Recent work linked the RNA-binding protein UNR to a number of human pathologies although little is currently known about how UNR functions in human cells. This thesis aims to elucidate how UNR functions by, among other things, discovering novel UNR-interacting proteins and transcripts and proteins that are differentially expressed in the presence or absence of UNR. It is shown that UNR levels decrease with increasing cell confluency in cultured HeLa cells but that they increase with increasing confluency in the wild type TP53-containing U2OS cell line. UNR is shown to colocalise to stress granules with TP53 in arsenite-stressed HeLa cells. A number of novel UNR-interacting proteins were discovered in three cell lines (HeLa, U2OS and SaOS-2), including HUWE1, NARR, SQSTM1 and LDB1. GO-term overrepresentation analysis confirmed that UNR is an RNA-binding protein as ‘RNA binding’ and ‘poly(A) RNA binding’ were the top two overrepresented molecular function GO terms by p-value across each of the cell types. Less expected overrepresented GO terms pertained to selenium metabolism and the extracellular exosome. There was no evidence for conservation of UNR-interacting transcripts across the cell types but there were some similar significantly overrepresented GO terms among the respective UNR-interacting transcripts. These included terms pertaining to RNA and the nucleus. The most significant UNR-interacting transcript in HeLa cells was PABPC1 and that the PABP protein was also significantly upregulated following UNR knockdown in HeLa cells. ‘Poly (A) RNA binding’ was a significantly overrepresented GO term among proteins differentially regulated following UNR knockdown in HeLa and U2OS cells. ‘Adherens junction’ was another significantly overrepresented GO term using proteins that were higher in abundance in siUNR-treated HeLa cells and either higher or lower in abundance in either arsenite-stressed or unstressed U2OS cells. UNR was observed at cell-cell junctions in HeLa and U2OS cells by immunofluorescence microscopy.

The glucagon receptor (GCGR), a family B G protein-coupled receptor (GPCR), plays an important role in regulating blood glucose levels through its ability to bind the 29 amino acid peptide hormone, glucagon (GCG). Antagonising GCG action is a potential therapeutic option for reducing hepatic glucose production. However, GCG-based therapy is currently limited to acute emergency treatment of hypoglycemia in patients with type 1 diabetes (T1D) (Habegger et al., 2010). Further insight into GCGR-mediated signalling pathways and mechanism of activation may provide the basis for therapeutic development. We investigated the ligand stimulated GCGR signalling using multiple assays including those measuring cAMP accumulation, ERK1/2 phosphorylation and intracellular Ca2+ (Ca2+ i) mobilisation. Through site directed mutagenesis and FACS analysis for the investigation of cell-surface expression, we identified several important residues. They included K16812.49, L16912.50 , H17012.51 and T1722.45 of the ICL1 region (G1651.63 -T1722.45 ) as potential determinant in signalling bias at the GCGR through Gq/11-coupling, C1712.44 as a critical determinant of GCGR expression and R1732.46 as an essential residue for G protein-coupling. Helix 8 residue E4068.49 was implicated in maintaining GCGR in an inactive state. Finally, TM4 residues G2714.49 , L2774.55 and V2804.58 were found to plays an important role in successful translation and/or trafficking of GCGR and (Ca2+ i) mobilisation. The GCGR-mediated pERK1/2 response way found to be both Gq/11 and β-arrestin1/2 mediated, whereas it was independent of PKA or Gβγ-subunits. The GCG analogue TH-GCG, contrary to previous reports (Wakelam et al., 1986, Lenzen et al., 1990), was characterised as a partial agonist at the GCGR, inducing a robust cAMP response but fails to induce a detectable Ca2+ i or IP1 response. We also identified a RAMP2-dependent potentiation of the GCG stimulated cAMP response at the GCGR. The research described in this thesis has produced novel data that contributes to a clearer understanding of GCGR pharmacology.

