Theses - Physiology, Development and Neuroscience
Permanent URI for this collection
Browse
Recent Submissions
Item Embargo Hepatic and Extra-hepatic Metabolism in NAFLD and the Role of Hepatocyte Oxygen SensingHolzner, LorenzNon-alcoholic fatty liver disease (NAFLD) is a growing healthcare challenge, affecting ~30% of the global population, however there are currently no specific treatments approved for the disease. A better understanding of pathophysiological mechanisms is required, including the close links between NAFLD and metabolic, cardiovascular and chronic kidney disease. Hypoxia-inducible factor 2α (HIF2α) accumulates in the livers of NAFLD patients and mouse models, and is a regulator of lipid metabolism. In this thesis, I investigated whether hepatocyte-specific deletion of *Epas1* (encoding HIF2α) protects against NAFLD, and whether this is associated with changes in mitochondrial and lipid metabolism. To investigate this, wild type mice and mice with a hepatocyte-specific deletion of *Epas1* were fed a high-fat, high-fructose, high-cholesterol diet (GAN diet) for 28 weeks and hepatic pathology and metabolism were assessed alongside measures of cardiac, renal and whole-body health and metabolism. Hepatic *Epas1* deletion did not protect against NAFLD, or GAN diet induced hyperglycaemia and hyperlipidaemia, but did ameliorate GAN induced hyperinsulinaemia. Moreover, hepatic *Epas1* deletion did alter hepatic mitochondrial respiration and expression of fatty acid oxidation (FAO) genes. Independent of diet, hepatic *Epas1* deletion was associated with accumulation of two specific sphingomyelin species, SM 41:1 and SM 42:2. GAN feeding also induced cardiac dysfunction, as assessed in Langendorff perfused hearts, as well as cardiac steatosis and accumulation of ceramides. Hepatic *Epas1* deletion did not protect against this, but was instead associated with cardiac dysfunction independent of diet, as well as accumulation of diacylglycerols, ceramides, and again, SM 41:1 and 42:2. Hepatic *Epas1* deletion did ameliorate cardiac sympathetic dominance in GAN fed mice. Similarly, GAN feeding induced renal steatosis, possibly due to lower FAO capacity and higher expression of renin. Again, hepatic *Epas1* deletion did not prevent this but may have worsened steatosis, and was associated with higher expression of angiotensin II type 1 receptor. Overall, hepatic *Epas1* deletion did not protect against NAFLD, but was instead associated with cardiac dysfunction and accumulation of potentially lipotoxic species, as well as higher renal expression of components of the renin-angiotensin system. Underlying mechanisms remain unclear, but programming by developmental anaemia in hepatic *Epas1* knockout mice may play a role.Item Open Access Anatomical and Electrical Characterisation of The Human Cochlea Towards the Improvement of Cochlear Implant PerformanceSwords, Chloe; Swords, Chloe [0000-0002-0431-4491]Cochlear implants (CIs) are transformative treatments that provide 'electronic hearing' to individuals with severe-to-profound hearing loss. This condition affects 1.2 million people in the UK, with 18,000 using a CI. However, the effectiveness of CIs is limited by the fundamental principles of cochlear function, particularly its spiral shape and inherent electrical conductivity. This is notwithstanding its small size and location deep within the temporal bone. This issue, known as 'current spread', presents challenges for accurate electrical stimulation and signal transmission. Understanding CI stimulus spread and its correlation with patient- and implant-dependent factors is difficult due to the inaccessibility of the inner ear and the current lack of a validated physical in vitro testing model in humans. Therefore, there is a need for a new modelling approach that can provide clinical insights. This research aims to develop anatomically and electrically accurate models of the human cochlea to examine the impact of CI design and stimulation types on current spread patterns towards the cochlear modiolus. First, cochlear anatomy was modelled in normal morphology cochleae. A template was developed and validated for segmenting cochleae from high- and low-resolution computed tomography (CT) imaging. Shape analysis techniques enabled the examination of shape variability in 83 normal morphology cochleae. Insights revealed sex-specific differences, with female cochleae being smaller and more tightly coiled than male cochleae. Additionally, it was suggested that two cochleae from the same patient might differ in size. In Chapter 3, the first 3D characterisations of three forms of inner ear malformations (IEM) (incomplete partition type-II and III, cochlear hypoplasia type-II) were produced from high-resolution histopathology. The location and number of spiral ganglion neurons (SGNs) were characterised. These findings highlighted important differences in IEM morphology and SGN location/quantity compared to normal morphology cochleae, which is relevant for CI surgeons and audiologists. Next, the creation and validation of two cochlear models is described. Both models are capable of predicting extracellular current spread patterns near the target neural elements. The goal of dual cochlear modelling is to understand better the factors affecting current spread within the cochlea and to develop testable hypotheses for systematic patient-by-patient evaluation. Dual modelling allows for cross-comparison of results, providing reassurance about accuracy. The first model measures current spread in ex vivo human cadaveric cochleae after characterising the anatomical variations in over 80 specimens. The second model is a 3D-printed cochlea. It mimics the electrical characteristics and duplicates the current spread experiments in 3D-printed replicas of the cadaveric cochleae. Finally, six 3D-printed models with densely and regularly spaced micro-recording wires were used to systematically assess the impact of different stimulation modes on electrode array placement within the cochlea. This work suggests a trade-off between achievable stimulation levels and current focusing. Notably, arrays positioned closer to the modiolus demonstrated greater current-focusing ability and higher stimulation levels, with preliminary evidence indicating that current-focusing multipolar modes might be most effective for tightly coiled cochleae implanted with perimodiolar arrays. Overall, this thesis enhances our clinical understanding of normal and malformed cochlear anatomy and creates two validated models of CI electrical stimulus spread.Item Embargo The Role of Parvalbumin Interneurons in Mouse Primary Visual Cortex during Visual Discrimination and Perceptual LearningKukovska, LiliaParvalbumin-expressing (PV) cells are the most common class of inhibitory interneurons in the rodent cortex and play important roles in balancing cortical circuit activity and learning (Xu et al., 2010). PV cells are suggested to mediate feedforward inhibition and control gain modulation (Markram et al., 2004; Tremblay et al., 2016). Although increasing their activity may improve discrimination (Lee et al., 2012), findings are inconsistent and the role of PV cells has been under debate (Atallah et al., 2012; El-Boustani et al., 2014; Wilson et al., 2012). The primary visual cortex (V1) has an early and a late phase of activity, likely reflecting bottom-up (feedforward) and integration of top-down (feedback) sensory processing (Lamme and Roelfsema, 2000; Wyatte et al., 2014). The feedforward sweep alone is sufficient to enable discriminations in mice (Resulaj et al., 2018), however, it is not known how PV cells contribute to this early processing and whether both easy and difficult discriminations are equally impacted by changes in inhibition. Perceptual learning increases the selectivity of V1 pyramidal (Pyr) and PV cells (Poort et al., 2015), and the emergence of PV-Pyr ensembles has been reported (Khan et al., 2018). Thus, PV cells may modulate Pyr cell activity differently depending on the stage of learning. However, such interactions have only been examined in a naïve V1 network and have not yet been studied after extensive behavioural training on a visual task. Plasticity following visual perceptual learning is thought to manifest in lower-level areas like V1 due to the high ‘specificity’ for the trained features (Fiorentini and Berardi, 1980; Schoups et al., 1995). Generalization and transfer of learning with simpler stimuli (McGovern et al., 2012), changes in higher-level representations, and influences from attentional feedback (Szpiro and Carrasco, 2015), decision-making areas (Diaz et al., 2017), and the oculomotor system (Kwon et al., 2013), challenges this view. Indeed, mice are extremely flexible in generalizing the task rule and adapt their behaviour over days to successfully discriminate previously inexperienced stimuli using a categorization strategy (Goltstein et al., 2021; Reinert et al., 2021). However, due to the complexity of perceptual learning likely affecting several brain areas and multiple levels of processing, it is important to have a full understanding of the flexibility of V1 representations and how the network adapts to reflect these changes over shorter timescales. Therefore, the thesis aims to investigate: 1) How does manipulating the strength and timing of PV cell activation in V1 affect visual discriminations of varying difficulty? 