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Peroxisomes represent crucial subcellular compartments for human life and health. They are remarkably dynamic organelles which respond to stimulation by adapting their structure, abundance, and metabolic functions according to cellular needs. Peroxisomes can form from pre-existing organelles by membrane growth and division, which results in peroxisome multiplication/proliferation. Growth and division in mammalian cells follows a well-defined multi-step process of morphological alterations including elongation/remodeling of the peroxisomal membrane (by PEX11β), constriction and recruitment of division factors (e.g. Fis1, MFF), and final membrane scission (by the dynamin-related GTPase Drp1) (Chapter 1). Although our understanding of the mechanisms by which peroxisomes proliferate is increasing, our knowledge on how the division/multiplication process is linked to extracellular signals is limited, in particular in humans. The classical pathway involved in peroxisome proliferation is mediated by a family of ligand-activated transcription factors known as peroxisome proliferator activated receptors (PPARs) (Chapter 1). This project focused on identifying novel signaling pathways and associated factors involved in peroxisome proliferation in humans. In this study, a cell-based peroxisome proliferation assay using the HepG2 cell model with spherical peroxisomal forms has been developed to investigate different stimuli and their ability to induce peroxisome proliferation (Chapters 2 and 3). In this system, peroxisome elongation has been used as the read-out for peroxisome 4 proliferation. We also showed that the number of peroxisomes increased after division of elongated peroxisomes indicating peroxisome proliferation. Different stimuli, such as fatty acids, PPAR agonists and antagonists, have been used in this study. PPAR agonists and antagonists had no stimulatory or inhibitory effect on peroxisome elongation in our assay, suggesting PPAR-independent regulatory processes. However, arachidonic acid and linoleic acid were able to induce peroxisome elongation, whereas palmitic acid and oleic acid were not effective. These findings indicate that general stimulation of fatty acid β-oxidation is not sufficient to induce peroxisome elongation/proliferation in HepG2 cells. Moreover, mRNA expression levels of peroxismal genes have been monitored during a time course in the HepG2 cell-based assay by qPCR. This analysis shows a regulation of expression of peroxins during peroxisome proliferation in human cells and suggests differences in the regulation pattern of PEX11α and PEX11β. In Chapter 4, motif binding sites for transcription factors in peroxisomal genes were analyzed. An initial map of candidate regulatory motif sites across the human peroxisomal genes has been developed (Secondment at the University of Sevilla, Spain with Prof. D. Devos). This analysis also revealed the presence of different transcription factor binding sites in the promoter regions of PEX11α and PEX11β, supporting different regulatory mechanisms. Based on the computational analysis, PEX11β contained a putative SMAD2/3 binding site suggesting a novel link between the canonical TGFβ signaling pathway and expression of PEX11β, a key regulator of peroxisome dynamics and proliferation. 5 Addition of TGFβ to HepG2 cells cultured under serum-free conditions induced elongation/growth of peroxisomes as well as peroxisome proliferation supporting a role for TGFβ signalling in peroxisomal growth and division (Chapter 5). Furthermore, to demonstrate that this induction is through a direct effect of TFGβ on the SMAD binding site found in PEX11β, we performed functional studies using a dual luciferase reporter assay with PEX11β wild type and mutated promoter regions (Secondment at Amsterdam Medical Center, Netherlands with Prof. H. Waterham). Whereas luciferase activity was induced by TGFβ stimulation with the PEX11β wild type promoter, mutation of the SMAD binding site abolished activation. In summary, this study revealed a new signaling pathway involved in peroxisome proliferation in humans and provided a tool to monitor peroxisome morphology and gene expression upon treatment with defined stimuli. Furthermore, I contributed to a study revealing that ER-peroxisome contacts are important for peroxisome elongation (Chapter 6). Our group identified peroxisomal acyl-CoA binding domain protein 5 (ACBD5), ACBD4 and VABP as a molecular linker between peroxisomes and the ER (Costello et al., 2017). Motif analysis of ACBD4 and ACBD5 promoter regions revealed that unlike PEX11β, these genes do not contain a binding site for SMAD, suggesting they are not co-regulated. Also, ACBD4 and ACBD5 do not share any common transcription factor binding sites suggesting different regulation. An interesting binding motif within the ACBD4 promoter is a glucocorticoid receptor binding site. In our study, we found potential glucocorticoid response elements (GRE) in other peroxisomal genes encoding β-oxidation enzymes. This may suggest an important role for glucocorticoid receptors in activating expression of peroxisomal genes resulting in the stimulation of fatty acid breakdown and energy production.

