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In the mammalian bloodstream, the protozoan parasite Trypanosoma brucei undergoes differentiation from proliferative slender forms to arrested, transmissible, stumpy forms. This transition is associated with extensive cytological and metabolic changes that promote survival in the tsetse midgut, and also influences infection dynamics within the mammalian host. A number of genes involved in this transformation were recently identified using an RNAi library screen for resistance to pCPTcAMP, a membrane-permeable cyclic AMP analogue that induces differentiation. These molecules, referred to here as posST (positive mediators of STumpy formation), were thereafter validated to regulate the slender to stumpy transition, with many of them apparently comprising part of a signal transduction and effector pathway. However, it is unknown how these proteins act in relation to one another or are ordered within the pathway. To this end, null mutants were created for several posST components in differentiation-competent pleomorphic trypanosomes and, in this genetic background, other members of the predicted pathway expressed to test their ability to restore stumpy formation. Analysis of distinct combinations has been used to build a preliminary pathway structure model for the signalling events underlying trypanosome quorum sensing. In addition, phosphoproteomic analysis of two null mutants has revealed downstream signalling effects of two posST kinases, MEKK1 and YAK. A similar extragenic suppression approach was also applied to explore the interaction between the identified drivers of stumpy formation and the target of rapamycin kinase, TOR4, which has previously been shown to act as a negative regulator of stumpy formation in monomorphs. Dual ablation of TOR4 and posST components revealed insight into the intersection of stumpy-promoting and stumpy-inhibiting pathways. Finally, a chemical-genetic approach was used to investigate the posST pathway using two differentiation-inducing compounds: the previously studied E667, and GKI7, newly identified from a kinase inhibitor set. RNAi lines for different posST components were tested for their ability to undergo development in the presence of these compounds. An RNAi library screen using GKI7 identified putative new mediators of stumpy formation.

WNT lipoglycoproteins (WNTs) modulate a plethora of cellular functions through the activation of the family of frizzled receptors (FZDs). Deregulation in components of the WNT signalling pathways is often observed in human cancers and associated with uncontrolled proliferation and metastasis. Frizzled receptor 6 (Fzd6), one of the ten human FZDs, is frequently overexpressed in cancer, but its role in tumorigenesis is still unclear. In this study we investigated the role Fzd6 in breast cancer. We found that expression of Fzd6 predicts distant relapse in patients with localised breast cancers, particularly in those bearing the triple negative subtype. Using a loss of function approach, we demonstrated that Fzd6 is important to regulate motility and invasion of breast cancer cells in vitro and in vivo. Indeed, Fzd6 regulates the tropism of breast cancer cells the bone, liver and heart of mice. Mechanistically, we found that Fzd6 signalling activates the small GTPase Rho and is important in the organisation of the fibronectin matrix. Both Rho and fibronectin have been previously implicated in the development of metastasis in different systems. All together, these results demonstrate that Fzd6 is an important driver of metastatic spread and a predictive marker of metastatic relapse in breast cancer patients. Fzd6 could therefore be used as a biomarker and target in metastatic breast cancer.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with a dismal prognosis. Few patients present with disease amenable to resection and chemotherapy is not curative. The incidence of ICC is rising worldwide and new therapeutic approaches are urgently required. Notch signalling is critical for the embryological development and regeneration of the biliary tree in the mammalian liver. Dysregulation of Notch is known to drive tumorigenesis in a range of solid and haematological malignancies and the aim of this work was to define its contribution to the pathogenesis of ICC. Transgenic overexpression of Notch1 has been described to result in the formation of biliary lineage tumours in the liver. I have used resected human tissue, a chemically-induced model of ICC in rat and a novel transgenic murine model in which the tumour suppressor p53 is conditionally deleted from biliary epithelia, to demonstrate that endogenous Notch signalling is acting via the Notch3 receptor to drive tumorigenesis. I use multiple independent methods of Notch3 blockade to establish that Notch3 promotes epithelial cell survival and self-renewal in ICC and demonstrate that Notch3 inhibition significantly attenuates tumour growth in vivo. My data suggest that Notch3 promotes activity through the PI3K/AKT cell survival cascade via a mechanism independent of the effector of canonical Notch, RBPJκ. Given the significant toxicity associated with gamma-secretase inhibitors these findings offer a novel and specific target for further investigation and future therapeutic development in ICC.

