Activation of sex steroid receptors is a key event in the regulation of uterine function. The expression of sex steroid receptors such as Oestrogen and Progesterone have been well characterised in normal human endometrial tissue. Expression of the androgen receptor (AR) has not been well characterised. The characterisation of AR in human endometrium is important to determine the role of androgens in physiological uterine events such as implantation and menstruation. The term "Intracrinology" refers to the ability of a peripheral target tissue to synthesise sex steroids. The expression pattern of metabolising enzymes determines a tissues steroidogenic potential. Key sex steroid metabolising enzymes include the 17β-hydroxysteroid dehydrogenases (17β -HSD), 5α-reductases (5αR), 3β-hydroxysteroid dehydrogenases (3β -HSD) and Aromatase. The objectives of this thesis were thus to determine in human endometrium; 1. The spatial and temporal expression of androgen receptor mRNA and protein in endometrium across the menstrual cycle. 2. The expression of critical androgen metabolising enzymes across the menstrual cycle - 17β -HSD type 2 mRNA and protein; 17β -HSD type 5 mRNA; 3β -HSD type 1 and 2 mRNA; and 5αR types 1 and 2 mRNA. 3. The spatial and temporal localisation, in endometrium exposed to high dose intra-uterine levonorgestrel, of AR mRNA and protein; 17β -HSD2 mRNA and protein; 17β -HSDS mRNA; 3β -HSD1 and 2 mRNA; and 5αRl and 2 mRNA. Endometrial biopsies from both normal endometrium and endometrium exposed to high dose intra-uterine levonorgestrel were subjected to optimised immunohistochemical protocols to determine the spatial and temporal immunolocalisation of the androgen receptor and 17β -HSD2. In-situ hybridisation (ISH) techniques were employed to localise AR mRNA in full thickness endometrial biopsies. Taqman real time RT-PCR examined the temporal variation in mRNA expression for AR; 17β -HSD2 and 5; 3β -HSD1 and 2; and 5aRl and 2 in normal endometrium and endometrium exposed to high dose intra-uterine levonorgestrel. The results determined that AR is expressed in the endometrial stromal compartment with down regulation of AR protein and mRNA in the late secretory phase. This localisation was confirmed with ISH data. Endometrium exposed to high dose intra-uterine levonorgestrel exhibits a significant decrease in stromal AR protein immunoreactivity when compared with proliferative endometrium. Temporal variations in expression of steroid metabolising enzymes were studied. Significantly elevated levels of 17β -HSD2 are expressed in the glandular compartment of mid secretory phase endometrium. Endometrium exposed to high dose levonorgestrel exhibited high levels of endometrial 17β -HSD2 protein in the first month after insertion of a LNG lUS, associated with high levels of 17β -HSD2 mRNA expression in endometrial tissue at the three month time point. Thus, increased levels of 17β-HSD2 would indicate the potential for lowered intracellular oestradiol levels at this time (since this enzyme converts oestradiol to the less potent oestrogen, oestrone). Thereafter, levels of 17β-HSD2 protein and mRNA significantly decline. 17β -HSD5 mRNA is also significantly increased in the mid secretory phase. The peri-menstrual period is associated with significant elevations in mRNA levels for both 3β-HSD enzymes and 5αR 2. 5αR 1 is significantly elevated in the mid-cycle phases. In endometrium exposed to high dose intrauterine levonorgestrel, an increased expression of 3P-HSD and a reduced expression of 5aR 2 are noted. In conclusion, AR had been localised to the endometrial stromal compartment with a significant down regulation noted in the late secretory phase. The expression pattern of metabolising enzymes identified in this study is consistent with the secretory phase human endometrium possessing significantly greater steroidogenic potential than the proliferative phase. Furthermore, the available literature suggests that secretory endometrium is a significant source of androgen production. Data has been published that implicates androgens as having an important role in physiological events such as implantation. However, the precise role and regulation of androgens, the androgen receptor and the metabolising enzymes in human endometrium requires further study Novel data is also described here regarding the effect of high local doses of progestogen on local tissue androgen receptor and sex steroid metabolising enzymes. Such data suggests a potential role for progestogens in influencing the uterine environment that may lead to novel interventions for the problematic breakthrough bleeding suffered by progestogen only contraceptive users.