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.

Toll-like receptors (TLR) are a family of pattern recognition receptors crucial for pathogen pattern recognition. Upon activation, TLRs induce innate immune responses such as cytokine production. However irregular TLR activities can provide fatal, hence fine tuning of the TLR induced responses are necessary. The TLR mediated immune responses are controlled by the positive/negative regulation of TLR signalling pathways, relocation of TLR proteins and modulation of TLR transcription. Systematic analyses of the agonist-induced transcriptional changes of TLRs were shown for the first time in my thesis. In my experiments, I have shown that each agonist induced a unique pattern of TLR transcription. Following PAM stimulation, mRNA levels of the cognate TLR1/2 increased whereas mRNA levels of the cross-regulating TLR4, 7/8/9 reduced in both cell lines and splenic macrophages from different mice strains. Through investigation of the signalling pathways responsible for mediating such TLR transcriptional changes, I then discovered the balancing effect between NFÎoB and MAPK signalling pathways. PAM induced TLR transcriptional changes were controlled by the additive and/or antagonistic interference between MAPK signalling cascades, ERK, JNK, P38 and NFÎoB signalling pathways. This was the first time that signalling synergy between MAPK and NFÎoB pathways were shown. Furthermore, PAM induced transcription of TLR1 and TLR8 may be partially regulated by the indirect feedback mediated by protein production. Importantly, the maintenance of the basal TLR mRNA expression also required activation of both MAPK and NFÎoB signalling pathways. In addition, signalling control for TLR transcription induced by different agonists (PAM vs. LPS) or in different species (chicken vs. mice) was compared. LPS induced transcriptional changes of the cross-regulating TLR1/2 and 3 but not the cognate TLR4 in RAW cells. The LPS induced TLR transcriptional changes required activation of a combination of MAPK and NFÎoB signalling pathways which shared both similarities and differences to the PAM induced signalling activation. In chicken, PAM induced more potent signalling activation, regulating the TLR transcriptional changes at a lower concentration than in mice. Overall, this thesis demonstrates that the transcriptional regulation of TLRs is complex, mediated by the coordination between MAPK and NFÎoB signalling pathways. These studies have significant implications in providing detailed insight of TLR transcriptional regulation which plays an important role in the regulation of TLR mediated innate immune responses. Please watch the following videos that I made for: A short introduction about TLR regulation - https://youtu.be/LTDdEZ3S97o A short explanation about TLR signalling - https://youtu.be/51IY5XhdJR8.

The PI3K/mTOR signalling pathway plays a major role in biology and disease. Therefore, effective inhibitors that target proteins of this pathway have been developed. However, acquired resistance of cancer cells is a prevalent phenomenon that limits the durable response of these compounds. It is becoming apparent that experimental approaches for comprehensive biochemical analysis contribute to understand the complex mechanisms that confer drug resistance, and advances in largescale technologies including genomic sequencing and proteomics allow unprecedented molecular coverage without being biased for specific genes/cellular pathways. Initially, the phenotypic response of sensitive and resistant cells to the absence or presence of a PI3K inhibitor (PI3Ki), as well as other kinases, was examined. This study revealed that PI3Ki-resistant cells experience extensive phenotypic changes upon withdrawal of the PI3Ki from the culture media. The regulation of the proteome and phosphoproteome of sensitive and PI3Ki-resistant cells grown with or without the PI3Ki was analysed by shotgun mass spectrometry-based label-free quantitative technology. This analysis demonstrated that the proteomes and phosphoproteomes of drug-resistant cells are remodelled conditional to the presence of PI3Ki, and that the levels of enzymes with metabolic roles are modulated in resistant cells. Functional analysis of the metabolism of cells capable to survive in absence of PI3K/mTOR activity demonstrated that the bioenergetic activity of these cells is contingent on the presence of the selection drug. The complete set of protein-coding regions of the genome (exome) of sensitive and PI3Ki-resistant cells was then sequenced. This study unveiled common alterations in exome regions across PI3Kiresistant cell lines, as well as a degree of genomic heterogeneity between them. Lastly, the impact of lactic acid, a metabolic product, on a defined signalling network of the MCF7 breast cancer cell line was analysed. This study described the capacity of this metabolite to change the activity of signalling network branches.

