Dissertations / Theses on the topic 'Signalling pathway'

The Multiple Endocrine Neoplasia type 2 syndromes are dominantly inherited cancer syndromes of tumour formation (affecting the thyroid C-cells, the adrenal chromaffin cells and the parathyroids) and developmental abnormalities. Germline activating mutations in the ret proto-oncogene, encoding a receptor tyrosine kinase (RTK), have previously been shown to predispose to MEN 2. In order to understand how Ret mutations cause MEN 2, it is important to understand the biology of Ret, including the signal transduction pathways recruited by both the normal and mutant Ret receptors. I have attempted to identify signalling proteins activated by Ret using two approaches. Firstly, the yeast 2-hybrid system was used to screen an expression library with the intracellular domains of both wild-type and MEN 2B Ret, in order to identify interacting proteins. The unreliability of the screen may have resulted from the Ret fusion proteins used being capable of weak activation of the 2-hybrid reporter genes in the absence of an interacting partner. Secondly, protein tyrosine phosphatases (PTPs) were chosen as candidates to interact with Ret. The population of PTPs expressed in tissues affected by MEN 2 (adrenal gland and a C-cell tumour cell line) was examined by degenerate RT-PCR of conserved phosphate domains. Five PTPs (PTPγ, LAR, PTPβ, SHP1 and SHP2) whose expression were detected were chosen as potential modulators of Ret and studied further. SHP1 and Ret expression studies on neural cell lines indicated that the PC12, Neuro 2A and SH-SY-5Y cell lines might be suitable for further signalling studies on SHP1 and Ret. NT cells would also be suitable if an efficient method of transfection could be found.

NF-κB is a transcription factor which is central to the regulation of genes involved in inflammatory and immune responses. Understanding the operation of NF-κB and its associated intracellular signalling pathway is essential in order to control a wide range of chronic inflammatory diseases, including asthma and rheumatoid arthritis. Abnormalities in the pathway are present in a variety of human cancers and may also affect the pathogenesis of Alzheimer's disease. Computational modelling of the signalling pathway is necessary to overcome the practical limitations of biological experiments and to facilitate a more comprehensive understanding of the system. The thesis begins by outlining existing understanding of the NF-κB signalling pathway, which in conjunction with a review of modelling methods is used to inform a different approach to model the pathway, using computational agents to represent individual molecules and receptors. The agent-based model is tested with well-understood chemical reactions before being used to describe the pathway. This provides a good appreciation of the system as a whole, offering a detailed description of events at every step in the pathway and allowing investigation of stochastic, spatial and structural parameters. The modelling process and simulation help to provide a prediction about the role of actin filaments of the cytoskeleton in regulating the unstimulated pathway; this is quantitatively validated by biological experiment. The effect of cell shape on the pathway is also investigated.

Introduction: Rhabdomyosarcoma (RMS) remains one of the most challenging tumours in paediatric oncology, accounting for around 5% of all malignant paediatric tumours. Of the two major subtypes of RMS, embryonal and alveolar, the latter portends a poorer clinical outcome. Canonical Wnt signalling pathway is an important evolutionarily conserved signalling pathway that is required for muscle development and embryonal somite patterning. β-Catenin is a potent nuclear transcriptional activator and is the central effector of the canonical Wnt signalling pathway. Interestingly, constitutional activation of Wnt signalling also promotes tissue invasion and metastasis in various tumours. Aims: This study aims to characterise the canonical Wnt/β-catenin signalling in paediatric RMS to assess its functional relevance to the tumourigenesis of RMS and to investigate if modulation of this pathway could provide a therapeutic target for RMS. Results: When we evaluated the immunohistochemical expression of β-catenin in the paraffin-embedded tissues derived from 44 RMS patients, we found positive expression in 26 cases. There was positive expression of β-catenin in the cytoplasm or the cell membrane of alveolar (9/14) and embryonal RMS (15/30); nuclear staining was only seen in two embryonal RMS cases. Next, we assessed β-catenin expression by immunoblot analysis in four RMS cell lines - RD and RD18 (embryonal); Rh4 and Rh30 (alveolar). We were able to demonstrate expression of major canonical Wnt proteins in all cell lines that included: β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked and LRP-6. To assess the functional significance of these proteins, we incubated the RMS cell lines with human recombinant Wnt3a to stimulate the Wnt signalling pathway. Thereafter, by using cellular fractionation and immunofluorescence experiments, we demonstrated change in the phosphorylation status of β-catenin, stabilisation of its active form and its nuclear translocation. By employing a TOP/FOP flash reporter gene assay, we showed a T-cell factor/lymphoid-enhancing factor (TCF/LEF)-mediated transactivation. In addition, we found a significant decrease in the proliferation rate of the alveolar RMS cells after Wnt3a stimulation. This decrease in proliferation rate was thought to be due to the concomitant activation of myodifferentiation as seen by the immunoblot expression of myogenin, MyoD1 and myf5. Our data indicates that the major regulatory proteins of the canonical Wnt/β-catenin signalling are expressed in RMS and that functional activation of this pathway, at least in a subset of RMS, may represent a novel therapeutic target.

We identified a synthetic hexapeptide that blocks Magnaporthe grisea appressorium formation, in artificial hydrophobic surface. The results suggest that peptides interfere with surface recognition. M. grisea non pathogenic pth1 mutants were complemented by N. crassa orthologous gene suggesting that the biochemical function of pth1 has not evolved specifically to play a role in appressorium development.

Ad5 and Ad12 inhibit ATR-dependent Chk1 phosphorylation. Ad5 E4orf3 promotes the relocalization of the MRN complex in order to inhibit Chk1 activation during infection, whereas Ad12 E4orf3 is unable to inactivate MRN by this method. Here we show that Ad12 inhibits Chk1 phosphorylation by targeting TopBP1, Timeless and Tipin for degradation. We have determined that Ad12 E4orf6 associates with the cellular Cul2-containing ubiquitin ligase to promote TopBP1 degradation. We have shown that Ad5 and Ad12 differentially activate Cullin-containing ubiquitin ligase complexes during infection. Furthermore, we have also determined that Ad12 E4orf3 promotes the degradation of Timeless and Tipin in an Ad12E1B-55k/E4orf6-independent, and Cul2-dependent fashion, during infection. Previous research from our laboratory identified WDR62 as possible E1B-55K interacting protein. The second aim of this study was to expand our current knowledge of this protein and determine its role during infection. Here we show that E1B-55K interacts with WDR62 in vivo and colocalizes with it at centrosomes. We also provide evidence to show that WDR62 functions in the cellular DNA damage response. Indeed, cells depleted of WDR62 by RNA interference resulted in a UV-sensitive phenotype, defects in ATR activation and G2/M checkpoint control, as well as displaying supernumerary centrosome during mitosis.

The pheromone mating pathway in baker’s yeast Saccharomyces cerevisiae enables this organism to initiate a developmental response upon detection of a mating pheromone. Stimulation of the Ste2 receptor by an external trigger molecule in one of the haploid mating cells of yeast MATa, results in the transcriptional induction of a subset of yeast genes. The aim of this study is to convert this system into a readily measurable response, to function as a biosensor. It is crucial for the improved system to be exclusive from the wild-type response. A successful improved system should be able to detect other peptides, such as peptide markers in diseases. An invivo luciferase activity assay utilizing a PFUS1:LUC construct, was developed to report the activation of the pathway. Deletions of both FAR1 and SST2 genes were proven to provide an increased signal to noise ratio. As an attempt to decouple Ste2 and its ligand, three α-factor analogues; N3G, E7 and C3G, were found to stimulate the mating pathway in decreasing order. A modified Ste12 (Ste12mod) transcription factor coupled with a modified pheromone response element (PREmod2) partnership was also designed. A library of Ste12mod was constructed by random mutagenesis of the N-terminal DNA-binding domain. The mutant Ste12 plasmids were transformed into a far1 sst2 ste12 strain complemented with pFUS1(mod2)K. Screening for mutants with an increased resistance to G418 resulted in 17 unique mutations. Further screening by mating and luciferase assays narrowed down to one mutation (F45I) that responds to modified PRE. However, this strain successfully mated with the wild-type, which warrant further modifications and screening to achieve a full orthogonal system. The results in this study not only provided more insight to the physical interaction between Ste12 and PRE, but also the possibility of uncoupling a pathway in an already developed system.

