Anatomy and Neuroscience - Theses
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ItemA detailed study of the deep and superficial venous systems of the anterior torso: clinical implications for the deep inferior epigastric artery perforator flap( 2015)Background Anecdotally, problems with venous outflow in deep inferior epigastric perforator (DIEP) flaps remain an unsolved problem. A comprehensive review of the entire DIEP literature as well as meta-analysis of the pooled data revealed that although venous complications were relatively common, these complications were poorly categorized and documented, and sporadically reported. Furthermore, an understanding of the relevant anatomical aetiology of these problems was not available, as hampered by the lack of high quality studies of venous anatomy of the anterior abdominal wall. I therefore set out to define the problems in DIEP flaps by performing an extensive literature review and meta-analysis, and thereby reclassify venous complications in DIEP flaps, as well as provide an anatomical explanation of the observations from the literature review and meta-analysis, by introducing a novel technique of studying the venous architecture of the torso in fresh and embalmed human cadavers. Previously, there have been few studies of the venous anatomy – due largely to the technical challenges involved, but also partly because the arterial anatomy was more immediately important to the survival of skin flaps used in reconstructive surgery. With maturing understanding of flap arterial anatomy, venous complications have now become one of the most common problems still remaining when raising flaps. There was therefore a need for greater anatomical understanding of venous architecture in order to explain the aetiology of these problems, and to refine flap design to reduce their occurrence. Materials and Methods 47 hemiabdomens and 24 hemi-chests from 26 separate cadavers were used in this study. All were preserved by freezing, but unembalmed. All veins in the borders of the specimens were cannulated, and veins were then initially primed with 6% hydrogen peroxide, and then injected with a lead oxide mixture and serially radiographed before and during dissection. Results The above method demonstrated venous anatomy of the anterior torso in unprecedented detail reproducibly. In particular, the anterior abdomen can be divided into four vertically oriented superficial venous territories corresponding to the superficial inferior epigastric and superficial circumflex iliac veins bilaterally. These territories communicate across the midline in anatomically fixed positions and patterns. There are separate deep venous territories corresponding to the vertically oriented venae comitantes of the deep inferior epigastric and deep circumflex iliac vessels – these territories do not directly correspond to the superficial venous territories, but communicate with them through two different types of venous perforators – small caliber venae comitantes, and large caliber venae communicantes. Venae communicantes were most frequently found periumbilically, and there were usually one to three of such connections in each hemiabdomen. 15.7% of venae communicantes were observed to not be accompanied by an artery of >1mm diameter as they perforated the deep rectus fascia. Conclusions My original contribution to knowledge was to introduce a novel technique enabling comprehensive retrograde venous perfusion in cadavers, and thereby to provide a description of the venous network in the anterior abdominal wall in unprecedented detail. This method has also demonstrated that superficial veins of the anterior torso have fixed patterns with specific tributaries in anatomically constant positions (subject to skin mobility) which were previously undescribed. Comparing these patterns to flap designs, particularly that of transverse lower abdominal flaps such as transverse rectus abdominis myocutaneous (TRAM) and DIEP flaps, has provided an anatomical explanation for various patterns of venous congestion described in the literature, as well as methods of pre-empting the reoccurrence of such problems in the future when raising abdominal flaps for reconstruction. A new classification for staging flap necrosis, which links the extent to necrosis to recommendations for further management was also presented based upon observations from a comprehensive literature review. These patterns and stages of flap necrosis, where caused by venous congestion, can be linked to disruption of the venous microarchitecture within the flap.