Macrophages are sentinels present in most tissues of the body, where they recognise and respond to biological dangers. Recognition and uptake of particles is mediated through phagocytic receptors which upon activation induce appropriate responses. These responses need to be tightly regulated in order to destroy pathogens but prevent uncontrolled inflammation. Phagocytosis is an evolutionarily conserved process required for host defence and homeostasis. During phagocytosis, particles are recognised by cell surface receptors that trigger rearrangement of the actin cytoskeleton and internalization of the bound particle into a de novo, membranous organelle known as the phagosome. Regulation of phagocytosis and phagosome maturation can be achieved through changes in transcription/translation and differential recruitment of proteins but also through their non-translational modifications. Here I explored the role of ubiquitylation in the phagosome biogenesis of Interferon-gamma (IFN-ɣ) activated macrophages. Ubiquitylation is a diverse, reversible post-translational modification which is not only involved in protein degradation but also in vesicle trafficking and immune signalling. My data shows that phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ. I applied a targeted AQUA peptide approach by which we quantified ubiquitin chain linkage peptides from phagosome samples by PRM. This data shows that all chain linkages apart from M1/linear chains are present on phagosomes. Furthermore, IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, I characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. I demonstrated that most ubiquitin chains are on the cytoplasmic site of the phagosome enabling an interaction of ubiquitin chains with cytosolic proteins such as Rab7. Rab7 a major regulator of vesicle trafficking could be shown to be ubiquitylated on phagosomes. I further showed that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation and RNF115 is responsible for most of the increase of K63 polyubiquitylation of phagosomal proteins. Knock-down of RNF115 promotes phagosome maturation and induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome and disruption of this pathway induces pro-inflammatory responses in macrophages. In conclusion, this is the first study showing unbiasedly that ubiquitylation plays an important role in vesicle trafficking to the lysosome.

ABSTRACT There is an increasing incidence of neoplasms in the kidney and a poor prognosis for patients who are diagnosed with advanced or metastatic kidney cancer. Renal cell carcinoma (RCC) constitutes the most prevalent form of kidney neoplasm in the adult population. Although surgery or cryoablation are successful curative treatments for localized RCC, improved diagnostic methods facilitating early detection and characterization of renal tumours may enable more effective use of less invasive treatments, especially for metastases. Currently there are no suitable tumour markers available for diagnostic, prognostic or predictive purposes. By increasing our knowledge of the underlying molecular characteristics of RCC, we may be able to identify molecular pathways involved in tumour growth and metastasis, and this knowledge may expedite the development of targeted therapies, and may identify useful markers of RCC development and progression. This thesis aimed to identify new genes involved in resistance to cancer therapy in RCC and to analyse their incidence and test their function in human RCC tissue, using immunohistochemistry in a large cohort of patients with RCC and paired normal tissue, and in vitro models. The lack of induction of apoptosis in RCCs by conventional cancer therapies such as chemotherapy, immunotherapy or radiation is central to their resistance to treatment. If apoptotic pathways that are activated in successful treatments were identified, they might be used for targets in future therapies. The hypothesis tested in this thesis was that genes or proteins involved in the molecular control of apoptosis, identified from RCC cell culture models and RNA microarray, will be useful for molecular profiling in human RCCs of different subtypes, as markers of those specific subtypes of the RCCs, as indicators of prognosis of the diagnosis, or as targets for future therapy regimens. The broad aims of this project were: To establish a model in which significantly increased apoptosis in RCCs in cell culture could be correlated with alterations in apoptotic pathway genes, to investigate the functional significance of some of those genes; and to maximise information gained from these basic experimental laboratory studies on new apoptosis genes in RCC development and progression by determining their expression patterns in tissue microarrays (TMAs) generated from different subtypes of human RCCs. The specific aims of the project were: 1) To establish an RCC cell culture model with high levels of induced apoptosis for RNA analyses using microarray to identified apoptotic genes that are novel in RCC investigations; 2) To describe the role and functional significance of some of the novel RCC apoptosis pathway genes using molecular investigations, including silencing RNA techniques; and 3) To use TMAs and immunohistochemistry to determine