We analysed immediate-early local and systemic responses to the perception of Pi in Pi-deplete plants or suspension cells. In Pi-deplete plants or cells, RNA abundance of Pi starvation-induced (PSI) genes is very rapidly downregulated in response to Pi or phosphonate, a non-metabolic analogue of Pi. This indicates that Pi perception, not its metabolism is the crucial event in Pi-signalling. We proposed a model for local Pi signalling in which the perception of Pi controls the stability of negative regulators of downstream responses.  Systemic signalling was examined in a split-root system. Pi perception also initiates systemic Pi signalling. The slow systemic translocation of Pi demonstrates that Pi itself is not the systemic signal. To access on the relationship between different nutrient metabolic and signalling pathways, the effects of varying carbon (C) and nitrogen (N) availability on phosphate (Pi) starvation responses and on plant growth were examined. Increased levels of C enhance PSI gene expression responses in roots and shoots, while elevated nitrogen or cytokinin (CK) treatments suppress these responses strongly in roots only. Elevated nitrogen or CK availability shifts shoot-root mass ratios in favour of shoots and alters cellular Pi concentration in both tissues, while increased carbon specifically promotes root growth. Our data indicate that the carbon-nitrogen balance informs growth control networks controlling shoot-root mass ratios. Altered allocation of growth potential by these two nutrients is not dependent on Pi nutritional status and is dominant over Pi starvation-induced growth responses. To identify genes involved in Pi signalling, a genetic screen with Pi starvation responsive reporter (luciferase) line was also performed. A candidate mutant was identified and initial analysis showed it is likely to be a signalling mutant.

Type two diabetes affects 5% of the world's population and is increasing in prevalence. A key precursor to this disease is insulin resistance, which is characterised by a loss of responsiveness to insulin in liver, muscle and adipose tissue. This thesis focuses on understanding insulin signalling using the 3T3-L1 adipocyte cell model. Computational modelling was used to generate quantitative predictions in the signalling pathways of the adipocyte, many of which are mediated by enzymatic reactions. This study began by comparing existing enzyme kinetic models and evaluating their applicability to insulin signalling in particular. From this understanding, we developed an improved enzyme kinetic model, the differential quasi-steady state model (dQSSA), that avoids the reactant stationary assumption used in the Michaelis Menten model. The dQSSA was found to more accurately model the behaviours of enzymes in large in silico systems, and in various coenzyme inhibited and non-inhibited reactions in vitro. To apply the dQSSA, the SigMat software package was developed in the MATLAB environment to construct mathematical models from qualitative descriptions of networks. After the robustness of the package was verified, it was used to construct a basic model of the insulin signalling pathway. This model was trained against experimental temporal data at 1 nM and 100 nM doses of insulin. It revealed that the simple description of Akt activation, which displays an overshoot behaviour, was insufficient to describe the kinetics of substrate phosphorylation, which does not display the overshoot behaviour. The model was expanded to include Akt translocation and the individual phosphorylation at the 308 and 473 residues. This model resolved the discrepancy and predicts that Akt substrates are only accessible to Akt localised in the cytosol and that PIP3 sequestration of cytosolic Akt acts as a negative feedback.

Wnt signalling is a critical cellular communication pathway controlling cell fate in all metazoan organisms. Timely activation of this pathway is crucial to coordinate development, control homeostasis of adult tissues, and to avoid cancer. Wnt signal transduction depends primarily on the activities of three multiprotein complexes; the 'degradasome', which targets the central effector β-catenin for degradation in the absence of Wnt; the 'signalosome', which is assembled by Dishevelled upon Wnt-receptor binding to inactivate the degradasome, thus allowing β-catenin to accumulate; and the 'enhanceosome', which captures β-catenin, granting it access to target genes and relieving their transcriptional repression by Gro/TLE. Many of the components of these complexes have now been identified, but details of their regulation, and in particular the mechanisms by which they are switched on and off, remain poorly understood. The majority of this thesis is concerned with the mechanism by which β-catenin relieves the transcriptional repression imposed upon Wnt target genes, and thereby activates the Wnt 'transcriptional switch'. In Chapter 2, I present data showing that apposition of Gro/TLE and UBR5, a HECT E3 ubiquitin ligase, by β-catenin promotes Gro/TLE ubiquitylation, earmarking it for extraction by the VCP/p97 ATPase and ultimately leading to inactivation of its repressive function. In Chapter 3, I present the results of a different, ongoing study to identify the mechanism by which a cytoplasmic negative regulator, Naked, acts to interfere with the function of Dishevelled, promoting the switching of signalosomes and the termination of canonical Wnt signalling. These findings advance our understanding of the mechanisms by which the Wnt signalling pathway is switched on and off, and suggest new targets for therapeutic intervention in Wnt- driven cancers.

The secret to the specificity of the ubiquitin-proteasome system lies in the protein-protein interaction domains of the diverse group of E3 enzymes. WWP2 is one such E3 enzyme, and the relevant protein-protein interaction domain is the WW domain. WWP2 has four WW domains which are used to interact with proline-rich motifs found in the sequences of the Smad signalling proteins that propagate or inhibit the TGFβ pathway, and in so doing, allows WWP2 to regulate its Smad targets. WWP2 has three isoforms that are known to participate in regulation of TGFβ signalling, but, even amongst isoforms of the same E3, they exhibit different specificities for components of the pathway. The reason for this is unknown, but it is likely to be due to the different domain composition of WWP2, since each of the isoforms has a different combination of WW domains. The aim of this thesis is to investigate the structure of the domains of WWP2, and to explore how this relates to the selectivity of different isoforms in the TGFβ pathway. Overexpression of recombinant WWP2 domains in a bacterial host, and affinity and sizeexclusion chromatography have been used to produce pure, high concentration protein samples. Both NMR spectroscopy and crystallography have been used in an attempt to elucidate the structure of WWP2 domains. NMR spectroscopy, the more successful of the two approaches, has allowed the elucidation of the structure of the fourth WW domain of WWP2. By observing ligand interaction using NMR, the binding site of WW4 is revealed and the substrate preference of WW4 and WW3 domains is observed on a molecular level. Evidence of phospho-regulation of substrate selectivity is presented, and a structural basis for this selectivity is proposed. In addition, a further layer of complexity is added to the WWP2 isoform-mediated regulation of the TGFβ signalling pathway, as a new isoform is discovered.

Mutations in Hedgehog (Hh) pathway genes such as Patched1 are of established importance in Basal cell carcinoma (BCC). However, BCC is unusual because it has indolent behaviour and rarely metastasises. Hh pathway gene mutations are rare in cancers with more typical malignant behaviour, leading to the hypothesis that the pathway plays an important role in BCC but has limited importance in cancers with typical malignant behaviour. This has been addressed in three ways. First, evidence that superficial BCC is a clonal proliferation driven by alterations at the Patched1 locus was sought. Second, evidence of Wnt pathway activation in BCC was sought by looking for nuclear accumulation of -catenin, because Wnt is a putative Hedgehog pathway target gene. Third, to determine whether the Hh pathway was important in a cancer with typical malignant behaviour, breast cancer was studied. In superficial BCC, microdissection of individual tumour nests and analysis for LOH in six cases using three microsatellite markers at the Patched1 locus was performed. Only one of the microsatellites was successfully employed. In four of the cases, all nests showed no LOH and in two cases, all nests lost the same allele. Concordant patterns of allelic loss/retention within cases represented strong evidence of clonality. In addition, the two cases with LOH suggested that clonal proliferation in superficial BCCs might be driven by Patched1 alternations. Accumulation of nuclear -catenin was found in 20 out of 86 BCCs and had a significant relationship with proliferation, indicating that the Hh pathway may act through its target genes. Alternative factors influencing -catenin distribution were sought: no relationship between nuclear -catenin and expression of E-cadherin was seen and no -catenin mutations in eight cases were found. Four breast cancer cell lines and HBL100, derived from normal mammary epithelium, showed substantially lower expression of Hh pathway target genes than BCC and were unresponsive to the pathway ligand, Sonic Hh, suggesting that the pathway is not important in this tumour. However, a relationship was found between the expression of Patched1 and in vitro invasion, the importance of which is uncertain. In conclusion, these results support the hypothesis that the Hh pathway is important in BCC but not in cancers with typical malignant behaviour.

