Anatomy and Neuroscience - Theses
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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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ItemSuppressors of oncogenic Cbl in the Drosophila eye.( 2018)Cbl is an E3 ligase, and downregulates several cellular signalling pathways, in this role by targeting receptor tyrosine kinases for endocytosis. Mammalian Cbl was first identified as the full-length isoform of v-Cbl, a C-terminal truncated dominant negative oncogene that permits binding of v-Cbl to Cbl targets but does not facilitate their ubiquitination. This results in constitutive activation of the receptor tyrosine kinase. In this thesis, I used a Drosophila analogue of v-Cbl, named Dv-cbl. GMR>Dv-cbl had been used prior to the commencement of this study to screen for modifiers of its rough and overgrown eye phenotype using the Gene search system, a transposon-based inducible expression system. In this study, a subset of the suppressors of the GMR>Dvcbl phenotype from that screen, and two other representative lines were further investigated. The published interactions and functions of the genes implicated by the GS lines are discussed and a method of suppression of the GMR>Dv-cbl phenotype by each line is suggested. In the published work presented in this thesis, the Akap200 expressing lines EP2254 and GS2208 were further studied. Expression of Akap200 in EP2254 was confirmed via mRNA in situ hybridisation, and its ability to also suppress the Ras85DV12 phenotype was confirmed. The ability of EP2254 to suppress GMR-Dv-cbl and sev-Ras85DV12 coexpression was confirmed. When GMR-Dv-cbl and sev-Ras85DV12 are coexpressed, a phenotype that is greater than the cumulative phenotype of each would suggest arises. In fact, GMR-Dvcbl (where Dv-cbl was directly driven from the GMR promoter) was used instead of GMR-Gal4, UAS-Dv-cbl (GMR>Dv-cbl) as the coexpression of GMR>Dv-cbl and sev- Ras85DV12 results in lethality, and coexpression of GMR-Dv-cbl and sev-Ras85DV12 did not. Alone, each has a mildly rough eye. I showed that EP2254 was able to suppress this phenotype and that this suppression was partially independent of apoptosis. The endogenous function of Akap200 in the Drosophila eye was then investigated. An mRNA in situ hybridisation experiment showed that endogenous Akap200 is present in the eye disc, and a series of immunohistochemical stains showed that Akap200 was expressed in a subset of photoreceptor cells. Knockdown of Akap200 using RNAi lines showed that endogenous Akap200 was having a modifying effect on the GMR>Dv-cbl phenotype, as knockdown of Akap200 enhances the GMR>Dv-cbl phenotype. A recent study suggests that Notch is protected from internalisation by Cbl by Akap200, which is consistent with the results in this thesis.
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ItemAncient DNA investigations at Samtavro, Republic of Georgia( 2016)The Samtavro cemetery, used from the Late Bronze Age through to the Early Middle Ages, is the largest known ancient burial ground in the Republic of Georgia and the Caucasus. Centrally located at the crossroads of major continents and trading networks, archaeological investigations have revealed a diverse assemblage of artefacts and cultural practices, including Greek, Roman, Persian, and Hunnic influences. In order to gain insight into the origin, affinity and legacy of the Late Antique (~300-500AD) Samtavro population, we sequenced whole mitochondrial genomes (mitogenomes) from 17 individuals buried in 14 tombs. In order to test whether tombs were being used to bury family members together, we sampled more than one individual from three different tombs. We find high mitochondrial DNA (mtDNA) diversity with all individuals from different tombs (n=14) carrying unique mtDNA haplotypes. Identical mtDNA haplotypes were found in two of the three tombs from which two individuals were sampled, probably reflecting the co-burial of kin. Fifteen individuals were included in a principal components analysis (PCA) of haplogroup composition and fourteen individuals in a multidimensional scaling plot (MDS) of Slatkin’s Fst values. These analyses show a strong west Eurasian influence with close affinity to populations from the Caucasus, Middle East and Europe. All (n=17) mitochondrial lineages belong to West Eurasian haplogroups suggesting minimal, if any, impact of ancient nomads of the Eurasian steppe. Fourteen individuals were included in a shared haplotype analysis which revealed that more than half (8/14=57%) of the hypervariable I (HVSI) haplotypes from Samtavro were previously reported in modern day Georgians. Similar haplogroup composition, low Fst values and a high haplotype sharing between ancient Samtavro and modern day Georgians suggests an autochthonous origin of the Samtavro population followed by a high degree of population continuity in the region over the last 1,500 years. However, the mtDNA represents just a single lineage precluding a fuller understanding without the co-analysis of nuclear and Y chromosomal genetic diversity.
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ItemTaxonomy of Proconsul: an issue of species numbers or sexual dimorphism?( 2015)I have investigated the alpha taxonomy of Miocene primate genus Proconsul by performing measurements on photographs of fossil dental material obtained from museum sources. My aim was to assess levels of variation, and thereby evidence for sympatric species, by comparing results with those obtained from sex-matched samples of extant hominoids. The holotype of the initial species described, Proconsul africanus, came from a mainland site in western Kenya. Additional examples of the genus were obtained subsequently from adjacent sites and from islands within Lake Victoria. By 1951, three species of progressively-increasing size were recognised, seemingly present at both island and mainland localities. However, subsequent investigations questioned whether, instead of two sympatric species at a particular site, particularly Rusinga Island, size disparities noted reflected the presence of a single sexually dimorphic species. I addressed this debate by comparing results of measurements of 144 Proconsul specimens with those from sex-matched samples comprising 50 specimens each of four extant primate genera: Pan, Gorilla, Pongo and Hylobates/Symphalangus. The protocol consisted of measuring occlusal views of adult molars to obtain linear measurements and cusp areas. By selecting samples on a 1:1 sex-matched basis for extant groups, and matching these against those for Proconsul, I minimised the potential that variations within the Proconsul material reflected sexual dimorphism within one species. I have investigated the situation relevant to each of two clusters of primate fossil sites, respectively on Rusinga and Mfangano Islands (Area 1) and at mainland sites around Koru (Area 2). The study has addressed a null hypothesis: that at each Area, only one Proconsul species was present from the relevant time-horizon. Analyses confirmed that levels of variation within Proconsul, at each of Areas 1 and 2, outweighed those within extant hominoids. More than one species of Proconsul existed contemporaneously at each cluster of Miocene fossil sites.
