Anatomy and Neuroscience - Theses
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ItemNeural plasticity and gene-environment interactions in the PLC-?1 knockout mouse(University of Melbourne, 2007)
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ItemFlamingo/Starry Night in embryonic abdominal sensory axon development of Drosophila( 2008)The seven-pass transmembrane atypical cadherin, Flamingo (also known as Starry Night) is evolutionally conserved in both structure and function in vertebrates and invertebrates. It plays important roles during the establishment of planar cell polarity (PCP) of epithelial tissues and during the development of axons and dendrites in both peripheral and central neurons. This thesis looks at the role of Flamingo/Starry Night in axon growth and guidance in the embryonic abdominal peripheral nervous system (PNS) of Drosophila. It describes the expression pattern of Flamingo in the PNS and its environment. A combination of single cell labelling and immunohistochemical techniques was used to define the effect of mutations in flamingo as well as several genes coding for potential Flamingo interaction partners. Rescue- and over-/mis-expression experiments featuring targeted expression of either a wild type version or mutant versions of flamingo provide information on the cellular and molecular mechanisms by which Flamingo regulates sensory axon development. Loss of Flamingo function results in a highly penetrant axon stall phenotype. Both sensory and motor axons frequently halt their advance early along their normal trajectories. Flamingo appears to mediate an axon growth promoting signal upon contact of sensory growth cones with specific early intermediate targets. Expression of Flamingo in sensory neurons is sufficient to rescue the mutant sensory axon phenotype. This rescue is at least partially independent of most of the extracellular region of the Flamingo protein. While Flamingo was previously found to have homophilic adhesion properties in vitro and appears to function by a homophilic mechanism during the neurite development of several types of neurons, this study supports a heterophilic signalling mechanism by which Flamingo fulfils its role in abdominal sensory axon growth promotion.
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ItemEnteric serotonin interneurons: connections and role in intestinal movement( 2008)5-HT powerfully affects gastrointestinal function. However, the study of these effects is complicated because 5-HT from both mucosa and a subset of enteric neurons acts on multiple receptor subtypes in enteric tissues. The role of neural 5-HT has been difficult to isolate with current techniques. This thesis aimed to elucidate the role of 5-HT neurons in motility using anatomical and functional methods. In Chapter 2, confocal microscopy was used to examine over 95% of myenteric neurons in guinea pig jejunum, categorized neurochemically, to identify neurons that received anatomically-defined input from 5-HT interneurons. The data showed that cholinergic secretomotor neurons were strongly targeted by 5-HT interneurons. In another key finding, excitatory motor neurons were surrounded by 5-HT terminals; this could provide an anatomical substrate for the descending excitation reflex. Subgroups of ascending interneurons and neurons with immunoreactivity for NOS, were also targeted by 5-HT interneurons. Thus, subtypes of these neurons might act in separate reflex pathways. Despite strong physiological evidence for 5-HT inputs to AH/Dogiel type II neurons, few contacts were identified. In Chapter 3, the confocal microscopy survey was extended to the three other interneuron classes (VIP/NOS and SOM descending interneurons; calretinin ascending interneurons) of guinea pig small intestine. A high degree of convergence between the otherwise polarized ascending and descending interneuron pathways was identified.
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ItemCloning and characterisation of gripe: a novel interacting partner of e12 during brain development( 2002-10)The mammalian cerebral cortex is a remarkable product of brain evolution, and is the structure that most distinctively delineates the human species from others (Northcutt and Kaas, 1995; Rakic, 1988). Neurons in the adult brain are organised into cytoarchitectonic areas, defined by distinct biochemical, morphological and physiological characteristics (Rakic 1988). Remarkably, this complex structure is generated from a simple neuroepithelium. What are the signalling mechanisms that direct neuron formation and subsequent functional-parcellation of the cerebral cortex? Key to the study of this process is an understanding of neuronal fate determination. Available evidence demonstrates an intrinsic programming potential by neuronal progenitors within subdomains of the developing cerebral cortex that is instructive for proper corticogenesis. These regional domains are demarcated by expression of certain transcription factors, including members of the Helix-Loop-Helix (HLH) family of proteins. The HLH family of transcription factors are key contributors to a wide array of developmental processes, including neurogenesis and haematopoiesis. These factors are thought to exert their regulatory influences by binding to cognate promoter-DNA sequences as dimers. While studies in mice have convincingly demonstrated that neurogenic HLH proteins such as NeuroD (Lee et al., 1995; Miyata et al., 1999; Liu et al., 2000) and Mash1 (Casarosa et al., 1999) are intimately involved in neuronal fate determination and terminal differentiation, the role of the ubiquitously expressed HLH protein, E12, in mammalian neurogenesis remains ambiguous. Originally discovered as an important regulator of lymphopoiesis, expression studies revealed its widespread expression in proliferative zones of multiple nascent organs of the embryo, including the developing cerebral cortex; implying a role for E12 in development of the nervous system. Since the function of E12 is, in part, coded by its capacity for protein dimerisation, a search was undertaken for binding partners in developing mouse brain, and using a yeast 2-hybrid assay. Yeast 2-hybrid prey libraries were constructed using complementary DNA (cDNA) isolated from embryonic mouse forebrain tissue at early (embryonic day e11.5) and peak (e15.5) stages of neurogenesis. Screening of these libraries for binding partners to an E12 bait resulted in cloning of HLH factors, such as Mash1, NSCL and Id2. Importantly, a novel binding partner, named GRIPE, was cloned as a novel GAP Related Interacting Protein to E12. GRIPE binds to the HLH region of E12, and may require E12 for nuclear import. Furthermore, GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidently detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were reconfirmed through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein whose dimerisation with E12 has important consequences for cells undergoing neuronal differentiation. A model is proposed to suggest how neurogenic HLH proteins that dimerise to E12 may promote signalling cascades driving early neuroblast differentiation.