Energy homeostasis is controlled in the hypothalamus. Hypothalamic tanycytes are a type of glial cell that lie in the wall of the third ventricle, between ventricular CSF and the key nuclei controlling feeding, and are potentially linked to this regulation. Tanycytes have been found to express sweet taste receptors; similar receptors exist for amino acids and could be present in tanycytes. Therefore, the aims of this work were to understand the tanycyte amino acid signalling process, determine the impact of diet on tanycyte function, and link tanycytes to energy homeostasis. Using ratiometric Ca2+ imaging, I showed that tanycytes responded to amino acids; the responses also involved ATP release via pannexin 1 and CalHM1 from tanycyte cell bodies and down their processes towards the parenchyma. As IMP increased the amplitude of tanycyte responses to some amino acids, one of the receptors is Tas1r1/Tas1r3. The Tas1r1 subunit was expressed in mouse tanycytes, and Tas1r1-null mice showed sex-specific altered responses to arginine and lysine. mGluR4 antagonist MAP4 reduced the responses to alanine, and mGluR4 was shown to be present in mouse tanycytes. Tas1r1/Tas1r3 and mGluR4 are therefore two of the receptors responsible for tanycyte amino acid signalling. Fasting and essential amino acid deprivation increased tanycyte sensitivity to alanine. Essential amino acid-deprived diet also reduced the intake of an alanine-enriched drink. While amino acid-imbalanced diets did not have strong effects on mouse metabolism over 24 hours, increasing dietary alanine content reduced feeding and increased metabolism in the light phase. These results demonstrate for the first time that tanycytes can detect amino acids, and that they do so via two different pathways, similarly to taste receptor cells in the tongue. Tanycytes can send ATP signals to inform the hypothalamus about nutrient availability. The data also show that tanycyte amino acid sensitivity is diet-dependent. High alanine food has a satiating effect, which could be partially mediated by tanycytes, although a direct link cannot be confirmed at this stage.

G protein-coupled receptors (GPCRs) elicit an ability to activate multiple downstream signalling pathways. It is becoming evident that for many GPCRs, agonists are able to activate several of these pathways, each to differing extents; a phenomenon termed pathway bias, or biased agonism. Here, work is presented quantifying biased agonism for the: adenosine A1 and A2A receptors, as well as the calcitonin-like receptor (CLR). For the adenosine receptors, novel selective, and non-selective, agonists are identified and characterised. Further, the extent of biased agonism is determined for A1R agonists with respect to their abilities to positively and negatively regulate cAMP production, mobilise intracellular Ca2+ and activate ERK1/2. The activity of triazoloquinazoline compounds against the A2AR is validated, identifying 3 to be selective. Further investigations into the ability of triazoloquinazolines to mediate cAMP production and ERK1/2 activation uncovers each tested agonist to be biased towards activating ERK1/2, at the A2AR. A characterisation of the effects of receptor activity modifying proteins (RAMPs) upon signalling from the CLR is presented: quantifying the extent of biased agonism, with respect to the ability of RAMP-CLR heterodimers to: mediate cAMP production and inhibition, as well as mobilise intracellular Ca2+, uncovering this to be a Gαq/11-mediated process. Further, through applying a saturation mutagenesis approach to the CLR, a potential interaction is identified between intracellular loop 1 (ICL1) and helix 8, which is broken upon receptor activation, further identifying ICL1 to be a region of the CLR responsible for influencing G protein specificity. Ultimately, these findings relating to both adenosine and CLR-based receptors uncovers further evidence of biased agonism at GPCRs, which may have potential implications upon improving the efficacy and safety profiles of novel pharmaceutics targeting these clinically relevant GPCRs.

Clathrin-mediated endocytosis (CME) plays a central role in fundamental processes such as synaptic vesicle recycling, receptor recycling, signalling and development. CME begins with clathrin assembly on the plasma membrane, facilitated by adaptor proteins. This process forms an endocytic vesicle that allows transport of cargo into the cell, and is followed by clathrin disassembly through the action of different adaptor/accessory proteins. A large number of different adaptor and accessory proteins are recruited during CME, in a spatially and temporally ordered manner. Although our understanding is growing as to the roles of individual adaptor proteins, we still do not understand the way in which some adaptors interact with clathrin or the molecular details of their interactions with one another in the presence of clathrin. Clathrin adaptor proteins contain short, linear clathrin-binding motifs, which form the basis of their interaction with the four distinct sites on the clathrin N-terminal domain (TD). An adaptor protein with tighter binding or more numerous clathrin binding sequences could displace one with weaker or fewer binding elements. This raises the question of whether adaptor proteins compete for binding to clathrin or whether they can bind simultaneously. Using certain biochemical and biophysical techniques in vitro and purified WT and mutant adaptor proteins, I have shown the complex ‘multiple TD linking effect’ of epsin 1 via the cooperative action of its two clathrin box motifs and unstructured region. Using the newly developed SPR/IAC (2-injection) method, I explored competition between five purified structurally and functionally diverse adaptor proteins when simultaneously binding to clathrin TD. I have shown how the complex structure of epsin 1 causes competition with β-arrestin 1 for clathrin TD binding. Such competition is observed between espin 1 and auxilin 1 as well, which reveals information about the mechanism of disassembly. However, β2-adaptin and auxilin 1 demonstrate no such competition.

