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<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

Conjugation of the Human Immunodeficiency Virus Transactivator of Transcription (TAT) to active proteins allows transport into the intracellular environment. This feature can be harnessed to deliver combinations of reprogramming factors (RFs) such as c-Myc, Oct4, Klf4 and Sox2 into somatic cells to derive induced pluripotent stem cells (iPSCs). For this project, TAT-fusion proteins including four TAT-conjugated RFs (TAT-RFs) have been produced and purified. All four TAT-RFs can bind specific DNA sequences. Bioactivity was tested in live cells using a novel assay based on an engineered fibroblast cell line that can be induced to express RFs by doxycycline and subsequently generate iPSCs. To test each TAT-RF, reprogramming was blocked by transient silencing of a single RF by siRNA and rescued by the corresponding TAT-RF. The results of this assay suggested that TAT-Klf4 was bioactive in cells; however, definitive evidence could not be obtained for other RFs.

Central to the age-associated decrease in immune system function, characterised by the increase in the frequency and severity of infections and autoimmune diseases, is the decrease in production of naïve T cells by the thymus. This results from the targeted degeneration or involution of the thymus with age. One of the principal causes of involution is the loss of organisation and functionality of the thymic epithelium, which confers the primary function of the organ via interactive regulation of T cell development. Although the mechanisms that govern the deterioration of the thymic epithelium are poorly understood, a number of recent reports indicate that the transcription factor, Foxn1, is required to maintain this compartment in the postnatal thymus. Thus, the first aim of this study was to precisely profile Foxn1 expression levels in aging postnatal thymic epithelial cells. The second aim was to investigate the effects of upregulating Foxn1 in the aging thymus, which was achieved using a novel, regulatable Foxn1 mouse model generated during this study. In this study I show that Foxn1 is expressed at different levels in different postnatal thymic epithelial cell (TEC) sub-populations suggesting a dosage-dependent mode of action for Foxn1. Additionally, using two experimental approaches, I show that Foxn1 expression decreases with age in TECs, supporting the current data that implicate the loss of Foxn1 as a potential cause of thymus involution. Next, I generated a tissue-specific, regulatable Foxn1 mouse model that allowed me to modulate Foxn1 expression in the postnatal thymus. Firstly, using this model, I show that thymus involution can be prevented by the up-regulation and maintenance of Foxn1 expression from the onset of involution. Thymi that up-regulated Foxn1 were overtly larger and exhibited greater cellularity in both the thymocyte and epithelial compartments compared to age matched controls. Additionally, the larger TEC compartment contained a higher proportion of functional and proliferating TECs that upregulated a panel of genes involved in TEC development and function. Next, I show that Foxn1 up-regulation in aged, involuted thymi is sufficient to partially reverse involution, as shown by an increase in TEC organisation and intrathymic T cell numbers. While other strategies that promote thymic rebound or reversal have been reported, including cytokine treatment or sex steroid ablation, these approaches are complicated by side effects and toxicity. Hence, I propose a novel model for immune reconstitution through the regulation of Foxn1 expression in the postnatal thymus.

In Drosophila melanogaster the visceral mesoderm (VM) develops during embryogenesis in a process where myoblasts become specified to generate two distinct cell types, the founder cells (FCs) and the fusion competent myoblasts (FCMs) that consequently fuses. The cell specification is dependent on cell signaling mediated by the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb), how this further sets up different identity programs that drive myoblasts to differentiate into FCs and FCMs is still not well understood. We have analysed whether the Midkine (MDK)/Pleiotrophin (PTN) homologues in Drosophila, Miple1 and Miple2 activate the Alk RTK in vivo. Earlier results from cell culture experiments suggested that vertebrate MDK/PTN is capable of activating ALK, findings that have become controversial with other studies showing contradictory results. We wanted to use Drosophila that have conserved homologues of both MDK/PTN and ALK, to address the question in vivo. We analysed the contribution of Miple in Alk dependent developmental processes such as visceral mesoderm (VM) specification during embryogenesis and in body size regulation of adult flies. Specification of VM as well as body size are not effected by loss of Miple proteins, and over expression of Miple proteins do not effect VM specification or body size. All together we conclude that there is no evidence that Miple1 or Miple2 can activate Alk in vivo. We found that loss of Miple protein effect the median lifespan of the fly which is reduced, interestingly the over expression of Miple proteins can promote an increased median life span in Drosophila. We have also analysed how Alk RTK signaling regulates the Gli-like transcription factor Lame duck (Lmd) in vivo on a post-translational level. It has already been reported that Lmd plays an essential role in specification of FCMs in the somatic mesoderm during embryogenesis. We detect Lmd protein exclusively in FCMs of VM in control embryos, but in Alk mutants Lmd protein is present in all cells of VM and opposite to this when Alk is activated in all cells in VM by over expression of Jeb this results in total loss of Lmd protein. This suggests that Alk signaling is regulating Lmd, and we additionally show that Lmd persist in FCMs in mutants where VM is specified but where myoblast fusion do not occur, supporting that Alk activity in FCs is regulating the downregulation of Lmd in FCMs upon fusion. Finally we have characterised the Rap1GEF C3G in vivo in Drosophila. In cell culture systems, the GTPase Rap1 has been identified to mediate Alk signaling and that this is regulated by the GEF C3G and interestingly the Drosophila C3G is expressed in the FCs of VM. We generated deletion mutants of C3G which exhibit semi-lethality and reduced life span, but no defects in visceral mesoderm development during embryogenesis. Instead we detected distinct phenotypes in somatic muscles of 3rd instar mutant larvae, with detachment and mistargeting of muscles, which effect localisation of integrins. We suggest that Drosophila C3G regulates Rap1 via inside out signaling of integrins which in turn effects cell adhesion in vivo in Drosophila larval muscles.

