Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.

This thesis concentrates on a detailed field and structural analysis of the Podolsko complex, Moldanubian unit, Bohemian Massif, complemented by a microstructural study and analysis of magnetic susceptibility (AMS). The Podolsko complex occupies the footwall of a major Variscan normal shear zone and is juxtaposed against the southern to southeastern margin of the Central Bohemian Plutonic complex. During the field work, more than 160 outcrops were examined, the AMS samples were taken at 25 stations, and samples for microstructural studies were taken from 12 localities. On the micro-scale, leucocratic migmatites contain abundant garnet grains which may represent relics of an earlier (ultra-)high preassure metamorphic phase. Retrogression is obvious in other samples of biotite migmatites of the Moldanubian Variegated unit. The retrogression is marked by the presence of sillimanite and chlorite. The main tectonometamorphic event in the Podolsko complex is extensive migmatization coeval with formation of pervasive flat-laying fabric. This is corroborated by the AMS study which indicates concordant steep to flat-laying magnetic and mesoscopic foliations striking NNW-SSE. The AMS also shows that the subhorizontal N-S to NNW-SSE trending magnetic lineations in the Podolsko complex correspond to those in the...

<p>The kidney's major role in filtration depends on its high blood flow, concentrating mechanisms, and biochemical activation. The kidney's greatest strengths also lead to vulnerability for drug-induced nephrotoxicity and other renal injuries. The current standard to diagnose renal injuries is with a percutaneous renal biopsy, which can be biased and insufficient. In one particular case, biopsy of a kidney with renal cell carcinoma can actually initiate metastasis. Tools that are sensitive and specific to detect renal disease early are essential, especially noninvasive diagnostic imaging. While other imaging modalities (ultrasound and x-ray/CT) have their unique advantages and disadvantages, MRI has superb soft tissue contrast without ionizing radiation. More importantly, there is a richness of contrast mechanisms in MRI that has yet to be explored and applied to study renal disease.</p><p>The focus of this work is to advance preclinical imaging tools to study the structure and function of the renal system. Studies were conducted in normal and disease models to understand general renal physiology as well as pathophysiology. This dissertation is separated into two parts--the first is the identification of renal architecture with ex vivo MRI; the second is the characterization of renal dynamics and function with in vivo MRI. High resolution ex vivo imaging provided several opportunities including: 1) identification of fine renal structures, 2) implementation of different contrast mechanisms with several pulse sequences and reconstruction methods, 3) development of image-processing tools to extract regions and structures, and 4) understanding of the nephron structures that create MR contrast and that are important for renal physiology. The ex vivo studies allowed for understanding and translation to in vivo studies. While the structure of this dissertation is organized by individual projects, the goal is singular: to develop magnetic resonance imaging biomarkers for renal system. </p><p>The work presented here includes three ex vivo studies and two in vivo studies:</p><p> </p><p>1) Magnetic resonance histology of age-related nephropathy in sprague dawley.</p><p>2) Quantitative susceptibility mapping of kidney inflammation and fibrosis in type 1 angiotensin receptor-deficient mice. </p><p>3) Susceptibility tensor imaging of the kidney and its microstructural underpinnings. </p><p>4) 4D MRI of renal function in the developing mouse. </p><p>5) 4D MRI of polycystic kidneys in rapamycin treated Glis3-deficient mice.</p> / Dissertation