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ItemAnatomy of the cutaneous lymphatics of the limbs and the lower trunk and its implications in the surgical treatment of lymphoedema( 2015)Background A more detailed understanding of the anatomy of the cutaneous lymphatic system is required in order to preserve its function during various procedures and to reconstruct it following its disruption. There is minimal data in the literature regarding the radiographic anatomy of the cutaneous lymphatics of the lower torso and the depth of the collectors in relation to the subcutaneous tissue layers in this region and the limbs. Materials and Methods The laboratory investigations were performed in two parts. First, the method previously described at our lab was refined and used to study the cutaneous lymphatic drainage of 8 anterior hemi-abdomen/upper thigh and 2 lower back specimens from 4 fresh human cadavers. In the second project injection, microdissection, radiographic, and histologic studies to define the course of lymphatics in the various layers of the subcutaneous tissues of 16 upper limbs and 16 lower limbs from 15 human cadavers were performed. Results The upper and lower abdominal collectors originated at the umbilical and midline watershed areas in a subdermal plane by the union of dermal precollectors. They were thin-walled and translucent and their diameter ranged between 0.2 and 0.8 mm. In the lower abdomen the depth of the collectors gradually increased as they coursed towards the groin. They eventually pierced Scarpa’s fascia before draining into the superficial inguinal nodes located deep to this layer. The transition from supra- to infra-Scarpa’s fascia plane occurred within 2-3 cm of the inguinal ligament in 95% of the collectors. The collectors of the lower back originating at the midline watershed area pierced the superficial fascia halfway between the posterior midline and the mid-axillary line before draining into the superficial inguinal nodes. In the upper thigh the collectors of the ventromedial bundle constituted the majority of the superficial collectors. They were deeper and thick-walled, and consistently drained into two large nodes inferolateral to the saphenous bulb. The local collectors of the thigh were more superficial and thin-walled, and drained into the nodes located superolateral to the saphenous bulb along the superficial circumflex iliac vein. In the second study five layers were consistently identified in the integument of all the upper and lower limb specimens, namely: (1) skin, (2) subcutaneous fat, (3) superficial fascia, (4) loose areolar tissue, and (5) deep fascia. Layer 2 was further divided into superficial (2a) and deep (2c) compartments by a thin, transparent, horizontal septum (layer 2b). The main superficial veins and the superficial nerves coursed in layer 4. The lymphatic collectors were found in layer 2c and layer 4. Conclusion The detailed radiographic anatomy of the cutaneous lymphatic collectors of the abdominal wall and the lower back is presented for the first time and the presence of Sappey’s midline and umbilical watershed areas is confirmed. When harvesting the groin lymph node flap for vascularised lymph node procedure, the medial and the inferior border of the dissection must be limited to the lateral border of the femoral artery and the groin crease, respectively, to minimize the likelihood of iatrogenic lower limb lymphoedema. The use of a numeric system to describe the subcutaneous tissue layers is recommended to improve meaningful communication among surgeons and anatomists. In lymphovenous anastomosis, indocyanine green lymphography is an unreliable method for identification of the superficial collectors of the thigh. This study identifies the medial proximal leg below the knee, the dorsum of the wrist over the anatomical snuffbox, and the volar proximal forearm as suitable areas for locating superficial collectors with nearby matching size veins. Results revealed that the rational for planning some surgical procedures appear to be based on false premises. For example in the vertical medial thigh lift operation choosing a dissection plane superficial to the great saphenous vein is unlikely to preserve the collectors of the ventromedial bundle. Also In the four cadavers studied, preserving Scarpa’s fascia during abdominoplasty would not preserve the lower abdominal collectors and prevent lymph collection.