Wnt morphogens control embryonic development and adult tissue homeostasis. Deregulation of the Wnt signalling pathway leads to human diseases. This thesis covers work on two antagonists of the Wnt signalling pathway, Wnt inhibitory factor 1 (WIF-l) (Chapters 1 to 5) and secreted Frizzled-related protein 1 (sFRP-l) (Chapters 1 and 6). In vertebrates the N- terminal WIF domain of the six-domain WIF-1 and the cysteine-rich domain of the two- domain sFRP-1 bind to Wnt proteins and inhibit signal transduction. My human WIF domain crystal structure reveals a novel binding site for phospholipid. Two acyl chains of the phospholipid extend deep into the domain while the lipid head group is surface exposed. Biophysical and cellular assays, combined with structure-guided mutagenesis, indicate a WIF domain Wnt-binding surface proximal to the lipid head group, but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The crystal structure of the six-domain WIF-l reveals EGFs I-V wrapped-back to interface with the WIF domain at EGF Ill. Binding studies locate a heparan sulfate proteoglycan-binding site in EGFs II-V, consistent with highly conserved, positively charged residues on EGF IV. My human sFRP-1 cysteine-rich domain crystal structures reveal a conserved hydrophobic homodimer interface, conformationally variant N- and C-termini, and a conformationally invariant, putative Wnt-binding site. The site is located proximal to a positively charged surface region, which is consistent with the observed sFRP-1 cysteine-rich domain binding to heparin and heparan sulfate. The combination of heparan sulfate proteoglycan- and Wnt-binding properties suggests a model for WIF-1 and sFRP-1 localisation, and signalling inhibition, within Wnt morphogen gradients. The findings described in this thesis provide insights into the medically-important mechanisms of Wnt signalling pathway.

The Notch signalling pathway is important in development and differentiation of a diverse range of both embryonic and adult tissues. There is now strong evidence implicating aberrant Notch signalling in the pathogenesis of T-cell acute lymphoblastic leukaemia (T-ALL), with over 50% of paediatric patients having activating mutations in NOTCH-1. This thesis aims to explore several aspects of the Notch pathway in both T-ALL and acute myeloid leukaemia (AML). Chapter one summarises the published data on the Notch signalling pathway itself, addressing the basic understanding of Notch activation through cell-to-cell interaction, as well as the mechanisms through which it is regulated. The role that Notch signaling plays in normal haematopoiesis is also discussed. Chapter three addresses the incidence and characteristics of NOTCH-1 mutations in a cohort of adult patients with T-ALL in comparison to the published study of paediatric T-ALL, as well as in a cohort of patients with infantile leukaemia and AML. Secondly, the prognostic significance of NOTCH-1 and FBXW7 mutation status of adult T-ALL patients treated on the UKALLXII was investigated. Thirdly, a novel mutation affecting the LNR domain of NOTCH-1 is reported. Chapter four includes data quantifying NOTCH-1 mutation level in T-ALL patients, as well as the stability of NOTCH-1 mutations at presentation and relapse. These data indicate NOTCH-1 mutations commonly occur as secondary events in leukaemia pathogenesis, and suggest widespread clonal heterogeneity in T-ALL. Chapter five explores the functional and prognostic consequences of a novel alternatively spliced isoform of the CSL transcription factor in AML, which was termed CSL-TREX (for TRuncates Exon X). The functional consequences of expressing CSL-TREX in CD34+ cells, in luciferase reporter assays and co-immunoprecipitation experiments with NOTCH-1 is reported. Chapter six summarises the overall implications of these findings to T-ALL and AML, and the future directions of research in this area.

Chapter 1 Provides an overview of drug discovery with particular emphasis on library selection and hit identification methods using virtual based approaches. Chapter 2 Gives an outline of the bone morphogenetic protein (BMP) signalling pathway and literature BMP pathway modulators. The association between the regulation of BMP pathway and cardiomyogenesis is also described. Chapter 3 Describes the use of ligand based virtual screening to discover small molecule activators of the BMP signalling pathway. A robust cell based BMP responsive gene activity reporter assay was developed to test the libraries of small molecules selected. Hit molecules from the screen were synthesised to validate activity. It was found that a group of known histone deacetylase (HDAC) inhibitors displayed most promising activity. These were evaluated in a secondary assay measuring the expression of two BMP pathway regulated genes, hepcidin and Id1, using reverse transcription polymerase chain reaction (RT-PCR). 188 was discovered to increase expression of both BMP-responsive genes. Chapter 4 Provides an overview of existing cannabinoid receptor (CBR) modulating molecules and their connection to progression of atherosclerosis. Chapter 5 Outlines the identification and optimisation of selective small molecule agonists acting at the cannabinoid 2 receptor (CB₂R). Ligand based virtual screen was undertaken and promising hits were synthesised to allow structure activity relationship (SAR) to be developed around the hit molecule providing further information of the functional groups tolerated at the active site. Subsequent studies led to the investigation and optimisation of physicochemical properties around 236 leading to the development of a suitable compound for in vivo testing. Finally, a CB₂R selective compound with favourable physicochemical properties was evaluated in vivo in a murine inflammation model and displayed reduced recruitment of monocytes to the site of inflammation.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in the majority of triple negative breast cancer patients (TNBC). However, the molecular determinants behind their limited response to EGFR-targeted therapies are poorly understood. Here, both the acute and chronic responses of TNBC to the EGFR-targeted therapy, cetuximab (CTX), have been investigated. The expression of EGFR has been analyzed in a cohort of 2000 breast cancer tumours from the public dataset as well as in a panel of breast cancer cell lines. Furthermore, the response of TNBC cell lines to CTX has been investigated using conventional biochemical methods. Finally, a comprehensive transcriptomic profiling of an acquired CTX-resistant TNBC model by RNA sequencing has been performed to understand the molecular determinants of acquired CTX resistance. The results confirmed that EGFR is highly expressed in TNBC in comparison to non-TNBC breast cancer tumours and cell lines, which was associated with adverse clinical outcomes. Targeting EGFR in TNBC cell lines using CTX failed to completely inhibit the EGFR signalling pathway and was associated with an increase in ADAMs-mediated release of endogenous EGFR ligands, EGF and TGFα. Inhibition of ADAMs (ADAM10 and ADAM17) significantly enhanced the anti tumour efficacy of CTX both in vitro and in vivo. Furthermore, transcriptomic profiling of the acquired CTX-resistant TNBC cell line (MDA-MB-468CR) revealed an activation of several key oncogenic pathways and genes, including the TGFβ/BMP pathway. Blocking BMP receptors (BMPRs) restored the sensitivity of resistant cells to CTX treatment. Collectively, current findings offer alternative strategies that could enhance the CTX response in TNBC. We further reported that simultaneous targeting of both EGFR and BMPR pathways could overcome CTX resistance, which might have important implications for the treatment of TNBC.