Escherichia coli diarrhoea is the most important source of mortality in piglets. The most frequently isolated strain in enterotoxigenic E. coli diarrhoea is F4ab/ac. Recent studies in South Africa reported non-fimbrial strains such as PAA and EAST-1 to be prevalent. The objective of the study was to determine whether there are breed differences among pigs with respect to E. coli adhesion phenotypes and correlate them to polymorphisms at selected candidate genes in the South African population. A total of 225 pigs aged 3-12 weeks of the imported (Large White, Landrace and Duroc), local and crossbreds, were sampled from the Eastern Cape and Limpopo provinces of South Africa and genotyped for PCR-RFLP polymorphisms at four candidate genes associated with E. coli F4ab/ac resistance/susceptibility. These genes were Mucin 4 (MUC4), Mucin 13, (MUC13), Mucin 20 (MUC20) and Transferrin Receptor (TFRC). The TFRC and MUC13 genes were less polymorphic, the C allele was close to fixation and the homozygous CC genotype was the most frequent in all three pig populations. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for the TFRC locus. The g.8227G>C polymorphism in MUC4 segregated in all three breeds and the marker was moderately polymorphic. There was a significant difference (P <0.05) in genotypic distribution amongst breeds for MUC4.The g.191C>T polymorphism in MUC20 segregated in the local and crossbred pigs and was close to fixation in the imported pigs. There was a significant difference (P <0.05) in allelic and genotypic distribution amongst breeds for MUC20, which was moderately polymorphic. There was a reduction in heterozygosity in both the TFRC and MUC13 loci, although MUC4 and MUC20 genes had higher heterozygosity levels. The MUC4 gene had a negative FIS value, indicating outbreeding at this locus. The MUC20, MUC13 and TFRC genes had a positive FIS value, indicating inbreeding at these loci. Overall, the studied population was outbred. Imported pigs in TFRC and MUC20 deviated from Hardy-Weinberg equilibrium (HWE). All breeds were in HWE at the MUC4 and MUC13 genes. There was no linkage disequilibrium observed amongst the analysed loci. iv A total of 109 piglets of three breeds (Large White, indigenous and crossbred) aged 3-5 weeks, were investigated for the susceptibility to E. coli F4, PAA strains and EAST-1 toxin. Adhesion tests were conducted on pig intestinal cells, which were viewed under a phase contrast microscope. Three phenotypes were identified as, adhesive, weakly adhesive and non-adhesive. There was a significant association (P <0.05) between breed and level of adherence of the F4 and PAA strains. Highest frequencies of adhesion phenotypes were observed in the indigenous pigs for both F4 and PAA E. coli strains. Large White pigs had the lowest frequency of non-adhesion in F4 and PAA E. coli strains. The F4 strain had a higher (P <0.05) level of adherence compared to PAA and EAST-1 in Large White pigs. Age of pigs had a significant effect on the level of E. coli adherence in indigenous and crossbred pigs (P <0.05). Adhesion of F4 and EAST-1 was higher in weaned indigenous and crossbred pigs, respectively, than in suckling piglets. There was no significant difference between F4 adhesion and the genotypes at all four candidate genes genotypes. The study showed that both imported and local pig populations carry receptors and are susceptible to F4, PAA and EAST-1 E. coli infections. Indigenous pigs were less susceptible than Large White to E. coli infection. Although polymorphic and segregating in the populations, the MUC4 g.8227G>C and MUC20 g.191C>T mutations were not associated with the adhesion phenotypes and cannot be used in the selection of susceptible animals. / M.Sc.Agric. University of KwaZulu-Natal, Pietermaritzburg 2013.

Better understanding of pyroclastic density current (PDC) dynamics is one of the key volcanological focuses, as PDCs represent one of the most life-threatening volcanic hazards. PDCs associated with explosive collapse calderas are difficult to observe and examine directly, and thus research of internal architecture of calderas and their PDC deposits is focused on extinct and partly eroded volcano-plutonic systems. Such a case is the Late-Carboniferous Altenberg-Teplice caldera in NW Bohemian Massif, which exposes a large body of ignimbrites (deposits of the PDC) called Teplice rhyolite (an intra-caldera fill). This body is well exposed on the southern flank of the Krušné hory/Erzgebirge Mts., mainly its members: Teichweg, Lugstein-Pramenáč, Vlčí kámen-Medvědí vrch and Přední Cínovec. As these ignimbrites appear macroscopically isotropic, I employed the Anisotropy of magnetic susceptibility (AMS) in order to quantify their internal structure. A total of 1232 specimens from 63 sampling stations were analyzed for the AMS, complemented by susceptibility vs. temperature variations and petrographic observations. Obtained AMS data, carried by a mixture of paramagnetic ferrosilicates and low-Ti titanomagnetite, indicate various processes recorded in ignimbrites. The relatively oldest and moderately welded Teichweg...