2) How does the influence of PV cells on the local V1 network change with experience? 3) How does V1 rapidly represent previously untrained orientations given the flexibility of mice to generalize their task knowledge? To address these questions, I used a simple go/no-go orientation discrimination task and a combination of optogenetics to activate PV cells and two-photon imaging to record the activity of V1 cell populations. First, I found that performance improved for easy discriminations but only when PV cells were stimulated during the early phase of V1 activity. This was supported by changes in V1 neuron activity, where selectivity to the rewarded stimulus increased following PV cell activation. Second, I identified that PV cells exert global effects on the V1 population through divisive inhibition, both before and after learning. However, when taking into consideration stimulus relevance, the inhibition exerted by PV cells after learning was orientation-dependent. Third, I found that V1 representations are remarkably flexible, adapting the same processes that affect trained orientations to novel, but perceptually similar, orientations thus enabling mice to perform accurate discriminations. Taken together, these findings increase our understanding of the role of PV cells and V1 in visual discrimination and perceptual learning.Item Embargo Progesterone Regulation of the GnRH Pulse GeneratorWall, EllenKisspeptin neurons in the arcuate nucleus (ARN) represent the gonadotrophin- releasing hormone (GnRH) pulse generator responsible for driving the pulsatile release of GnRH and luteinising hormone (LH) secretion. In a healthy menstruating woman, following the mid-cycle ovulation, progesterone (P4) levels dramatically increase and exert a negative feedback action upon pulsatile LH secretion necessary for maintaining normal fertility. In mice, the ARN kisspeptin neurons display synchronised patterns of firing approximately every hour in diestrus and, similar to humans, this slows dramatically during the luteal phase of the cycle. However, the exact mechanism by which P4 negatively feeds back to regulate pulsatile LH secretion remains unknown. Given that ARN kisspeptin neurons express the progesterone receptor (PR), I hypothesised that this may be the site at which P4 negative feedback occurs. I first assessed the profile of circulating P4 levels in mice at 8-hour intervals throughout the estrous cycle using liquid chromatography-mass spectrometry (LC-MS). The concentration of P4 was found to peak at 6 PM on proestrus (21.94 ng/mL) and was at its lowest at diestrus 10 AM (0.42 ng/mL). I next assessed the changes in *Pgr* mRNA expression within ARN kisspeptin neurons throughout the cycle using RNAscope. This revealed that *Pgr* mRNA levels in kisspeptin neurons remain unchanged across the cycle. Building on these findings, I next assessed the impact of circulating P4 on the activity of the GnRH pulse generator using ARN kisspeptin neuron GCaMP fibre photometry in freely behaving mice. Diestrous mice equipped for monitoring the activity of the GnRH pulse generator were injected with either 4 or 8 mg/kg P4 and kisspeptin neuron synchronisation episodes (SEs) were monitored for 24 hours. Both doses resulted in a significantly decreased frequency of SEs compared to the vehicle, which persisted for 6 hours. To establish the site of action of P4, I then undertook a GCaMP fibre photometry study in which 25 or 50 ng/mL of P4 was infused directly into the ARN. Surprisingly, this had no effect on the GnRH pulse generator activity. My next approach to establish the role of PR in kisspeptin neurons involved using CRISPR-Cas9 gene editing to selectively knockdown PR in ARN kisspeptin neurons in adult mice. This achieved a selective knockdown of approximately 42% of PR in ARN kisspeptin neurons. This was not found to have any substantial effect on the GnRH pulse generator activity, pulsatile LH secretion, or estrous cyclicity. However, it partially mitigated the inhibitory actions of peripheral P4 treatment on kisspeptin neuron SEs. Unexpectedly, the major phenotype of mice with ARN kisspeptin neuron PR knockdown was that of substantial obesity due to increased appetite. In summary, my findings suggest that P4 exerts its inhibitory actions on ARN kisspeptin neurons through temporally regulated PR-dependent and PR-independent pathways. The partial mitigation of P4’s inhibitory actions on the GnRH pulse generator activity following PR knockdown in the ARN kisspeptin neurons, along with the observed obesity phenotype, highlights the multifaceted role of PR in this neuronal population. These insights further our knowledge of the specific roles of ARN kisspeptin PR and could be pivotal for the development of new therapeutic strategies for reproductive and metabolic disorders, such as polycystic ovarian syndrome (PCOS).Item Embargo Marmoset emotional regulation during adolescence and adulthood, with a particular focus on risk-taking and the role of the subgenual anterior cingulate cortex in motivation and affective biasesLynn-Jones, TaylorAdolescence is a critical period during animal development, characterised bywidespread neural and behavioural changes, including increases in exploratory behaviour and risk-taking. These behavioural and neurobiological changes are particularly evident in primates, where social groups and relationships are complex, and maturation of the prefrontal cortex (PFC) is prolonged, lasting from childhood into early adulthood. The PFC is responsible for a wide range of higher-order functions including decision-making, reward valuation, and social cognition. In humans, adolescence is the age range in which a significant proportion of psychiatric disorders first appear (Kessler et al., 2005), so it is theorised that adolescence may be a time during which individuals are particularly vulnerable to stressors that could interfere with the development of PFC subregions and produce psychiatric symptoms (Arnsten, 2004). A region of the PFC that has been highly correlated with psychiatric disorders, specifically mood disorders, is the subgenual anterior cingulate cortex (sgACC). Overactivation of sgACC in humans is observed in depression, and reducing neuronal activity in sgACC using deep brain stimulation alleviates depressive symptoms (Mayberg et al., 2005). However, the mechanisms underlying the functional development of PFC subregions like sgACC and behavioural changes during adolescence are not well understood, indicating a need for animal models. During my PhD, I have used the common marmoset (Callithrix jacchus) to investigate how exploratory behaviour changes during adolescence, as well as the role of sgACC in behaviours that are impaired in depression, specifically motivation in adolescents and adults and generation of positive affective biases in adults. My first experimental chapter uses adult marmosets to look at the role of sgACC in affective biases, specifically, if DREADDs-mediated sgACC overactivation impairs the animals’ ability to show a positive bias toward a stimulus associated with high value food reward. I developed a novel touchscreen-based task to assess affective bias in marmosets, in which during the week animals are trained to associate two stimuli with either a high or low food reward, and their response bias between the stimuli is assessed with a preference test at the end of the week. Both behavioural and cardiovascular data were analysed. There is a subtle behavioural effect of sgACC overactivation to reduce positive bias toward the highly rewarded stimulus, and overactivation also seems to blunt cardiovascular arousal to high value reward, such that the more blunted the cardiovascular arousal to high reward, the greater the reduction in positive bias at preference test. This project expands on previous work from the lab looking at the role of sgACC in appetitive arousal and anticipatory anhedonia, but looks specifically at behavioural output, which is particularly useful for understanding biological mechanisms of depression symptoms. My second and third experimental chapters investigate the effect of DREADDs-mediated overactivation of sgACC on motivation in adult and juvenile marmosets, the first such experiment to be conducted on juvenile animals in the lab. DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) are a cutting edge technique that allows for minimally-invasive brain manipulations via a single surgical infusion of viral vectors that induce long-term expression of either an excitatory or inhibitory DREADDs receptor into a brain region of interest. Previously in the lab, the role of sgACC in motivation in adult marmosets had been assessed with direct pharmacological overactivation using in-dwelling cannulae. My results corroborate these findings for DREADDs-mediated sgACC overactivation and identify changes in