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ItemThe microvascular supply of locoregional flaps in head and neck reconstruction: revealing the anatomical bases for selected clinical applications( 2015)Reconstruction of defects of the head and neck remains a challenge to even the most experienced reconstructive surgeon. Tissue loss or destruction may follow ablation of tumour, trauma or infection as well as congenital anomalies. Efforts to provide basic functions such as oral continence, corneal protection, social interaction and a robust barrier from the environment are subject to the constant battle to provide well vascularised, resilient yet pliable tissue as inconspicuously as possible. To achieve success, the surgeon must possess a detailed understanding of the microvascular anatomy of their chosen reconstructive technique. To this end, the microvascular anatomy of three emerging techniques was explored using ex vivo injection studies, radiographic imaging and clinical correlation. Ex vivo cadaveric dissection studies followed non-destructive contrast-enhanced computer tomographic angiography. Further, in vivo computer tomographic angiography was used to correlate ex vivo findings with clinical reality. Finally, a brief pilot series of clinical cases demonstrated clinical utility and implementation of translational research. The superior thyroid artery perforator flap presents a new technique exploiting the often redundant anterior cervical skin as a locoregional, pedicled flap amenable for reconstruction of lateral and lower facial defects. The arterial and venous circulation of the superior thyroid perforator flap was examined in a cadaveric dissection study, plus in vivo computer tomographic angiography and finally in a brief pilot series of clinical cases. The arterial supply was consistently found to be a direct cutaneous perforator from the superior thyroid artery, with venous drainage via the anterior jugular veins. The supraclavicular flap was investigated to further clarify the microcirculation of this technique, previously neglected due to concerns of poor reliability. This study utilised cadaveric angiographic techniques to describe the pattern of microcirculation. Here, a series of small calibre choke anastomoses and reliance on interperforator and retrograde flow was found to influence the reliability of this flap. Consequently, we recommend strict adherence to maximal flap dimensions and consideration to include multiple venous anastomoses in order to reduce the chance of flap necrosis. Further investigations into microvascular communications in head and neck reconstruction focussed on the most complex surgical endeavours in the region- vascularised tissue allotransplantation. This field remains in experimental stages with heavy reliance on preoperative cadaveric studies to better prepare and understand the surgical requirements. Further to the technical difficulties, total flap failure would rendering the patient with no facial integument or skeletal structure and exposure of vital deep structures, bringing with it a real risk of death. This study used computer tomographic angiography to provide increased detail of the communications of the facial artery within the three-dimensional allotransplant. The facial artery was found to perfuse the ipsilateral and contralateral facial tissue including Le Fort III segment via a network of deep-to-superficial and cross-midline anastomoses. Whilst this suggests that a single unilateral facial artery may be sufficient to supply such an osteocutaneous allotransplant, risk aversion should prevail and the authors recommend including additional anastomoses to reduce the chance of potentially fatal vascular compromise of the allograft. This work provides advancement in the current understanding of head and neck microvasculature and targets three potentially valuable techniques to illustrate how this understanding can improve patient outcomes.
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ItemRegulation of SOX9 in mammalian sex determination and differentiation( 2015)Mammalian testis development is initiated by the key sex-determining gene, SRY, which together with SF-1, activates SOX9 expression. Activation occurs through the testis-specific enhancer of SOX9 core element, TESCO. Human mutations in these genes or their regulatory regions can disrupt testicular development, leading to disorders of sex development (DSDs) which are congenital conditions resulting in atypical chromosomal, gonadal or anatomic sex. Around 70% of 46,XY DSDs remain unexplained genetically. This thesis investigates several aspects of SOX9 regulation in male development. Firstly, the evolution of a novel sex-determining mechanism with altered Sox9 regulation in unusual mammals lacking SRY is described. Secondly, genotype-phenotype correlation was discovered between mutant SF-1 activity on TESCO and DSD severity. Thirdly, the identification and characterisation of novel testicular and genital tubercle SOX9 enhancers mutated in DSDs are described. The sex determining mechanism of mammals Ellobius lutescens and Ellobius tancrei that lack Sry and a Y chromosome has remained elusive. A 14 bp deletion in TESCO was discovered in these species, resulting in the loss of a highly conserved SOX/TCF binding site. Introduction of the 14 bp deletion into mouse TESCO led to elevated TESCO activity in in vitro reporter assays. Increased TESCO activity may therefore form the basis for the loss of Sry from this species, since consequent increased SOX9 levels would render Sry dispensable for testis development. Mutations in SF-1 are associated with highly variable reproductive phenotypes in 46,XY DSD. The mechanism underlying the broad spectrum of phenotypes and the precise mechanism of SF1 action that fails in DSDs are unknown. Twenty SF-1 mutants identified in 46,XY DSD patients were examined for their ability to activate TESCO in transactivation