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 Å resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

Campylobacter jejuni colonizes the intestinal mucosa of all food-producing animals and humans (Newell & Fearnley, 2003) and is the leading cause of human bacterial gastroenteritis worldwide (Blaser, 1997; Friedman et al., 2000). It is unclear exactly how C. jejuni induces disease outcome in humans, however, a number of factors have been implicated in the pathogenesis of this organism, including colonization and adherence, invasion and translocation, toxin production, LOS and capsule structures, flagella and motility, protein glycosylation and chemotaxis. Chemotactic behaviour has been demonstrated in C. jejuni (Hugdahl et al., 1988) and previous studies have shown the importance of chemotactic motility to the ability of C. jejuni to colonize and cause disease (Takata et al., 1992; Yao et al., 1997). However, the mechanisms controlling the C. jejuni chemotaxis signalling pathway remain largely unknown.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Prostate cancer is the leading form of male cancer in western countries. However, despite a great deal of interest from the scientific community, the cause still remains elusive. What is clear, is that androgenic signalling is important within prostate cancer progression, with early cancer under androgenic control and androgen regulated genes, such as PSA, commonly used for patient prognosis and progression. 3',5'- cyclic adenosine monophosphate (cAMP) is an important second messenger signalling molecule, which has been extensively studied within the field of oncogenesis. cAMP signalling effects are transduced via its effector molecules, PKA and EPAC, and the temporal and spatial control of cAMP dynamics are orchestrated by phosphodiesterase enzymes (PDEs). This signalling pathway has been implicated in a wide range of cellular processors such as, cellular differentiation, transcription, proliferation, apoptosis and learning and memory. In addition, it has also been linked to steroid receptor signalling such as oestrogen, progesterone, and androgen receptors. PKA can be expressed as 2 forms (PKA-I and PKA-II) depending on the regulatory unit isoform content of the enzyme. In the field of cancer research, PKA-I and PKA-II expression profiles have been shown to be linked to the phenotype of the cancer, i.e. overexpression of PKA-I is linked to a more proliferative cancer. Furthermore, other protein intermediates within the cAMP signalling pathway, including PKAC isoforms (PKA catalytic unit) and PDEs, change with respect to isoform expression and regulation during cancer progression. In this thesis, I have described my original research in which I endeavoured to understand the role of several components of the cAMP signalling axis in human prostate cancer. I have profiled the isoforms of PKA in androgen sensitive and insensitive prostate cancer, identifying isoforms that are dysregulated in the androgen independent disease stage. In particular, I found a down regulation of PKARIIβ and PKACβ isoforms in AI prostate cancer compared to AS. The function of these isoforms are not well categorised in prostate cancer. Due to the higher expression of these isoforms in AS cells, I went on to scrutinise these isoforms in relation to their interaction with androgenic signalling. Here, I have uncovered previously undiscovered aspects of PKA signalling, including the direct interaction of PKARIIβ and PKACβ isoforms with the androgen receptor (AR), and that PKA can phosphorylate the AR at site S791. Further analysis of this putative phosphorylation site would be beneficial to isolate a functional output. In addition, following previous work in the laboratory that identified PDE4D7 as a potential biomarker in prostate cancer, I have investigated novel-PDE4D7 controlled signalling pathways using cutting edge techniques. This data allows a better understanding of the mechanism behind the alteration of PDE4D7 driven signals and their significance during prostate cancer progression. Given more time and scrutiny, changes in these identified novel-PDE4D7 controlled signalling pathways, such as N-MYC, could show sites for exciting study into prostate cancer progression.

This project highlights the important role of cell signalling pathway during tooth regeneration. Biomaterials can be designed to activate relevant cell signals for the purpose of dental repair and tooth regeneration. Based on the results in the present project, strategies directly targeting cell signalling pathway may provide new approaches for periodontal regenerative tissue engineering.

This project contributes to our understanding of bone cell biology and sheds light on the potential therapeutic application of Notch signalling pathway on bone-related diseases. The thesis was a step forward in answering how bone cells communicate with each other and determinate their own fates. It provides the first evidence demonstrating Notch signalling is critical in bone cell functions.

The mechanism of body and organ size control is an unsolved puzzle. Initially characterized in Drosophila melanogaster, the Salvador/Warts/Hippo (Hippo) signalling pathway, highly conserved throughout evolution, defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis, and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. Previously Willin/FRMD6 has been proposed as the human orthologue of Expanded and, to date, little is known about the functional role of Willin in mammalian cells. My study elucidated the mechanism by which Willin antagonizes the transcriptional co-activator YAP. In MCF10A cells, Willin ectopic expression antagonizes YAP-induced epithelial-mesenchymal phenotypes via YAP Ser127 phosphorylation site. Loss of Willin expression attenuates MST1/2, LATS1, and YAP phosphorylation promoting YAP's oncogenic transformation activity in vitro, as analysed by its ability to display epithelial-to-mesenchymal transition (EMT) features. These biological outputs are YAP dependent. These data support the involvement of Willin in the regulation of the mammalian Hippo signalling activity by activating the core Hippo pathway kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but the functional consequences of these biochemical changes have not been previously addressed. I showed that in MCF10A cells, loss of KIBRA expression displays EMT features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the Ser 127 phosphorylation site and I showed that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Willin/FRMD6 was first identified in rat sciatic nerve, which is composed of Schwann cells and fibroblasts. To elucidate the function of Willin in the mammalian sciatic nerve, I showed that Willin is predominantly expressed in fibroblasts and that its expression activates the Hippo signalling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These evidence show that Willin modulates sciatic nerve fibroblast activity, indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.

Prohibitin (PHB) is a highly conserved, multi-functional protein that acts as a tumour suppressor and androgen receptor (AR) corepressor. We found that downregulation of PHB by androgens is required to drive androgen-dependent prostate cancer cell growth. This effect can be mimicked through the use of PHB siRNA, whereby reduction in PHB protein levels enhances cancer cell and xenograft tumour growth. Conversely, PHB overexpression results in rapid accumulation of prostate cancer cells in G0/G1. The potential importance of PHB in prostate cancer development merits investigations into possible androgen-mediated mechanisms of PHB regulation. We observed significant reduction in endogenous PHB mRNA and protein levels, and a 20% decrease in transfected PHB promoter activity, following androgen treatment. We have also demonstrated interaction between AR and the proximal PHB promoter. These results suggest that AR may directly regulate levels of its own corepressor. In addition, we have shown that mutagenesis of residues with potential importance for PHB post-translational modification alters PHB subcellular localisation and permits PHB to function as a coactivator of AR. Recently, PHB has been identified as a potential target of microRNA 27a (miR-27a). Knockdown of miR-27a in gastric cancer cells increased PHB mRNA and protein expression (Liu et al., 2009). We therefore hypothesised that miR-27a may function as an 'oncomiR' in prostate cancer through PHB suppression. It was established that androgen-mediated downregulation of PHB can be attributed largely to miR-27a, since we found that: 1) MiR-27a expression leads to reduced PHB mRNA and protein levels through association with PHB 3'UTR, resulting in enhanced expression of AR target genes, PSA and TMPRSS2, and increased prostate cancer cell growth. These effects can be abrogated through miR-27a binding site mutation, anti-androgen treatment, exogenous PHB expression, or addition of miR-27a anti-sense inhibitor oligo (ASO) 2) MiR-27a is upregulated by androgens and downregulated by anti-androgens in prostate cancer cells. This represents a novel mechanism of PHB regulation, and implies that miR-27a may function as an "oncomiR" in prostate cancer. Hence, decreasing miR-27a levels may provide a potential thereapeutic strategy for the treatment of prostate cancer. Upon investigation of mechanisms of miR-27a regulation by androgens, a complex and novel mechanism of regulation was found: AR associates with the miR-27a promoter, leading to increased polycistronic transcription of miR-27a and its cluster partners, miR-23a and miR-24-2. Thus AR exhibits an element of regulation at the level of transcription. However, all three mature miRs show accumulation upon androgen treatment in the absence of transcription, whilst pri-miR levels are increased following AR silencing. These data thus suggest that AR signalling accelerates processing of primiR-23a27a24-2 in addition to its transcriptional role, increasing levels of mature miR-27a and its cluster partners. In contrast, anti-androgen treatment blocks processing of the pri- miR, reducing mature miR-27a levels and thus increasing PHB expression. We propose that AR- mediated post-transcriptional regulation of miR-27a occurs during Drosha-mediated primiR- 23a27a24-2 processing, since activity of a primiR-23a27a24-2-specific Drosha processing reporter is reduced by androgen treatment. GSK3B inhibition led to reduced miR-27a biogenesis, suggesting that GSK3B kinase activity may be involved in androgen-enhanced Microprocessor primiR-23a27a24-2 cleavage. I have also demonstrated that Drosha is transcriptionally androgen regulated through AR association with the Drosha promoter but this occurs too late to mediate androgen-regulated miR- 27a accumulation. It was also found that miR-27a targets the receptor tyrosine kinase EGFR, which has multiple cell signalling functions in cancer. Taken together, these data demonstrate that PHB is a target of oncomiR-27a in prostate cancer and that PHB is regulated by androgen both at the levels of transcription and translation, mediated by several related pathways: 1. Androgen binding to AR facilitates association with potential AREs within the PHB promoter, repressing gene transcription 2. Androgen signalling leads to upregulation of miR-27a, through increased transcription and enhanced Drosha-mediated primiR processing, which then associates with PHB 3'UTR, resulting in PHB translational inhibition. 3. Post-translational modification of PHB by phosphorylation and/or acetylation is vital in determining PHB subcellular localisation and corepressive function. We identify a novel mechanism of androgen-mediated post-transcriptional regulation of miR maturation, generating a positive feedback loop by which AR maintains its own activity by reducing corepressor activity. Additionally, miR-27a ASO may represent a potential novel therapeutic for prostate cancer.