motivation across adolescence with DREADDs-mediated overactivation of sgACC. My fourth experimental chapter focuses on changes in marmoset exploratory behaviour from infancy to late adolescence, assessed using a novel object test. Novelty- and sensation-seeking can often be risky, and these behaviours are known to peak during human adolescence (Steinberg et al., 2017). Risk -taking has also been associated with negative mental health outcomes later in life (Smout et al., 2020). My work shows that risk-taking and novelty-seeking in marmosets also peaks during adolescence, indicating that these animals are a good model of human adolescent neurodevelopmental trajectories (Sawiak et al., 2018), but also of behavioural changes during adolescence.Item Open Access From genomics to function: Canine genetics highlight novel mechanisms of obesity and related traitsMcClellan, AlyceObesity is an increasingly prevalent complex disease with burdens on both canine and human health. It is a multifactorial disease with many environmental and biological risk factors and is highly heritable in humans, pointing towards a role for genetics. Obesity heritability is also observed in dogs where breeds show different risk of obesity. Despite the negative health consequences that result from obesity in dogs, research into its causes is limited. However, thanks to the genetic architecture of dogs, shared environment between dogs and humans, and the shared risk factors and comorbidities, the canine model offers opportunity to study obesity. Investigations in this thesis aim to elucidate genetic influences in canine obesity and the functional mechanisms conferring these to better understand obesity in both dogs and humans. The hypothalamic melanocortin receptors (MCR) are central to the control of energy homeostasis across species. The first investigations in this thesis aimed to better understand the role of canine MCR genes in obesity. Missense variants MC3R p.M320I and MC4R p.V213F were detected at varying frequencies across the breed groups. The loss-of-function mutation, MC3R p.M320I, was common in Labrador retrievers. Carrying the mutation was associated with delayed onset of puberty and a decrease in adiposity and weight. The effect of MC3R p.M320I on weight was different in obese and non-obese dogs, supporting a role of MC3R in regulating the boundaries of weight. Carrying MC4R p.V213F had inconsistent results on adiposity and weight in different breeds. Evidence suggested this may be due to variable genetic background. Using *in vitro* signalling assays, the MC4R p.V213F variant was shown to have a small loss-of-function effect. Next, variants predicted to impact protein function within known hypothalamic genes were identified in beagles. These studies provide valuable insight into the hypothalamic control of energy homeostasis in canine obesity and its conserved roles between species. To better understand the genetic basis of obesity across different dog breeds, a genome wide association study (GWAS) for food motivation was performed in a large cohort of mixed ancestry dogs. Although no genome wide significant associations were identified, the nominally significant loci were narrow and six were prioritised. Relevant candidate genes were identified in these regions. This included the gene coding for ghrelin, a hormone known to regulate appetite. In addition, a region of high significance in this GWAS overlapped with one previously associated with breed average obesity risk. In this region was the R-spondin2 gene (RSPO2), well known in canine genetics for harbouring an insertion which causes the furnishings hair phenotype. Furnished dogs were identified as having an increased risk of diabetes, hypothesised to result from poor adipose expansion. To identify novel functional mechanisms of obesity, the results of an additional canine obesity GWAS were explored which had identified DENN domain containing 1B (*DENND1B*), a protein involved in trafficking. Cross-species evidence that genomic variation in human *DENND1B* alters expression in the hypothalamus suggested it may regulate MC4R signalling. *DENND1B* and *MC4R* were then shown to be expressed in the same hypothalamic cells using murine single cell RNA sequencing data. Subsequently, overexpression of DENND1B *in vitro*, showed a reduction in MC4R downstream signalling and an increase in receptor internalisation, consistent with an increase in obesity risk. This represents the discovery of a novel mechanism in the central regulation of energy homeostasis. DENND1B was also implicated in adipose function. Overexpression of DENND1B in murine pre-adipocytes resulted in reduced glucose uptake highlighting possible pleiotropic effects of DENND1B in obesity mechanisms. The results of this thesis showcase the benefits of studying complex disease in dogs and provide novel insights into the genetic basis of canine obesity. They also contribute to an improved understanding of the mechanisms important in energy homeostasis across species.Item Open Access Cell Fate Decisions in the Developing LungHughes, TessaThe epithelium of the adult lung can be broadly segmented into two different categories: a bronchiolar region consisting of conducting airways which are responsible for conducting air towards the alveoli, and an alveolar region where gas exchange takes place (Herriges and Morrisey, 2014). During embryogenesis, the airways are first laid down through an iterative process of branching morphogenesis, which terminates at the alveoli. Throughout gestation the lungs are filled with fluid, but at the moment of birth this fluid is expelled and lungs must be competent to function for gas exchange and enable breathing (Szoták-Ajtay et al., 2020). Research has suggested that a multipotent progenitor population of epithelial cells resides in the distal tip of the developing lung (Nikolić and Rawlins, 2017) and is capable of differentiating into both bronchiolar and alveolar cells. Clarifying which tip cells differentiate to form the different lung compartments, and what factors promote lineage-restriction and competence loss are questions that remain to be addressed. Exposure to glucocorticoids has previously been demonstrated to accelerate lung maturation (Laresgoiti et al., 2016). However the changes in tip cell fate decisions and lung morphology induced by glucocorticoids are not well understood. To address these questions I have taken a clonal lineage-tracing approach to label lung tip epithelial progenitors. A range of tamoxifen doses were administered to two different mouse models, representing either a biased (tip cells only) or unbiased (random) labelling approach, to ensure an appropriate number of initial cells were being labelled. Different conditions and protocols were tested to image entire lung lobes. A protocol where wholemount immunostaining is combined with CUBIC-1A clearing, and imaged on a confocal microscope has been most successful. Using these optimised conditions I have imaged entire E18.5 embryonic mouse lung lobes following lineage-labelling. I have developed an image analysis pipeline to assess cell fate outcomes in the context of the overall branching structure in both the biased and unbiased lineage tracing models. Embryonic mouse lungs exposed to glucocorticoids in drinking water from embryonic day 12 - 15 showed larger, more robust alveolar regions with increased expression of mature alveolar markers. Those exposed by intraperitoneal injection at embryonic day 13 showed swelling of the distal regions, increased lumen space, and decreased branching. To explore the mechanism of glucocorticoid activity in human lungs, organoids were derived from late-stage (17 post-conception weeks or older) embryos and treated with a variety of glucocorticoids or other maturation reagents. These showed varying levels of expression of alveolar type II markers by both immunohistochemistry and quantitative reverse transcription polymerase chain reaction. These glucocorticoid exposures were repeated in mouse lung explants, which demonstrated that the maturation effect of glucocorticoids is achieved through multiple signalling mechanisms, affecting a variety of cell types throughout the lung. My results will facilitate future studies of whole organ clonal analysis and of branching structure morphology. They also provide deeper insight into the morphology changes that occur during dexamethasone treatment, and provide potential avenues to explore the signalling mechanism by which glucocorticoids activate lung maturation.Item Controlled Access Charting the hippocampal cognitive map: Investigating the emergence of grid cell and place cell firing patterns in novel and familiar environmentsKrstulovic, MarinoHippocampal place cells comprise the main building blocks of the cognitive map which is used to encode our surroundings quickly and rapidly. Prominent theoretical models argue that place cells emerge via a linear summation of different grid cells from the medial entorhinal cortex. However, recent experimental evidence shows place cells emerging in novel environments without grid cells. The experimental framework