Meal pattern has been identified as a factor influencing the thermic effect of food (TEF) and metabolic status, and therefore health. This thesis investigated the effects of an irregular meal pattern, with a controlled energy intake, in normal-weight (n=11) and obese with insulin resistance (n=9) females over a 14-day period. Measurements were made of the TEF, circulating glucose, insulin, lipids concentration and appetite regulation, using a crossover design. The irregular intervention period led to a significant reduction in the TEF following a test drink consumption in both normal-weight and obese participants. Glucose iAUC responses to the test drink measured over 3h were higher after the irregular compared with the regular intervention with no difference in the insulin response in the normal-weight study. In the obese study, glucose responses were unaffected by the regular and irregular intervention periods, whilst there was a main effect of meal pattern in insulin responses. In the normal-weight study, fasting GLP-1 decreased after both interventions. In contrast, fasting GLP-1 increased after both interventions in the obese study. Furthermore, in the obese study, the regular intervention produced a higher GLP-1 iAUC compared with the irregular intervention, but there were no such effects in the normal-weight study. The normal-weight study showed that fasting PYY was lower after the interventions compared with before. Moreover, iAUC for PYY increased after the interventions compared with before. However, there were no significant differences in fasting and iAUC PYY responses between the two interventions in the obese study. A regular meal pattern appears to be associated with greater TEF, which might result in more favourable energy balance for weight maintenance. Also, it is likely that a regular meal pattern improved insulin sensitivity in healthy normal-weight females. Therefore, a regular meal pattern could be a lifestyle factor that may promote an individual’s health.

New strategies are urgently needed to increase physical activity participation in the increasingly sedentary population to combat the rising rates of obesity and metabolic disease. The aim of this thesis was to provide evidence that practical high-intensity interval training (HIT) strategies can remove many of the major exercise barriers for obese individuals and people with type 1 diabetes that could potentially increase physical activity participation. Secondly, this thesis aimed to provide mechanistic evidence to explain the physiological effectiveness of HIT as a means to reduce the risk of cardio-metabolic disease. In Chapters 4 and 5, 32 obese adults with at least 3 additional cardiovascular disease (CVD) risk factors completed one of three 12-week training programmes 3x/week: Home-HIT (n=9); Laboratory-based supervised HIT (Lab-HIT; n=10) or home-based moderate intensity continuous training (Home-MICT; n=13). Changes in V ̇O2peak, insulin sensitivity, body composition, flow-mediated dilation (FMD) and aortic pulse wave velocity (PWV) were assessed. Muscle biopsies were taken to assess changes in capillarisation, mitochondrial density, intramuscular triglyceride (IMTG) content and eNOS and GLUT4 protein expression using quantitative immunofluorescence microscopy. Adherence and compliance (Home-HIT 96±3% & 99±1%; Home-MICT 88±4% & 100±0%; Lab-HIT 97±1% & 100±0%, respectively) to training did not differ between groups. Training increased V ̇O2peak and Matsuda insulin sensitivity index (P < 0.05). BMI, body fat percentage and visceral fat decreased (P < 0.05). FMD increased and aortic PWV decreased in each group (P < 0.05). Immunofluorescence microscopy revealed increased capillarisation, mitochondrial density, IMTG content and eNOS and GLUT4 protein expression (P < 0.05). In Chapter 6, 14 people with type 1 diabetes completed a randomised counterbalanced crossover design whereby continuous glucose monitoring was used to assess glycaemic control and risk of hypoglycaemia following a single bout of HIT and moderate-intensity continuous training (MICT) on separate days, compared to a non-exercise control day (CON). In Chapter 7, 14 people with type 1 diabetes (n=7 per group) completed six weeks of HIT or MICT 3x/week and the effect on glucose control and markers of cardio-metabolic health were measured. Chapter 6 showed no difference in the time, incidence or severity of hypoglycaemia over the 24-hour or nocturnal period between the CON, HIT and MICT days. In Chapter 7, six weeks of HIT or MICT improved V ̇O2max by 14% and 15%, respectively and aortic PWV by 12%, with no difference between groups. Therefore, Chapters 6 and 7 demonstrate that HIT is an effective exercise strategy for people with type 1 diabetes that reduces the two major barriers of lack of time and fear of hypoglycaemia. Finally, in Chapter 8, eleven previously sedentary individuals with type 1 diabetes completed 6 weeks of Home-HIT. Blood glucose was monitored before, immediately and 1h after all of the exercise sessions. Perceptions of the program along with attitudes towards exercise, barriers to exercise and previous experiences of exercise were evaluated using an online survey. Training session adherence was 93±2%, with participants achieving their target HR in 99±1% of sessions. Blood glucose was not different from baseline immediately or 1h post HIT exercise. Training increased V ̇O2peak by 8% (P=0.015), but blood pressure was unchanged (P=0.445). The qualitative data showed that the Home-HIT programme was positively received with many benefits. In conclusion, this thesis provides strong evidence that HIT can reduce major barriers to exercise and potentially increase exercise participation in these at-risk populations. Furthermore, Home-HIT was shown to be an effective strategy to improve a wide range of physiological markers indicative of improved cardio-metabolic health. Importantly, Home-HIT not only reduced traditional barriers to exercise, but also the key barrier in people with type 1 diabetes, fear of hypoglycaemia. As such, Home-HIT may represent an effective strategy to improve health in obese individuals with elevated CVD and people with type 1 diabetes by increasing exercise participation. Future research should investigate the effects of Home-HIT on a larger scale using larger cohorts and longer training periods using large-scale randomised controlled trials.

