Anatomy and Neuroscience - Theses
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ItemThe Angiosome Concept Revisited( 2019)Introduction The angiosome concept was originally described in 1987. The principles of this theory codify our understanding of the vasculature of the human bodyand underpins much of modern plastic surgery. However, with the advent of perforator flaps, and particularly large fasciocutaneous flaps such as propeller flaps, its validity has been questioned, especially with regard to the number of perforator territories that can be captured safely when a flap is raised. This thesis aims to address the criticisms of the angiosome concept, with a view to demonstrating its validity and applicability to perforator flaps. Methods Initial investigations comprised a study of the effect of injection pressure on the radiographic appearance of the cadaveric vasculature, in order to demonstrate the validity of the technique in distinguishing types of interperforator anastomosis. This involved whole lower limb radiographic contrast injections followed by plain radiography or computed tomography to explore the correlation between anastomotic type and superficial cutaneous nerves. These studies were followed by selective dissection and injection of cutaneous perforating arteries in different regions of the body, and a review of archival injection studies, to compare morphology of perforator territories. Results High injection pressure was found to result in an increase in filling of a perforator’s peripheral network, without significantly altering the calibre of the perforator trunk or main branches. Whole limb injection studies demonstrated that large calibre true anastomoses were found along the course of, but often separate to, the superficial cutaneous nerves and veins of the leg. Cross sectional imaging demonstrated that true anastomoses tended to be found in an intermediate plane in the superficial fascia. Thermographic imaging studies correlated well with computed tomographic angiography (CTA), suggesting that it may be used as an alternative for preoperative imaging. Comparative studies of perforators demonstrated great variability in perforator territories throughout and within regions around the body, though some generalisations could be made: large calibre true anastomoses tended to be found along the course of superficial cutaneous nerves and veins, which run longitudinally in the limbs, and circumferentially in the trunk, widely spaced perforators have larger territories, and where a perforator territory is small, adjacent territories tended to be large. Discussion These results demonstrate that cadaveric injection studies do not distort the vasculature and are a valid technique for distinguishing types of interperforator anastomosis. They also cast doubt on the suggestion that flaps may be able to capture additional territories by 'selective hyperperfusion’. The finding of large calibre true anastomoses along the path of superficial cutaneous nerves account for the observation that large fasciocutaneous flaps may be safely performed in the leg, as they take advantage of existing ‘freeways’ of true anastomoses. Selective injection of perforators reinforced the clinical territory principle of the angiosome concept: that the number of territories that may be safely captured in a flap is a single territory distal to a line of choke anastomoses, with a greater number of territories perfused in sequence in the presence of true anastomoses. The idea of ’the functional perforator angiosome’ is introduced, to help conceptualise the territory which may be safely perfused by a given perforator. Applications of the angiosome concept are explored, including perforator flap design, surgical incision placement, and allotransplantation. Conclusion This thesis demonstrates the continued validity and applicability of the angiosome concept in the era of perforator flap surgery.
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ItemThe role of neuronal TrkB in central nervous system myelination( 2019)Myelin, the specialised membrane surrounding many axons in the nervous system, is vital for the normal sensory and motor function as well as high order function such as learning and memory. Despite the importance of myelin in neural development and functions, little is known whether neuronally expressed molecules can promote the central nervous system (CNS) myelinating process. Through selectively deleting the expression of neurotrophin receptor TrkB in neurons in vivo (TrKB cKO), I have found that neuronal TrkB is essential for oligodendroglial cell production and lineage progression in multiple CNS regions including the lumbar spinal cord white matter tracts, cerebral cortex and corpus callosum and importantly de novo myelination, independent of axonal number and calibre in vivo. I have performed ultrastructural analysis of myelinated axons and found that TrkB cKO mice have significantly fewer myelinated axons compared to control mice during early postnatal development, indicative of delayed initiation of myelination. The analysis of myelin thickness via G ratio has revealed thinner myelin membranes in TrkB cKO mice compared to littermate control mice during development, persisting to late adulthood (n=3 mice/genotype/timepoint). This hypomyelinating phenotype has resulted in impaired myelin function, as evident by reduced optomoter responses in TrkB cKO mice compared to control mice. Moreover, I have found that TrkB cKO mice have significantly fewer oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes in both white and grey matter regions throughout postnatal development and into adulthood compared to littermate controls. A 24-hour EdU labelling experiment has demonstrated that neuronal TrkB expression is required for oligodendroglial production. Interestingly, the overall number of astrocytes and microglia has remained unchanged after neuronal TrkB deletion, suggesting a specific effect upon oligodendroglial lineage cells and myelination. In addition, assessment of neuronal morphology revealed no significant difference in overall dendritic branching including the number of primary and secondary processes. Taken together, results of this PhD study identify that TrkB is a novel neuronal signal that instructs oligodendroglial lineage development and de novo myelination within different neural circuits, indicating a new mechanism that underpins nervous system plasticity.