whether RCC subtypes can be distinguished by protein expression profiles of selected new apoptosis pathway genes. The thesis is presented as a literature review (Chapter 1), materials and methods (Chapter 2), four original research segments (Chapters 3 to 6), and finally a segment that summarises the results and presents future directions (Chapter 7). The first of the original research Chapters (Chapter 3) addressed a question “can apoptosis in RCCs be induced in cell culture to such a level that apoptotic pathways may be analysed?” Two RCC cell lines (ACHN and SN12K-1) were treated with IFN-a (500IU/mL), radiotherapy (20 Gy) or dual therapy of these two treatments. Apoptosis was quantified using microscopy and morphological characteristics and verified using enzymatic labelling of cells. The ACHN cell line, treated with the dual therapy and analysed at 24 hours, had a significantly increased level of apoptosis (p<0.05) compared with the non-therapy treated controls, and negligible mitosis. The increased expression or activation of at least some known apoptotic pathway genes (Bcl-2, Bax, caspase-3, 8 and 9, and p53) was verified in this model. The ACHN/dual therapy model was selected for further study. The second of the original research Chapters (Chapter 4) addressed the question “what apoptosis-regulating genes are significantly different in the apoptotic ACHN cells?” An RNA microarray assay (112 apoptosis-related genes) was carried out using RNA extracted from treated and non-treated ACHN cells and analysed for alterations of at least 1.9-fold in transcript levels. 21 genes had upregulated transcript levels in the treated cells, and one had down-regulated transcript levels. A search of the literature revealed three gene families with altered transcript levels in the treated RCCs that were novel: the TNF receptor-associated factor (TRAF), caspase recruitment domain (CARD) and cell death-inducing DFF-45 effector (CIDE) gene families. Representative members of these families were then investigated for protein expression alterations. The results for one particular gene, TRAF1, indicated it might be worthy of further study in modulation experiments (Chapter 6). The next research chapter (Chapter 5) asked the question “since the ACHN cells express TRAF-1 and have increased TRAF-1 with increasing apoptosis, what happens to the levels of mitosis and apoptosis in these cells when TRAF-1 expression is knocked down?” Silencing RNA (siRNA) techniques were used to knock down TRAF-1 in the ACHN RCC cell line using the same model as was described in Chapter 3. Successful knock down was gained after 72 hrs of transfection with a commercially-available siRNA against TRAF-1. These cells were then treated with the radiation, IFN-α or dual therapy treatments. In the cells with TRAF-1 knock down, there was significantly less apoptosis and more mitosis than was seen in the non-transfected cells. These results indicate that TRAF-1 does play a functional role in induction of apoptosis in RCCs and is worth investigating further in targeted therapies. The final of the research Chapters (Chapter 6) looked at the molecular distinctions among ccRCC, papillary, collecting duct, chromophobe and unclassified types of RCC. Molecules that might distinguish one from another would be valuable clinically, and it would be especially valuable if these molecules could also be targeted for therapeutic benefit. By knowing the action of these molecules in the apoptotic pathways used by RCCs when they do regress or die during cancer therapies, we might be able to devise new targeted therapies for RCCs. This research Chapter asked the question “could molecular profiling with an array of apoptotic pathway genes, novel to investigations in RCCs, provide information that would distinguish the subtypes?” TMAs prepared from 121 RCC and paired normal patient samples, where available, were investigated. Most RCC samples were ccRCC. Antibodies against selected members of the TRAF), CARD and CIDE gene families were selected from the RNA microarray data. These genes (TRAF1, TRAF3, TRAF4, inhibitor of caspase-activated DNAase/ICAD and nucleolar protein-3/NOL3) were analysed in TMAs using immunohistochemistry and digital scanning or bright field microscopy and graded scales of intensity and distribution, blinded to sub-type of RCC. After microscopy, scores for subtypes of RCC were compared with their normal tissue. Significant differences were found for TRAF1 and NOL3. The results indicate TRAF1 and NOL3 have potential for improving outcome or diagnosis in RCCs. In summary, there are no effective treatments against metastatic RCC, and no suitable grade or stage-defining biomarker for metastatic RCC subtypes. One of the main reasons for therapy resistance in RCCs is their inability to use or activate apoptotic pathway molecules. By investigating the reasons for RCC resistance to cancer therapies, we may be able to improve both diagnosis and treatment strategies. By defining the mechanisms and pathways of resistance to therapy-induced apoptosis, and developing methods to manipulate the pathways to cell death to defeat therapy resistance, we will have a better chance to develop successful markers of RCC subtypes and also design new and successful RCC therapies.