of this thesis is guided by the Field-boundary interaction model which argues that place cells may play a more fundamental role in shaping the emerging grid cell spatial pattern in novel environments. We show that over a three-hour novel environment trial place and size-matched grid fields display similar field size convergence dynamics. This is to be expected if place cells contribute to grid cell formation. However, grid cells also show an initial grid-like spatial pattern, as well as similar convergence rates across modules, indicating that their convergence is governed by internal entorhinal neural network properties. Overall, it can be hypothesized that the influence of place cells on the emerging grid cell pattern has been underestimated. Field-boundary interaction model also predicts that boundaries will influence nearby emerging grid and place fields. In large familiar environments with different western boundary configurations, we establish that Compression has a limited range, while Expansion has a more global effect on the grid cell spatial pattern. In both configurations place cells form multiple fields which show large and heterogeneous shifts throughout the environment. Lastly, we explore how multi-field place cells emerge in familiar and novel virtual reality 1D tracks. Comparable to real environment studies, on shorter tracks place cells mostly from single fields, while on longer tracks majority of place cells produce multiple fields. On a novel track single field cells gradually converge until stability, in contrast to multi-field cells which display non-monotonic convergence dynamics. Overall, the results of this thesis further our understanding of the emergence of the hippocampal cognitive map.Item Open Access Sleep oscillations and their temporal association in neocortices in miceLi, YuqiExperiences during wakefulness are replayed during sleep to consolidate the relevant information. At the circuit level, this process is thought to be reflected by the synchronised cortical activity during non-rapid eye movement (NREM) sleep. This cortical synchronisation is described by slow wave activity (0.5 - 4Hz) and the emergence of spindles (10 - 16 Hz). Slow waves and spindles can be recorded locally in one cortical area or globally throughout the cortex. They are often temporally associated with hippocampal ripples (80 - 250 Hz). Our understanding of how local and global sleep oscillations are shaped by various experiences and learning is incomplete, as the majority of the studies examine single modalities with a focus in the prefrontal cortex (PFC). Therefore, this thesis aims to compare the sleep oscillations in PFC and somatosensory cortex (S1) in baseline and upon experiences. I conducted three sets of experiments. First, I recorded local field potential during baseline sleep in PFC and S1. In agreement with previous reports, I showed that PFC spindles preferentially followed slow waves, while S1 spindles were similarly probable to occur before and after slow waves. In hippocampus, ripples preferentially occurred outside slow waves in PFC, while this observation was weaker in S1. Next, I investigated how experiences shaped sleep oscillations and their temporal associations in PFC and S1 by conducting a PFC dependent appetitive Y-maze and a S1 dependent object exploration task. Task performance enhanced slow wave activity and sigma activity in both cortices, while learning of Y-maze was predicted by a closer temporal association between slow waves and spindles in PFC. In contrast, my preliminary results on the object exploration task suggested that temporal association of oscillations was modified in S1 but not in PFC. Last, I attempted to characterise the mechanism of brain oscillation coordination. Acting as a communication hub between PFC and hippocampus, midline thalamus has the potential of synchronising neocortical oscillations and hippocampal ripples. Using optogenetic tools, I performed a sustained inhibition of midline thalamus and observed an increased peak frequency of spindles in PFC and enhanced sigma oscillations in S1, while temporal association of oscillatory events was differentially modulated in PFC and S1. My findings indicated that during baseline sleep, the sleep oscillations in PFC and S1 exhibited distinct temporal association with each other. The change in the temporal association of oscillations was confined to the previously engaged cortex during task performance. Additionally, the midline thalamic activity contributed differently to the oscillations in these cortical areas. Together, these results highlighted a different temporal association of oscillatory events in S1 to what has been reported in PFC, with stronger responsiveness to recent sensory experience. Future experiments could be conducted to manipulate oscillatory strengths and temporal association between oscillatory events to establish the causal relationship.Item Embargo Mechanical mapping of lineage boundaries in the developing Xenopus laevis embryoMcGinn, RossStem cells are capable of both self-renewal and differentiation into a range of other cell types and must regularly make decisions between these fates. While we have a good understanding of how the chemical environment influences these decisions, there is mounting evidence that stem cells integrate mechanical and chemical signals *in vitro* when making fate decisions. Specifically, substrate stiffness has been shown to be an important regulator of stem cell fate, though we currently know little about the contribution of viscosity, and *in vivo* nvestigations are still missing. In this project I used atomic force microscopy (AFM) to investigate the viscoelasticity of the developing *Xenopus laevis* embryo and compared mechanical maps to fate maps of the developing embryo found in literature, along with my own lineage tracing. First, I determined that the presence of the vitelline membrane obscures the mechanical properties of the embryo below, and so must be removed, and that the AFM could reliably measure embryonic tissue up to an angle of 21° from horizontal. I then measured the stiffness and viscosity of three distinct embryonic regions: the vegetal pole, the animal pole, and the equator, during early gastrulation. I found the vegetal pole to be the stiffest and most viscous region, followed by the equator and then the animal pole. The vegetal pole also had the largest viscous component contribution, followed by the animal pole and then the equator. I found that the embryo stiffness is increasing over time, while the viscosity remains constant, allowing me to apply a correction to past and future measurements that remove the effect of embryonic aging on the stiffness. To determine the role of the mechanical environment in stem cell lineage commitment during development, I labelled two single cells with fluorescent dextrans at the 8-cell stage, then tracked their descendants and measured mechanical maps across the lineage boundaries. I found that primarily endoderm cell regions were both stiffer and more viscous than ectoderm cell regions, and that this endoderm region was more elastic dominated than the ectoderm region. I found a clear correlation between local mechanical properties and stem cell fate choice/lineage restriction during embryonic development *in vivo*, and my future work will focus on expanding these investigations to look for a causal relationship. I also found evidence that the viscous component of biological tissue should be considered when investigating the impact of the mechanical environment on stem cells. A better understanding of how stem cell decisions are regulated *in vivo* could lead to advances in stem cell treatments for disease or illness, as well as increased control over stem cells in the laboratory.Item Embargo Exploring the Role of Long Non-Coding RNA in Neural Stem Cell Reactivation from Quiescence: A Functional Genomics ApproachJudge, LeiaGiven the proposed role of long non-coding RNA (lncRNA) in fine-tuning large-scale genomic responses to stimuli and cell-fate transitions, previous members of the Brand lab compared genome-wide changes in DNA polymerase II occupancy at lncRNA loci during Drosophila melanogaster neural stem cell (NSC) reactivation from quiescence. Through doing this, ten novel quiescence-associated lncRNAs were identified. One such lncRNA, pron, was found to be strongly upregulated in reactivating NSCs compared to quiescent NSCs. pron is localised to multiple nuclear puncta, suggesting that it might act in trans to regulate gene expression during reactivation from quiescence. Using a mutant line expressing a truncated, non-functional lncRNA molecule, pron was subsequently found to regulate the timing of NSC reactivation from quiescence. Building on these findings, the work in this thesis aims to deepen our understanding of the role of lncRNA molecules in regulating NSC quiescence and reactivation in Drosophila melanogaster and to specifically understand how the candidate lncRNA pron regulates gene expression during this process. I characterised the expression and potential function of NSC-associated lncRNAs