Molecular and morphologic studies were used to determine the evolution, classification and differentiation of Nymphaea odorata. Molecular analyses of the nuclear internal transcribed spacer (ITS) region, the chloroplast trnL-F region, and inter-simple sequence repeat (ISSR) markers determined the variation present between and within two species of Nymphaea. The ITS region resulted in a phylogeny depicting strong separation between species (N. mexicana and N. odorata) and some separation between N. odorata's subspecies. The ITS region contained polymorphisms, which upon SAHN clustering and principle coordinate (PCOA) and minimum spanning tree (MST) analyses produced groups similar to the clades in the ITS phylogeny. Sixteen accessions were chosen for trnL-F analysis, where a subspecies-specific molecular marker was found. In most accessions the marker confirmed the original subspecies classification. Molecular analyses using ISSRs characterized among population variation in N. odorata and N. mexicana using five primers. ISSR markers among populations were highly variable within a species and were used in UPGMA, PCOA and MST analysis, which resulted in separation between the subspecies. Both univariate and multivariate analyses were performed on quantitative and qualitative morphological characters. An analysis of variance resulted in six morphological characteristics that were statistically significant (P< 0.05), the majority being leaf blade characteristics. Multivariate statistics of principle component analysis and discriminate analysis resulted in groups for each subspecies, both emphasized the importance of quantitative leaf blade characteristics. Overall, both morphology and molecular characteristics supported the classification of subspecies for ssp. odorata and ssp. tuberosa, due a lack of strong segregation of characteristics. / Master of Science

Krebs entsteht durch das Zusammenspiel von Keimbahn- und somatischen Mutationen sowie Umweltfaktoren. Für Fortschritte in Prävention, Früherkennung und Behandlung von Krebs ist es essenziell die phänotypischen Folgen dieser Mutationen und die Rolle von Umweltfaktoren bei der Steigerung des Krebsrisikos zu verstehen. In dieser Arbeit untersuche ich zunächst die Auswirkungen von Mutationen in einfachen Sequenzwiederholungsregionen (SSRs) auf das Zellwachstum in einem Hefemodell. In einem Mutationsakkumulationsexperiment in Stämmen mit defektem Mismatch-Reparatursystem zeige ich, dass die Störung von MutSβ zu einer erhöhten SSR-Mutationsrate führt, insbesondere bei SSR-Loci die länger als 8 bp sind. Meine Ergebnisse legen nahe, dass MutSβ hauptsächlich für die Reparatur an längeren Repeat-Loci verantwortlich ist. Schließlich zeige ich, dass SSR-Mutationen meist geringfügige, negative Auswirkungen auf das Zellwachstum haben. Als Nächstes untersuche ich die Auswirkungen von Zigarettenrauch auf das Transkriptom von zugänglichem Atemwegsgewebe am Beispiel des Nasenepithels von gesunden Freiwilligen und Patienten mit Verdacht auf oder diagnostiziertem Lungenkrebs. Ich stelle fest, dass Gene und biologische Funktionen, die durch das Rauchen beeinträchtigt werden, bei Patienten langsamer auf ein gesundes Ausgangsniveau zurückkehren. Zudem zeige ich, dass Patienten durch anhaltende, Rauch-assoziierte Immunveränderungen gekennzeichnet sind. Schließlich präsentiere ich einen innovativen Lungenkrebs-Klassifikator, der durch die Berücksichtigung der nasalen Genexpression von Rauch-assoziierten Genen eindeutig bessere Ergebnisse erzielt als ein Modell, das ausschließlich auf klinischen Informationen basiert. Damit belege ich das Potenzial der Genexpression des Nasenepithels zur Verbesserung der Risikostratifizierung auf Bevölkerungsebene anhand eines nicht-invasiven Tests. / Cancer is initiated and sustained by the interplay of germline and somatic mutations and environmental factors. Understanding the phenotypic consequences of mutations and the role of environmental factors in increasing cancer risk is key to improving cancer prevention, early detection, and treatment. In this thesis, I first take advantage of a yeast model to investigate the effects of mutations in simple sequence repeat regions (SSRs) on cell growth. I describe a mutation accumulation experiment conducted in strains with a deletion of the MutSβ gene, a component of the mismatch repair system (MMR). I show that abrogating MutSβ function leads to an increased SSR mutation rate, with a bias towards deletions. I also report a drastic increase in mutation rate in SSR loci longer that 8-bp, suggesting MutSβ is primarily responsible for repair at longer repeat loci. Finally, I show that many SSR mutations have small deleterious effects on cell growth. Next, I investigate the effects of cigarette smoke on the transcriptome of an accessible airway tissue, nasal epithelium, in a cohort of healthy volunteers and patients with suspected or diagnosed lung cancer. I find that smoke injury response is strikingly different in healthy individuals and clinic patients, with genes and biological functions affected by smoking showing a slower reversal to healthy baseline level in clinic patients. I find persistent smoking-associated immune alterations to be a hallmark of the clinic patients. Finally, I show that a lung cancer classifier including nasal expression of smoke-injury-associated genes performs better than a model based exclusively on clinical information, providing evidence for the potential of nasal epithelial gene expression to improve population-level risk stratification with the use of a non-invasive test.