The Hedgehog signalling pathway is an essential developmental pathway present in all bilaterians that is involved in embryogenesis, body patterning and stem cell homeostasis. Dysregulation of the Hh pathway leads to various kinds of cancer, such as basal cell carcinoma and medulloblastoma. Smoothened (SMO), a Frizzled-type G-protein coupled receptor (GPCR), is the essential transmembrane signal transducer within the Hh pathway, conveying the signal from the upstream transmembrane protein, Patched1 (Ptc1), to the downstream intracellular proteins. The mechanisms by which SMO transmits the Hh signal from the extracellular environment, through the plasma membrane and to the intracellular proteins are not known. In this thesis, I present my work into the structural and functional characterisation of the extracellular and transmembrane domains (TMD) of human SMO in order to better understand the molecular mechanisms of its signal transduction. The extracellular region of SMO contains a highly conserved cysteine-rich domain (CRD) and a linker domain (LD). I present the first crystal structure of the CRD, LD and TMD of SMO, which is also the first crystal structure of a GPCR with a large functional extracellular domain. This structure revealed a domain architecture for SMO that enables regulation of its transmembrane domain by its extracellular domains. It also revealed a cholesterol molecule bound to the CRD, which we subsequently determined to be a new endogenous small-molecule agonist for SMO. I present five further structures of SMO bound to different small molecule agonists and antagonists. Together, these structures demonstrate that the position of the CRD relative to the TMD reflects the activation state of SMO. We also generated nanobodies against the extracellular region of SMO in order to stabilise its conformation. These studies not only improve our understanding of the workings of a key transmembrane protein within a fundamental signalling pathway but will also aid efforts to develop better therapeutics for an important cancer target.

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Systemic sclerosis (SSc) is immune-mediated fibrotic disease of unknown aetiology. Among the dominant pathogenic manifestations of SSc belong vascular changes, production of autoantibodies, activation of innate and adaptive immune responses and fibrotic processes. Transforming growth factor beta (TGF-β) has been identified as a central profibrotic factor stimulating fibroblasts to produce collagen. There are, however, a number of other mediators involved in the pathogenesis of SSc. Mutual activation and amplification of these molecules and their cascades may be a central mechanism of the SSc pathogenesis. Hedgehog (Hh) canonical signalling pathway plays an important role in the development and progression of fibrotic diseases. Expression of Hh target genes can be regulated through a canonical or non-canonical signalling cascade. The non-canonical activation of GLI transcription factors by TGF-β has not yet been investigated in SSc. The substantial part of this thesis is focused on the study of the mutual interaction of TGF-β and Hh signalling pathway. In vitro analysis confirmed TGF- β/SMAD3 dependent activation of GLI2 in dermal fibroblasts. Fibroblasts specific knockout of GLI2 prevented the development of experimental fibrosis in vivo. Combined targeting of canonical and non-canonical Hh...