assays in vitro. Fifteen of the twenty mutants showed reduced SF-1 activation on TESCO, eleven with abnormal subcellular localisation. Fourteen SF-1 mutants were predicted in silico to alter DNA, ligand or cofactor interactions. A positive genotype-phenotype correlation was observed with the extent of reduction in TESCO activation being proportional to the severity of patient phenotypes. These data implicate aberrant SF1-mediated transcriptional regulation of SOX9 in 46,XY DSDs. The lack of TESCO mutations in 46,XY DSD patients prompted a search for additional testicular enhancers that may be functioning in humans. Array comparative genomic hybridisation analysis of isolated DSD patients revealed a 78 kb minimal sex determining region (RevSex) far upstream of SOX9 that was duplicated in 46,XX and deleted in 46,XY DSDs. It was postulated that RevSex contains a gonadal enhancer and the most highly conserved sub-region within RevSex, called SR4, was subjected to further analysis. However, SR4 was neither responsive to sex determining factors in vitro nor active in the gonads of transgenic mice, suggesting that SR4 may not be functioning as a testicular enhancer. SR4 transgenic mice showed reporter activity in the genital tubercle, the primordium of the penis and clitoris, a previously unreported domain of Sox9 expression. SOX9 protein was detected in the genital tubercle, notably in the urethral plate epithelium, preputial glands, ventral surface ectoderm and corpus cavernosa. SR4 may therefore function as a Sox9 genital tubercle enhancer, mutations of which could possibly lead to hypospadias, a birth defect seen in the DSD patients in the RevSex study. However, conditional ablation of Sox9 in the genital tubercle using Shh-Cre/+;Sox9flox/floxmice revealed no genital tubercle abnormalities, possibly due to compensation by similar Sox factors. SR4 activity and the observed SOX9 expression pattern suggest that SR4 may be functioning as an enhancer that drives Sox9 expression in the genital tubercle. These findings suggest a novel role for Sox9 in the direct regulation of external genitalia development. The work described in this thesis on the genetic regulation of SOX9 has shed light on the molecular mechanisms that control mammalian sex determination and differentiation. This research has enabled a better understanding of the mechanism of evolutionary adaptation and speciation, as well as the molecular aetiology of DSDs.
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ItemThe role of the TAM receptors in CNS myelination and demyelination( 2015)Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The TAM family of receptor-tyrosine kinases, comprising Tyro3, Axl and Mertk, is widely expressed in the CNS and has been shown to be critical in the outcome of both toxin-induced, as well as inflammatory demyelination. In addition, previous work has demonstrated a direct impact of Gas6-mediated TAM signalling in the regulation of myelination. However, our understanding of the contributions of each individual receptor in these processes remains poor. In order to potentially utilise TAM receptor functions in the development of new therapeutics, it is of crucial importance to dissect these contributions as pan activation of all three receptors may have undesired off-target effects. The aim of this thesis was therefore to provide further insight into which TAM receptor transduces the pro-myelinating effects of Gas6 and which receptors may be important in limiting demyelination. Using electron microscopy, I found that while deletion of Gas6 only results in a mild, non-statistically significant reduction in developmental myelination, Tyro3 deficiency significantly impairs initiation of this process. In vitro data suggest that Tyro3 expressed on oligodendrocytes is required for normal myelination and that this receptor is required for Gas6-mediated enhancement of myelination. Oligodendrocytes deficient in Tyro3 display a reduction in the activation of Erk1, a signalling molecule involved in the induction of myelin gene expression, suggesting that the effects of Tyro3 upon myelination may be mediated at least in part by Erk1. I also could demonstrate that Tyro3 deficiency alone is not sufficient to significantly alter cuprizone-induced demyelination. This is also true for heterozygous microglia-specific Mertk deletion, indicating that homozygous deletion may be required to unravel potential effects of this receptor in experimental demyelination. Finally, injection of Axl or Mertk activating antibodies did not alter EAE disease course which may have been due to detrimental effects probably caused by antibody-mediated hyperactivation of the immune system. In summary, the data presented in this thesis describe for the first time that Tyro3 is a regulator of CNS myelination and that this is regulated by its expression on oligodendrocytes, possibly to an extent through Erk1 activation. Neither Tyro3 deletion nor partial deletion of Mertk in microglia alone affected cuprizone-induced demyelination. Finally, my findings suggest that TAM activating antibodies may not be ideal for therapeutic activation of these receptors in inflammatory conditions.