Haemophilus parainfluenzae (Hp) and H. influenzae (Hi) are closely related members of the Pasteurellaceae family and are common commensal bacteria of the human nasopharynx. Whilst Hi is frequently implicated in meningitis, otitis media and respiratory tract infections, reports of pathogenic behaviour by Hp are very rare. Lipopolysaccharide (LPS) is a key component of the Gram negative cell wall, and its structure influences the ability of Haemophilus to interact with the host and evade immune clearance. A better understanding of the differences in LPS structure between Hi and Hp could help to ascertain which parts of the molecule are important for commensal and pathogenic behaviour. Hi LPS comprises lipid A, a conserved oligosaccharide inner core, and an oligosaccharide outer core that differs between strains. The latter is partly phase variable by the slipped strand mispairing during replication of DNA repeat tracts within several LPS biosynthesis genes. Very little was known about LPS in Hp so we investigated its biosynthesis and structure in a panel of 20 Hp carriage isolates. Using PCR, DNA sequencing and Southern analysis we demonstrated that Hp possesses homologues of the Hi lipid A and inner core LPS synthesis genes and a few of the genes for outer core synthesis; however, homologues of the Hi phase variable outer core genes were largely absent and did not contain repeat tracts. The results of immunoblotting and collaborative structural analysis were consistent with this data. Phosphocholine, a phase variable Hi LPS epitope that has been implicated in otitis media, was found to be absent in Hp LPS due to the lack of four genes required for its biosynthesis and incorporation. The introduction of these genes into Hp led to the phase variable addition of phosphocholine to the LPS, indicating that there is no fundamental reason why Hp could not use a similar mechanism of variation to Hi if it was advantageous to do so. SDS-PAGE data suggested the presence of O-antigens (repeated chains of sugars) in many of the Hp strains, an unusual feature for Haemophilus, and all of the strains were found to contain a potential O-antigen synthesis locus. Each locus encodes homologues of several glycosyltransferases in addition to either the Wzy polymerase- or ABC transporter-dependent mechanisms of O-antigen synthesis and transport. Comparisons of wild type and isogenic mutant strains showed that the O-antigen enhances resistance to complement-mediated killing and appears to affect adhesion to epithelial cells in vitro. Hp is a successful commensal organism but lacks the flexibility of adapting its LPS using repeat-mediated phase variation, potentially limiting its range of host niches.

Species identifications have been historically difficult in the economically important spruce budworm (Choristoneura fumiferana) pest complex. Morphological, ecological, behavioural, and genetic characters have been studied to try to understand the taxonomy of this group, but diagnostic character states differ in frequency rather than being complete replacements between each species. I developed a morphology-based character system that focuses on forewing colour components (Chapter 2), as well as eight simple sequence repeats (SSRs, also referred to as microsatellite markers) (Chapter 3). I tested these along with a 470 bp region of COI mitochondrial DNA (mtDNA) (Chapter 2, 4) to determine their congruence with putative species that were identified by adaptive traits (larval host plant, length of larval diapause, larval and adult morphology, pheromone attraction, distribution). The morphometrics system was effective for identification of the five species tested, with only slight overlap between C. fumiferana and C. biennis. MtDNA distinguished C. fumiferana and C. pinus pinus, but the remaining species shared haplotypes. SSRs distinguished four species (C. fumiferana, C. pinus pinus, C. retiniana, C. lambertiana) but the remaining four species that were included in this survey (Chapter 4) remained mixed within two populations. There was evidence for hybridization between several species pairs. I also conducted a detailed study (Chapter 5) in Cypress Hills, an isolated remnant coniferous forest in western Canada, where identifying individuals from the Choristoneura fumiferana complex has been impossible due to the unusual ecogeographic characteristics of the area. I integrated data on behaviour, ecology, morphology, mtDNA, and SSRs, comparing Cypress Hills populations to those from other regions of North America to determine which species they resembled most. I delimited at least three populations, resembling C. fumiferana, C. occidentalis and C. lambertiana. Adult flight phenology, along with pheromone attraction, were identified as major isolating mechanisms between these populations. My studies highlighted the importance of integrative taxonomy for understanding species boundaries. Their patterns of differentiation suggest that spruce budworm species have recently diverged via natural selection in spite of some gene flow. Overall, this work is intended to contribute to more accurate identification of specimens and a better understanding of the evolutionary processes that drive speciation. / Systematics and Evolution

No description available.

The Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is one of the important insect pests of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and other grasses. To date, there are four RWA biotypes identified in South Africa. The virulent biotypes emerged, partly due to climate change and new genetic variations within populations of RWA; hence there is a need to improve host-plant resistance, as an effective control measure. Bread wheat (Triticum aestivum L.) accession Cereal Introduction (CItr) 2401 is known to be resistant to all RWA biotypes worldwide. The goal of this study was to use a backcrossed near-isogenic line (NIL) BC5F5 mapping population, developed from a cross between CItr 2401 and susceptible Kavkaz, to identify and validate single nucleotide polymorphism (SNP) markers linked to the resistance phenotype in CItr 2401. This was achieved by (i) conducting a preliminary study that evaluated the suitability of simple sequence repeat (SSR) markers previously reported in literature for discriminating stacked RWA resistance genes and, (ii) employing SNP markers for the first time in a RWA resistance study as a future alternative to the widely used SSR markers. None of the tested SSR markers showed potential use in marker-assisted selection (MAS). The mapping population was phenotypically evaluated for RWA resistance using the four South African biotypes, viz. RWASA1, RWASA2, RWASA3 and RWASA4. Analysis of variance (ANOVA) showed significant (P<0.001) differences of genotypes after confirming the normality of residuals and homogeneity of variance. The Illumina iSelect 9,000 wheat SNP platform was used to genotype the two crossing parents and a selection of 24 NIL genotypes from the mapping population. Eight SNP markers found to be linked to the phenotype were converted to breeder-friendly and high-throughput Kompetitive allele-specific polymerase chain reaction (KASP) markers. The designed KASP markers were validated on the two crossing parents, the 24 NIL sent for SNP genotyping, on the mapping population and on the preliminary study genotypes for their effectiveness. The KASP assays developed in this study will be useful for stacking the RWA resistance from CItr 2401 with other Dn genes effective against the RWA. / Life and Consumer Sciences / M. Sc. (Life Sciences)