Carbon catabolite repression (CCR) is a mechanism by which micro-organisms preferentially utilize more easily metabolizable carbon sources in comparison to less easily metabolizable carbon sources. It prevents the organisms from unnecessary expenditure of energy and enables them to exploit the nutrients in appropriate manner. It represents a complex system of gene regulation. The main aim of this study was to study the intracellular localization of proteins involved in CCR including CreA, CreB, CreC and CreD in A. nidulans in repressing and derepressing conditions. The major regulatory protein involved in CCR in A. nidulans is CreA. It is a DNA-binding repressor, but very little is known about the molecular events that allow CreA function to result in appropriate regulation in response to carbon source. To determine the amount and localization of CreA in different carbon sources, strains were made over-expressing GFP and HA tagged CreA. Western analysis showed that high levels of full length CreA can be present in cells that show normal responses to carbon catabolite repression, whether they are grown in repressing or derepressing media. Hence the amount of CreA is similar in both the conditions and thus degradation of CreA is not a key step in carbon catabolite repression. Fluorescence microscopy studies have shown that CreA is in the nucleus under repressing and derepressing carbon conditions and this is not affected by the absence of CreB or CreD, the other important proteins in A. nidulans. Thus mere localization of CreA in nucleus is not sufficient to cause carbon catabolite repression and there is some modification process involved for CreA to act as a repressor protein in CCR. CreB is a deubiquitinating protein and CreC is a protein containing five WD 40 repeats, a putative nuclear localization signal (NLS) and a proline rich region and both the proteins are present in the cell in a complex. CreB was localized using strains that over-expresses GFP tagged CreB and fluorescence microscopy. CreB is present mainly in the cytoplasm in both repressing and derepressing conditions. Moreover, intracellular localization of CreB is unaffected by the presence or absence of CreD. However, the amount of CreB was higher in a creD+ background as compared to a creD34 mutant background, implying that the presence of CreD affects the amount of CreB in the cell. CreC was localized by using strain that over-expresses YFP tagged CreC and it is also present mainly in the cytoplasm. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p from S. cerevisiae. CreD is proposed to be involved in ubiquitination process in CCR in A. nidulans. Localization studies have shown that CreD is present throughout the cell in a punctate pattern with more in the cytoplasm than in the nucleus. CreB and CreD co-localize in some regions of the cell whereas in other regions either CreB or CreD is present. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346526 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Most terrestrial plants make use of beneficial symbiotic associations to obtain nutrients (eg. nitrogen (N) and phosphorous (P)) from fungi in exchange for photoautotrophic carbon. However, plant parasitism (defined here as the ability of certain plants to parasitize other living material) has evolved in the plant kingdom and such plants obtain some, or all, of their nutritional needs from a host, which is severely negatively impacted by the parasite. While the physiological adaptations are well studied, the underlying molecular and biochemical mechanisms of plant parasitism remain largely unknown. As a first approach, a biochemical blueprint of primary metabolites present within parasitic plant species was constructed. The metabolomes of nineteen parasitic plants, ranging from hemi- and holoparasitism to mycoheterotrophism, were profiled via gas chromatography mass spectrometry (GC MS) based technology and targeted spectrophotometric assays. Based on these analyses, three important observations were made. First, parasitic plants were severely carbon deprived, despite being successful in colonizing and exploiting their hosts. Second, the levels of organic acids participating in mitochondrial respiration decreased and certain amino acids and soluble protein content increased. This suggests that parasitic plants utilize alternative respiratory substrates to compensate for a limitation in carbon supply. Third, although characterized by reduced carbohydrate pools, minor sugars normally not associated with plant metabolism, dominated the soluble sugar pool. The presence and significance of one of these sugars, namely turanose (α-D-glucopyranosyl-(1→3)-α-D-fructofuranose), was further investigated. Turanose biosynthetic reactions could be demonstrated in Orobanche minor extracts. Protein purification and mass spectrometry identification suggested that turanose biosynthesis occurred uniquely in parasitic plants. Future work will elucidate the functional significance of turanose metabolism in plant parasitism. Taken together, this study significantly contributes to our understanding of plant parasitism through development of metabolic signatures associated with distinct parasitic classes. These biochemical profiles highlighted several important strategies and alternative metabolic pathways that are either expressed or constitutively activated during parasitism. This knowledge broadens the scope of using parasitic plants in several biotechnological applications or as a novel research tool to address fundamental questions in plant science. / AFRIKAANSE OPSOMMING: Meeste landelike plante maak