resort due to their stability toward most resistance mechanisms. However, we recently observed the emergence of carbapenemase-producing Enterobacteriaceae. The aim of the present study consisted to explore (i) the epidemiological situation of carbapenem-resistant Enterobacteriaceae isolated in North Lebanon between 2008 and 2012, (ii) the identification of the resistance mechanisms, and (iii) the characterization of pathogenicity and genetic background of the corresponding strains. We observed a 4-fold increase in the prevalence of Enterobacteriaceae exhibiting decreased susceptibility or resistance to carbapenems in the clinical isolates collected at hospital during 2008-2012. The prevalence of fecal carriage of carbapenemase-producing Enterobacteriaceae was estimated to 1.5% in healthy children of the community. OXA-48 was the only carbapenemase identified among non-redundant isolates which spread to seven species. E. coli and K. pneumoniae were the main represented species. The OXA-48-encoding gene (blaOXA-48) was carried by three novel IncL/M plasmids of 49 kb, 63 kb and 84 kb. However, 67% of E. coli strains encoded blaOXA-48 chromosome-mediated. The sequencing of the previously mentioned genetic structures by high -throughput approaches showed that they are the product of genetic rearrangements involving the Tn21-like transposon, the insertion sequence IS1R and a novel composite transposon designed Tn6237. The chromosomal insertion of blaOXA-48 was due to the acquisition of element Tn6237 leading consequently to the transposition of 20 kb plasmid fragment into different sites of E. coli chromosome. The pathogenicity profile of K. pneumoniae strains showed that they did not belong to highly virulent capsular genotypes K1 and K2, but harbored factors promoting virulence and host colonization. E. coli isolates belonged to different phylogroups, including phylogroups B2 and D known to contain the extra-intestinal pathogenic strains. An E. coli strain was characterized by a broad accumulation of pathogenicity islands (n=8). In addition, this strain induced an unusually high lethality in a mouse model of sepsis. In conclusion, the acquisition of the blaOXA-48 gene is linked to the spread of related IncL/M plasmids, which are marked by a plasticity leading to a chromosomal location of blaOXA-48 and probably promoting its persistence. It leads to a multiclonal diffusion of K. pneumoniae and especially E. coli potentially highly virulent. This association between E. coli and the carbapenemase OXA-48 is worrying because E. coli represent a putative important reservoir and a pathogen agent responsible for frequent infections that can be a life threatening.

Characterizing the genetic contributors to complex disease traits will inevitably require consideration of haplotypic phase, the specific alignment of alleles on a single homologous chromosome. In population based studies, however, phase is generally unobservable as standard genotyping techniques provide investigators only with data on unphased genotypes. Several statistical methods have been described for estimating haplotype frequencies and their association with a trait in the context of phase ambiguity. These methods are limited, however, to diploid populations in which individuals have exactly two homologous chromosomes each and are thus not suitable for more general infectious disease settings. Specifically, in the context of Malaria and HIV, the number of infections is also unknown. In addition, for both diploid and non-diploid settings, the challenge of high-dimensionality and an unknown model of association remains. Our research includes: (1) extending the expectation-maximization approach of Excoffier and Slatkin to address the challenges of unobservable phase and the unknown numbers of infections; (2) extending the method of Lake et al. to estimate simultaneously both haplotype frequencies and the haplotype-trait associations in the non-diploid settings; and (3) application of two Bayesian approaches to the mixed modeling framework with unobservable cluster (haploype) identifiers, to address the challenges associated with high-dimensional data. Simulation studies are presented as well as applications to data arising from a cohort of children multiply infected with Malaria and a cohort of HIV infected individuals at risk for anti-retroviral associated dyslipidemia. This research is joint work with Drs. S.M. Rich, R.M. Yucel, J. Staudenmayer and A.S. Foulkes.