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ItemRegulation of newborn neuron survival and the inflammatory cell response after traumatic brain injury by Suppressor of Cytokine Signalling-2 (SOCS2)( 2015)Every year, millions of people fall victim to traumatic brain injury (TBI) globally. Despite the large prevalence of this condition and continued research efforts, strategies for its treatment are lacking. The discovery of adult neurogenesis, a process by which new neurons are generated in the adult brain under normal physiological conditions, has opened doors for research into treatments whereby endogenous neural precursor cells (NPCs) may be encouraged to aid neuronal repair following TBI. The two neurogenic regions of the adult brain are the sub-granular zone (SGZ) of the dentate gyrus and the sub-ventricular zone (SVZ) of the lateral ventricles. The brain has been shown to have an inherent capacity to enhance endogenous neurogenesis in these regions in response to TBI. Further, SVZ-derived NPCs are able to migrate to ectopic cortical injury sites after experimental TBI. However, despite this potentially neuroregenerative response, the large majority of injury-induced newborn neurons do not survive to become mature functional cells. Further research is required to better understand the molecular mediators of neurogenesis in the adult brain, in order to harness its therapeutic capacity after TBI. Among the currently identified mediators of endogenous adult neurogenesis is the Suppressor of cytokine signalling-2 (SOCS2). SOCS2 overexpressing (SOCS2Tg) mice show an enhanced survival of newborn adult hippocampal neurons. Also, cultured cortical neurons overexpressing SOCS2 display enhanced neurite outgrowth. Therefore, this thesis aimed to further explore the role of SOCS2 in endogenous adult neurogenesis under both non-injury and injury conditions. In the SOCS2Tg mouse, SOCS2 is overexpressed in all cells of the body including all cells and regions of the brain. Therefore, the improved newborn adult hippocampal neuron survival observed in these mice may have been a function of an altered microenvironment rather than due to neuron-specific SOCS2 overexpression. In an adult WT mouse brain, SOCS2 is expressed at greatest levels in the hippocampal dentate gyrus and CA3 region. Therefore, in Chapter 3, to help establish whether or not there is a newborn hippocampal neuron-specific role for SOCS2, adult hippocampal neurogenesis was examined in SOCS2 null (SOCS2KO) mice, in which the dentate gyrus would likely be the most affected by SOCS2 loss. To examine immature neuron (neuroblast) generation and newborn neuron survival, SOCS2KO mice were administered EdU for 7d to label proliferative NPCs. At 8d newly generated neuroblasts were quantified following doublecortin and EdU co-labelling. At 35d matured newborn neurons were quantified following NeuN and EdU co-labelling. No significant differences were present in neuroblast generation at 8d between genotypes, however at 35d SOCS2KO mice had reduced numbers of mature newborn neurons. The previously defined role for SOCS2 in regulating neurite outgrowth was hypothesised to be important in newborn adult hippocampal neuron development and therefore affected in SOCS2KO mice. No differences in newborn adult hippocampal neuron dendritic tree morphology were found between SOCS2KO and WT mice. However, SOCS2KO dendrites did have a higher density of mushroom morphology spines. This study highlighted a potential role for SOCS2 in regulating newborn adult hippocampal neuron maturation processes, which may be important for early newborn neuron integration required for survival. Given the enhanced newborn neuron survival previously established in SOCS2Tg mice under non-injury conditions, these mice were examined in this thesis for injury-induced SVZ-derived adult cortical neurogenesis. It was hypothesised that SOCS2Tg mice would have improved newborn neuron survival near the injured cortex. Adult SOCS2Tg mice were subjected to a mild (Chapter 4) or moderately-severe (Chapter 5) controlled cortical impact TBI and administered EdU for 7d after TBI to label proliferative cells. At 35d post-mild TBI, no EdU+NeuN+ newborn neurons were observed near the cortical lesion in SOCS2Tg and WT mice. However, SOCS2Tg mice displayed a greatly enhanced number of injury-induced EdU+CD11b+ macrophages/microglia compared to WT mice. Injury-induced astrogliosis, quantified from EdU+GFAP+ co-labelled cells, also displayed a similar pattern but with much smaller cell density. Injury-induced oligodendrogenesis, quantified from EdU+Olig2+ co-labelled cells, showed to genotype differences. Moderately-severe TBI mice were also administered erythropoietin (EPO) for 7d post-injury together with EdU to aid newborn neuron survival. At 35d after moderately-severe TBI, newborn neurons were observed near the cortical lesion but in similar numbers for both SOCS2Tg and WT mice, with or without EPO treatment. EdU+CD11b+ and EdU+GFAP+ cell density was not different between genotypes at 35d after moderately-severe TBI. The same was true for EdU+Olig2+ cells. Motor function testing revealed an improved motor function recovery in SOCS2Tg mice with or without EPO treatment, whereas WT mice showed improved motor function only after EPO treatment. Further, at 7d after moderately-severe TBI, the time point of peak motor function deficit for WT mice, SOCS2Tg mice had a smaller brain lesion area and an increased number of M2-like anti-inflammatory macrophages/microglia surrounding the lesion. This study suggested a novel role for SOCS2 in modulating neuroinflammatory processes, potentially by having an anti-inflammatory effect in a cortical brain injury environment. Overall, this thesis provides further evidence for the role of SOCS2 in regulating adult hippocampal neurogenesis and has also revealed a potential mechanism by which SOCS2 may support newborn neuron maturation. Further, this thesis presents a novel and potentially beneficial role for SOCS2 in regulating TBI-induced neuroinflammation. These findings have important implications in the search for the developing novel therapeutic strategies for the treatment of TBI and other neurodegenerative diseases.