Combined anisotropy of magnetic susceptibility (AMS) and crystallographic studies were applied on a neogenne lava flow, for which we know the supposed flow path. Samples were studied under microscope, the minerals were analysed on microprobe, the orientation of olivine crystals was determined via EBSD and magnetic properties were studied. AMS data acquired from samples collected from representative outcrops of lava flow show weak preferred orientation of magnetite-ulvöspinel. EBSD analysis suggests only slight orientation of plagioclase in one sample. Analysed composition of olivine corresponds with mathematical model for eruption temperature and crystallization succession. Rootless cone (disorderly breccia cone) in lava body was found and desribed in the abandoned Machův lom quarry.

Better understanding of pyroclastic density current (PDC) dynamics is one of the key volcanological focuses, as PDCs represent one of the most life-threatening volcanic hazards. PDCs associated with explosive collapse calderas are difficult to observe and examine directly, and thus research of internal architecture of calderas and their PDC deposits is focused on extinct and partly eroded volcano-plutonic systems. Such a case is the Late-Carboniferous Altenberg-Teplice caldera in NW Bohemian Massif, which exposes a large body of ignimbrites (deposits of the PDC) called Teplice rhyolite (an intra-caldera fill). This body is well exposed on the southern flank of the Krušné hory/Erzgebirge Mts., mainly its members: Teichweg, Lugstein-Pramenáč, Vlčí kámen-Medvědí vrch and Přední Cínovec. As these ignimbrites appear macroscopically isotropic, I employed the Anisotropy of magnetic susceptibility (AMS) in order to quantify their internal structure. A total of 1232 specimens from 63 sampling stations were analyzed for the AMS, complemented by susceptibility vs. temperature variations and petrographic observations. Obtained AMS data, carried by a mixture of paramagnetic ferrosilicates and low-Ti titanomagnetite, indicate various processes recorded in ignimbrites. The relatively oldest and moderately welded Teichweg...

Doctor of Philosophy / Department of Plant Pathology / Frank F. White / Plants have evolved sophisticated mechanisms to protect against microbial invaders of which resistance (R) genes are an important component. R genes mediate specific recognition of pathogens possessing cognate avirulence (avr) gene products, which leads to the induction of plant defense responses and the arrest of pathogen ingress. In contrast to numerous examples of R gene–avr interactions, the susceptible interaction is less well examined. Recent studies on rice and wheat indicate that host resistance to pathogens also involves genetic variability in dominant traits for susceptibility. Xanthomonas oryzae pv.oryzae (Xoo) causes bacterial blight disease in rice, a serious threat in the major rice growing regions of Asia. The pathogenicity of Xoo depends on the translocation of a cocktail of effector proteins into rice cells by a type III secretion system. The family of transcription activator like (TAL) effectors is the one of the most intriguing due to their eukaryotic features and function as major virulence determinants. The specificity of TAL effectors is determined by the nearly identical repeat units at the center of each protein. The major virulence determinant of the strain PXO99A is PthXo1, which hijacks the transcription of the host susceptibility (S) gene Os8N3, an allele of recessive resistance gene xa13. The strains that overcome xa13-mediated resistance harbor alternate major TAL effectors including PthXo2, PthXo3 and AvrXa7. Alternate effectors do not induce Os8N3. This study identified the alternate S gene Os11N3, which is dependent on the effectors AvrXa7 and PthXo3. The effectors bind to specific elements in the proximal promoter regions of the respective S genes and act as transcriptional activators. Our results indicate that rice–Xoo interactions involve gene-for-gene susceptibility to bacterial blight in addition to gene-for-gene resistance.