Early studies of the tomato ethylene signalling network using yeast two-hybrid screen previously identified three novel proteins (IntCR22, 242 and 266) that could interact with a putative ethylene kinase LeCTR2 (Lin et al., 2003). In this study, it has been demonstrated that IntCR22 is a cytoplasmic UDP-glycosyltransferase and IntCR266 is a chloroplast metallo-proteinase homologue to the Arabidopsis FtSH5/VAR1, whereas IntCR242 encodes a novel chloroplast protein with a C-terminal histidine-rich domain. In order to gain more insight into the tomato ethylene signalling mechanism, the sub-cellular localization and protein-protein interactions of the tomato ethylene signalling components have been investigated by fluorescent protein labelling and yeast two-hybrid experiments. Three tomato ethylene receptors (ETR1, NR and ETR4) and a downstream regulator EIN2 have been found in the endoplasmic reticulum (ER). Three putative downstream MAPKK kinases (CTRs) could interact with the C-terminus of the ethylene receptor possibly on the cytoplasmic side of the ER, whereas a novel ethylene signalling component GREEN-RIPE was located in the Golgi. It was therefore concluded from the localization study that IntCR242 and IntCR266 were false positives from the yeast two-hybrid screen and could not interact in vivo with the ethylene signalling components. The results presented in this study, in line with previous ethylene research suggest a possible involvement of the plant endomembrane system in the ethylene signalling network. However, the question as to how the ethylene signal moves from the ER localized receptors to promote activation of genes for the transcription factors within the nucleus remains unsolved.

Introduction. The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are common in Western Europe (200-400 cases /100,000) and associated with substantial morbidity, although mortality is now low. There is presently a great unmet need for novel therapeutics in IBD as present agents are limited by lack of efficacy, toxicity and poor patient acceptance. Recent findings from genome-wide association studies (GWAS) have characterised the genetic architecture of CD and UC. Defects in innate and adaptive immunity have been clearly established, and substantial novel insights into disease pathogenesis have been gained. Over 30 genes / loci are now associated with CD; a number of these, along with a few specific loci, are also associated with UC. The hedgehog (HH) signalling pathway is critical to gastrointestinal development and plays key roles in intestinal and immune homeostasis. Furthermore, in addition to well described roles in tumorigenesis, it is evident that recapitulation of embryonic HH signals play critical roles in response to acute and chronic inflammatory challenge in diverse tissues. Aims. The main aims of the work presented in this thesis were to characterise the expression of key HH signalling components in the healthy and inflamed human intestine, establish whether germline variation in HH genes is associated with IBD and describe the in vitro responses of intestinal epithelial cells to pathogen associated molecular patterns. The WNT pathway, antagonised by HH in the intestine, and two HH target genes (NKX2.3 and CCL20) were also analysed for evidence of association with IBD. Methods. Expression of HH and WNT signalling components was described by immunohistochemistry and microarray analysis in healthy controls (HC), CD, UC, and non- IBD inflamed terminal ileal and colonic samples. Gene-wide haplotype-tagging studies were performed for GLI1 in Scottish, English and Swedish CD and UC, and Scottish early-onset colo-rectal cancer, IHH in Scottish IBD, NKX2.3 in Scottish and UK IBD, and CCL20 in Scottish, Swedish and Japanese IBD. Evidence for association of all HH (n=13) and WNT (n=27) signalling genes in CD was established by analysis of UK GWAS data and metaanalysis from UK, French/Belgium and N American studies. The effect of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on HH signalling was assessed in colonic epithelial cells (SW480). The effect of HH pathway agonists and antagonists on NFκB activity and cytokine expression was analysed in SW480 cells and peripheral blood mononuclear cells (HC and IBD patients) in vitro. Results. The expression of HH pathway ligand is present in the intestinal epithelium and the pathway response network in the lamina propria demonstrating the paracrine nature of HH signalling in the intestine. Immunohistochemical studies and microarray analysis demonstrates that HH pathway activity is decreased in all forms of colonic inflammation studied in man. Variation in Glioma-associated oncogene homolog 1 (GLI1), a key HH transcription factor located at 12q13 (IBD2), was associated with IBD (p<0.0001), UC (p<0.0001) and to a lesser extent CD (p=0.03) in Scotland, a finding replicated in English IBD and UC. This association was attributed to a non-synonymous SNP (rs2228226C→G) with pools odds ratio of 1.194 in meta-analysis of over 5000 individuals from Scotland, England and Sweden (p=0.0002). There was association of this SNP with early-onset colorectal cancer, but of borderline significance (p=0.05). The variant protein (Q1100E) is 50% less active than wild-type protein in vitro. IHH was not associated with CD or UC. Preliminary evidence was produced for association at SUFU (10q24; p=0.005), a GLI1- binding protein, and at the WNT3 / WNT9B locus (17q21; p=0.0005). MDP stimulation of colonic epithelial cells decreased HH pathway activity. Exogenous HH increased expression of CCL20. CCL20 promoter polymorphisms were associated with UC in Japanese patients (p=0.018) but not in Scotland or Sweden. NKX2.3 was associated with IBD in Scotland (UC>CD), but there was insufficient power for fine-mapping of causative variants. Conclusions. Multiple lines of evidence presented here demonstrate that the HH signalling pathway is involved in IBD pathogenesis. In key complementary in vivo studies (conceived by CWL; conducted in collaboration with the Gumucio lab in Ann Arbor) we have demonstrated that Gli1+/- mice develop early, severe colitis with high mortality in response to acute inflammatory challenge. Furthermore, lamina propria antigen presenting cells are identified as the key HH target cells. With HH agonists and antagonists in extensive preclinical and early clinical testing, these studies have real potential to translate into novel therapeutics for patients with IBD.

Von Hippel–Lindau (VHL) is the E3 ubiquitin ligase targeting hypoxia-inducible transcription factor-alpha (HIF α) for proteasomal degradation. The crucial function of VHL in response to hypoxia and cellular oxygen sensing are well established, owing to the use of genetic tools through knockout and knockdown that inactivate VHL. However, the functional consequences of specifically interrupting the interaction between VHL and HIF α remain to be elucidated. The development of a chemical probe that unambiguously blocks the VHL:HIF α interaction downstream of HIF α hydroxylation by prolyl hydroxylase domain (PHD) enzymes, would address biological questions about VHL molecular targets and functional consequences of disrupting the interaction. Here, small molecules inhibiting the VHL:HIF α interaction were shown for the first time to stabilise HIF α and elicit HIF transcriptional activity in cells. The most potent VHL inhibitor identified is VH298. VH298 is potent, cell-permeable, selective, and not toxic at the concentration required for HIF α stabilisation. Further characterisation shows that VHL inhibitor exclusively induces HIF-dependent changes in global gene and protein expression, demonstrating the specificity of the inhibitor. VHL protein level was found to increase in the presence of VHL inhibitor, which in turn promotes the degradation of HIF α in prolonged inhibition. The work herein characterises the VHL inhibitor as a chemical probe of the hypoxia signalling pathway with great potential to address biological questions regarding the roles and regulation of VHL. The VHL inhibitor is a unique tool due to its on target selectivity and specificity in inducing HIF activity, without affecting HIF-independent response, and exerts its effect further downstream than PHD hydroxylation. This work provides a foundation and cellular proof of concept for future studies evaluating therapeutic potential of VHL inhibitor in diseases, such as chronic anaemia, ischaemia, and inflammation-driven diseases.