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ItemThe KH-domain protein Psi fine-tunes transcription of developmental genes to pattern cell and tissue growth in Drosophila( 2019)Mammalian FUBP-family proteins play roles in both transcription and RNA processing, by binding single stranded nucleic acids via their KH domains. Particularly, in vitro mammalian tissue culture studies suggest that FUBP1 drives MYC transcription in response to growth stimuli. MYC is a key regulator of growth and cell cycle progression essential for normal development. Furthermore, expression of MYC is aberrantly upregulated in approximately 70% of all cancers. Understanding the mechanisms that control MYC expression may lead to insights into how MYC dysregulated in diseases such as cancer. However, the significance of the transcriptional control by FUBP1 proteins at the MYC promoter and genome-wide has remained unclear in the context of development in whole organisms. Mammalian studies have been complicated by the fact that mammals have three FUBP-family proteins that bind overlapping genomic targets. However, in Drosophila, the three mammalian FUBP proteins are represented by one ortholog, Psi. Due to a high degree of structural similarity between FUBP1 and Psi proteins, we hypothesized that Psi will also regulate transcription of MYC and other targets. We have taken advantage of the reduced functional redundancy in Drosophila to demonstrate that an essential in vivo function of Psi is control of cell and tissue growth. Analysis of published Co-IP-mass spectrometry screens positioned Psi in an interactome predominantly comprised of RNA Polymerase II transcriptional machinery, particularly the transcriptional Mediator (MED) complex, a known sensor of developmental signalling inputs. Our work indicates that a key transcriptional target of the Psi/MED network that impacts tissue growth is Myc. During development of the wing disc epithelial tissue, Psi was required to promote Myc expression in response to activated PI3K pathway signalling. In addition to Myc, Psi binds to multiple transcriptional targets in the wing epithelium in vivo. Intersection of RNA-seq to detect differentially expressed genes following Psi depletion, and wing-specific direct binding sites identified genome-wide by Targeted DamID, revealed a developmental patterning signature for Psi including Wnt, Notch and TGF-beta cell fate determinant pathways. Moreover, analysis of direct Psi targets identified several novel growth regulators, including the TGF-beta signalling regulator tolkin (tok) and the CD36 scavenger receptor-related epithelial membrane protein (emp). Together our data suggest Psi integrates cellular signalling inputs to directly modulate transcriptional outputs and fine-tune development.