Gradient echo (GRE) imaging, a magnetic resonance imaging (MRI) technique that is sensitive to changes in the magnetic susceptibility property of tissues, has recently revealed significant signal heterogeneity in white matter (WM) at high magnetic field B0 ≥ 3T. Various aspects of the underlying white matter microstructure have been linked to the observed contrast between white matter regions. This thesis investigates the origins of the observed differences in GRE signal behaviour. We proposed an explicit multi-compartmental model of WM that incorporates realistic representation of the geometry and magnetic susceptibility of the underlying microstructure that can be used to study the effects of WM microstructural changes on GRE signal characteristics. In particular, we looked at the apparent transverse relaxation rate (R2*) and the resonance frequency, as well as their respective deviations from mono-exponential decay and linear phase evolution. Next, we investigated the effect of WM fiber orientation on GRE signal using healthy human volunteers at 3T by correlating the GRE signal from different WM regions with WM fiber orientation information. Using literature-based parameters, we demonstrated that the geometric model predicted similar trends. Lastly, we studied the effect of myelin on GRE signal using a cuprizone mouse model at 7T . An ex vivo study was used to correlate GRE signal in fixed mouse brain with normalized myelin stain intensity. Simulated GRE signal from hypothetical scenarios of demyelination were then compared with the experimental results. R2* and resonance frequency were then used in an in vivo longitudinal study to track myelin changes during demyelination and subsequent remyelination.

Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2010. / Thesis presented in partial fulfilment of the requirements for the degree Master of Medical Science (Human Genetics) at the University of Stellenbosch. / Bibliography / ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) remains the leading cause of death worldwide by a single infectious agent, despite significant advances in biomedical sciences. The idea that host genetics plays a role in the development of disease was proposed by Haldane in 1949. The observation that only 10% of immunocompetent individuals develop disease while others are able to successfully contain it, further suggests that host genetics plays an important role. TB is thus a complex disease, with the causative bacterium, Mycobacterium tuberculosis, host genetic factors and environment all contributing to the development of disease. To date several genes have been implicated in TB susceptibility, albeit with small effect. Genome-wide association studies (GWAS) offer the means to identify novel susceptibility variants and pathways through their ability to interrogate polymorphisms throughout the genome without being limited by our understanding of the immune processes involved in TB infection and disease progression. TB and sarcoidosis are both granulomatous diseases, and we therefore hypothesized that the genes and their associated variants identified in recent GWAS conducted in West Africa for TB, and Germany for sarcoidosis, could alter susceptibility to TB in the South African Coloured (SAC) population. In the sarcoidosis GWAS, ANXA11 was shown to alter susceptibility to sarcoidosis; whereas in the TB GWAS, CADM1 was found to alter susceptibility to TB. This study tested the association with TB of 16 polymorphisms in 5 potential TB host susceptibility genes in the SAC population. A well designed case-control study was employed, using the TaqMan® genotyping system to type the various polymorphisms. Any polymorphism that was found to be significantly associated with susceptibility to TB was then subjected to further analysis to determine the functional effect of the polymorphism. Promoter methylation patterns were also investigated in ANXA11 as another mechanism to elucidate its role in TB susceptibility. A 3’ UTR ANXA11 polymorphism was found to be strongly associated with susceptibility to TB, including 3 haplotypes. The gene expression analysis identified differential transcriptional levels between individual with the different genotypes, with individuals homozygous for the A-allele exhibiting a 1.2-fold increase in gene expression relative to those homozygous for the G-allele. Methylation analysis however found no differences between cases and controls. In addition, 16 novel polymorphisms were also identified, 15 of which occurred in the 3’UTR of ANXA11. The mechanism of action of ANXA11 in TB susceptibility is hypothesised to be in the area of endocytosis, autophagy or apoptosis. A weak association was noted with one of the 5’ UTR polymorphisms of CADM3, which did not hold up to further analysis in the GWAS study, and no functional work was therefore done. This work facilitates our understanding of the role of host genetics in susceptibility to TB and adds to the growing amount of information available. Proper understanding of the role that host genetics plays in TB susceptibility could result in better treatment regimens and prediction of individuals who are at a greater risk of developing TB, a disease that still kills millions of individuals annually. / AFRIKAANSE OPSOMMING: Tuberkulose is verantwoordelik vir meer sterftes as enige ander aansteeklike siekte, ten spyte van die voortuitgang wat die Biomediese Wetenskappe tans beleef. In 1949 het Haldane voorgestel dat die genetiese samestelling van die gasheer ‘n rol speel in vatbaarheid vir aansteeklike siektes. Vir tuberkulose word hierdie aanname gesteun deur die feit dat slegs 10% van individue wat geïnfekteer word aktiewe simptome ontwikkel, terwyl 90% die siekte suksesvol sal afweer. Tuberkulose is dus ‘n komplekse siekte wat veroorsaak word deur Mycobacterium tuberculosis, maar wat beïnvloed word deur genetiese sowel as omgewingsfaktore. Verskeie gene is al geïdentifiseer wat ‘n rol speel in vatbaarheid vir tuberkulose, tog is hul invloed betreklik klein. Genoom-wye assosiasiestudies (GWAS) bied unieke geleenthede vir die identifisering van nuwe polimorfismes wat genetiese vatbaarheid kan beïnvloed. Hierdie tegniek kan die hele genoom fynkam, sonder dat enige vooropgestelde idees oor die immuunrespons teen tuberkulose ‘n invloed sal hê. Tuberkulose en sarkoïdose is albei siektes wat die vorming van granulomas veroorsaak. Verskeie gene met hul geassosieerde variante is geïdentifiseer in ‘n onlangse GWAS, wat gefokus het op populasies in Wes-Afrika en Duitsland. Ons hipotese was dat die polimorfismes wat in hierdie studie geïdentifiseer is, ‘n invloed kan hê op genetiese vatbaarheid vir TB in die Suid-Afrikaanse Kleurlingbevolking (SAK). Die sarkoïdose GWAS het bevind dat ANXA11 vatbaarheid vir die siekte beïnvloed, terwyl CADM1 in die tuberkulose GWAS geïdentifiseer is. Die studie het die assosiasie tussen 16 variante en tuberkulose vatbaarheid ondersoek in die SAK populasie. Die variante strek oor 5 potensiële tuberkulose vatbaarheidsgene. Goedbeplande pasiënt-kontrole assosiasiestudies is gedoen en die polimorfismes is gegenotipeer deur gebruik te maak van die TaqMan® genotiperingsisteem. Enige polimorfisme wat beduidend met tuberkulose geassosieer was, is verder geanaliseer om die moontlike funksionele invloed daarvan te bepaal. Promotormetileringspatrone van ANXA11 is ook geanaliseer, om ‘n addisionele meganisme in tuberkulose vatbaarheidheid te ondersoek. Na genotipering van die polimorfismes is ‘n 3’ UTR ANXA11 variant geïdentifiseer wat beduidend met tuberkulose vatbaarheid geassosieer was. Drie haplotipes is ook geïdentifiseer. Geenuitdrukkingsanalise het aangedui dat verskille in transkripsie vlakke voorkom in individue met verskillende genotipes. Individue wat homosigoties was vir die A-alleel het ‘n verhoging van 1.2 in geenuitdrukking gehad, relatief tot individue wat homosigoties was vir die G-alleel. Metileringsanalise het egter geen verskil aangedui tussen pasiënte en kontroles nie. Addisioneel, is 16 nuwe variante ontdek, waarvan 15 in die 3’UTR van ANXA11 geleë was. Die meganisme waarmee ANAX11 genetiese vatbaarheid vir tuberkulose beïnvloed, blyk in die area van endositose, apoptose of outofagie, te wees. ‘n Swak assosiasie is gevind vir ‘n 5’ UTR variant van CADM3 en is nie verder opgevolg in die GWAS nie. Gevolglik is geen funksionele studies op hierdie polimorfisme gedoen nie. Hierdie studie dra by tot ons kennis oor die rol wat die genetiese samestelling van die gasheer speel in vatbaarheid vir tuberkulose. Indien die rol van mensgenetika in tuberkulose vatbaarheid korrek verstaan word, kan behandeling van die siekte verbeter word en kan individue wat ‘n hoër risiko loop om tuberkulose te ontwikkel geïdentifiseer word.