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ItemA morphological characterisation of central neural pathways to the kidney( 2005-04)This study was undertaken to locate and characterise the neurons in the central nervous system that project to the kidney. In particular, the aim was to illustrate and characterise the neural link between regions in the hypothalamus known to influence renal function and fluid balance, and nerves known to innervate the kidney.
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ItemProthymosin alpha, a gene differentially expressed in CD34+ cells( 2004)Haemopoietic stem and progenitor cells from bone marrow and cord blood are well characterised with respect to their phenotype, growth in clonal assays, responsiveness to cytokine stimulation, receptor profile and their ability to sustain multilineage engraftment of receptive hosts in animal models of transplantation and of course, clinically in the treatment of some haemopoietic and immunological disorders. It is generally accepted that cells bearing the CD34+ phenotype are enriched for the most primitive of haemopoietic stem cells that possess the cardinal features of self-renewal and multipotency. However, the molecular mechanisms, the spectrum of expressed genes that give rise to the physical characteristics of haemopoietic progenitor cells are not well understood. Furthermore, although CD34+ cells from different sources (bone marrow, cord blood, mobilised peripheral blood) share many common features, there are also significant differences. (For complete abstract open document)
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ItemInherited epilepsy syndromes in multiplex families( 1988)The epilepsies are a common group of disorders where genetic factors are known to be important. The challenging search for genes for the idiopathic epilepsies has been difficult and, on commencing this study, no genes for idiopathic epilepsy had been found. The common idiopathic epilepsies, which follow complex (polygenic or multifactorial) inheritance, have been the subject of genetic linkage studies around the world to establish their chromosomal localization. These studies have met with limited and controversial success to date. This may be in part due to the fact that these studies have relied on collections of a number of small families. Such samples are likely to be genetically heterogeneous making linkage analysis difficult. My research has been based on the alternative approach of studying large multiplex families where epilepsy is likely to follow single gene inheritance. This approach has two advantages. First, providing the assumption of a single major gene is correct, the genetic inter-relationships of clinically defined epilepsy syndromes can be determined. Second, large multiplex families with carefully characterized epilepsies are the ideal substrate for molecular genetic analysis to search for epilepsy genes as they are unlikely to be confounded by genetic heterogeneity. I studied many large families with multiple affected individuals (often more than 20) with a history of seizures. This involved study of all available family members, including affected and unaffected individuals, to establish if there was a history of seizures. Detailed electro-clinical characterization of each individual’s epilepsy syndrome was performed using a validated seizure questionnaire, as well as general medical history and neurological examination. Eye witness accounts of seizures were sought as well as previous medical records. Results of investigations such as imaging studies and previous electroencephalographic (EEG) studies were obtained. All available individuals underwent EEG studies, including sleep studies where possible. Genealogical research was carried out where necessary to clarify family relationships for later molecular genetic analysis. My work has resulted in the description of three new autosomal dominant partial epilepsy syndromes expanding the group of idiopathic partial epilepsies, and one new inherited generalized epilepsy syndrome. The most common of the three new partial epilepsies, Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE), is characterized by childhood onset of clusters of violent nocturnal motor seizures. Seizures are often undiagnosed or misdiagnosed as sleep disorders such as nightmares or night terrors, psychiatric diagnoses such as hysteria, or movement disorders. In the largest Australian family where I identified 27 affected individuals, our molecular genetic collaborators (Professor Grant Sutherland, Adelaide) mapped the gene to chromosome 20q13.2. Using a candidate gene approach, together with Dr Ortrud Steinlein (University of Bonn), they identified a mutation in the cx4 subunit of the neuronal nicotinic acetylcholine receptor in this family. This was the first gene described for an idiopathic epilepsy. The second new partial epilepsy syndrome, Autosomal Dominant Rolandic Epilepsy with Speech Dyspraxia (ADRESD), was described in a single Australian family. In this disorder, typical rolandic seizures occur in midchildhood accompanied by centro-temporal spikes on EEG. In this family, epilepsy followed autosomal dominant inheritance and was associated with