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ItemImpact of urbanisation on population’s health in Mtskheta region – Georgia, from Late Bronze Age to Early Middle Ages( 2019)Abstract The emergence of urbanisation is considered to be an important period in human history as it is associated with an increase in population density, diversification of subsistence activites, and the establishment of ancient cities, often with poor sanitation. Each of these factors contributed to the spread of disease and a change in diet. Numerous investigations suggest that the impact of early urbanisation on population health and diet is not homogeneous everywhere but varies based on the particularities of the region in question. The Caucasus is one such region. Located between the Black and Caspian seas it has always been chracterized by vibrant cultural dynamism. It acted as a frontier between urban societies of the ancient Near East and the nomadic populations of the ancient Eurasian steppes. As such this is an excellent location to study the effect of urbanisstion on health and diet. This dissertation focuses on paleopathological examinations of skeletal materials from the region of Caucasus, specifically the Greater Mtskheta territories located in the central part of the south Caucasus. The anthropological material from Greater Mtskheta forms one of the largest collections in the region. Human skeletal materials from 13 cemeteries, from three important time-periods were examined: the Late Bronze/Early Iron Age pre-state society with rural-type settlements (15th–6th centuries BC); the Antique period with the emergence of urbanisation and the establishment of the Iberian kingdom (5th century BC–4th century AD); and the Early Middle Age period with developed urbanisation and the collapse of the Iberian kingdom (4th–7th centuries AD). To observe the changes in health and diet throughout these different periods, pathological conditions such as porotic hyperostosis and dental diseases—caries, periapical lesions, antemortem tooth loss and calculus—were examined. Besides these pathologies, molecular analysis of dental calculus was done for these three periods. Statistically, there were no significant differences in lesions and the degrees of activity of porotic hyperostosis. But there was a significant trend in prevalence of porotic hyperostosis across time-periods and between sex. This indicated that deterioration in health conditions during the transitional periods was gradual and did not severely affect the population. The incidences of caries, periapical lesions, antemortem tooth loss and calculus throughout the periods may be associated with changing diet. An insignificant decrease of caries lesions during the urbanisation period may suggest diversification of food production and less dependence of grain consumption, rich in hydrocarbonates, which are responsible for caries. A statistically significant difference in the oral microbiome in pre-and urbanised-state societies may be associated to an increase of frequency of porotic lesions on the skulls and the rise of abscesses over time. The decrease and low incidences of caries throughout the periods may be associated with the absence of specific bacteria in the oral microbiome of the ancient population of Georgia. This study is part of a growing body of research into the impact of early urbanisation on population health and diet. Using certain pathological changes on human skulls, together with analysis of dental diseases, and combining these with the study of oral microbiomes, it contributes to future research on similar topics. This is particularly relevant for the Caucasus, where this type of research has not yet been conducted.
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ItemInvestigating the role of Seizure related gene 6 family proteins and their BACE shed products at excitatory synapses: impacts on motor and cognitive function( 2019)Correct wiring of the brain during development is a complex process that involves the precise spatial and temporal expression of a vast number of genes and proteins. Excitatory synapses in the central nervous system are formed primarily onto specialised protrusions of the dendrite called dendritic spines. During development, synaptic contacts mature into dendritic spines through a process that requires stabilisation and strengthening of the synaptic contact. Strengthening of neuronal connections is not only important for synaptic maturation during development but is also generally assumed to be the cellular process underlying learning and memory in the adult. Seizure related gene 6 (Sez6), Sez6 Like (Sez6L) and Sez6 Like 2 (Sez6L2) comprise a family of homologous proteins widely expressed throughout the brain and Sez6 knockout (KO) mice revealed a role for Sez6 in dendritic arborisation and the development of excitatory synapses. Additionally, Sez6 family proteins have been linked to neurodevelopmental disorders, with which synaptic dysfunction is commonly associated. Therefore, the broad aim of this project was to further characterise the role of Sez6 family proteins, and their shed ectodomains, in synaptic structure and function and additionally investigate their role in motor and cognitive function in the mouse. To elucidate the functions of Sez6 family proteins in the central nervous system, wild-type (WT) and Sez6 triple knockout (TKO) mice, which lack all three Sez6 family proteins, were used. Compared to WT, Sez6 TKO mice were found to have reduced spine density in the hippocampus and dendritic spines were shifted to more immature morphologies in the somatosensory cortex. Additionally, synaptic function was found to be altered in the prelimbic cortex. Sez6 TKO mice had impaired motor learning and motor coordination from as early as 6 weeks old. Cognitive testing revealed Sez6 TKO mice had enhanced stress responsiveness, impaired working and spatial short-term memory but intact spatial long-term memory in the Morris water maze that was accompanied by a reversal deficit. Sez6 family members are substrates of the Alzheimer’s protease, beta-amyloid precursor protein cleaving enzyme 1 (BACE1), and are cleaved proximal to the transmembrane domain to produce a shed ectodomain. The contribution of the shed Sez6 family protein ectodomains to cognition, and the potential for BACE inhibitor treatment of Alzheimer’s disease to be associated with cognitive side-effects as a result of impaired processing of other non-amyloid precursor protein substrates, was assessed by chronically treating WT and Sez6 TKO mice with a BACE inhibitor. In both WT and Sez6 TKO mice, BACE inhibition did not significantly impact cognition or anxiety-related behaviour in the behavioural paradigms utilised. WT mice treated with BACE inhibitor were, however, found to be hyperactive on the elevated open field and this effect was not observed in treated Sez6 TKO mice suggesting a role for the shed ectodomains of Sez6 family proteins in regulating activity levels. The results presented in this thesis further our understanding of the role of Sez6 family proteins in synaptic structure and function and contribute to the body of evidence that links Sez6 family proteins to the cognitive and motor deficits observed in patients with neurodevelopmental disorders, such as autism and schizophrenia.