>Magister Scientiae - MSc / The incidence of type 2 diabetes (T2D) is persistently increasing globally. T2D is associated with pancreatic β cell dysfunction and insulin resistance in peripheral tissues such as the liver and skeletal muscle. Skeletal muscle is the major site for insulin stimulated glucose uptake. Maintenance on a gestational high fat diet may programme insulin resistance. Programming is induced by the exposure of organisms to either a stimulus or insult during foetal and/or early neonatal life and alters offspring physiology and metabolism. The aim of the present study was therefore to investigate the effects of maternal diets, varying in fat content, on neonatal hepatic and skeletal muscle gene (mRNA) and protein (immunoreactivity) expression of proximal insulin signalling factors: insulin receptor alpha (IRα), insulin receptor substrate 2 (IRS2) and phosphoinositide 3-kinase-p110 alpha (PI3K-p110α), and to assess the therapeutic potential of Aspalathus linearis extract after high fat programming. Pregnant rats were randomised into groups maintained on diets with varying fat proportions: 10% (control), 20% (20F), 30% (30F) and 40% (40F) fat as energy throughout gestation. Neonatal liver and skeletal muscle were collected to determine the proximal insulin signalling expression profiles of the target factors: IRα, IRS2 and PI3K-p110α. Quantitative polymerase chain reaction (qPCR) was applied to determine mRNA expression of these target insulin signalling factors. Immunostaining of the target proteins in the liver and skeletal muscle was performed followed by relative quantification with image analysis software. Further, Aspalathus linearis (Al) extract was orally administered to mothers during gestation in the 10% (Control-Al) and 40% (HFD-Al) diets at a dose of 150 mg/kg. Body weight, food intake and blood glucose concentrations were monitored throughout gestation in mothers. Maternal diets, varying in the percentage of fat content, showed no significant effect on neonatal hepatic IR and IRS2 mRNA expression. However, hepatic PI3K mRNA expression was elevated in 30F neonates compared to 20F neonates. Skeletal muscle IR and PI3K mRNA expression were reduced in the 30F and 40F neonates compared to 20F neonates. There was reduced hepatic IRα immunoreactivity in 40F neonates compared to control and 20F neonates. Further, skeletal muscle IRα immunoreactivity was significantly reduced in 30F and 40F neonates compared to control neonates. Therefore foetal high fat programming reduced IRα in both the liver and skeletal muscle which may impair proximal insulin signalling in these glucose recipient organs. Aspalathus linearis had no effect on maternal serum insulin and glucagon concentrations. In addition, maternal caloric intake, body weight and organ weights (liver, brain and pancreas) were not altered amongst the groups. Further, HFD-Al neonates were heavier than control neonates. In conclusion, Aspalathus linearis, at a dose of 150 mg/kg, had neither harmful nor ameliorative effects in pregnant mothers fed high fat diet during gestation. In addition, Aspalathus linearis treatment had no ameliorative effects on neonates from mothers fed high fat diet throughout gestation.

The primary aim of my PhD thesis was to identify and characterise novel modulators of intracellular signalling during early vertebrate development. The first phase of my thesis was to design and execute a large-scale gain of function screen in order to identify novel modulators of various important signal transduction pathways during early Xenopus development. From this screen I identified twenty novel of growth factor signalling. In the second phase of my PhD study, I concentrated on the characterization and mode of action of one of the genes I identified in the screen; namely fezf2. I showed that Fezf2 regulates neurogenesis in the diencephalon by locally promoting Wnt signalling through repression of lhx2 and lhx9. Notably, this investigation on the function of fezf2 not only revealed the previously undiscovered role of fezf2-mediated Wnt regulatory mechanism during diencephalon development, but also confirmed our in vivo screening approach in identifying potential regulators of signalling pathways. To the end, my PhD project has provided me with a fruitful journey of discovery, which started with the design and execution of a large-scale screen, ending with the detailed characterization of a factor involved in the modulation of signalling and forebrain development. This study is has broadened our understanding of how intracellular and extracellular signals are integrated during embryonic development process, which forms an interactive network ultimately resulting in appropriate cell differentiation, organ formation, and regional patterning.