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We are currently in the midst of a revolution in ageing research, with several dietary, genetic and pharmacological interventions now known to modulate ageing in model organisms. Whilst it has been known for almost 100 years that dietary restriction (DR) extends lifespan across wide evolutionary distances, the mechanisms through which it acts are still unknown. Using three different recombinant inbred ILSXISS mouse strains, which vary in response to DR; from lifespan extension to lifespan shortening, my PhD has sought to identify the mechanisms involved in DR-induced lifespan extension. Ultimately by exploiting the genetic heterogeneity of these mouse strains may help identify the mechanisms through which DR acts to slow ageing. During my PhD I (1) examined how these animals respond metabolically to DR, (2) determined the impact of DR on mitochondrial function, as mitochondrial dysfunction is a well characterised hallmark of ageing, and DR is known to attenuate age-related mitochondrial dysfunction, and (3) investigated proteostasis in these mice using isotopic labelling. A number of metabolic changes were found to be important to lifespan extension with DR, namely maintenance of gonadal white adipose tissue (WAT) stores and increases in brown adipose tissue (BAT). Conversely, large losses of WAT was associated with lifespan truncation following 40% long-term DR. Surprisingly, enhanced glucose homeostasis was not found to be a prerequisite to lifespan extension with DR. Hepatic mitochondrial dysfunction associated with reduced lifespan of TejJ114 mice under 40% DR, but similar dysfunction was not apparent in skeletal muscle mitochondria, highlighting tissue-specific differences in the mitochondrial response in ILSXISS mice to DR. Increased proteostasis as measured by the newprotein/newDNA ratio, was increased following short-term DR, highlighting increased synthesis of cytoplasmic proteins in the skeletal muscle as important to DR-induced lifespan extension, however results were both tissue and protein specific. The evidence produced in this thesis strongly suggests that numerous aspects of metabolism and mitochondrial function are associated with lifespan shortening, and that the inability for metabolic adaptability may be detrimental to lifespan. This thesis helps to elucidate the impact of genotype on key hallmarks of DR and highlights the importance of utilising both genders and genetically heterogeneous murine strains in order to understand the shared features of slowed ageing.

The immune system relies on the correct spatial and temporal positioning of cells in order to function; cells need to be able to move throughout the circulatory system to survey for pathogens, to migrate from their resident sites in tissues when they sense infection or injury to alert other cells, or to migrate to the site of damage or infection to help mobilise a response. These functions often involve chemokines, small cytokines that signal through chemokine receptors, which are G-protein coupled receptors expressed on the cell membrane. Different chemokines are regulated differentially and can be associated with certain tissues or developmental processes, meaning the suite of receptors expressed by each cell type determines which tissues it is capable of entering, and the precise location it takes up once inside the tissue. Atypical chemokine receptors are a class of chemokine receptors that do not initiate the downstream signalling pathways typical of a G-protein coupled receptor, as they do not recruit intracellular G-proteins, and generally don't induce cell migration. Instead, these receptors are thought to function mainly as chemokine scavenging receptors, internalising and destroying their ligands before rapidly recycling to the cell surface. In this way, the levels of chemokines in the body are prevented from becoming oversaturated thus dampening the ability of cells to respond to signals. ACKR3 and ACKR4 are two atypical chemokine receptors that are expressed on endothelial cells and keratinocytes in the skin. Here, I have studied their expression on cultured human lymphatic endothelial cells and keratinocytes, and modulation in response to immune stimuli on these cells using a combination of qPCR and immunofluorescent staining. These strategies revealed that ACKR3 and ACKR4 are expressed on cultured LECs and keratinocytes and may be differentially regulated by both cell types in response to inflammatory stimuli including bacterial (LPS) and viral (Unmethylated CpG DNA) signatures. Although chemokine scavenging activity could not be detected on these cells, these findings suggested a role for ACKRs 3 and 