gebruik van voordelige simbiotiese assosiasies met swamme om voedinsgtowwe (bv. stikstof (N) en fosfor (P)) van hulle te verkry in ruil vir koolstof geproduseer deur die plant. Plant parasitisme (gedefinieer hier as die vermoë van sekere plante om ander lewende materiaal te parasiteer) het ontwikkel in die planteryk waar hulle sommige, of al hul voedings stowwe van 'n gasheer plant ontvang, wat erg negatief geraak word deur die parasiet. Terwyl die fisiologiese aanpassings goed gebestudeer is, is die onderliggende molekulêre en biochemiese meganismes van plant parasitisme steeds grootliks onbekend. As 'n eerste benadering, was hierdie projek geïnisieer om 'n biochemiese bloudruk op te bou van primêre metaboliete teenwoordig in parasitiese plante. Die metabolome van negentien parasitiese spesies, wat wissel van hemi - en holoparasiete tot mikoheterotrofiese plante, is ondersoek deur gas chromatografie – massa spektrometrie (GC MS) gebaseerde tegnologie en geteikende spektrofotometriese toetse. Gebaseer op hierdie ontledings was drie belangrike waarnemings gemaak. Eerstens, parasitiese plante was erg koolstof arm, ten spyte daarvan dat hulle suksesvol is in die aanhegting en ontginning van voedingstowwe vanaf gasheer plante. Tweedens, die vlakke van organiese sure wat deelneem aan mitochondriale respirasie het afgeneem, terwyl sekere aminosure en oplosbare proteïen inhoude toegeneem het. Dit dui daarop dat parasitiese plante gebruik maak van alternatiewe respiratoriese substrate om te vergoed vir 'n beperking in koolstof aanbod. Derde, alhoewel parasitiese plante gekenmerk word deur verminderde koolhidraat inhoude, het skaarse suikers wat normaalweg nie verband hou met plant metabolisme nie, hulle oplosbare suiker inhoud oorheers. Die teenwoordigheid en betekenis van een van hierdie suikers, naamlik turanose (α -D -glucopyranosyl-(1→3)-α-D-fructofuranose), was verder ondersoek. Die sintese reaksie van turanose kan gedemonstreer word in Orobanche hederae uittreksels. Proteïen suiwering en massa spektrometrie identifikasie het voorgestel dat turanose biosintese uniek plaasvind in parasitiese plante. Toekomstige werk sal aandui wat die betekenis is van turanose metabolisme in plant parasitisme. Saamgevat het hierdie studie aansienlik bygedra tot ons begrip van plant parasitisme deur ontwikkeling van metaboliese handtekeninge wat verband hou met onderskeie parasitiese klasse. Hierdie biochemiese profiele beklemtoon verskeie belangrike strategieë en alternatiewe metaboliese paaie wat óf uitgedruk of konstitutief geaktiveer word tydens parasitisme. Hierdie kennis verbreed die omvang van die gebruik van parasitiese plante in verskeie biotegnologiese toepassings of as 'n nuwe navorsings instrument om fundamentele vrae in plant wetenskap aan te spreek.

Species possess characteristics that are considered adapted to burning and these allow them to outcompete species and dominate in fire prone environments. It has therefore been proposed that fire might have played a critical role in the observed expansion of the grasslands, during the late Miocene. The aim of this study was (i) to investigate whether plant response to fire was a result of physiology or (ii) whether it was due to phylogenetic history. This was achieved by doing a pair-wise comparison between Panicoideae (and Panicoideae) and non-Panicoideae (Danthonioideae and Aristidoideae) species. Pre-fire characteristics, that would enhance fire frequency and assist with plant recovery after burning, were compared across phylogenies and photosynthetic type. Post fire plant recovery was then followed in a field and pot comparison which examined the re-growth of the leaf canopy area, leaf mass, above-ground biomass and the cost of this to the below-ground biomass. The pre-fire characteristics showed both a photosynthetic and phylogenetic response. It was found that the species showed a greater canopy death during winter and had a lower moisture content than the species. These characteristics would potentially contribute towards a larger fuel load in the species. However, the comparison of the dead standing biomass at the end of winter and the below-ground biomass, showed a phylogenetic response with the Panicoideae having a proportionally larger dead standing biomass and below-ground biomass than the non-Panicoideae. These results suggest that not only did the Panicoideae have a larger potential fuel load but that they also shunted carbon below-ground, enabling a fast recovery after being burned. The post-fire results were more strongly determined by phylogeny than by photosynthetic type. The Panicoideae recovered faster and more completely than the non-Panicoideae grasses, possibly contributing to their success and expansion under conditions of increased fire frequency. Although recovery of the and Panicoideae were similar, frequently burnt grasslands are dominated by the Panicoideae. Hence, this dominance cannot be explained by differences in their fire responses and may be determined by the post-fire environmental conditions that potentially advantage species possessing the photosynthetic pathway. Panicoideae dominance is limited to mesic environments where fire is the likely driver of grassland expansion while more arid environments are dominated by non-Panicoideae species. Representative species from these non-Panicoid subfamilies showed poor recovery after fire. This suggests that factors other than fire were the likely drivers of these xeric grassland expansions. The ability of these subfamilies, and particularly the species, to cope with drought remains a likely selective mechanism that requires further research.