AGAMOUS-Like 15 (AGL15) encodes a MADS-domain transcription factor that is preferentially expressed in the plant embryo, and may function as a regulator in embryonic developmental programs. A number of direct downstream targets of AGL15 have been identified, and while some of these target genes are induced in response to AGL15, others are repressed. Additionally, direct target genes have been analyzed that exhibit strong association with AGL15 in vivo, yet in vitro, AGL15 binds only weakly. Taken together these data suggest that AGL15 may form heterodimers, or ternary complexes with other proteins, thus modulating the specificity and function of AGL15 in planta. Yeast two-hybrid screens were undertaken to identify novel proteins able to interact with AGL15, and a number of interesting and potentially biologically important AGL15-interacting partners are reported here. These include members of a histone deacetylase complex, a COLD SHOCK DOMAIN (CSD)-containing protein, a Khomology domain/CCCH type zinc finger containing protein, a bZIP transcription factor, a homeobox-leucine zipper protein, a LATERAL ORGAN BOUNDARIES (LOB) domain containing protein, and an Agenet domain containing protein. Interactions between AGL15 and other MADS domain factors that are expressed in embryonic tissue, including SEPALLATA 3 (SEP3) have also been indentified. The regions of AGL15 that mediate interactions with the aforementioned proteins were mapped, and the capacity of these proteins to interact with other plant MADS-domain proteins tested. It is reported herein that AGL15 interacts with members of the SWI-INDEPENDENT 3/HISTONE DEACETYLASE (SIN3/HDAC) complex, and that AGL15 target genes are also responsive to an AGL15 interacting protein that is also a member of this complex, SIN3 ASSOCIATED POLYPEPTIDE OF 18 KD (SAP18). AGL15 can repress transcription in vivo, and a region essential to this repressive function contains an LxLxL motif that is conserved among putative orthologs of AGL15. What is more, the aforementioned motif mediates the association of AGL15 with SAP18 in yeast two-hybrid assays, thus providing a possible mechanism for explaining how role AGL15 regulates gene expression via recruitment of a histone deacetylase complex.

Natural and synthetic heterocyclic cations that bind to the DNA minor groove have demonstrated effectiveness as therapeutic agents for cancer, parasitic and viral diseases, as well as powerful probes for use to extend our fundamental understanding of DNA molecular recognition. Crystal and NMR structures with a variety of minor groove binding compounds have shed light on the structural varieties of these systems, the important solvent molecules in the complexes, and the induced fit effects for binding of both DNA and the bound small molecule. Topics of specific importance in DNA recognition are the development of a greater variety of cell-permeable minor groove agents that have increased DNA binding sequence selectivity. In this dissertation, the structural and energetic basis of the interaction between DNA and minor groove binders has been systematically investigated. A set of powerful and complementary biophysical methods have been used: gel electrophoresis with ligation ladder assay, circular dichroism, mass spectrometry, surface plasmon resonance and isothermal titration calorimetry have been applied to determine the binding stoichiometry, binding affinity, kinetics and thermodynamics, and also the structural influence that minor groove binders can have on DNA. The results of several minor groove complexes clearly show that based on DNA sequences, minor groove binders can have multiple binding modes and consequently affect the geometry of DNA minor groove and the overall DNA curvature in distinct manners. In addition, the binding enthalpy of a minor groove binder is essentially salt concentration and binding mode independent. Besides the investigation of DNA-minor groove binder complex, the binding and inhibition of transcription factor PU.1 has also been studied. The highly positive charged PU.1 targets DNA by inserting an α-helix in the major groove of the 5’-GGAA-3’ site, and displays a strong salt concentration dependency. A set of minor groove binders have been rationally designed based on the high-affinity DNA sequence for PU.1 to target the flanking sequences of the 5’-GGAA-3’ site. They display a structure-related PU.1 inhibition efficacy. This work demonstrates that minor groove binders are capable of modulating PU.1 by targeting the opposite groove and supports future efforts to develop agents for other transcription factors.

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