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ItemInvestigating the role of Gpr62 in oligodendrocyte development and central nervous system myelination( 2015)Myelination is a highly regulated process in the vertebrate nervous system whereby glial cells wraps axons with insulating myelin in order to allow rapid electrical conduction and metabolic support, a process which is fundamental for proper axon function and survival. Current research in the central nervous system (CNS) has focused on factors affecting oligodendroglial differentiation, yet the molecular interactions that occur post-differentiation between the oligodendrocyte and other CNS cell types during developmental myelination and myelin maintenance are still unclear. This thesis investigates the role the orphan G-protein coupled receptor (GPCR) Gpr62, as it relates to oligodendrocyte development and myelination. First, in silico analysis in conjunction with in situ hybridization showed that Gpr62 is specific to and highly expressed in the CNS, specifically within myelinating oligodendrocytes. Moreover, Gpr62 expression was shown to be regulated by myelin regulatory factor (MyRF), a key transcription factor regulating expression of major myelin genes. Secondly, a germline knockout mouse strain of Gpr62 was investigated. Gpr62 knockout mice showed no significant affect on myelin thickness, myelin gene or protein expression, initiation of myelination, or oligodendrocyte formation relative to their wildtype littermates, all of which were assessed in the developing, adult, and aging brain. An RNA-sequencing analysis revealed few differentially expressed genes between the knockout and wildtype controls, which were not validated in independent cohorts. Interestingly, the RNA- sequencing revealed several differentially expressed genes in close proximity to the Gpr62 locus. Further analysis indicated that these differentially regulated genes proximal to the Gpr62 locus were the result of Sv129-inherited DNA from the ES cells used for targeting, a result with significant implications for other germline knockout lines generated in the same fashion. Finally, forced viral expression of a tagged version of Gpr62 using an AAV virus demonstrated for the first time the cellular localization of the Gpr62 protein, showing expression within the oligodendrocyte extensions and myelin sheath, expressed along the adaxonal space. Collectively, these results indicated that Gpr62 is a novel oligodendrocyte-specific GPCR located along the myelin sheath, yet it is dispensable for myelin development and maintenance. Thus, failure to identify a role for Gpr62 may be the results Gpr62 having a function in the oligodendrocyte outside of developmental myelination. Indeed, the specificity and regulation of Gpr62 expression in oligodendrocytes by MyRF, and its localization within the myelin sheath, suggests Gpr62 may play a role in the oligodendrocyte that has yet to be explored.
