The aetiological diagnosis of recessive non-syndromic hearing loss poses a challenge owing to marked heterogeneity and the lack of identifying clinical features. The finding that up to 50% of recessive non-syndromal genetic hearing loss among Caucasians was due to mutations in GJB2, the gene encoding Connexin 26 (Cx26) was a breakthrough, whose value as a diagnostic tool has been limited by the significant variation in the prevalence of deafness genes and loci among population groups. The significant association of the GJB6-D13S1830 deletion among individuals with one mutant GJB2 allele highlighted the need to explore population specific genetic mutations for NSHL. Although data from Sub-Saharan Africa is limited, reported studies found a high prevalence of R143W GJB2 mutation among Ghanaian, the 35delG mutation in 5 out of 139 Sudanese and a low prevalence of GJB2 variations among 385 Kenyan deaf children. The mutation spectrum of Waardenburg Syndrome (WS) in Africans has not been documented. During a visit to a School for the Deaf in the Limpopo Province of South Africa in 1997, it was noted that a high number of students came from Nzhelele sub-district. All had childhood onset hearing loss with no associated anomalies or disorders. The question arose as to whether there was a high-risk area for deafness in the Limpopo Province and what the aetiology of this hearing loss was.The main aim of this study was to investigate the role of GJB2, the GJB6-D13S1830 deletion, and the four common mitochondrial mutations, A1555G, A3243G, A7511C and A7445G, in the African hearing-impaired population of Limpopo province in South Africa, and to identify the mutation spectrum of the deafness genes found. The type and degree of hearing loss in this hearing impaired population would also be assessed. Secondly, this study sought to identify the mutations in a sibling pair with 2 clinical WS and to use the findings in a future study to establish the mutation spectrum of WS in the African population of the Limpopo province and of South Africa in general. The study was designed as a two phase study, in which phase 1 was used for hypothesis formulation and phase 2 was for hypothesis testing. While phase 1 was a descriptive retrospective case study, phase 2 was a combination of sample survey and prospective descriptive case study. In phase 1, demographic data of 361 students in two schools of the deaf in the Limpopo province was analyzed for evidence of areas of high risk populations for deafness in the province. In phase 2, a group of 182 individuals with genetic non-syndromic hearing loss (NSHL) and two siblings with clinical WS from two schools for the Deaf in the Limpopo Province of South Africa were investigated. A thorough clinical examination, audiological evaluation and urinalysis were done. Mutational screening was carried out in all 184 subjects using genomic DNA using single-strand conformation polymorphism (SSCP), multiplex polymerase chain reaction (PCR), and direct sequencing for GJB2, and Restriction Fragment-Length Polymorphism (PCR–RFLP) analysis for GJB6, and SSCP, hetero-duplex analysis, and direct sequencing of the first 8 exons of PAX3 and all of MITF for Waarenburg syndrome. Data analysis was by geographical mapping, frequency tables, tests of association with calculation of odds ratios, and binary logistic regression analysis using STATA and GIS mapping systems. The results indicate that there seem to be areas of genuine populations at risk for hearing loss in the Limpopo province of South Africa, namely Mutale and parts of Makhado and Thulamela municipalities. In Thulamela (NP343) wards 11-15, 26-30 and 31-35, and in Mutale (NP 344) wards 6-10, together accounted for 67 (18%) of participants in phase 1, and 33 (18%) of the participants in phase 2 of the study. Mutale municipality in the Vhembe 3 district gave with a projected prevalence of at least 13.14 deaf children per 100,000 African population attending the local school for the deaf. The observed hearing loss is a genetic, non-syndromic form, which is mainly severe and severe to profound, although without any clear defining configuration or shape. It is a stable, non-progressive and prelingual form of hearing loss, implying that this may be a recessive form of deafness. No identifiable environmental confounding factors or associations were identified. The deafness is not linked the common known auditory gene mutations in GJB2, the GJB6-D13S1830 deletion, or the common mitochondrial mutations A1555G, A3243G, A7511C and A7445G. Severe and profound levels of hearing loss were found in 22.8% and 75% of the cohort respectively, with the majority exhibiting flat (70.1%) or sloping (23.4%) audiograms that were commonly symmetrical (81.5%). However, as indicated, there was no clear pattern in the audiological findings overall. None of the 184 hearing impaired individuals exhibited any of the reported disease causing mutations of GJB2, including 35delG. There was, however, a high prevalence of two variants, the C>T variant at position g.3318-15 and the C>T variant at position g.3318-34, occurring in 21.4% and 46.2% of the deaf cohort respectively. The same variants were found to occur in 35% and 42.6% of a normal hearing control group (n = 63) respectively, indicating that these variations are polymorphisms. In three subjects (1.63% of the cohort), a T>A homozygous variation at position g.3318-6 was detected. Its significance in the causation of NSSNHL is yet to be determined. The GJB6-D13S1830 deletion was not detected in any of the participants. None of the four mitochondrial mutations screened for were found. 