and further built upon our understanding of the diverse roles played by lncRNA in vivo. Specifically, using traditional genetic techniques I confirmed that pron acts in trans to regulate the timing of NSC reactivation from quiescence. Given its nuclear localisation, I sought to better understand the molecular function of pron during reactivation at the genomic level and characterise the role of potential target genes. To do this, I used RNA-DamID to identify in vivo targets of pron. I correlated this genome-wide binding data with differential gene expression and chromatin accessibility, using a combination of single-cell RNA sequencing and chromatin accessibility DamID, to better understand the role of pron in vivo. I also identified specific RNA-binding protein and DNA-RNA triplex-forming sequences within pron and used these to inform pron function. In pron mutant NSCs, I identified genome-wide changes in the expression and accessibility of genes associated with Hippo signalling, ribosome biogenesis, mitochondrial energetics and cell cycle regulation—key processes implicated in NSC reactivation. This work demonstrates that pron acts in trans to regulate the expression of a suite of genes involved in orchestrating NSC reactivation, strengthening the evidence for functional roles for lncRNAs in vivo. Positionally orthologous, or syntenic, transcripts of pron were identified in several species, including humans (PROX1-AS1) and mice (Prox1os), indicating a potential evolutionarily conserved function of CR31386, and suggesting the potential importance of lncRNAs in integrating genomic signals during significant cellular events.Item Embargo Cellular Morphodynamics Reveals Divergent Mechanisms of Gastrulation in InsectsBattistara, MargheritaDuring embryonic development, dynamic cellular rearrangements collectively result in tissue morphogenesis. One fundamental morphogenetic process is tissue internalisation, of which the earliest occurrence drives mesoderm gastrulation in a wide variety of organisms. In both the fly Drosophila melanogaster and the beetle Tribolium castaneum, the mesoderm is specified ventrally by conserved genetic factors as an epithelial sheet that buckles inwards, forming a tube that subsequently collapses. While the overall morphogenesis of the mesoderm appears analogous between the two species at the tissue level, the level of conservation of the cellular behaviours driving this process remains elusive. To address this gap, I developed a morphodynamics pipeline to segment and analyse 3D cell shapes in an unbiased and quantitative framework. This enabled me to mathematically describe the coordinated cell shape changes responsible for mesoderm morphogenesis in Drosophila. These transformations are linked to known cellular processes that take place sequentially: apical constriction of tall columnar epithelial cells, cell shortening, mitosis and cell spreading, together contributing to a collective epithelial-mesenchymal transition. In contrast, the application of my pipeline in Tribolium embryos revealed asynchronicity in cellular behaviours driving mesoderm invagination and epithelial-mesenchymal transition. By complementing my morphometric analysis with high-resolution live imaging and staining of cytoskeletal components, I identified two distinct phases of mesoderm internalisation in Tribolium. First, the mesoderm is specified as a cuboidal monolayer which undergoes a wave of semisynchronous cell divisions. Strikingly, at this stage a significant portion of mesodermal cells individually ingress via out of plane divisions and, in small part, cell extrusion. Internalised cells adopt a mesenchymal phenotype, whilst cells that have remained in the epithelium plane apically constrict and collectively fold inside. The tube then collapses and cells adopt a spread mesenchymal phenotype, which coincides in shape distribution with Drosophila mesenchymal cells. The differences between these species in mesodermal cell shape trajectories leading to the mesenchymal state can be ultimately attributed to two factors: the epithelium sheet's initial height, and the timing of cell division, either prior or subsequent to tissue folding. My study provides a generalised framework for extracting, analysing and comparing three-dimensional cell shapes from microscopy images of tissues, further offering adaptability to other systems. In the context of mesoderm gastrulation in Drosophila and Tribolium, it brings new insights to our understanding of conserved and divergent mechanisms underlying tissue internalisation. While apical constriction appears to orchestrate collective cell invagination via tissue folding in both insects, in Tribolium cells can individually ingress via out of plane mitosis and undergo a sudden transition from an epithelial to a mesenchymal phenotype. Out of plane divisions are a relatively underexplored phenomenon in developmental biology, whilst spindle misorientation in epithelia has been correlated with pathologies such as cancer and microcephaly. My study opens the door for exploring the mechanisms triggering out of plane divisions and their mechanical effects on tissue morphogenesis in an accessible organism.Item Open Access Amylin, tissue hypoxia and metabolic dysregulation in diabetic heart and liverKnapton, AliceType II diabetes mellitus (T2DM) is a growing burden, with the global prevalence rising from 4.7% of the adult population in 1980 to 8.5% in 2014. Owing to systemic metabolic changes in T2DM and myocardial and hepatic insulin resistance, the diabetic heart and liver are exposed to different levels of metabolites. Amylin, or islet amyloid polypeptide (IAPP), is a 37 amino-acid peptide hormone, that is co-secreted with insulin from pancreatic β-cells in response to nutrient stimuli. Amylin dyshomeostasis occurs in T2DM. Initially, as peripheral insulin resistance develops, β-cells compensate by increasing insulin production. As proinsulin and proIAPP are co-secreted, this also results in an increase in proIAPP production. Consequently, during the hyperinsulinaemic stages of T2DM, hyperamylinaemia also occurs. Human amylin then has the propensity to form oligomers, fibrils, and aggregates in T2DM patients, forming deposits in pancreatic islets and in extra-pancreatic peripheral organs, e.g. the blood vessels and parenchyma of kidneys, the heart, and the brain. Rodent amylin does not have this propensity to form aggregates. Therefore, amylin is an aspect of T2DM that is unexplored in many rodent models. The work presented in this thesis aimed to further our understanding of the impact of amylin dyshomeostasis on metabolic dysfunction in the diabetic heart and liver. In a diabetic rat model expressing human amylin (HIP rats), amylin aggregates were deposited in pancreatic islets, kidney, and cardiomyocytes, in a similar fashion to patients with T2DM. These amylin deposits stimulated hypoxic signalling, with elevated HIF2α levels being observed. Oxidative and energetic stress was also provoked. This resulted in downstream mitochondrial dysfunction and metabolic changes, including a lower Electron Transport Chain (ETC) capacity, and decreased fatty acid oxidation (FAO). In the livers of the HIP rats, the presence of human amylin resulted in an elevated capacity of complexes within the mitochondrial ETC. This enhanced ETC activity was associated with increased supercomplex formation. This was accompanied by an elevation in FAO. These responses are typical of an acute stress response to hypoxia in the liver. To understand the impact of hypoxia on metabolism against a background of T2DM, a milder rat model of T2DM, generated through high-fat feeding and streptozotocin injections, expressing only non-amyloid forming rodent amylin, was exposed to hypoxia. In these rats, there was a trend towards hypoxia lower respiratory capacity in the LEAK state and OXPHOS state supported by the fatty acid-derived substrate, octanoyl carnitine, as well as substrates for complex I&II respiration, and complex II alone. In conclusion, amylin aggregates are deposited around and internalised within cardiomyocytes in HIP rats. HIP rats then exhibit a reduced cardiac respiratory capacity and a reduction in the capacity for fatty acid oxidation. The metabolic perturbations that are unique to the hearts of HIP rats, may be due to amylin aggregate deposition causing tissue hypoxia, supported by our observations of elevated HIF2α expression and hypoxia signalling. In the liver, the presence of human amylin in HIP rats resulted in an elevated cardiac respiratory capacity and rate of fatty acid oxidation, which was associated with elevated mitochondrial supercomplex formation.Item Open Access Exploring a Nuclear Insulin Receptor Signalling Pathway in Drosophila Neural Stem CellsBrace, MaireThe insulin receptor (InR) is a central regulator of metabolism, growth, and proliferation in both humans and *Drosophila*. Integrating a range of signals, InR coordinates pleiotropic, tissue‐specific outputs. Neural stem cells (NSCs) give rise to the vast range of cell types that comprise the central nervous system (CNS) in a manner highly regulated