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ItemThe role of Frizzled-7 in gastrointestinal biology( 2015)The tissues of the gastrointestinal tract; oesophagus, stomach, small and large intestines, are lined by a simple columnar epithelium. An important function of the epithelium is to balance the exchange and absorption of nutrients that are used by cells of the body, whilst simultaneously preventing the entry of potentially harmful pathogens that can induce infection, inflammation and/or cellular transformation. As such, the unrelenting mechanical, chemical and biological stresses stimulate the epithelium of the gastrointestinal tract to undergo constant renewal, as way of eradicating damaged cells. The ability of the epithelium to continually renew is driven by adult stem cells that reside in defined niches throughout the gastrointestinal tract. Despite knowledge of their existence, only over the last decade has the identity and molecular pathways that regulate gastrointestinal stem cells been revealed. Originally discovered from its link to cancer and the genesis of colorectal cancer (CRC), the Wnt signalling pathway, the molecular events of which are detailed in chapter 1, was one of the first signalling pathways shown to play a fundamental role in the regulation of gastrointestinal stem cells and in malignancies of the gastrointestinal epithelium. As such, manipulation of the Wnt pathway is an attractive means to modify the behaviour of gastrointestinal stem cells that can be exploited for regenerative purposes or in the treatment of gastrointestinal cancer. However, prior to the commencement of this thesis, the role of the transmembrane receptors responsible for transducing Wnt signals in gastrointestinal biology, the Frizzled receptors, was poorly defined. The aims of this thesis are to identify the function of Frizzled-7, one of ten receptors of the Frizzled family, in gastric epithelial homeostasis, intestinal regeneration and examine therapeutic benefit of targeting Frizzled-7 for treating gastric cancer. Findings from this thesis provide greater insight into the contribution and function of a specific Frizzled receptor in several gastrointestinal contexts. In chapter 1, I provide an overview of the literature pertaining to gastrointestinal stem cells, the Wnt signalling pathway and the role of Wnt signalling in gastrointestinal homeostasis, regeneration and cancer. In chapter 2, I investigate the role of Frizzled-7 in the homeostasis of the gastric epithelium. I demonstrate that conditional deletion of Frizzled-7 in the gastric epithelium triggers a rapid repopulation of Frizzled-7 deficient cells with wild-type cells, which suggests Frizzled-7 is required for gastric homeostasis. Furthermore, targeted deletion of Frizzled-7 from a known population of gastric stem cells does not trigger epithelial repopulation, demonstrating a requirement for Frizzled-7 in a yet-to-be identified population of gastric epithelial cells. These findings draw close parallels to our recently published observations in the small intestine, demonstrating the critical role for Frizzled-7 in transmitting essential Wnt signals in stem cells of the gastric epithelium. The biological process of intestinal regeneration following injury is contingent on robust Wnt signalling and stem cell activity. In chapter 3, I examine the requirement of Frizzled-7 in intestinal regeneration following irradiation, a well characterised model of intestinal regeneration. Compared to wild-type mice, Frizzled-7 knockout mice have impaired intestinal regeneration, characterised by diminished proliferation, decreased Wnt signalling and an increase in cell cycle inhibition. Interestingly, the integrity of the intestinal epithelium is eventually restored in Frizzled-7 knockout mice, which is considered due to an increase in Paneth cells, which are known to facilitate intestinal regeneration following injury. Similar to the intestine, activating mutations to components of the Wnt pathway are commonly observed in gastric cancer and associated with poor prognosis. In chapter 4, I explore the potential therapeutic benefit of Frizzled-7 inhibition in two independent mouse models of intestinal-type gastric cancer and human-derived gastric cancer cells. Conditional deletion of Frizzled-7 from gastric tumours is sufficient to reduce tumour growth with accompanied decreased angiogenesis. Furthermore, significant reductions in tumourigenicity are observed following targeted inhibition of Frizzled-7 in human gastric cancer cells. Collectively, these data demonstrate that Frizzled-7 is rate-limiting for the growth of gastric tumours, which reveal the therapeutic benefit of targeting Frizzled-7 in the treatment of intestinal-type gastric cancer. Finally, in chapter 5, I bring together the primary conclusions from each study and discuss their relevance with respect to the importance of Wnt signalling in gastrointestinal biology. Collectively, the findings from this thesis demonstrate and highlight a pivotal role for Frizzled-7 in regulating and maintaining gastrointestinal stem cells in dynamic gastrointestinal contexts; homeostasis, regeneration and cancer, which have direct translation to the treatment of gastrointestinal malignancies.