4 These results indicate that GJB2 is not a significant deafness gene in the African population of the Limpopo Province of South Africa and that significant genes for non-syndromic recessive hearing loss in this population are yet to be found. The geographical clustering of deafness found in this study, combined with the lack of identifiable common associated clinical features among the subjects of this study (excluding the WS sibling pair), suggests that these subjects have a genetic recessive non-syndromal type of hearing loss. In the context of historical and cultural evidence of consanguinity in this population, a founder effect cannot be ruled out. A rare mutation, R223X, previously identified only once out of 470 WS patients, was identified in the PAX3 gene among the WS sibling pair. A novel silent change GGG>GGT at amino acid 293, was also identified. These identical findings document, for the first time, a molecular defect in WS in an African sibling pair, and confirm WS Type I in this family, which could be found in other WS type I South Africans in the Limpopo Province of South Africa. The current study demonstrated that parents of genetically hearing impaired children in these areas are able to detect hearing loss at an early age, with over 60% suspecting their children’s hearing loss below 6 months of age. A child-centered management model encompassing all the areas relevant to childhood deafness/hearing impairment, which takes into consideration the prevailing logistical and financial constraints of the available healthcare system, is proposed. The implementation of this model requires a paradigm shift from the current fragmented model of service delivery to a cohesive patient-centered approach, based on concrete data from appropriate community based research, in which all the relevant parties communicate and share resources. 5 It would achieve the goals of early detection and intervention, as well as inclusive education for all. The relevant health and education policies are already in place and the posts funded. Equitable implementation of these policies would require appropriate community based research, as well as improved communication and consultation between the various stakeholders to ensure an efficient and affordable quality healthcare service for all hearing impaired South Africans.

The overall aim of this research was to improve the understanding of the ecology and diversity of Trichoderma species within the soil and rhizosphere of onion (Allium cepa L.) and potato (Solanum tuberosum L.) under intensive management in New Zealand. The indigenous Trichoderma population was measured in a field trial at Pukekohe over a three year period under six different crop rotation treatments. The treatments included two continuous onion and potato rotations (intensive), two onion/potato mixed rotation (conventional), and two green manure rotations (sustainable). Results showed that Trichoderma populations were stable in both the rhizosphere and bulk soil (1.5 x 10² to 8.5 x 10³ CFU g⁻¹ ODS). The planting and incorporation of an oat (Avena sativa L.) green manure in the sustainable rotations positively increased Trichoderma colony forming unit (CFU) numbers in the rhizosphere soil from 3.4 x 10² to 2.5 x 10³ g⁻¹ ODS. A Trichoderma species identification method was developed based on colony morphology. Representative isolates were verified using restriction fragment length polymorphism (RFLP) and DNA sequencing. The method allowed for rapid and reliable identification of isolated Trichoderma species. Five species were identified in the Pukekohe soil: T. asperellum, T. atroviride, T. hamatum, T. harzianum and T. koningii. Results showed identical species diversity between the rhizosphere, rhizoplane and bulk soil. The species did not strongly compete between each other for the rhizosphere ecological niche and differences in species proportions seemed to be caused by environmental factors rather than the rotation treatments. The incorporation of oat green manure in pots did not significantly promote the indigenous Trichoderma population size and diversity in the rhizosphere of onion plants up to 4 months old. The identified species were the same as in the field trial. The incorporation of onion scale residues was shown to result in low Trichoderma and high Penicillium CFU numbers and a reduction in plant size. Additionally, the