4 in the inflammatory response. Further experiments in vivo explored the expression and modulation of ACKR3/CXCR4 and ACKR4 on epithelial cells including lymphatic endothelial cells, keratinocytes and vascular endothelial cells in the spleen. Flow cytometry was used to examine tissues both at rest and after inflammation (Aldara-mediated psoriasis model, or TPA-mediated contact hypersensitivity model) and investigate the regulation of ACKR3/CXCR4 or ACKR4 in response to these stimuli. Key findings included the strong overlap and differential regulation of ACKR3 and CXCR4 in response to TPA in the infundibulum subset of keratinocytes. Additionally, lymph nodes of Ackr4-/- mice were significantly enlarged after repeated treatments with Aldara. This appeared to be due to CCL19 dysregulation, but adoptive transfer suggested that there was no defect in leukocyte homing in these mice. This suggested an as yet undetermined response to enhanced CCL19 bioavailability that did not prevent the correct migration of leukocytes to secondary lymphoid organs. Overall, these experiments suggested that ACKR3 and ACKR4 are modulated in response to several inflammatory stimuli both in vivo and in vitro, and that the modulation of homeostatic chemokines can play a role in the response to inflammatory events. This was particularly important in the context of skin inflammation, where inflammatory chemokines, CXCR4 and ACKR2 have all been implicated in severity and duration of inflammatory events, but few studies have yet described the potential contributions of ACKR3 or ACKR4.

Many DNA repair pathways have been documented in Trypanosoma brucei but less attention has been paid to damage tolerance, a reaction in which lesion bypass is needed, in particular to ensure continued genome replication. Such bypass is promoted by translesion DNA polymerases (TLS Pols). T. brucei has ~15 TLS polymerases candidate genes, only two of which have been functionally examined to date. Understanding the roles provided by TLS Pols could reveal new aspects of T. brucei biology. Here, I examine the activities of TLS Pol Nu (PolN), TbPolZ and TbPolQ (HelQ) in bloodstream cells. RNAi against TbPolN results in slowed growth after ~24 hours, which is associated with altered DNA content, changed cell morphology and sensitivity to DNA damage. Surprisingly, growth and morphology defects are reduced after ~48 hours, without apparent RNAi reversion. In addition, depletion of the protein seems to lead to an aberrant distribution of the chromosomes, as visualised by telomere fluorescent in situ hybridization. TbPolN epitope tagging demonstrates a discrete localisation of the protein at the periphery of the nucleus in the absence of damage, with a more widespread, but non-uniform localisation after damage. EdU labelling and γH2A analysis after TbPolN knockdown reveal a decrease in proliferating cells, which accumulate nuclear DNA damage. Finally, we show that TbPolN interacts with a nuclear putative non-canonical PolyA polymerase. Taken together, these data suggest TbPolN may be involved in T. brucei nuclear DNA maintenance. RNAi of TbPolZ (zeta) did not impair growth but resulted in increased sensitivity to methyl methanesulphonate (MMS) damage and UV radiation, suggesting a possible role in the response against both genotoxic agents. Generation of TbPolZ null mutants confirmed that the protein is non-essential and plays a role in genotoxic damage repair. Surprisingly, TbPolZ epitope tagging not only showed a nuclear signal, but a mitochondrial signal was also detected. These data were supported by immunoprecipitation, where mitochondrial proteins were obtained as potential interaction partners. These data suggest a contribution of TbPolZ to both nuclear and kinetoplast genome maintenance. Targeted RNAi of the third putative TLS-related factor, TbHelQ, was unsuccessful. Despite this, sequence analysis of the protein indicates that its current annotation as a PolQ homologue is inaccurate, since the predicted protein is not a joint polymerase-helicase like in other eukaryotes, but only a putative helicase. Hence, it is suggested it should be renamed TbHelQ. Immunoprecipitation and colocalisation analyses indicate a possible role of TbHelQ in homologous recombination, given the potential interaction of the factor with BRCA2 and other factors involved in this repair process. Notably, the predicted interactome of TbHelQ differs from that of TbPolN, suggesting discrete functions in T. brucei. Taken together, these data reveal widespread and variant functions of three putative TLS DNA polymerases in the parasite genome biology, suggesting a possible role in the maintenance of genome integrity in T. brucei.

