published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

No description available.

Neuroblastoma is a heterogeneous pediatric malignancy originating from the developing sympathetic nervous system, with poor long-term survival for high-risk patients (~40%). About half of advanced neuroblastomas harbor high-level amplification of the MYCN gene, and these tumors show few, if any, additional driver lesions. Despite significant increase in the body of knowledge of genetics in neuroblastoma, all the high-risk patients follow similar therapeutic procedures and little advancement has been made on molecular target based therapies. The major challenge is to dissect the complexity and heterogeneity of these tumors to find driver genes and activated pathways that are essential for the survival of these cancer cells. We used an integrated systems biology approach to define the core regulatory machinery responsible for maintenance of an aggressive neuroblastoma phenotypic state. In the first part of the thesis, I will discuss our computational approach to decipher the tumor heterogeneity by subtype classification, followed by identification of master regulator protein modules for three distinct molecular subtypes of high-risk neuroblastomas, which were validated in a large independent cohort of cases. We propose that such modules are responsible for integrating the effect of mutations in upstream pathways and for regulating the genetic programs and pathways necessary for tumor state implementation and maintenance. The second part of the thesis is focused on experimental validation of putative master regulators in the subtype of neuroblastomas associated with MYCN amplification. By using RNAi screening followed by experimental and computational analyses to elucidate the interdependencies between the top master regulators, we identified TEAD4-MYCN positive feedback loop as a major tumor maintenance mechanism in this subtype. While MYCN regulates TEAD4 transcriptionally, TEAD4 regulates MYCN through transcriptional and post-translational mechanisms. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally orchestrate 70% of the subtype-specific gene expression signature. TEAD4 gene expression was associated with neuroblastoma patient survival independently of age, tumor stage and MYCN status (P=2.1e-02). In cellular assays, MYCN promoted growth and repressed differentiation, while TEAD4 activated proliferation and DNA damage repair programs, the signature hallmarks of MYCN-amplified neuroblastoma cells. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk neuroblastoma pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. The critical role of the core master regulator module in controlling tumor cell viability, both in vitro and in vivo, and its clinical relevance as a prognostic factor highlights TEAD4 as a novel and highly effective candidate target for therapeutic intervention. In this thesis, we demonstrate that interrogation of tumor specific regulatory networks with patient-derived gene expression signatures can effectively elucidate molecular subtypes as well as the core transcriptional machinery driving subtype specific hallmarks. This approach enables identification of oncogenic and non-oncogenic dependencies of high-risk neuroblastoma and is applicable to other tumor subtypes.

Begonia is one of the ten largest plant genera and is found throughout the tropics. I have used Begonia section Gireoudia to study the genetics underlying vegetative diversity in tropical herbaceous plants. Section Gireoudia is a large Central American group. The section is remarkably diverse in morphology and habitat preference. It ranges from wet rainforests to seasonally dry forests. I have investigated variation in morphological, anatomical and ecophysiological differences for 21 species in Begonia section Gireoudia. Based on the observed variation, species in Begonia section Gireoudia form a complex and unique group that stands out from currently analysed taxa in the global scale of variation on the basis of leaf function and resource use strategy traits as well as their peculiar leaf anatomy. Traits directly related to leaf function such as photosynthesis and stomatal conductance has very low values which overlap with those of CAM and aquatic plants. Values for traits indicative of resource use such as leaf mass area (LMA) and leaf dry matter content (LDMC) are also very low in Begonia when compared with the values observed globally. The trait- trait correlations across the species in section Gireoudia were also investigated and revealed patterns in micromorphology and ecophysiology. Some of the traits measured are correlated with each other in apparently straightforward, well charaterised biological relationships e.g., the variation among Begonia species in stomatal conductance and net assimilation rate are positively correlated. On the other hand, the linkage of high Amass with high Nmass which is in large part the result of a direct causal relationship, has been observed at the global scale but this relationship is not significant in Begonia section Gireoudia. I examined B. plebeja and B. conchifolia, two very closely related though ecologically divergent species from Meso-America, in more detail. I detected significant differences between the species for a number of phenotypic variables which may be related to their habitat preferences. This suggested that environmental conditions have driven divergent evolution of phenotypic traits for these two species. Using a mapping population generated from hybrids between these two species I was able to examine the genetic basis of these differences. This revealed that although some traits (such as anthocyanin accumulation) appear to be under simple genetic control, most of the variation between species has complex genetic inheritance patterns. I used QTL analysis to identify significant QTLs for 20 physiological, anatomical and morphological traits which varied between these two species. Leaf shape traits appear to be largely influenced by a few loci of large effect, making these good potential targets for further analysis. The study also identified clusters of coincident QTLs for different correlated traits identifying pleiotropic genes or suites of linked loci.