Tgf-β ligands induce cell cycle arrest in a variety of mammalian epithelia, including in the prostate. Genetic deregulation of the downstream canonical Smad-dependent pathway is an early well-studied event in tumorigenesis, yet, in prostate cancer such mutations are rare, leaving unexplained how Tgf-β represses prostate cell proliferation. Here, we adopted a variety of pharmacological and genetic approaches to dissect the pathways controlling proliferation in mouse prostate organoids. We found that Egf signalling is a potent proliferative stimulus through the concomitant activation of both Pi3k/Akt and Mapk/Erk pathways. However, the autocrine release of the Tgf-β family ligand Activin A has a dominant role over Egf-induced proliferation by promoting the non-canonical Tak1-p38α/β axis, which leads to Mapkapk2, p16, p21, and Stat1/2 activation and cell-cycle arrest. Bypass of the proliferation barrier can spontaneously occur upon long-term culture and it is associated to aberrant Activin A signalling and DNA replication stress. Finally, orthotopic transplantation of adapted organoids into immunocompetent hosts, leads to aberrant outgrowth and the emergence of prostatic intraepithelial neoplasia (PIN). Overall, our experiments unveil how Activin A limits mouse prostate progenitor cells proliferation and provide a rationale for the frequent MAP3K7 (TAK1) and ACVR2A (Activin A type II receptor) deletions observed in human primary prostate cancers (20% in the TCGA 2015 dataset).

Cystic fibrosis (CF) is one of the most common genetic diseases in Europe and North America. Chronic bacterial infections with Pseudomonas aeruginosa (P. aeruginosa) are common among CF patients and are associated with increased disease progression among patients. While inflammation is considered to be a key driver of lung function decline, the precise mechanisms at play have remained unclear. The objective of this thesis was to evaluate the role of inflammatory signalling components that result in host cell death during respiratory infections observed in CF. First, I investigated the differences in inflammatory mechanisms and cytokine expression induced by P. aeruginosa isolated from early versus chronic infections in CF. I found that early respiratory isolates of P. aeruginosa from CF patients induced inflammasome signalling, cell death, and IL-1β expression by THP-1 macrophages, yet little expression of other proinflammatory cytokines. However, P. aeruginosa isolates from chronic infections induced relatively less THP-1 macrophage inflammasome signalling, cell death, and IL-1β expression but greater production of other cytokines. Using laboratory reference strains and various mutants of P. aeruginosa, I validated how due to their inability to induce early and extensive host cell death, isolates from chronic infections are able to induce sustained levels of proinflammatory cytokines, which may contribute to the pathogenesis observed in CF. I then investigated one specific virulence factor identified among clinical P. aeruginosa isolates, the effector protein ExoU. ExoU is known to induce rapid host cell death and has previously been described to be an inhibitor of caspase-1, limiting IL-1β secretion in immune cells. Using relevant laboratory reference strains, I have shown that ExoU is able to induce IL-1β expression at lower multiplicities of infection or at earlier time points than described in previous reports when infecting THP-1 macrophages and NuLi-1 bronchial epithelial cells. Through immunoblotting and the use of relevant inhibitors, it was found that this observed difference could be partially dependent on the activation of various caspases, including ones that induced canonical and non-canonical inflammasome activation. Overall, this described work adds to our understanding of respiratory infections observed among CF patients and could shed light on possible therapeutic options to reduce disease progression.