Despite being translationally null, erythrocytes contain miRNA at concentrations equal to nucleated cells, meaning that of the miRNA found in peripheral blood ~ 99 % is located in red cells. Despite this huge abundance, the literature currently regards erythrocytic miRNA (e-miRNA) as redundant. Data from Hamilton lab challenges this notion as it shows that e-miRNA are stable, maintain a catalytic potential, and have sequence profiles similar across species, indicating conservation. This thesis investigates what the potential function of emiRNA could be, both within erythrocytes and in other cells. To develop hypotheses, RISC-protein interactions were evaluated unearthing the appearance of an apparently post-translationally modified AGO2; what this modification is, remains to be defined. As part of a homeostatic hypothesis, the idea that e-miRNA may act as a molecular signal and regulate the transcriptome of phagocytosing cells was evaluated. However, it was discovered that e-miRNA are lost as RBCs age, potentially through vesicularisation. The differences between RBC vesicles that occur in vivo and those that occur when blood is stored ready for transfusion were evaluated. This highlighted a potential artefact within the literature with regards to what is termed as “microvesicles” (MVs). The clinical concern regarding storage MVs was evaluated through the generation of a phago/endocytosis model, and consistent with recent publications, no mal-effect was observed. This portion of the study highlighted a secondary structure within macrophages that is not a phagolysosome, but does process internalised RBC-MVs.

Haematopoietic stem cells (HSCs) have a huge clinical relevance as they are regularly used in bone marrow transplants worldwide. This therapy has profound potential to alleviate diseases of the blood and immune system, where others are ineffective. However, HSCs cannot currently be cultured long term ex vivo, as they rapidly differentiate or senesce. Hence, genetically matched donors must often be found for transplant and studies of HSCs require costly animal models. Mimicking the microenvironment of the bone marrow, in which they reside, by incorporating supportive stromal cells including mesenchymal stem cells (MSCs), has the potential to overcome these limitations. This project aimed to create an HSC-permissive MSC spheroid culture system using magnetic nanoparticles and a collagen gel. An existing spheroid system was optimised for HSC-MSC co-culture and then characterised to assess the potential for HSC support. MSC spheroids were more quiescent, and expressed higher levels of HSC-supportive genes such as nestin and CXCL12. Subsequently, additional bone marrow cell types were introduced to the model to mimic vascular and endosteal areas of the bone marrow niche. HSC behaviour within these models was investigated. MSCs, endothelial cells, and osteoblasts exhibited gene expression changes in line with those predicted from examination of the physiology of endosteal and vascular regions of the bone marrow: i.e. higher activity at the vascular niche (model including endothelial cells), and lower activity at the endosteum (model including osteoblasts). However, gene transcription and phenotypic analyses of HSCs following culture within the bone marrow models produced more inconclusive results. Hence, further optimisation of the conditions of the model and repetition of results presented here are required to develop the system so that it truly mimics physiological bone marrow. The development of such a model has many applications, including in drug discovery, modelling disease states, and probing haematopoietic functions.

To understand cell biology in detail, we must explore subcellular organization in 3D and locate proteins at high resolution. To achieve this, the most popular approach is to use two complimentary imaging techniques; light and electron microscopy. Combining these techniques by correlative or correlated light and electron microscopy (CLEM) allows cellular events to be observed first by fluorescence microscopy and then the same event can be tracked and visualised at high-resolution using electron microscopy. However, a current challenge is to develop probes to precisely visualize proteins in cells using this method. This thesis introduces FerriTag, a new genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron- dense ferritin particle that can specifically label target proteins rapidly and efficiently using rapamycin-induced heterodimerization. The processing protocol described for CLEM is simple yet robust and can potentially be used for tagging any protein-of- interest. FerriTag is easily distinguished from background in electron micrographs due to its high signal to noise ratio and also provides a labelling resolution of 10 ± 5 nm. These qualities make FerriTag an ideal probe for CLEM. FerriTag can be used to perform contextual nanoscale mapping of protein location relative to a subcellular structure. This was utilised to study the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits, providing insight to the roles played by HIP1R, clathrin and actin during clathrin-mediated endocytosis. FerriTag offers great potential for future CLEM applications and will be a useful discovery tool for cell biology.