The glycine decarboxylase complex (GDC) of Succharomyces cerevisiae composed of four subunits (P, H, T and L) and plays an important role in the interconversion of serine and glycine and balancing the one-carbon unit requirements of the cell. It also enables the cell to use glycine as sole nitrogen source. This study was concerned with characterising the molecular mechanism of transcriptional regulation of the GCVgenes encoding the subunits of the GDC. The important findings of this work can be summarised as follows: i) Transcription of the GCV genes are regulated by glycine and rich nitrogen sources, which are mediated by different cis-acting elements. The LPDl gene did not show a glycine response since its transcriptional regulation is distinct from that of the other genes encoding the GDC subunits. ii) Glycine analogues or serine did not affect expression of GCV2, and therefore glycine probably needs to be metabolised to effect the glycine response of the GCV genes. iii) The repression of the GCV2 gene expression by rich nitrogen sources is mediated by a sequence between -227 and -205 of GCV2, and NCR-regulatory mutant studies showed that repression is not directly controlled by the known NCR system. iv) The glycine response of GCV2 is mediated by a motif (the glycine regulatory region; GRR; 5'-CATCN7CTTCTT-3') with CTTCTT at its core. Additional sequence immediately 5' of this motif (between -310 to -289) plays a minor role for the gene's full glycine response. v) The GRR of the GCV genes can mediate the glycine response by either activation or repression, indicating that the transcription factor(s) mediating the glycine response is/are dual-functional in nature. vi) Studies of GCV2 gene expression using different regulatory mutants showed that expression of the gene is further modulated by other transcription factors such as and Baslp which are distinct from the glycine response and possibly involved in setting up the basal expression level. vii) I n vitro studies of the GRR-protein interaction revealed THF affects the affinity of the DNA-binding protein(s) for the GRR. The importance of THF in regulation of the GCV2 gene was also shown in vivo using a foll mutant that is unable to synthesise any folates. THF or a C1-bound derivative of it acts as a ligand for the transcription factor, thus influencing transcription of the GCV genes in the appropriate physiological manner. viii) Using heparin-Sepharose chromatography fractions, four complex formations (complex I to IV) were observed with the GRR. The protein responsible for one of these was separable from the others. EMSA profiles using the GRR of the GCVI and GCV2 genes (in the presence or absence of THF) were very similar, indicating that these genes bind the same proteins and are regulated in a similar manner. ix) Mutation of the CTTCTT motif within the GRR caused significant reduction in in vitro DNA-protein complex formation, however, THF addition overcame this reduction. x) Only complex II formation was observed with a DNA fragment spanning -322 to -295, and THF affected this complex formation. xi) Footprinting analyses of complex I revealed that the binding protein protected the GRR of the GCV2 gene from DNaseI activity. This protein is an excellent candidate for the glycine response regulatory protein. Titration experiments using EMSA showed that this protein can dimerise. A preliminary genome-wide analysis of the S. cerevisiae transcriptome was carried out using miniarray membrane hybridisation. This investigated the global transcriptional changes within the cell in response to the addition of glycine into the medium. Identification of genes related to various cellular processes including onecarbon metabolism gave an insight into the regulation of the cellular metabolic flow, especially that of one-carbon metabolism. The results indicated that: xii) Glycine is transported into mitochondria to be used as substrate for the GDC which (with mitochondria1 SHMT) produces serine that is subsequently utilised for the various one-carbon metabolic pathways, such as methionine synthesis and purine synthesis. xiii) A gene of unknown function (YER183C) which showed homology to the gene for human 5,lO-CH-THF synthetase was identified from gene-array analysis to be upregulated on glycine addition, indicating the protein encoded by this gene may be involved in balancing the metabolic flow between methionine and purine synthesis when THF pools are disturbed by glycine addition. xiv) Addition of glycine to the medium also triggers the expression of other metabolic genes related to amino acid biosynthetic pathways and that of many other genes which are not directly related to one-carbon metabolism. This may be due to prolonged culturing with glycine in the medium resulting in altered expression of genes mediated by one or more secondary factors. These may reflect an adaptive response rather than a direct consequence of glycine induction. On the basis of the above data, a model for the mechanisms regulating glycine response is presented.