Haematopoiesis is the process to produce haematopoietic stem cells (HSCs), haematopoietic progenitors (HPCs) and terminally differentiated cell types. In the adult, HSCs resided in bone marrow while in the embryo, haematopoiesis occurred sequentially in several niches including yolk sac, aorta-gonad-mesonephros (AGM) region, placenta and fetal liver. The AGM region is the first place where HSCs arise in vivo and therefore should provide important factors to induce haematopoiesis. The mouse embryonic stem cells (mESC) system is a powerful platform to mimic the development process in vitro and is widely utilized to study the underlying mechanisms because they are pluripotent and can be genetically manipulated. A novel co-culture system has been established by culturing differentiating mESCs with primary E10.5 AGM explants and a panel of clonal stromal cell lines derived from dorsal aorta and surrounding mesenchyme (AM) in AGM region. Results of these co-culture studies suggested that the AM-derived stromal cell lines could be a potent resource of signals to enhance haematopoiesis. Molecular mechanism involved in haematopoiesis is a key research direction for understanding the regulation network of haematopoiesis and for further clinical research. A series of studies have demonstrated involvement of the Notch signalling pathway in haematopoiesis during development but with controversial conclusions because of the difference of models concerning various time windows and manipulating populations. This project aimed to investigate the role of Notch signalling pathway during haematopoiesis in the AGM environment. We analyzed the expression of Notch ligands in AGM-derived stromal cells with or without haematopoietic enhancing ability. No correlation was observed between ligand expression and haematopoietic enhancing ability in stromal cell lines or between Notch activity in EBs and haematopoietic enhancing ability. We demonstrated that inhibition of the Notch signalling pathway using the γ-secretase inhibitor could abrogate Notch activity in both mES-derived cells and the haematopoietic enhancing AM stromal cell line. To better understand the involvement of the Notch signalling pathway in a more specific spatial-temporal environment, we established a co-culture system of haemangioblast like cells (Flk1+) with one of AM region derived stromal cell lines with haematopoietic enhancing ability . We found that the AM stromal cell line could enhance Flk1+ derived haematopoiesis as assessed by haematopoietic colony formation activity and production of CD41+cKit+ progenitor cells. Based on the issue that the inhibitor could potentially affect both the ES cells and stromal cells, we carried out genetic approaches to overexpress or knock down Notch signalling pathway in this Flk1+/AM co-culture system. Interestingly, it was found that when Notch activity was enhanced in Flk1+ cells, the production of haematopoietic progenitors was inhibited and the number of cells expressing the pan-haematopoietic marker CD45 was reduced. By using the inducible dominant negative MAML1 system to knock down Notch activity, it was found that the haematopoiesis in the Flk1+/AM co-culture system was not affected, which could be accounted for the low Notch activity in this system. These results supported the hypothesis that the Notch signalling pathway plays a role in modulating Flk1+ derived haematopoietic differentiation within the AGM microenvironment.

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s and old age is the strongest risk factor for developing PD. PD has traditionally been seen as a motor disorder, but its non-motor symptoms such as dysautonomia, sensory dysfunction, sleeping problems and neuropsychiatric features equally add to the disease burden. There is no cure for PD and this is probably a reflection of our poor understanding of the disease pathogenesis. One way of tackling this is to focus on the small, but significant number of PD patients with a family history compatible with Mendelian autosomal inheritance (10-15%). Hereditary and sporadic PD share important clinical and neuropathological features, and there is reasonable hope that dissecting molecular pathways of PD gene products will have more general implications for the pathophysiology of PD associated neurodegeneration and help device new treatment strategies. Mutations in the FBXO7 gene have recently been shown to cause an autosomal recessive early onset Parkinsonian-pyramidal syndrome and FBXO7 has been designated as PARK 15 (Di Fonzo et al., 2009). FBXO7 is a member of the F-box protein family, which functions as the variable subunit of Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complexes and as such dictate substrate specificity. The canonical outcome of ubiquitylation is proteasomal degradation and my working hypothesis is that FBXO7 may be involved in protein quality control in the brain. A perturbation thereof may be a first step towards FBXO7 dependent disease. At the time of starting with my PhD project, little was known about the molecular function of FBXO7 and how mutations in FBXO7 result in neurodegeneration. In order to learn more and dissect the signalling pathway of FBXO7 I have used tagged stable overexpression cell lines of the FBXO7 wildtype as well as human disease mutant proteins for tag-pulldowns followed by mass-spectrometry to identify interacting partners and possible substrates. With this approach I have been able to confirm the interaction between FBXO7 and its core SCF E3 ligase partners as well as some of the previously reported interacting partners. I have been able to show that not only the FBXO7 wildytpe protein, but also all of the so far reported human disease mutants are able to assemble into an SCF complex. Hence, my fist conclusion is that the human disease mutants do not exert their pathogenicity by SCF complex disruption. Next, a knock-in (KI) mouse model of one of the pathogenic FBXO7 mutations (R378G) was generated and evaluated by molecular and biochemical approaches as well as motor and behaviour phenotyping. In particular, I have used the Fbxo7 mouse model for extensive proteomic screens to identify wildtype (wt) and KI Fbxo7 interactors: endogenous Fbxo7 immunoprecipitations from mouse brain lysates and subsequent fingerprint mass-spectrometry; differential whole proteome: ex vivo differential dimethyl labelling of wt and KI brain samples, and Fbxo7-dependent ubiquitinome analysis: quantitative di-GLY capture proteomics combining in vivo SILAC labelling with antibody-based affinity enrichment of “di-GLY remnant motifs”- containing peptides prior to proteomic profiling of the wild-type in comparison to the homozygous R379G Fbxo7 KI ubiquitinome in MEF lysates. The di-GLY remnant motif is the signature peptide of ubiquitinylated protein sites at peptide level after tryptic digestions. Some of my findings are: • For the first time I show that endogenous Fbxo7 actually assembles into an Skp1-Cullin1-Fbxo7 complex and that the pathogenic R378G does not disrupt SCFFbxo7-KI complex formation in vivo. This is true for the Fbxo7 KI mouse model, but also for patient derived immortalized cell lines carrying the R378G FBXO7 mutation.• Endogenous Fbxo7 interacts with the Sumo E3 ligase complex RanBP2/ RanGAP1*Sumo1/Ubc9 complex. • In the differential enrichment of ubiquitylated protein species in SILAC labelled wild-type and homozygous R379G Fbxo7 KI MEFs, I have clearly identifies 2 highly conserved lysine residues, which are conserved amongst VDAC 1, 2, and 3 in mouse as well as human homologous, to be preferentially ubiquitinylated in a Fbxo7 wild-type background (in collaboration with Dr. Patrick Pedrioli, MRC Programme leader).• There is a significant difference in motor performance between wildtype and homozygous R379G KI Fbxo7 mice at 10 months of age (in collaboration with Dr. Steve Martin, Neuroscience Division, Dundee). • Furthermore, I have successfully set up an in vitro FBXO7 dependent ubiquitinylation assays.