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ItemTranscriptomic diversification along the monocyte-macrophage continuum.( 2019)Current models of innate immune responses describe hard wired, gene-centric signalling networks, with limited capacity to define the molecular mechanisms underpinning transcriptomic diversity. It is well established that a transcriptional spectrum of responses accompanies acute macrophage activation, however it is unclear whether this spectrum originates in monocytes and to what extent it continues throughout reinfection. The contribution of molecular mechanisms such as enhancers and alternative transcription start sites is also undetermined. Given the critical roles that myeloid cells play in directing acute infection and priming adaptive immunity, it is important the regulation of their responses be understood. This thesis employed bioinformatic analysis of Cap Analysis Gene Expression (CAGE) and microarray data to describe transcriptional diversity along the monocyte-macrophage continuum. Using CAGE to map transcription start sites for capped RNAs, this thesis has shown that pathogen-specific transcriptional diversification commences early in monocyte infection (Chapters Three and Four) and continues throughout acute macrophage infection (Chapter Five). Transcriptomic diversity during acute infection was the product of kinetic and pathogen-specific engagement of distinct transcription start sites. Engagement of multiple transcription start sites drove responsiveness by regulating expression amplitude in functionally focused inflammatory gene sets and diversifying secondary response networks via expression of distinct protein isoforms. Chapter Six extended these studies of acute infection, demonstrating that transcriptional phenotypes continue to diversify during reinfection. These findings highlight the importance of studying innate immune responses at the isoform level and prompt the need to revise current models of innate immune signalling, such that monocyte-macrophage biology should no longer be modelled as a series of static states, but rather, as a continually evolving continuum.
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ItemDevelopment of in vitro and in vivo models for the study of myelin plasticity( 2019)The central nervous system (CNS) constantly responds to changes in environmental stimuli by undergoing structural and functional modifications. Some stimuli induce persistent CNS changes which in turn underpin adaptive behaviours that enable individual animals to function in their unique environmental circumstances. This phenomenon, referred to as neuroplasticity, has been studied predominantly with respect to adaptive neuronal changes, and has focused primarily on synaptic changes and the molecular transduction mechanisms that mediate them. It is increasingly recognised, however, that glial cells can also be modified by external stimuli. Oligodendrocytes – the myelinating glia of the CNS which facilitate efficient nerve impulse conduction and support axonal metabolism – have also been demonstrated to undergo long term changes in response to environmental stimuli. Experience-dependent changes in oligodendrocyte number or myelination could underpin adaptive behaviours via modifications to neuronal metabolism and nerve impulse conduction. The emerging consensus is that stimulation – whether indirectly through modulating sensory, motor, or social experience, or directly through modulating neuronal activity – increases oligodendroglial lineage progression and myelin production. It has further been demonstrated that myelin plasticity is an axon-specific phenomenon whereby, when given the choice, myelin segments preferentially form on axons that are more highly active relative to those that are nearby but less active. The molecular mechanisms that mediate myelin plasticity are not well understood, and studies addressing this question have predominantly focused on the role of extracellular, pro-myelinating signals released by neurons in an activity-dependent manner. Comparatively little is known about the oligodendroglial intrinsic molecular transduction mechanisms that mediate myelin plasticity. This thesis aimed to develop a model system for studying myelin plasticity, including in particular to investigate the molecular transduction mechanisms that are triggered within oligodendroglia to mediate myelin plasticity. In developing such