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ItemStructural damage to the enteric nervous system following ischemia / reperfusion injury( 2012)Ischemia reperfusion (I/R) injury can occur in a number of organs, however the intestine is possibly the most susceptible to I/R damage. Intestinal I/R injury is treated as a life threatening injury as tissue necrosis can rapidly progress and the non specific clinical presentation of the disease makes it difficult to diagnose. In addition, I/R injury is an inevitable consequence of intestinal transplantation and, with an increasing success rate, the demand for intestinal transplantation for end stage organ failure patients has increased. The damage caused to the intestine following a period of I/R has been well documented for a number of cell types, and results have demonstrated mucosal sloughing, muscle degeneration, and the damage caused to enteric neurons. However, the level of damage inflicted on enteric glia following I/R remains unexplored, despite the knowledge that glia contribute to neuronal maintenance and survival and are involved in maintaining mucosal integrity. Through the use of immunohistochemistry, this study shows for the first time that major damage to enteric glia occurs 3 hr following I/R injury, earlier than the peak of neuronal damage, and that structural damage to glia, revealed by cytoskeletal disruption (glial fibrillary acidic protein, GFAP, disaggregation) and loss of S100B, is associated with changes to the enteric neuronal protein, Hu in neighbouring neurons. I was able to quantify these changes, and to determine if the GFAP, S100B or Hu were differentially affected following arterial (A), venous (V), or arterial plus venous (AV) occlusion. In addition, through histochemistry, this study also documented the structural damage to the mucosa following A, V and AV occlusion. My study has added to our knowledge on the damaging effects of intestinal I/R injury on enteric glia, and has identified an association between the damage observed in enteric glia and neighbouring neurons. These results highlights the possible importance of enteric glia in I/R and may be beneficial for future studies investigating neuroprotective strategies against intestinal I/R injury.
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ItemThe anatomical and clinical study of the posterolateral trunk in reconstructive surgery: a study of the deep circumflex iliac artery and gluteal artery perforators( 2011)Background: Perforator flaps are becoming increasingly popular methods of reconstruction. Its appeal lies in the predictability of tissue bulk and low donor site morbidity when compared to the more traditionally accepted musculocutaneous flaps. The deep circumflex iliac artery osteomusculocutaneous flap, first described in 1979, has been used in a variety of reconstructions. In particular, it is well documented for use in the hemi-mandible. Similarly, the superior and inferior gluteal musculocutaneous flaps have been used for various breast and perianal reconstructions. As perforator flaps, they are poorly understood and therefore, surgeons often avoid them. Both the deep circumflex iliac artery (DCIA) and superior and inferior gluteal artery perforator (SGAP and IGAP) flaps are located in the posterolateral region of the abdomen and pelvis. The skin overlying these flaps is hairless and both flaps are associated with well hidden donor scars. The perforators have versatile pedicle length, diameter and arc. A study was therefore undertaken to better understand the anatomy of the perforator associated with the DCIA perforator and the SGAP/IGAP with a view to improve preoperative assessment of appropriate patients and improving surgical outcome. Method: A combined cadaveric and clinical study of the vascular anatomy of the posterolateral trunk was undertaken. This included an ink and lead-oxide injection of cadavers to investigate the DCIA perforators. The lead-oxide specimen also underwent x-ray radiographic assessment of the perforators. A Computer Tomographic Angiography (CTA) assessment of 44 DCIA and 160 SGAP/IGAP flap perforators were also performed in patients who were undergoing preoperative CTA prior to breast reconstruction. The clinical study was a series of patients who underwent pre-operative imaging with a view to proceed to a DCIA or GAP perforator flap. Their pre-operative findings and post-operative outcomes were assessed. Appropriate institutional ethical approvals were obtained. Result: Both the cadaveric and CTA studies demonstrated that just over half of hemi-abdomens were suitable for DCIA perforator flaps. All patients studied had sizable SGAP perforators whilst 95% of patients had sizable IGAP perforators. All patients who underwent a planned DCIA, SGAP or IGAP perforator flap at one institute had pre-operative imaging and assessment of their perforators. Whilst just over half of these patients had suitable DCIA, all the patients had well sized SGAP or IGAP. Those with poor DCIA perforators underwent the traditional free fibula flap instead. Their outcomes were comparable with low morbidity and high flap survival rate. Conclusion: Both the DCIA perforator and SGAP/IGAP flaps are useful reconstructive options. The DCIA flap is useful for reconstruction requiring bone, soft tissue as well as skin coverage. The SGAP and IGAP flaps are viable options for reconstruction of variable amount of soft tissue and skin defects. With an understanding of their perforators and a detailed planning of the surgery with CTA, both these flaps can be performed safely and effectively. These posterolateral wall perforator flaps are associated with low donor site morbidity, a well hidden scar and with improved understanding of their anatomy; the DCIA and SGAP/IGAP perforator flaps are safe and sure option available to the reconstructive surgeon.