RESUMO: O corpo carotídeo (CB) é um pequeno órgão sensível a variações na PaO2, PaCO2 e pH. As células tipo I (células glómicas) do corpo carotídeo, as unidades sensoriais deste órgão, libertam neurotransmissores em resposta às variações dos gases arteriais. Estes neurotransmissores atuam quer em recetores pré-sinápticos, localizados nas células tipo I, quer em recetores póssinápticos, localizados nas terminações do nervo do seio carotídeo, ou em ambos. A activação dos recetores pré-sinápticos modula a atividade do corpo carotídeo, enquanto que, a activação dos recetores pós-sinápticos, de carater excitatório, desencadeia um aumento da frequência de descarga das fibras do CSN, com subsequente despolarização dos neurónios do gânglio petroso, e posterior despolarização de um grupo específico de neurónios do centro respiratório central, desencadeando, como resposta final, hiperventilação. Estes recetores pré- e pós-sinápticos podem ser classificados em ionotrópicos ou metabotrópicos, estando os últimos acoplados a adenilatos ciclases transmembranares (tmAC). O mecanismo exato pelo qual as variações dos gases arteriais são detetadas pelo CB não se encontra ainda completamente elucidado, mas tem sido sugerido que alterações nos níveis de cAMP estejam associadas ao mecanismo de deteção de variações de O2 e CO2. Os níveis de cAMP podem ser regulados através da sua via de síntese, mediada por dois tipos de adenilatos ciclases: tmAC sensível aos eurotransmissores e adenilato ciclase solúvel (sAC)sensível a variações de HCO3/CO2, e pela sua via de degradação mediada por fosfodiesterases. A via de degradação do cAMP pode ser manipulada farmacologicamente, funcionando enquanto alvo terapêutico para o tratamento de patologias do foro respiratório (e.g. asma, hipertensão pulmonar, doença pulmonar obstructiva crónica e apneia do sono), que induzem um aumento da actividade do CB.O trabalho descrito nesta dissertação partiu da hipótese de que a actividade do CB é manipulada por fármacos, que interferem com a via de sinalização do cAMP, tendo sido nosso objectivo geral, investigar o papel do cAMP na quimiotransdução do CB de rato, e determinar se a actividade dos enzimas responsáveis pela via de sinalização do cAMP é ou não regulada por variações de O2/CO2. Assim, a relevância deste trabalho é a de estudar e identificar possíveis alvos moleculares (sAC, isoformas de tmAC e PDE) com potencial para serem usados no tratamento de patologias relacionadas com o controlo respiratório. A primeira parte do presente trabalho, centrou-se na caracterização farmacológica da PDE4 no CB e em tecidos não quimiorecetores (e.g. gânglio cervical superior e artérias carótidas), e na observação do efeito de hipóxia aguda na acumulação dos níveis de cAMP, induzidos pelos inibidores de PDE, nestes tecidos. A quantificação de cAMP foi efectuada por técnica imunoenzimática (EIA), tendo sido elaboradas curvas de dose-resposta para os efeitos de inibidores, não específicos (IBMX) e específicos para a PDE2 e PDE4 (EHNA, Rolipram e Ro 20-1724), nos níveis de cAMP acumulados, em situações de normóxia (20%O2/5%CO2) e hipóxia (5%O2/5%CO2). A caracterização das PDE no gânglio cervical superior foi aprofundada, utilizando-se a técnica de transferência de energia de ressonância por fluorescência (FRET) em culturas primárias de neurónios, na presença de inibidores não específicos (IBMX) e específicos para a PDE3 e PDE4 (milrinone e rolipram, respetivamente). Foram igualmente estudadas, através de RT-qPCR, as alterações na expressão de PDE3A-B e PDE4A-D, no gânglio cervical superior, em resposta a diferentes percentagens de oxigénio. Na segunda parte do trabalho investigou-se a via de síntese do cAMP no CB em resposta a variações na concentração de HCO3/CO2. Em concreto, o protocolo experimental centrou-se na caracterização da sAC, dado que a sua actividade é regulada por variações de HCO3/CO2. A caracterização da expressão e regulação da sAC, em resposta a variações de HCO3/CO2 ,foi efectuada no CB e em tecidos não quimioreceptores periféricos (e.g. gânglio cervical superior, petroso e nodoso) por qRT-PCR. A actividade deste enzima foi caracterizada indirectamente através da quantificação dos níveis de cAMP (quantificação por EIA), induzidos por diferentes concentrações de HCO3/CO2, na presença de MDL-12,33-A, um inibidore da tmAC. A expressão das isoformas da tmAC no CB e gânglio petroso foi determinada por RT-qPCR. Adicionalmente, estudámos a contribuição relativa da tmAC e sAC no mecanismo de sensibilidade ao CO2 no CB. Para o efeito foram estudadas as alterações: 1) nos níveis de cAMP (quantificado por EIA) na presença de diferentes concentrações de HCO3/CO2 e ao longo do tempo (5-30 min); 2) na ativação da proteína cinase A (PKA, FRET baseado em sensores) em células tipo I do CB; e 3) na frequência de descarga do CSN (registos) na presença e ausência de ativadores e inibidores da sAC,tmAC e PKA. Por último, foi caracterizada a expressão e actividade da sAC nos quimioreceptors centrais (locus ceruleus, rafe e medula ventro-lateral) através de técnicas de RT-qPCR e EIA. A expressão das isoformas da tmAC foi aprofundada no locus coeruleus através de RT-qPCR. Por fim, comparámos a contribuição da tmAC e sAC nos níveis de cAMP no locus coeruleus em condições de normocapnia e hipercapnia.O nosso trabalho teve os seguintes resultados principais: 1) PDE4 está funcional no corpo carotídeo, artérias carótidas e gânglio cervical superior de rato, embora a PDE2 só se encontre funcional neste último; 2) Os efeitos dos inibidores de PDE nos níveis de acumulação de cAMP foram exacerbados em situações de hipóxia aguda no CB e artérias carótidas, mas foram atenuados no gânglio cervical superior; 3) No gânglio cervical superior, diferentes tipos de células apresentaram uma caracterização específica de PDEs, sugerindo uma subpopulação de células neste gânglio com funções fisiológicas distintas; 4) Embora todas as isoformas de PDE4 e PDE3 estivessem presentes no gânglio, a PDE3a, PDE4b e a PDE4d foram as isoformas mais expressas. Por outro lado, incubações de gânglio cervical superior, em diferentes percentagens de oxigénio, não alteraram (não regularam) significativamente a expressão das diferentes isoformas de PDE neste órgão; 5) a sAC encontra-se expressa e funcional no CB e nos quimiorecetores centrais estudados (locus coeruleus, rafe e medula ventrolateral). A sAC apresenta maior expressão no CB comparativamente aos restantes orgãos estudados, exceptuando os testículos, orgão controlo. Variações de HCO3/CO2 de 0/0 para 24/5 aumentaram os níveis de cAMP no CB e quimiorecetores centrais, tendo sido o aumento mais significativo observado no CB. Concentrações acima dos 24mM HCO3/5%CO2 não induziram alterações nos níveis de cAMP, sugerindo que a actividade da sAC se encontra saturada em condições fisiológicas (normocapnia) e que este enzima não desempenha qualquer papel na deteção de situações de hipercapnia; 6) No CB, a expressão das isoformas tmAC1, tmAC4, tmAC6 e tmAC9 é mais elevada comparativamente à expressão da sAC; 7) Utilizamos diferentes inibidores da tmAC (MDL 12-330A, 500μM, 2’5’-ddADO, 30-300μM, SQ 22536, 200μM) e da sAC (KH7, 10-100μM) para estudar a contribuição relativa destes enzimas na acumulação do cAMP no CB. Tanto a tmAC como a sAC contribuem para a acumulação dos níveis de cAMP em condições de hipercapnia. Contudo, existe um maior efeito destes inibidores nas condições de 12 mM HCO3/2.5%CO2 do que em condições de normocapnia e hipercapnia, sugerindo um papel relevante destes enzimas na atividade do CB em situações de hipocapnia; 8) Não se observaram variações nos níveis de cAMP em resposta a diferentes concentrações de HCO3/CO2 ao longo do tempo (5-30 min). O efeito inibitório induzido por ddADO e KH7 foi sobreponível após 5 ou 30 minutos de incubação em todas as concentrações de HCO3/CO2 estudadas; 9) Por último, verificou-se um aumento na frequência da descarga do nervo do seio carotídeo entre as condições de normocapnia e hipercapnia acídica. Ao contrário do KH7 (10μM), o 2’5’-ddADO reduziu significativamente a frequência de descarga do nervo, quer em condições de normocapnia quer de hipercapnia acídica. Contudo, não se verificou aumento na frequência de descarga do nervo entre normocapnia e hipercapnia isohídrica, sugerindo que a sensibilidade à hipercapnia no CB é mediada por variações de pH. Em conclusão, os resultados decorrentes deste trabalho permitiram demonstrar que, embora os enzimas que medeiam a via de sinalização do cAMP possam ser bons alvos terapêuticos em condições particulares, a sua actividade não é específica para o CB. Os resultados sugerem ainda que o cAMP não é um mediador específico da transdução à hipercapnia neste orgão. Contudo, os nossos resultados demonstraram que os níveis de cAMP são mais elevados em condições fisiológicas, o que sugere que o cAMP possa ter uma função homeostática neste orgão. Por último, o presente trabalho demonstrou que os aumentos de cAMP descritos por outros em condições de hipercapnia, não são observáveis quando o pH se encontra controlado. ------------------ ABSTRACT: The work presented in this dissertation was aimed to establish how specific is cAMP-signaling pathways in the CB mainly in different CO2 conditions and how O2 concentrations alter/drives the manipulation of cAMP signaling in the CB. The experimental studies included in this thesis sought to investigate the role of cAMP in the rat CB chemotransduction mechanisms and to determine whether the enzymes that participate in cAMP signal transduction in the CB are regulated by O2/CO2. We characterized the enzymes involved in the cAMP-signaling pathway in the CB (sAC, tmAC, PDE) under different O2/CO2 conditions. Our results demonstrated that many of these enzymes are involved in CO2/O2 sensing and while they may be useful in treating conditions with alterations in CO2/O2 sensing,they will not be specific to chemoreception within the CB: 1) PDE4 is ubiquitously expressed in CB and non-chemoreceptor related tissues and their affinity to inhibitors change with O2 tensions in both CB and carotid arteries, and 2) sAC and tmAC are expressed in peripheral and central chemo- and non-chemoreceptor tissues and their effect on cAMP levels do not change between normocapnic and isohydric hypercapnic conditions. Our results provide evidence against a specific role of cAMP as a mediator for O2 and CO2 chemotransduction in the rat CB and emphasized the role of pH in CO2 sensitivity of the CB. Furthermore, our results demonstrate that cAMP levels are maintained higher under physiological conditions, supporting recent finding from our lab, which all together suggests that cAMP has a homeostatic function in this organ.