presence of high levels (6.0 x 10⁵ CFU g⁻¹ ODS) of Penicillium CFU was negatively correlated with the presence of Trichoderma CFU. The effect of oat incorporation on Trichoderma saprophytic growth was also investigated in a soil sandwich assay and revealed no significant differences. A series of experiments indicated that onion extract obtained from dry onion scale residues had no antifungal activity against either Trichoderma or Penicillium and instead tended to promote their hyphal growth and sporulation. It also showed that competition between Penicillium and Trichoderma isolates was limited despite the ability of Penicillium to produce a wide range of inhibitory substances. Four indigenous Trichoderma species (T. atroviride, T. hamatum, T. harzianum and T. koningii) were shown to be rhizosphere competent in a split tube experiment over a 6 week period. The results of this experiment revealed that, the Trichoderma species clearly displayed differences in their ability to colonise the rhizosphere of young onion seedlings. Species such as T. koningii had the greatest rhizosphere colonising ability regardless of soil depth while T. harzianum displayed the weakest ability. Results also indicated that when inoculated as a mixture the four species competed with one another to colonise the rhizosphere. Overall, this research indicated that the studied crop rotation treatments and the use of oat as a green manure did not strongly promote indigenous Trichoderma populations. Species diversity was constant throughout the research with T. hamatum and T. koningii being the most frequently isolated species.

Inflammatory bowel diseases, (IBD), are a group of chronic disorders of the gastro-intestinal tract, and include Crohn’s disease (CD) and Ulcerative Colitis (UC). The pathogenesis is not known, but involves at least in part a loss of tolerance towards the commensal colonic microbiota. In this thesis, we show in animal models of CD and UC that the colonic mucosal blood flow increased compared to healthy animals. This blood flow increase is due to an up regulation of endothelial nitric oxide synthase (NOS). Further, we show in the UC model that the thickness of the firmly adherent colonic mucus layer increased compared to healthy animals. This increase is due to an up regulation of inducible NOS in the epithelium. Both the blood flow and mucus thickness increase appear to be protective mechanisms.  We demonstrate that the firmly adherent colonic mucus layer acts as a partial barrier towards luminal bacteria. In the UC model, this barrier is destroyed, causing increased bacterial translocation. The adhesion molecule P-selectin was up regulated in the UC model, leading to increased interactions between leukocytes and the endothelium, but also increased interactions between platelets and the endothelium. This indicates that not only leukocytes, but also platelets are involved in colonic inflammation. The addition of the probiotic bacterial strain Lactobacillus reuteri prevented disease by normalizing P-selectin levels and endothelial interactions with leukocytes and platelets. Lactobacillus reuteri also decreased bacterial translocation over the epithelium. In summary, this thesis highlights the importance of colonic barrier functions, and investigates the role of the microbiota in experimental IBD.

The aim of this thesis was to provide a picture of the trypanosomosis and drug resistance prevalence in Eastern Province of Zambia, to understand the underlying factors of drug resistance (drug use habits), to improve the diagnosis of trypanosomosis in livestock and finally, to improve the diagnosis of isometamidium resistance in T.congolense. After an introductory part where available trypanosomosis and trypanocide resistance diagnostic methods are described and discussed, the body of the thesis is divided in two main sections. In the first section are presented the results of a cross-sectional and a longitudinal epidemiological survey describing the geographical distribution of trypanosomosis cases, of resistant isolates and of cattle treated with isometamidium chloride. The results of the monitoring of unsupervised treatments of cattle with isometamidium by farmers and veterinary assistants with the Isometamidium-ELISA technique are also presented. The second section describes the development of two new diagnostic methods, the first one allowing the diagnosis of trypanosome infections with high sensitivity and specificity through semi-nested polymerase chain reaction and restriction fragment length polymorphism. This is the first report of a pan-trypanosome PCR test (a single PCR test for the diagnosis of all important pathogenic trypanosomes of cattle). The second new method that was developed allows the diagnosis of isometamidium resistant T.congolense strains by PCR-RFLP. This is the first report of a PCR based diagnostic test of trypanocide resistance in T. congolense.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

No description available.