speech dyspraxia and cognitive impairment. Clinical anticipation was found suggesting the possibility of a triplet repeat expansion as the genetic basis of this disorder. This family may provide a means to finding a gene for the most common epilepsy syndrome of childhood, benign rolandic epilepsy. The third new autosomal dominant partial epilepsy syndrome is Familial Partial Epilepsy with Variable Foci (FPEVF). Also described in a single Australian family, this disorder is intriguing as partial seizures originate in different parts of the cerebral cortex in different family members. In this family, our molecular genetic collaborators have evidence suggestive of linkage to chromosome 2. Febrile Seizures Plus" (GEFS+). Families with GEFS+ are common but have previously escaped classification as many members have mild forms of generalized epilepsy or febrile seizures alone and occasional individuals have severe refractory epilepsies. Penetrance of the disorder is often quite low. GEFS+ is delineated here in a large Victorian family here and subsequently, our Canadian molecular genetic collaborators have found linkage to chromosome 2. The four new inherited epilepsy syndromes described here can be recognized clinically allowing early diagnosis, appropriate management, prognostic and genetic counselling. Clinical characterization has been successful in laying the foundation for molecular genetic research which has resulted in the definite or suggestive genetic linkage in 3 disorders and the discovery of the first genetic defect in an idiopathic epilepsy. Identification of epilepsy genes will yield new insights into the neurobiology of the epilepsies and may lead to targeted anti-epileptic therapies and ultimately prevention of some types of epilepsy.
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ItemA study of depression in Huntington's disease( 2008)Huntington’s disease (HD) is an inherited neurodegenerative disorder that is caused by a mutation of a single gene, huntingtin. The disease is more commonly known for the characteristic choreiform movements that develop in the later, more advanced stages of the disease. However, cognitive deficits and psychiatric symptoms are frequently observed prior to the onset of the motor symptoms. Little is known about the pathological bases for the neuropsychiatric features which include increased irritability and heightened aggression. Depression affects 30-50% of HD patients and is the most commonly diagnosed psychiatric symptom. This is proportionally higher than in the general population and it is possible that inherent pathological changes in the HD brain render a HD-gene positive individual more susceptible to depression. Using a variety of behavioural tests, the R6/1 transgenic mouse model of HD was found to display altered responses reflective of depression-related behaviour, indicating that the HD mutation confers a genetic susceptibility for developing depression. The behavioural alterations were more robust in female HD mice reflecting a possible sex-dependent manifestation of the depression symptoms in the human HD population that has yet to be investigated. The onset and rate of progression of HD is strongly influenced by the environment and the development of depression is similarly impacted upon by environmental factors (e.g. stress, negative life events). The experimental paradigms of environmental enrichment and wheel-running slow the development of motor and cognitive symptoms in R6/1 HD mice and the present study reports that both paradigms also correct the depression-related behavioural phenotype. This study also found that HD mice had muted responses to two common classes of antidepressant drugs, highlighting the need for a detailed examination of the efficacy of drug treatments in HD patients. Depression susceptibility is linked to genetic variance in the human population and studies of gene candidates in mutant mice report the detection of behavioural phenotypes similar to the present study. The depression-related behavioural phenotype of the R6/1 HD model was found to be associated with early down-regulations in mRNA levels of the ii serotonin (5-HT) 1A and 5-HT 1B receptors in the cortex and the hippocampus. Additionally, female HD mice had reduced cortical 5-HT transporter gene expression. Collectively, these findings indicate that a disruption of serotonergic signaling in the HD brain contributes to the development of depression in HD. Brain-derived neurotrophic factor (BDNF) gene expression is down-regulated in the HD brain, however the expression pattern of exon-specific splice variants was previously unknown. This study reports that BDNF mRNA levels are reduced in the hippocampus by an early age but also reports that individual exon-specific transcripts are differentially down-regulated in males and females, although the functional relevance of this remains to be investigated. Overall, this study has demonstrated that the R6/1 transgenic mouse model of HD is ideal for further investigating the occurrence of depression in pre-motor symptomatic HD. It has also identified alterations in gene expression of key components of neuronal signaling which might be linked to the molecular basis of depression.