Nitric oxide (NO) negatively regulates platelets and impaired NO signalling can lead to arterial thrombosis. The source of platelet-derived NO is unclear with recent proposals of NO synthase (NOS) independent NO sources, such as S-nitrosothiols (RSNOs) and inorganic nitrate/nitrite. Sildenafil citrate, a phosphodiesterase 5 (PDE5) inhibitor, enhances NO/cGMP signals in cells expressing PDE5 such as platelets. The aims of this study were to investigate the antiplatelet properties of sildenafil, its mechanism of action and to determine the upstream sources of NO affecting platelet function. The functional effect of sildenafil was determined using a range of in vitro and in vivo platelet assays. The mechanism of action of sildenafil and upstream sources of NO/cGMP signals were assessed pharmacologically using established methods of in vitro and in vivo platelet aggregation. Bioconversion of nitrate to nitrite was determined using gas-phase chemiluminescence. The functional significance of NO/cGMP signalling events in platelets were investigated in vivo in W.T and eNOS-/- (a model of vascular dysfunction) mice. Sildenafil exerted an antiplatelet effect by enhancing transient NO/cGMP signals generated by platelets independent of NOS activity in vitro. Inhibition of proposed mechanisms of NO release from RSNOs did not modify the inhibitory effect of sildenafil suggesting that RSNOs did not mediate platelet NO/cGMP signals. Nitrite was able to drive inhibitory cGMP signalling events in platelets in vitro. Furthermore, nitrate inhibited platelet function in eNOS-/- mice in vivo following enhanced bioconversion to nitrite, potentially as a compensatory mechanism due to impaired NO signalling. In conclusion, inorganic nitrate/nitrite may critically regulate platelets following bioconversion to NO and dietary sources of nitrate/nitrite may generate compensatory NO during vascular disease. Furthermore, sildenafil may be beneficial in reducing the risk of platelet-driven cardiovascular disease by enhancing NO/cGMP signalling derived from both enzymic and inorganic sources and restoring impaired NO signalling during endothelial dysfunction.

Gastric cancer (GC) is the 4th most common cancer in the world. Although there is lots of information on the factors that cause GC such as Helicobacter pylori there is still much to learn about gastric carcinogenesis. In this thesis I aimed to develop: 1) Lgr5 antibodies to identify gastric stem cell and potential cancer stem cell populations. 2) New models of gastric cancer that might offer new insights into the cell of origin of gastric cancer and disease pathogenesis. The Wnt target gene LGR5/GPR49 (Leucine-rich repeat-containing G protein-coupled receptor 5) is a specific marker for stem cells in the small intestine, colon, hair follicle, mammary gland, kidney and stomach. Conditional deletion of APC, which activates Wnt signalling, exclusively in the Lgr5+ cells led to adenoma formation in the small intestine and stomach. This work suggested that stem cells were a likely cell of origin for cancer in these tissues. These important insights into Lgr5+ stem cells and cancer stem cells were obtained from experiments employing mouse models. However, currently the translation of these results to human stem cells and cancer are hampered by the lack of antibodies that recognize endogenous levels of Lgr5. Therefore I have generated antibodies targeting native human LGR5. Using colorectal cancer cells I have demonstrated that the Lgr5 antibodies I produced were able to identify endogenous Lgr5. Further optimization and characterization of the antibodies are needed from human primary samples but the antibodies I have produced could be very important tools for isolation and characterization of human Lgr5 + stem cell populations. Given Lgr5 has been shown to be a cell of origin for gastric adenoma in parallel I have also attempted to generate novel models of gastric cancer. These should allow me to address the contribution of Lgr5 and Wnt signalling to the initiation and progression of gastric cancers. To do this I have characterized and then used an inducible stomach Cre transgene to target deletion or activation of known oncogene and tumour suppressor genes to gastric epithelium: APC, TP53, E-cadherin, BRAF and ALK5. Although further characterization is required I have shown that BRAF mutation can lead to an intestinal type metaplasia, most likely to be Spasmolytic Polypeptide-Expressing Metaplasia (SPEM). Combined mutation of E-cadherin and TP53 led to mice develop stomach lesions that had similarities to diffuse type gastric cancer. Taken together in my thesis I have developed new tools to analyse Lgr5 and also generated some potentially exciting new models of gastric cancer. These should allow us to evaluate the role of Lgr5 and lead to a better understanding of gastric carcinogenesis.

With the advent of Tyrosine kinase inhibitors (TKI) as a therapy for Chronic myeloid Leukemia (CML), the patients now enjoy a life expectancy close to that of the general population. But some patients do get unresponsive to the TKI treatment over time due to several mutations in the kinase domain of the BCR-ABL fusion protein, which further leads to activation of multiple signaling cascades within the leukemic cell, helping it survive and proliferate. This project validates and optimizes a new method of In situ PLA that incorporates the usage of different padlocks and template oligos. Multiple cross-reactivity tests and interaction assays in multiple cancer cell lines will further optimize this system as a robust multiplex protein-protein interaction detection tool. Proteins associated with the MAP-K, PI3-K, and Jak-STAT signaling pathways were the main detection targets.

The modern age faces a progressive lengthening of life span, which is often associated with the onset of degenerative disorders such as Parkinson’s, Alzheimer’s and cancer. These diseases do not yet have a cure. The Hedgehog/Gli signalling pathway is one of the most promising pathways of cellular communication. Pharmacological modulation of this pathway would provide a remarkable approach for new potential therapeutics in regenerative medicine and cancer. Attention was focussed on coagulin L and withanolide F, two natural steroidal lactones featuring interesting reactive functionalities. The aims of this project were to investigate the chemical modifications of these natural products to prepare novel analogues for a structure-activity relationship study. 3β-Methoxy-pregnenolone is known as able to treat degenerative pathologies of the nervous system which could be implicated in Parkinson’s and Alzheimer’s diseases. The 3β-methoxy group also provides metabolic stability in vivo. Therefore, a library of steroidal analogues decorated differently on the right-hand side has been prepared by organometallic additions of heterocycles to the side-chain of this commercially-available steroid. The 14β-hydroxyl group is rarely found amongst withanolides, and no syntheses of withanolides with the cis-C/D ring rearrangement have been reported to date. Thus, the total synthesis of unnatural withanolide analogues based on the Hajos and Parrish ketone was investigated starting with readily available materials. Coagulin L is the major metabolite extracted at AnalytiCon Discovery from plant material. Hence, its conversion into the more active withanolide F was explored. Further elaboration of the left-hand side to provide additional novel compounds for our library of small molecules was also investigated.