in both space and time. With a comparatively simple CNS that nonetheless exhibits remarkable biological conservation, combined with superior genetic tractability, *Drosophila melanogaster* is an ideal model organism for studying the principles of neurogenesis and NSC biology. In *Drosophila*, insulin signalling is necessary and sufficient for the reactivation of NSCs from quiescence, a conserved state of mitotic dormancy. InR has been observed in the nucleus of a range of cell types *in vitro* for decades, but it is only more recently that the significance of this is becoming clear. Data reveal a novel nuclear arm of the insulin signalling pathway, in which InR associates with chromatin genome‐wide, strongly localizing to promoters and activating the transcription of relevant genes. This finding represents a growing paradigm shift, encompassing other receptor tyrosine kinases (RTKs), whereby direct nuclear signalling complements canonical membrane‐localised signal transduction. Given the complexity of insulin signalling and its dysregulation across an array of chronic human diseases, this nuclear pathway constitutes an exciting avenue for basic and translational science. Characterising the nuclear actions of InR might offer a more complete understanding of the longer‐term effects of insulin signalling. In the context of the CNS, it may offer a more complete understanding of NSC dynamics, which in turn may guide future therapeutic strategies for brain repair and difficult‐to‐treat malignancies. In this thesis, nuclear insulin signalling in *Drosophila* NSCs is studied via a variety of *in vivo* methods. Cyan fluorescent protein (CFP)‐tagged InR was expressed with the GAL4 system to examine its subcellular localization. InR‐CFP is present in the nucleus of *Drosophila* NSCs throughout CNS development. This localization pattern is not upheld in NSC progeny, where InR‐CFP exhibits strong membrane localisation. Next, the endogenous InR locus was tagged with green fluorescent protein (GFP) knock‐in using the CRISPR/Cas9 system. At endogenous InR expression levels, no clear nuclear localization was visible on confocal microscopy. However, the presence of nuclear InR was later confirmed with NanoDam. NanoDam, a recently published technology based on Targeted DamID, was used to profile the chromatin association of InR in NSCs *in vivo* genome‐wide. NanoDam was performed at endogenous levels of InR expression in the InR‐GFP background, and under misexpression conditions in the UAS‐*InR‐CFP* background. InR associates with chromatin genome‐wide in both genetic backgrounds. These data represent the first known finding that InR associates with chromatin in any cell type in *Drosophila*, and that InR associates with chromatin in NSCs in any organism. More genes are reproducibly and significantly bound in the endogenously tagged InR‐GFP background than the misexpression UAS‐*InR‐CFP* background, which may be due to higher expression levels in the latter background saturating physiological binding patterns and leading to more random interactions. Consistently, significantly more genes are bound in reactivating than quiescent NSCs; and consistently, InR binding is enriched in promoters. Amongst the most significantly bound genes in reactivating NSCs are genes with known roles in NSCs, signalling pathways, cell growth, and proliferation. Given the known role of canonical insulin signalling in the reactivation of quiescent NSCs, this work hypothesizes that nuclear insulin signalling contributes to this process. Exploring the transcriptomes of quiescent and reactivating NSCs with scRNA‐seq and intersecting this data with the NanoDam data revealed genes differentially expressed and bound by InR in reactivating NSCs. These constitute candidate genes that may act downstream of nuclear InR to mediate reactivation. They include genes with known roles in NSC development; genes that encode important RNA‐modifying proteins; and genes that modulate the activity of central signalling pathways known to affect NSC dynamics. Amongst these candidate genes, several stand out as being of particular biological interest. *Mettl3* encodes a conserved RNA methyltransferase. The m(6)A modification METTL3 catalyses is the most prevalent mRNA modification in vertebrates and influences mRNA stability, translational dynamics, and RNA pol II pause release. The latter is an important mechanism of enabling rapid, synchronous cell state changes, of which reactivation is a prime example. Preliminary data show that *Mettl3* knockdown is associated with a reactivation delay (Cian Doherty and Andrea Brand, unpublished), making METTL3 acting downstream of nuclear InR to facilitate reactivation an exciting model to investigate. *dachs*, a negative regulator of Hippo signalling constitutes another intriguing candidate gene; a switch between Hippo signalling and insulin signalling is vital for reactivation to occur. Dpp is an established signal for stem cell self‐renewal and prevents premature differentiation, and genes pertinent to its signalling, including the *tkv* receptor and *shn* transcription factor are also candidate genes. Overall, the findings support a model in which nuclear InR signalling serves as a point of integration of important NSC signalling pathways and orchestrates their cross‐regulation to fine‐tune the balance between pro‐quiescence and pro‐proliferation signals. The binding of a broad range of relevant genes and a resultant modest and combinatorial transcriptional activation was a picture also seen in mammalian cells. Future work will be necessary to validate the candidate genes and models proposed by this work and to elucidate the mechanisms of InR nuclear import and transcriptional activation. Some pertinent experimental avenues are suggested in the conclusions.Item Open Access Understanding the Genetic Basis of Obesity: Lessons from Man’s Best FriendWallis, Natalie; Wallis, Natalie [0000-0001-9543-3711]Obesity is an increasingly prevalent and complex disorder which poses a serious threat to canine and human health. Obesity is ultimately caused by caloric excess, and results from the complex interplay between environmental, behavioural, and genetic factors. Study of human obesity has been extensive whilst that in dogs has been limited. Past data on how biological risk factors impact canine obesity have been contradictory and the genetic basis of canine obesity is poorly understood. The heritability of human obesity is estimated at 40-70% and, despite considerable study, the majority of genetic variants responsible for this heritability are yet to be uncovered. Therefore, the understanding of obesity genetics across species is incomplete. In dogs, selective breeding and population bottlenecks simplify gene mapping for complex disease, offering the chance to unpick the ‘missing heritability’ of obesity in both dogs and humans. The studies of this thesis aim to better understand the development of canine obesity and gain insight into obesity genetics across species. First, biological risk factors for obesity were investigated in a population of British pet Labrador retrievers (n = 521), an obesity-prone dog breed. Linear regression was used to assess known and novel risk factors for canine obesity. Findings demonstrated a nuanced effect of known risk factors, with neutering and age having sex-dependent effects on obesity outcome. Chocolate coat colour was discovered as a novel risk factor for Labrador obesity. Some of these risk factors were mediated through an increase in food motivation, providing valuable insight into underlying mechanisms of action in canine obesity. Subsequently, a genome wide association study (GWAS) for obesity in Labrador retrievers was conducted. Multiple obesity-associated loci were identified, and regions of interest harboured candidate genes and variants. Candidate genes were interrogated using cross-species resources, and mechanisms of action hypothesised. Similarities between canine and human obesity means genetic associations in dogs can prioritise and identify obesity genes in human genomic studies. Syntenic human regions and genes were explored through GWAS and rare variant enrichment analyses in large scale human cohorts. Canine obesity loci of large effect served to highlight human loci implicated in common obesity, including genes *CDH8*, *CARD11* and *DENND1B*. Variants in *CSNK1A1* were also found to be associated with monogenic cases of severe, early onset obesity in humans. Carriers were pursued through familial investigation and deep clinical phenotyping. Further, a canine GWAS association of large effect at the *SEMA3D* locus provides strong orthogonal evidence of its importance in energy homeostasis. Canine GWAS summary statistics were used to construct a canine polygenic risk score method for obesity, something not previously achieved in dogs. Genetic scores for obesity predicted phenotypes in related but not unrelated dog breeds. Polygenic scores also provided insight into known within-breed variation in canine obesity risk, explaining associations with coat colour. Polygenic background was shown to influence