a model, two approaches were employed. First, an in vitro myelinating co-culture model was developed. A standard co-culture protocol was adopted and refined to produce robustly myelinating co-cultures of dorsal root ganglion (DRG) neurons and oligodendrocyte precursor cells (OPCs). To stimulate neuronal activity, both the hM3Dq pharmacogenetic and the channelrhodopsin-2 (ChR2) optogenetic techniques were explored. The pharmacogenetic stimulation technique was ineffective at driving DRG neurons to the levels of activity reportedly required for inducing myelin plasticity. In contrast, the optogenetic stimulation technique reliably drove DRG neurons to fire at the required frequency. Contrary to expectations, optogenetic stimulation did not increase myelin production in co-cultures, nor did it increase the propensity of myelin segments to preferentially form on optogenetically stimulated relative to control axons. The reasons for this are unclear, but are unlikely to be related to phototoxicity and are more likely to be explained by a negative effect of high ChR2 expression on myelination in these co-cultures. Second, an in vivo pharmacogenetic model was employed to drive activity of cortical neurons in juvenile hM3Dq transgenic mice. Contrary to expectations, there was no evidence for an activity-dependent increase in oligodendroglial lineage progression. The reasons for this are unclear, however they could relate to the young age of the animals in this relative to other studies of myelin plasticity or to the large population of neurons undergoing activity manipulation in this relative to other studies of myelin plasticity. The implications for glial plasticity, and for how it is studied, are discussed.
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ItemThe effect of genetic susceptibility and immunosuppressant treatment on mononuclear cell phenotype in multiple sclerosis( 2019)Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The immune system plays a significant role in the pathophysiology of the disease and research into the mechanisms that may be potentially contributing to the disease is essential to help advance knowledge in the field and ultimately seek better outcomes for MS patients. A key set of cells that are believed to play a role in MS immunopathology are mononuclear cells which consist of B-cells, T-cells, monocytes and their derivatives. The aims of this thesis was to assess the effects of 1) genetic susceptibility and 2) immunosuppressant treatment, on mononuclear cell phenotype in MS. The first aim involved interrogating a key co-stimulatory molecule, cluster of differentiation 40 (CD40), expressed on mononuclear cells which is known to play a fundamental role in the regulation of the humoral immune response. CD40 is a risk gene for the development of MS and I sought to assess the effects of the single nucleotide polymorphism (SNP) rs1883832 on the phenotype of CD40-expressing mononuclear cells. Utilising a range of bimolecular methods and samples taken from MS individuals and healthy matched-controls (HC), I showed that SNP rs1883832, particularly the ‘risk’ genotype (homozygous TT) was correlated with reduced soluble CD40 (sCD40) levels in HC, but not MS patients. I did not observe any phenotype-dependent differences in sCD40 levels between MS (in quiescent state) and during active demyelination compared to HC. Upon assessment of key cytokines produced by B-cell and monocytes, I observed elevated gene expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in the MS population linked to the CD40 risk TT-genotype. In the second component, I showed that the immunosuppressant drug Cladribine, is highly toxic to mononuclear cells, and that it down-regulates the expression of the key phagocytic marker MERTK at the protein level (preliminary data, study is ongoing). In summary, the data presented in this thesis suggests that the CD40 risk gene and the immunosuppressant Cladribine play roles in altering the phenotype in mononuclear cells. The understanding from this body of work provides some insights into how genetic susceptibility can drive changes in mononuclear cell phenotype which may impact on the modulation of co-stimulatory mechanisms in humoral immunity. Moreover, the findings from the Cladribine study may assist the field in the discovery of alternative mechanisms of the drug on mononuclear cells and therefore inform a refined approach to treatment of MS.