One-carbon metabolism, a network of enzymatic reactions involving the transfer of methyl groups, depends on B-vitamins as cofactors, folate as a methyl group carrier, and amino acids, betaine, and choline as methyl group donors. One-carbon metabolism influences many processes in cancer initiation and development such as DNA synthesis, genome stability, and histone and epigenetic methylation. To study markers of one-carbon metabolism and inflammation in relation to colorectal cancer (CRC) risk, we used prediagnostic plasma samples from over 600 case participants and 1200 matched control participants in the population-based Northern Sweden Health and Disease Study cohort. This thesis studies CRC risk with respect to the following metabolites measured in pre-diagnostic plasma samples: 1) folate, vitamin B12, and homocysteine; 2) components of one-carbon metabolism (choline, betaine, dimethylglycine, sarcosine, and methionine); and 3) three markers of different aspects of vitamin B6 status. In addition, this thesis examines three homocysteine ratios as determinants of total B-vitamin status and their relation to CRC risk. In two previous studies, we observed an association between low plasma concentrations of folate and a lower CRC risk, but we found no significant association between plasma concentrations of homocysteine and vitamin B12 with CRC risk. We have replicated these results in a study with a larger sample size and found that low folate can inhibit the growth of established pre-cancerous lesions. Using the full study cohort of over 1800 participants, we found inverse associations between plasma concentrations of the methionine cycle metabolites betaine and methionine and CRC risk. This risk was especially low for participants with the combination of low folate and high methionine versus the combination of low folate and low methionine. Well-functioning methionine cycle lowers risk, while impaired DNA synthesis partly explains the previous results for folate. We used the full study cohort to study associations between CRC risk and the most common marker of vitamin B6 status, pyridoxal' 5-phosphate (PLP), and two metabolite ratios, PAr (4-pyridoxic acid/(PLP + pyridoxal)) estimating vitamin B6 related inflammatory processes and the functional vitamin B6 marker 3-hydroxykynurenine to xanthurenic acid (HK:XA). Increased vitamin B6-related inflammation and vitamin B6 deficiency increase CRC risk. Inflammation was not observed to initiate tumorigenesis. Total B-vitamin status can be estimated by three different recently introduced homocysteine ratios. We used the full study cohort to relate the ratios as determinants of the total B-vitamin score in case and control participants and estimated the CRC risk for each marker. Sufficient B-vitamin status as estimated with homocysteine ratios was associated with a lower CRC risk. These studies provide a deeper biochemical knowledge of the complexities inherent in the relationship between one-carbon metabolism and colorectal tumorigenesis.

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The global DNA methylation pattern is erased and re-established during oogenesis and again in preimplantation (PI) embryo development. Understanding where these methyl groups come from and how the process of methylation is regulated is important, as disruptions could result in detrimental effects. The methionine cycle that produces the cellular methyl pool is linked to the folate cycle. The key enzyme linking theses cycles is Methylenetetrahydrofolate Reductase (MTHFR) which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Mthfr RNA and protein are present throughout mouse oocyte and PI embryo development, including the germinal vesicle, MII egg, 1-cell embryo, 2-cell embryo, morula and blastocysts. In MII eggs the protein appears to be heavier than in any other stage. This was reversed by treatment with Lambda Protein Phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. MTHFR was progressively phosphorylated beginning shortly after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing after egg activation using strontium chloride. Potential kinases responsible for the phosphorylation of MTHFR have been identified however not in oocytes or PI embryos. DYRK1A/1 and GSK3A/B have both been suggested to mediate the phosphorylation, however when inhibited showed no effect on the oocyte sample. An LC-MS/MS assay was attempted to measure the activity of MTHFR in wildtype and knockout mouse liver samples, however unsuccessful in the amounts needed to be used for comparison to oocytes. Overall, MTHFR is present in the developing stages of interest and is mediated in some capacity by phosphorylation modifications around the MII stage of development.

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