the penetrance of a well characterised canine *POMC* mutation that affects obesity risk by altering hypothalamic leptin-melanocortin signalling. Additionally, polygenic background affected how owner management practices impact obesity outcome in dogs. This thesis provides novel insight into canine and human obesity, including the identification of new obesity-related genes. An improved understanding of canine obesity risk factors is established and highlights areas for further investigation, particularly through intra-breed study. Findings demonstrate the benefits of studying complex disease in non-traditional animal models such as the dog. This work informs the treatment and prevention of obesity in both human and veterinary medicine.Item Embargo The role of polarisation in the first cell fate decision of the mouse embryoLamba, AdiyantA fundamental process in embryonic development is the first cell fate decision, when cells take on distinct lineage identities for the first time. In mammals, this separates embryonic inner cell mass (ICM) from extra-embryonic trophectoderm (TE) during pre- implantation development. In the mouse, the process is classically attributed to the consequences of apical-basal polarity, which forms at the 8-cell stage: those cells which retain the apical domain after cell divisions are specified as TE, and the rest as ICM. However, more recent research has shown that early molecular heterogeneities between cells before polarisation can also bias cell fate. The existence of different models calls into question the role of polarisation in the first cell fate decision. The first part of this study focuses on reconciling the polarity and heterogeneity models. It was previously thought that polarisation occurs only at the late 8-cell stage. By studying its timing in detail, it is possible to split cells into ‘normal polarising’ (NP) cells which polarise at the late 8-cell stage, and ‘early polarising’ (EP) cells at the early 8-cell stage, the latter found in approximately 20% of embryos. Although EP cells follow the canonical polarity pathway — involving the critical factors Tfap2c, Tead4 and RhoA — they have molecular and morphological differences from NP cells and are biased towards symmetric divisions and TE fate. Blastomeres with low activity of the arginine methyltransferase CARM1 prior to polarisation are known to be biased towards TE, and inhibition of CARM1, or overexpression of its substrate BAF155, increases the frequency of early polarisation. Thus, this study proposes that early heterogeneities influence cell fate by altering the timing of polarisation. The final part of this study addresses the ability to detect polarity. Tracking polarisation over time currently requires invasive fluorescence imaging. Here, artificial intelligence is used to detect whether an embryo is polarised from unstained images, after training based on bright-field movies annotated using the corresponding fluorescence channel. The resulting model has an accuracy of 85% for detecting polarisation, significantly outperforming human volunteers trained on the same data (61% accuracy). Taken together, this study advances our understanding of polarisation and its role in the first cell fate decision, while also providing a tool for further investigation.Item Embargo Identifying novel roles for basement membrane-associated proteins in the adult Drosophila intestinal epitheliumEldridge-Thomas, Buffy; Eldridge-Thomas, Buffy [0000-0003-4070-2827]Maintenance of a healthy epithelium relies on the functionality of resident stem and progenitor cells, which must divide and differentiate in a way that precisely meets tissue requirements. To achieve this, cells integrate an array of information, including chemical, mechanical and adhesion-based cues, which collectively provide the instructions to direct cellular behaviour. A potential source of such signals is the basement membrane, a thin extracellular matrix which underlines the basal side of all epithelia, and to which epithelial cells adhere via various receptors. Despite their ubiquity, basement membranes are understudied and there remains much to understand regarding how their components and receptors impact epithelia. Using the adult *Drosophila* midgut as a model system to investigate epithelial stem and progenitor cell behaviour *in vivo*, I searched for novel roles for basement membrane associated proteins in the intestinal epithelium. Integrins are major receptors used by cells to bind the extracellular matrix, and they recruit a number of intracellular proteins including the conserved mechanoeffector Vinculin. I contributed to a project investigating the role of Vinculin in the intestinal epithelium, where *vinculin* mutants show increased intestinal stem cell proliferation and accelerated progenitor cell differentiation. I used scanning electron and confocal microscopy to confirm that *vinculin* mutant midguts have elevated cell numbers and developed a workflow for quantifying proliferation and differentiation. Using transmission electron microscopy, I showed that *vinculin* mutant midguts do not have an abnormal basement membrane. This corroborated other observations which suggest that, in the context of intestinal cell production and differentiation, Vinculin functions at cadherin cell-cell junctions, not at Integrin cell-matrix adhesions, specifically in enteroblast progenitors. Here, Vinculin is required to keep the progenitor in a quiescent state and to suppress division of neighbouring intestinal stem cells. This work revealed that mechanical regulation at the contact site between stem cells and their progeny is used to control cell number and enhances understanding of how mechanical signals contribute to intestinal epithelial homeostasis. In a separate project, I identified the conserved transmembrane proteoglycan Syndecan as an essential protein for intestinal stem cell maintenance. Syndecan is a basement membrane receptor, with a plethora of other intra- and extracellular binding partners, that is dysregulated in multiple human diseases. I found that RNAi-mediated depletion of Syndecan from intestinal stem cells, but not from other intestinal epithelial cell types, causes loss of these stem cells. Without Syndecan, intestinal stem cells acquire abnormal cell morphologies and display cell division-associated defects. In addition, Syndecan-depleted intestinal stem cells develop large nuclear lamina invaginations, nuclear shape changes and acquire DNA damage. Ultimately, the vast majority of Syndecan-depleted intestinal stem cells are lost from the epithelium, via a combination of apoptosis and other mechanisms. My work found that Syndecan has negligible effects on major chemical signalling pathways, and Syndecan also seems not to act via two components of the Linker of Nucleoskeleton and Cytoskeleton complex in a potential mechanotransduction pathway to the nucleus. In parallel, I sought to investigate whether Syndecan is required in other somatic stem cell types. My collaborator, Dr Chantal Roubinet, found that Syndecan depletion from *Drosophila* neural stem cells causes abnormal nuclear size and shape; abnormal mitotic nuclear envelope remodelling and delayed cell division, indicating Syndecan plays a common role in stem cell behaviour. My work newly identifies Syndecan as a regulator of intestinal stem cell maintenance and finds a connection between this transmembrane protein and nuclear properties in multiple stem cell types. In future, uncovering Syndecan’s precise mode of action and its molecular partners in this model system may help provide a new framework to delineate its function in human disease.Item Embargo Cell Fate Decisions in the Early Mammalian EmbryoIwamoto-Stohl, LisaIn mammals, successful pre-implantation development leads to the formation of a tri-lineage structure known as the blastocyst, consisting of the epiblast, primitive endoderm and trophectoderm, which will give rise to the new organism, the yolk sac and placenta respectively. These three lineages must be established from the totipotent zygote via two successive cell fate decisions in the appropriate sequence, position and proportion, to generate a blastocyst capable of implantation and further development. In mammals this process has long been thought to be regulative, with cell-cell interactions flexibly determining the eventual fate of cell. This is in contrast to commonly studied non-mammalian embryos in which pre-patterning of the embryo, driven by spatially localised factors, is a common feature. However, early blastomeres of mouse embryos have been reported to have distinct developmental fates, potential and heterogeneous abundance of certain transcripts, prior to the first cell fate decision. Nevertheless, the extent of the earliest intra-embryo differences remains unclear and controversial. Utilizing single-cell proteomics by mass-spectrometry I show that 2-cell mouse and human embryos contain an alpha and a beta blastomere as defined by differential abundance of hundreds of proteins. Such asymmetrically distributed proteins include Gps1 and Nedd8, depletion or overexpression