Cancer cachexia is a common secondary pathology that develops in up to 80% of all late-stage cancer patients. Cachexia is highly deleterious, and leads to the irreversible loss of adipose and muscle tissue in affected patients. The aetiology of cachexia is poorly understood, in part due to the fact it is a highly complex multifactorial condition. Several factors and pathways associated with cachexia have so far been uncovered, but there is a lack of understanding of the key drivers of the pathology and integrated molecular mechanisms that regulate the progression of the disease. It is also unclear whether specific tumour characteristics predispose patients to developing cachexia, resulting in a dearth of early predictive markers for the pathology. As a result there are no convincing preventative or therapeutic options available for patients, and the condition represents an area of unmet clinical need. In this thesis we define a novel model for cancer cachexia in tumour-bearing Drosophila melanogaster larvae. We demonstrate tumour-bearing Drosophila larvae can recapitulate the key human cachectic phenotypes of muscle wasting and intramyocellular lipid droplet formation, as well as many molecular characteristics of the human pathology. The development of cachectic phenotypes in these larvae was dependent on tumour genotype, and not on tumour size, developmental delay, larval bloating, or starvation. These data suggested these animals represented a novel cancer cachexia model, suitable for use in the investigation of molecular mechanisms driving wasting. JAK/STAT signalling is implicated in the onset of cachectic wasting in human patients and model systems. We investigated the role of JAK/STAT signalling, and found that decreasing the activity of JAK/STAT signalling in wasting muscle was sufficient to rescue muscle wasting, and was unexpectedly accompanied by an increase in tumour size. We hypothesise that this may represent a previously unappreciated role of cachexia as a tumour suppressor as a component of the anti-tumour immune response. The data presented in this Thesis suggest tumour-bearing Drosophila larvae represent a novel cachectic model, one that is likely to prove suitable for use in the investigation of host-tumour interactions in the generation of cachexia.

Peripheral neuropathies (PN) represent a significant health burden, due to chronic and debilitating symptoms, that can be inherited or caused by everything from alcohol,chemotherapy and diabetes. PN are associated with both demyelination and axon degeneration, which play a fundamental role in their pathogenesis. The aim of this thesis is to identify novel mechanisms and/or potential therapeutics for protecting axon degeneration and promoting myelination. This is significant because there are currently no specific therapeutics, for PN, that have passed clinical trial successfully. Recent work in the Barnett lab has implicated heparan sulphate proteoglycans (HSPGs), as therapeutic targets in central nervous system (CNS) injury. HSPGs are able to bind a plethora of proteins, including chemokines and growth factors, where they facilitate binding to their corresponding receptors. We therefore hypothesised that HSPGs may have a regulatory role in PNS injury. To address this I data mined previously published microarrays for sciatic nerve (SN) injury. This principally identified the candidate heparanase (Hpse),which was up-regulated after injury in several different microarray analyses. Hpse is able to regulate HSPG signalling and is already implicated in several diseases including: cancer, diabetes and Alzheimer’s disease, making it an interesting candidate for further research. In order to question the role of Hpse in PNS injury we used two models of Wallerian degeneration:ex vivo SN injury model and an ex vivo neuromuscular junction (NMJ) injury model. Inhibiting Hpse acutely accelerated axon loss, while its exogenous treatment was protective. Furthermore we found that Hpse can regulate β-catenin protein levels and the transcription of genes, predicted to be regulated by β-catenin. This included Sox2, which led to an acceleration in Schwann cell dedifferentiation after in-jury. Recent evidence has linked Schwann cell dedifferentiation to early events in axon fragmentation. We also found that heparin sulphate mimetics significantly promote in vitro myelination, the mechanism for which requires further research. In conclusion, we have identified Hpse, an up-regulated enzyme after SN injury, in protecting axon loss during SN degeneration. Within the field, Wallerian degeneration is considered a useful model for studying the mechanisms behind axon loss in peripheral neuropathies, for which the role of Hpse warrants further investigation as a potential therapeutic target. Lastly we also identified heparin sulphate mimetics in promoting peripheral myelination, also making them potential therapeutic targets.