<p> The Notch signalling pathway is a lateral inhibition pathway that serves to limit the number of cells in a proneural cluster (a group of equipotent cells) that will adopt a neural cell fate during neurogenesis in Drosophila. The proper segregation of neural and epidermal progenitor cells during neurogenesis requires the expression of both the proneural genes and the neurogenic genes. Expression of proneural genes, such as achaete, gives cells the potential to commit to a neural cell fate. The neurogenic genes encode proteins that act in the Notch signalling cascade and are required for cell fate determination during Drosophila neurogenests. Notch and Delta are neurogenic genes that encode large transmembrane proteins. Interaction between the extracellular domains of Notch and Delta is thought to transmit a signal to the nucleus by way of the DNAbinding Suppressor of Hairless protein. In response to Notch activation Suppressor of Hairless is translocated to the nucleus where it activates the transcription ofthe neurogenic genes ofthe Enhancer of split complex (E(spl)-C). The products of the E(spl)-C are bHLH transcription factors. They possess a Cterminal tryptophan-arginine-proline-tryptophan (WRPW) motif that interacts with the product of another neurogenic gene, groucho. The groucho gene product encodes a protein containing a WD40 repeat element. When bound to Groucho, E(spl) bHLH proteins are able to repress transcription of proneural genes, such as achaete, thereby directing the cell to adopt a non-neural cell fate.</p> <p> A number of murine groucho homologues have been identified and named Grg's (Groucho related genes). Three full length Grg proteins have been identified which contain all five domains found in the Drosophila Groucho protein. Two short Grg proteins have also been identified which only contain one of the domains found in the full-length Grg proteins. A number of murine homologues of the Drosophila E(spl)-C have also been identified and named Hes (Hairy/Enhancer of split) proteins. Like the gene products of the Drosophila E(spl)-C, the Hes proteins are bHLH proteins containing a C-terminal WRPW motif. One of the Hes proteins, Hes3, is lacking a basic domain and therefore lacks the DNA-binding activity possessed by the other Hes proteins. </p> <p> Attempts were made to detect interactions between Grg and Hes proteins using co-immunoprecipitation techniques. The anti-WD40 antibody, which recognizes the long WD40-containing Grg proteins, was able to specifically immunoprecipitate 35S-labelled Grgl . This antibody was also able to recognize WD40-containing Grg proteins present in Pl9 cell extracts. However, attempts to co-immunoprecipitate radiolabelled Hesl and AMLlb proteins with Grg proteins present in P19 cell extract were unsuccessful due to the low affinity of the antiWD40 antibody and the background caused by the binding of the test proteins to Sepharose. A second method of co-immunoprecipitation was attempted using an HA-tagged Grgl fusion protein and a commercially available anti-HA antibody. The attempt to co-immunoprecipitate 35S-labelled Hesl with radiolabelled HAtagged Grg 1 was unsuccessful due to a high degree of background caused by Hesl binding to protein G Agarose. Using the Yeast Two-Hybrid interaction assay, the WD40-containing Grg proteins, Grgl and Grg4, were found to interact with Hesl. However, using the same assay WD40-containing Grg proteins were found not to interact with Hes3, which lacks DNA-binding activity. A Western blot was performed to determine if the Hes3 fusion proteins were being expressed in transformed yeast but none were detected. This may have been due to the poor affinity of the anti-GAL4 activation domain antibody. A similar Western blot demonstrated that the Grg proteins, fused to the GAL4 DNA binding domain, were being expressed in transformed yeast extract. The WD40-containing Grg proteins, Grgl and Grg4, were also found not to interact with AMLlb, a protein which contains a C-terminal VWRPY domain which is reminiscent of the Cterminal WRPW interaction domain found in Hes proteins and Drosophila E(spl) proteins. However, WD40-containing Grg proteins were able to interact with an AML 1 b mutant in which the VWRPY motif was mutated to VWRPW in the Yeast Two Hybrid assay. </p> / Thesis / Master of Science (MSc)

Yes / Heterozygous loss of function mutations in the type II bone morphogenetic protein receptor (BMPR-II), a member of the transforming growth factor (TGF-β) receptor family, underlie the majority of familial cases of pulmonary arterial hypertension (PAH). The TGF-β1 pathway is activated in PAH and inhibitors of TGF-β1 signaling prevent the development and progression of PAH in experimental models. However, the effect of currently utilized therapies on the TGF-β pathway is not known. Prostacyclin analogues remain the first line of treatment for clinical PAH. We hypothesized that these agents effectively decrease the activity of the TGF-β1 pathway. Beraprost sodium (BPS), a prostacyclin analogue selectively inhibits proliferation in a dose-dependent manner in mouse primary pulmonary arterial smooth muscle cells (PASMCs) harbouring a pathogenic BMPR2 nonsense mutation in both the presence and absence of TGF-β1 stimulation. This study demonstrates that this agent inhibits TGF-β1–induced SMAD-dependent and -independent signaling via a PKA dependent pathway by reducing the phosphorylation of SMADs 2 and 3 and p38MAPK proteins. Finally, in a monocrotaline (MCT)-induced rat model of PAH, which is associated with increased TGF-β signaling, this study confirms that treprostinil (TPS), a stable prostacyclin analogue, inhibits the TGF-β pathway by reducing SMAD3 phosphorylation. Taken together, these data suggest that prostacyclin analogues inhibit dysregulated TGF-β signaling in vitro and in vivo and reduce BMPR-II-mediated proliferation defects in mutant mice PASMCs. / The authors acknowledge financial support from the British Heart Foundation, United Kingdom (Programme Grant 1-2004-357 to R.C.T. and N.W.M.), a Heptagon Life Science Proof of Concept Fund (grants KCL24 and KCL25 to M.T.N. and R.C.T., respectively), and the Great Britain Sasakawa Foundation (grant B70 to M.T.N.)