This thesis examines how individual differences in social traits relate to behavioural and physiological responses to affective touch. Over the last few decades the functionality of C-Tactile afferents (CTs) have been investigated, with researchers positing that CTs function to signal the rewarding value of social tactile interactions. Here, by exploring the relationship between trait sociability and affective touch perception this social touch hypothesis is explored. In the first three studies, the role of sociability on the vicarious experience of affective touch was investigated. In study one; the aim was to determine how trait sociability affected an individual's vicarious experience of affective and discriminative touch. Here, individuals with the lowest number of autistic traits and theoretically the highest sociability were found to show the greatest sensitivity in their affective ratings of different velocities of touch, resulting in a significant quadratic relationship between non-CT-optimal and CT- optimal stimuli at CT-innervated locations. In study two the aim was investigate the vicarious experience of touch in young children. Children both with a diagnosis of ASD (here theorised to be one extremity of trait sociability) and without ASD observed the same videos depicting social touch. It was found that young children (aged 7-12) did not show the typically observed vicarious preference for CT-optimal velocity touch. Furthermore, there was no difference between children with an autism spectrum disorder (ASD) and typically developing children in their affective ratings of the observed touch. Study three again took the same sample of videos and used facial EMG to see whether the explicitly rated vicarious preference for CT-optimal over non-CT-optimal touch could be detected implicitly. It was found that observation of CT-optimal social tactile interactions did not elicit the same affective responses that have previously been reported in response to directly felt touch. This finding is perhaps consistent with the rather weak affective response elicited by touch in comparison to pain, for example. The fact that self-reported levels of empathetic concern correlated negatively with corrugator muscle activity, indicative of negative affect, in response to touch on CT innervated sites suggests individual differences in implicit affective response to touch are present. In study four, the aim was to determine whether individual differences in trait sociability affected implicit affective responses to first-hand experience of touch. Consistently, participants will low levels of autistic traits (high trait sociability) showed greater zygomaticus activity, indicative of positive affect, during evaluation of the touch they received than those with high levels of autistic traits. Stroking touch elicited little activity in the Corrugator, indicative of negative affect, in either group. Finally, study 5 used EEG to determine how the cortical activity related to fast conducting A-beta stimulation compared to later activity in response to slow conducting CTs. Specifically an ultra-late potential (ULP) was measured for CT-optimal stimuli. Furthermore, 30cm/s, which generates greater A-beta stimulation than 3cm /s, elicited a significantly greater p300 orienting response. Furthermore, there was a significant difference in the ULP peak amplitude between individuals with high and low levels of autistic traits suggesting differential patterns of activity. Taken together, these studies suggest that touch, including touch targeted to optimally activate CTs is indeed processed differently, both physiologically and behaviourally, by individuals with different levels of autistic traits, whether directly felt or vicariously experienced. It is hypothesised that these differences s reflect variation in sensitivity to the rewarding value of social stimuli. These studies provide some of the first evidence that individual differences in stable personality traits are associated with differential responses to social / affective touch.