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ItemOn type-1 interferons, neuro-inflammation and Alzheimer's disease( 2015)Alzheimer’s disease (AD) is the most common form of dementia worldwide. Hyper-phosphorylation of tau, leading to intracellular neurofibrillary tangles, and accumulation of the amyloid-β (Aβ) peptide, leading to formation extracellular plaques, are the two key brain proteinopathies associated with the progressive neuro-degenerative disease. To date, therapeutics targeting these pathologies have proven ineffective in the treatment of AD and highlights the need for new lines of investigation into disease mechanisms. Neuro-inflammation is evident in AD patients, comprising of enhanced gliosis surrounding Aβ deposits and pro-inflammatory cytokine load. This dysregulated innate inflammatory response is deleterious and facilitates neuro-degeneration. Identifying critical mediators controlling this neuro-inflammation will prove beneficial in developing anti-inflammatory therapies for the treatment of AD. The type-1 interferons (IFNs) are pleiotropic cytokines that control pro-inflammatory cytokine secretion and are master regulators of the innate immune response. This thesis carries the hypothesis that the type-1 IFNs play a critical role in the exacerbation of neuro-inflammation and actively contribute to the progression of AD. This thesis aimed to characterise the role of type-1 IFNs in the neuro-inflammatory response to soluble Aβ1-42 in CNS cell types, evaluate the effect of removing type-1 IFN signalling in the APPSWE/PS1ΔE9 mouse model of AD and hence identify a role for type-1 IFNs in the progression of AD. Soluble Aβ1-42 triggers a type-1 IFN neuro-inflammatory response in primary cultured neurons. Removal of type-1 IFN signalling (IFNAR1-/-) in these cultures attenuated pro-inflammatory responses to Aβ1-42 affording protection against neurotoxicity via attenuation of pro-apoptotic caspase-3 activation. The use of Myd88-/-, IRF7 and IRF3 knockdown cultures, critical in toll-like receptor-dependent signalling, identified that neurons utilise this receptor family to detect Aβ1-42 and initiate a neuro-degenerative type-1 IFN response. Primary IFNAR1-/- mixed astrocyte and microglial cultures display an attenuated type-1 IFN and pro-inflammatory cytokine response to Aβ1-42. These cultures adopt an anti-inflammatory and neuro-protective M2-like polarisation state, opposing the wildtype neuro-degenerative M1-like response to soluble Aβ1-42. To investigate the role of in vivo type-1 IFN signalling in the progression of AD, APPSWE/PS1ΔE9 mice lacking type-1 IFN signaling were generated. APPSWE/PS1ΔE9 x IFNAR1-/- were partially rescued from spatial learning and memory deficits as assessed by the Morris water maze. These mice displayed no alterations in amyloid plaque load but reduced soluble Aβ monomer concentrations were detected. The type-1 IFN response was attenuated in APPSWE/PS1ΔE9 x IFNAR1-/- mice displaying altered pro-inflammatory cytokine expression. Interestingly, cortical astrogliosis was elevated in these mice but microgliosis was attenuated. These microglial populations adopted a neuro-protective M2-like activation state, supporting our in vitro findings. Finally, a type-1 IFN signature was evident in the brains of human post-mortem AD patients Findings from this thesis identify the type-1 IFNs as key mediators of the neuro-inflammatory response in AD. This response is deleterious to disease progression and suggests that targeting type-1 IFN signalling may be therapeutically relevant for anti-inflammatory treatment of AD.
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ItemTAM signalling in CNS demyelination and multiple sclerosis( 2015)Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Involvement of the immune system in the pathogenesis of MS is a key feature of the disease, and an understanding of the mechanisms underlying how immune responses are shaped during CNS demyelination will provide insight into the development of new therapeutic strategies. The TAM (Tyro3, Axl, Mertk) family of receptor tyrosine kinases and their ligands Growth Arrest-Specific 6 (Gas6) and Protein S (ProS) have been shown to modulate many immunological processes important during central demyelination. The major aim of this thesis is to provide further insight into TAM biology in the context of both an animal model of inflammatory demyelination and human MS. By conducting a study examining MS patients and common genetic variations within TAM genes, I identified the MERTK gene as a novel MS susceptibility gene. Examination of plasma from MS patients revealed that levels of the TAM ligand PROS are decreased in MS and that low PROS levels are associated with increased MS disease severity. To interrogate the role of TAM signalling in modulating disease severity during inflammatory demyelination, I used the experimental autoimmune encephalomyelitis (EAE) animal model and observed major changes in TAM gene expression within the CNS and peripheral immune cells during EAE. Examination of Gas6-/- mice during EAE showed that absence of the TAM receptor ligand Gas6 results in both attenuated microglial/macrophage responses and disease severity during the effector phase of EAE. Conditional deletion of Mertk from dendritic cells (DC) resulted in worse disease during the effector phase of EAE. Stratification by sex revealed sexual dimorphism in TAM gene expression and also in the outcome of EAE in both Gas6-/- mice and mice with DC-specific deletion of Mertk. In summary, the data presented in this thesis suggest that the TAM family plays key roles in MS susceptibility and modulating innate immune responses during inflammatory demyelination, providing evidence for members of the TAM family as either markers of disease severity and/or therapeutic targets for the treatment of MS.