When tomato Craigella is infected with Verticillium dahliae Dvd-E6 (Dvd-E6), a tolerant state is induced with substantial pathogen load, but few symptoms. Unexpectedly, these plants are more robust and taller with Dvd-E6 behaving as an endophyte. Some endophytes can protect plants from virulent pathogens. This research was undertaken to improve understanding of the cellular and molecular nature of Verticillium tolerance in tomato, especially whether infection by Dvd-E6 can protect Craigella from virulent V. dahliae, race 1 (Vd1). To permit mixed infection experiments a restriction fragment length polymorphism (RFLP)-based assay was developed and used for differentiating Dvd-E6 from Vd1, when present in mixed infections. The results suggested that protection involves molecular interplay between Dvd-E6 and Vd1 in susceptible Craigella (CS) tomatoes, resulting in restricted Vd1 colonization. Further studies showed a dramatic reduction of Vd1 spores and mycelia. To examine genetic changes that account for these biological changes, a customized DNA chip (TVR) was used to analyze defense gene mRNA levels. The defense gene response was categorized into four groups. Group 1 was characterized by strong induction of defense genes followed by suppression. However, Vd1-induced gene suppression was blocked by Dvd-E6 in mixed infections. These genes included some transcription factors and PR proteins such as class IV chitinases and beta glucanases which are known to target fungal spores and mycelia. Experiments also were repeated with a Craigella resistant (CR) isoline containing a fully active Ve locus (Ve1+ and Ve2+). The biological results showed that the presence of the Ve1+ allele resulted in restricted Vd1 colonization and, in a mixed infection with Dvd-E6, Vd1 was completely eliminated from the plant stem. Surprisingly, there was no significant increase in defense gene mRNAs. Rather, elevated basal levels of defense gene products appeared sufficient to combat pathogen attack. To investigate functional effects of the genetic changes observed, an inducible RNAi knockdown vector for a defense gene (TUS15G8) with unknown function (pMW4-TUS15G8) as well as the Ve2 resistance gene (pMW-Ve2) was prepared as a initial step for future transformation analyses. Taken together the results reveal intriguing but complex biological and molecular changes in mixed infections, which remain a basis for future experiments and potential agricultural benefits. / Canadian Commonwealth Scholarship and Fellowship Plan

Part I. The aldosterone synthase gene (CYP11B2) polymorphism T-344C in blood pressure and left ventricular hypertrophy. BACKGROUND: Aldosterone is a key cardovascular hormone, it significantly influences volume, pressure and electrolyte balance. Aldosterone plays an important role in development of left ventricular (LV) hypertrophy and myocardial fibrosis. The aldosterone synthase gene (CYP11B2) is an important candidate gene region in essential hypertension. DESIGN AND METHODS: We assessed the influence of the T-344C polymorphism of aldosterone synthase - the rate-limiting enzyme in aldosterone biosynthesis - on the structure of the left ventricle in young normotensive men. The population included 113 normotensive mid-European Caucasian men aged 18-40 years (mean 27 +/- 5 years). We also studied the association of -344T/C polymorphism of the CYP11B2 gene with the presence and severity of hypertension in 369 individuals, of whom 213 were hypertensive patients (139 controlled hypertensive, 74 resistant hypertensive) and 156 were healthy normotensive subjects. The genotype was assessed using polymerase chain reaction with subsequent cleavage with restriction enzyme HAEIII (restriction fragment length polymorphism method) and visualization with ethidium bromide. Plasma renin activity (PRA) and plasma...