This thesis set out to explore oral sensory processing. Oral sensory processing extends beyond taste perception, the nerves that innervate the mouth and carry taste information to the brain also carry chemosensations, thermal sensations and somatosensations. While a great deal is understood about oral chemo and thermal perception, this thesis focuses on the not fully recognised oral somatosensory processes. A substantial amount of movement occurs within the mouth, from movement while speaking to chewing food. As food moves around the mouth, different oral receptors are activated and the consumption experience changes. Taste perception varies between individuals in a way that has led to the identification of the taster status genetic polymorphism of taster status where three taster groups (hyper-taster, taster, tolerant taster) with differing sensitivity to bitter tastes were identified. This sensitivity is further represented in anatomical differences with differing densities of fungiform papillae on the tongue. Using psychophysical methods and the taster status phenotype, this thesis examined if different regions of the tongue and mouth experienced different chemostimulant intensity and if dynamic touch changed the intensity perception of chemostimulants in chapter 4. This identified that different regions of the oral cavity experience chemostimulant intensity differently with the tip of the tongue being the most sensitive and the vermillion of the lower lip the least sensitive to sensation. Furthermore, whilst there was no main effect of touch on sensation intensity an interaction between touch type, taster status and oral locations was found when using 10-ppm capsaicin and Sichuan pepper. A dynamic touch on the lip with mint oil was also considered more intense than a static touch. Chapter 5 investigated the possibility that C tactile (CT) afferents were present in the lower lip, the structure of the lip skin widely suggests that CTs are not present but their regular use in the affective behaviour of lip-to-lip contact between individuals suggests otherwise. By applying the standardised psychophysical stroking approach to the lip, cheek and mucosa the classic psychophysical inverted U associated with CT like behavioural responses to touch was found on the cheek where CTs are known to be present as well as on the lower lip. This CT like response on the lip warrants further detailed investigation. Serotonin (5-HT) is widely associated with hedonic experiences and reduced 5-HT levels are a linked with depression and anhedonia. 5-HT is also a candidate neurotransmitter associated with taste transduction. Chapter 6 describes an acute tryptophan depletion (ATD) study that examined the peripheral and central effect of reduced 5-HT levels on taste perception. The primary findings highlight that tryptophan levels do not effect sweet, sour, salt and bitter taste detection ability. A significant difference in bitter taste intensity and pleasantness was identified with tryptophan depletion increasing the taste intensity and decreasing bitter pleasantness at suprathreshold concentration. An effect of taster status was identified in bitter intensity ratings with tolerant-tasters reporting a greater intensity of sensation in the tryptophan depletion session than in the control. During the course of the experimental phase of this thesis, it became clear that describing oral sensations was a difficult task. When asked to describe how sensations felt within their mouth in chapter 4, participants were unable to find words to describe sensations. Therefore, the final study in chapter 7 describes the development of a candidate oral lexicon to aid in describing mouth feel and oral sensations highlighting that the approach to lexicon development previously used to develop the McGill Pain Questionnaire and the Touch Perception Task can successfully be applied to the development of an Oral Lexicon.

Grounded in an integrated framework (Duda, 2013) that pulls from achievement goal theory (AGT; Nicholls, 1989) and self-determination theory (SDT; Deci & Ryan, 2000) framework, the current thesis presents the steps taken to develop and test the Multidimensional Motivational Climate Observation System (MMCOS) in sport. Almost exclusively, research examining the motivational coaching environment from an AGT and/or SDT perspective has relied on athletes’ self-reports of the environment. Few studies have used an observational methodology to rate objective features of the coaching environment as emphasized within AGT and SDT-based research, and no studies have integrated AGT and SDT to provide an objective assessment of the motivational environment. To address these limitations, the thesis begins by outlining the development and validation of the MMCOS in Chapter 2. In Chapter 3, the MMCOS is used to examine the relationship between observed and perceived dimensions of the coaching environment and subsequent relations with athlete motivation. Following this, the objectively rated motivational environment is examined in training and match contexts (Chapter 4). Finally, the MMCOS is employed to further understand the dynamics of the observed and perceived motivational coaching environment in an elite sport setting (Chapter 5). Findings from the present set of studies provide initial evidence for the reliability and validity of the MMCOS when utilized within different samples (i.e., grassroots and elite) and contexts. In the future, the MMCOS could be used alongside self-report measures and provide an alternative assessment of the coach-created motivational environment in sport, as well as contribute to the evaluation of coach-education programmes grounded in an AGT and SDT perspective.