of which in one blastomere of the 2-cell embryo impacts lineage segregation. Fascinatingly, halved mouse zygotes already display protein asymmetries, which resembles alpha and beta blastomeres, suggesting differential proteome localisation already within zygotes. I also find that beta blastomeres may have a greater developmental potential, and give rise to a blastocyst with a higher proportion of epiblast cells than alpha blastomeres. Human 2-cell blastomeres also partition into two clusters sharing strong concordance with clusters found in mouse, in terms of differentially abundant proteins and functional enrichment. This provides the first demonstration of intra-zygotic and inter-blastomere proteomic asymmetry in mammals that has a role in lineage segregation. In humans, this early period of development is prone to failure, with a third of human pregnancies estimated as being lost prior to implantation. A high incidence of aneuploidy is thought to be a major driver of pregnancy failure and understanding the behaviour of aneuploid cells is of great interest. As the blastocyst forms and implants, aneuploid cells may be eliminated through cell competition with diploid cells or show differences in their lineage segregation, impacting the composition and proportion of each lineage in the blastocyst, The mouse embryo does not have similar high intrinsic rates of aneuploidy, but reversine, a spindle assembly checkpoint inhibitor, can be used to recapitulate the human aneuploid embryo to some extent, and to observe the elimination of aneuploid cells. Here, I return to the human, utilising human embryonic stem cells and new integrated stem cell based embryo models, to characterise conserved aneuploid cell depletion in mouse and human stem cell co-cultures of diploid and aneuploid cells. Furthermore, I use stem cell lines harbouring specific aneuploidies to determine if specific aneuploidies confer differential ‘fitness’ and elimination rates. Overall my PhD has examined two questions regarding early mammalian development: 1) when do the cells of the embryo first become different to each other and how does this interplay with lineage segregation and 2) can a model of the mosaic aneuploid human embryo be generated to better understand the fate of aneuploid cells within the three lineages of the blastocyst.Item Controlled Access The role of ventromedial and dorsolateral prefrontal cortex in physiological and behavioural dysfunction in non-human primates, of relevance to mood and anxiety disordersBanai Tizkar, RanaThe research presented in this thesis investigates the causal role of area 25 of the subcallosal anterior cingulate cortex and area 46 of the dorsolateral prefrontal (dlPFC) cortex in depression- and anxiety-like behaviour in a non-human primate, the common marmoset. Depressed patients show hyperactivity in area 25, and hyperactivity and hypoactivity of area 46 in the right and left hemispheres respectively. Moreover, these two seemingly unrelated regions, one involved in emotion and visceral activities (area 25) and the other involved in cognition (area 46), have sparse direct anatomical connections yet their negative coupling is observed in the state of disease and after successful treatment. Furthermore, levels of the stress hormone cortisol, which is elevated in depression and anxiety, are positively correlated with activity within area 25. However, the causal role of cortisol in the activity of this region is unknown although it has been shown that area 25 expresses both mineralocorticoid and glucocorticoid receptors, which suggests elevated cortisol can directly modulate activity in this region. Three subsections investigating the above, form three chapters of this thesis. Chapter three focuses on the effect of overactivation and inactivation of area 25 in two behavioural tests assessing motivational and consummatory behaviour of relevance to the symptom of anhedonia, the loss of pleasure. In chapter four the effect of the local increase of cortisol in area 25 in three domains of motivational, anticipatory, and anxiety-like behaviour is assessed. In chapter five the effect of overactivation and inactivation of area 46 within dlPFC on anxiety-like behaviour and basal cardiovascular activity is investigated under four conditions of vehicle control, left hemisphere, right hemisphere, and bilateral manipulations, in order to assess reports of asymmetry in dlPFC function. Taken together, the findings provide evidence for bidirectional effects of area 25 manipulation on motivation, and consumption, albeit with a more complex relationship with reward value for the latter. Moreover, cortisol is shown to have a causal impact on area 25 function as manifested in anxiety-like behaviour and anticipatory anhedonia. Since the effects were observed only with a very short pre-treatment time, it implicates those cellular mechanisms known to underlie the rapid effects of cortisol. These effects were observed despite the low number of subjects (n = 4), however, whether the lack of effect on cardiovascular response is due to the low power could be investigated with more subjects. Finally, the results revealed that bilateral and left hemisphere inactivation of area 46 increased anxiety-like behaviour. However, to establish the role of area 46 in the central autonomic network, further investigation is required since ANOVA showed no effect of manipulation despite the significant effect observed with the Linear Mixed Effect Model. The current interpretation of the observed results is that the effect size is small and there are high individual differences, both of which can only be addressed with an increased number of subjects. In contrast, overactivity of the right hemisphere did not increase anxiety as implicated in the depression and anxiety literature. The evidence overall points to functional asymmetry within area 46. The opposing effects of area 25 and area 46 manipulations on anxiety-like behaviour reported here support the correlatory findings in humans for the negative relationship between subcallosal and dlPFC activity in human mood and anxiety disorders. When translating these preclinical findings to the clinical domain it should be noted that the reward used here, similar to many preclinical models, is a primary reward, which has an innate value essential for homeostasis. The neural circuit underlying primary and secondary rewards may vary and requires further investigation. Hence, these findings are only applicable with regard to primary rewards when translating to human studies.Item Open Access Neocortical functional network development at the microscale: impairments and mechanisms in Rett Syndrome.Dunn, AlexanderThe brain comprises intricate neuronal circuitry arranged in complex patterns. A key challenge in studying brain network topology is that microscopic networks at the microscale cannot be studied noninvasively in humans. It is particularly challenging to study microscale networks during early development in animal models as the brain is rapidly changing in size. This could have implications for deciphering how an individual can show apparently normal development before rapid decline. The neurodevelopmental disorder, Rett Syndrome, characterised by Mecp2 deficiency, is a pertinent example of this. In the present thesis, I aimed to thoroughly characterise early neocortical functional network development at the microscale and how this is disrupted in a Rett Syndrome mouse model, before examining some of the mechanisms involved. For translatability, I also tested the viability of using a graph theoretical approach in 3D, human tissue—cerebral organoids—for future research to study neocortical network development in health and disease. Micro-electrode array recordings of primary murine cortical cultures showed that Mecp2-deficient, Rett Syndrome model networks showed impairments in spontaneous spiking and bursting activity. They showed reduced global functional network connectivity and modularity. Simulations revealed deficits in computation of the balance of connection benefits/costs. Interestingly, Mecp2-deficient network deficits were seen as early as two weeks in vitro which precedes the putative onset of behavioural decline in this Rett Syndrome model (~8 weeks). Crucially, optogenetic suppression of parvalbumin-expressing inhibitory interneurons restored spatiotemporal spiking dynamics. Finally, human cerebral organoids did show complex topology and hub node features beyond that which could be explained by high firing rates at 180 days in vitro. To conclude, neocortical microscale functional networks mirror many aspects of macroscale network development, and several network features are disrupted in Rett Syndrome. Early cell type-specific intervention may restore network activity patterns in Mecp2-deficient networks. In the future, human cerebral organoids provide a promising complementary approach to animal models for future study of complex network topology in three-dimensional networks derived from human cells. Further work is required to establish whether they adhere to key principles and patterns seen in vivo.