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ItemRegulation of Drosophila Myc transcription by the single stranded DNA/RNA binding proteins Psi and AGO1( 2019)MYC has wide-ranging functions, with potential to amplify transcriptional output to activate cell growth, metabolism and cell cycle progression, thus driving oncogenic programs. As even small increases in MYC abundance are sufficient to drive the proliferative cell growth fundamental to tumour progression, understanding the molecular mechanisms controlling MYC expression will provide insight into mechanisms of MYC dysregulation in cancer. In mammalian in vitro and ex vivo systems, the KH domain single stranded DNA/RNA binding protein FUBP1 binds the Far Upstream Sequence Element (FUSE) in the activated MYC promoter to modulate transcription. Using genetic models, this thesis demonstrates that the sole FUBP family member in Drosophila, Psi, is essential for Myc transcription, cell and tissue growth in the wing epithelium, in vivo. Psi is not only required to maintain endogenous levels of Myc mRNA abundance, but depletion results in a significant increase in RNA Pol II activity on the Myc gene. Consistent with Psi regulating Myc at the level of transcription, we demonstrate Psi interacts physically and genetically with the transcriptional Mediator complex (MED), thus providing a mechanism for integration of developmental signals for patterning Myc transcription, cell and tissue growth in the wing imaginal disc epithelium. We further report physical and genetic interaction between Psi and AGO1, the RNA binding protein component of the RNA-induced silencing complex (RISC). AGO1 loss-of-function mutations restored growth in the Psi knockdown wing, suggesting negative roles for AGO1 in Psi-dependent cell and tissue growth. AGO1 depletion was not only sufficient to increase Myc mRNA and protein abundance in the wing, but also increased Myc function i.e. transcriptional activity as measured by increased abundance of Myc targets required for ribosome biogenesis and cell growth (e.g. Ribosomal Proteins, rRNA and RNA Pol I subunits). Myc knockdown returned AGO1-depleted nucleolar compartments to the normal range, demonstrating dependency of increased growth on Myc i.e. rather than direct effects of AGO1 on ribosomal components. Interestingly, the following observations suggest AGO1 represses Myc at the level of transcription: 1) significant AGO1 enrichment on Myc promoter by ChIP, 2) physical interaction between AGO1 and MED, and 3) that AGO1 depletion activates the Myc promoter and the increased Myc mRNA requires RNA Pol II transcriptional activity. Together, these observations suggest a novel role for AGO1 as a transcriptional repressor of Myc, which underlies the tumour suppressor behaviour observed for AGO1 in the Drosophila wing.
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ItemUsing induced pluripotent stem cells to model primary open-angle glaucoma( 2019)Glaucoma is a group of optic neuropathies that may be characterized by gradual degeneration of retinal ganglion cells (RGCs) and their axons leading to irreversible vision loss [1, 2]. Glaucoma is the second leading cause of blindness worldwide [3-5] and it is estimated that the number of people affected by the disease will reach 80 million by 2020, while more than 11 millions will be bilaterally blind [6]. In this project I focus on primary open-angle glaucoma (POAG), as it accounts for the majority of glaucoma cases worldwide. So far, multiple risk factors for glaucoma have been identified; however, the exact mechanism causing RGC loss in patients remains elusive. Furthermore, examination of RGCs affected in POAG is difficult pre-mortem due to their anatomical location. To overcome this problem, somatic cells can be reprogrammed into patient-specific induced pluripotent stem cells (iPSCs), which can be then differentiated into cell type of interest, i.e. RGCs. This PhD project consisted of several steps. First, I assessed the feasibility of transferring the iPSC culture into the automated platform. Using automation was essential to generate large number of samples required for analysis. The transition to automation was successful, as evidenced by maintenance of iPSC morphology, expression of pluripotency markers and ability to differentiate into derivatives of three germ layers. I also demonstrated that incorporating automation into human (h) iPSC culture allows standardization of maintenance and passaging procedures reducing inter-sample variability and human error. I subsequently used the platform to generate over 300 hiPSC lines for POAG modelling. In parallel, I optimized RGC differentiation protocol to obtain sufficient number of cells for their examination with single cell RNA sequencing (scRNA-seq). Next, iPSC-derived RGCs were subjected to scRNA-seq to gain in-depth information about transcriptomic differences between healthy controls and POAG patients. Understanding mechanisms underlying RGC function, maintenance of homeostasis and those conferring susceptibility to POAG is crucial to discover new therapeutic targets and commence the process of drug discovery.