The establishment of neuronal polarity constitutes a central phase in neuronal development and synaptogenesis. In my thesis, I study factors that regulate the development of neuronal polarity and its relationship with neurotransmitter receptor expression. For my experiments, I have investigated the development of sensory neurons from neonatal rat nodose ganglia in culture. Sensory neurons have a pseudo-unipolar morphology, do not extend dendrites, and are devoid of synaptic connections on their somata. However, nodose neurons form synapses de novo in cultures, and I show that the neurons have retained the ability to extend dendrites. Extrinsic factors control dendrite extension by these neurons: the ganglionic satellite cells inhibit the growth of dendrites and induce the neurons to develop a unipolar morphology. In the absence of satellite cells, nodose neurons establish a new multipolar morphology and, in response to nerve growth factor (NGF), extend several dendrites. However, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) do not induce the neurons to extend dendrites, but promote the expression of properties typical of nodose neurons in vivo. / As nodose neurons acquire a new dendrite-axonal polarity in the presence of NGF, they increase the density of functional neuronal nicotinic acetylcholine receptors (nAChRs) on their somato-dendritic domains. To learn more about the relationship between dendrites extension and nAChR gene expression, I have examined the changes in transcript levels of nAChR subunits in neonatal rat sympathetic neurons developing in culture. I show that the developmental pattern of nAChR subunit expression in the cultured neurons follows closely that of sympathetic neurons developing in vivo, with the exception of one specific subunit $ alpha sb7$. I show that the increase in $ alpha sb3$ mRNA levels correlates well with an increase in the density of functional nAChRs on the neurons. In addition, my results suggest that these increases are regulated by mechanisms intrinsic to neonatal sympathetic neurons. On the other hand, the changes in $ alpha sb7$ gene expression, which correlate with changes in $ alpha$-bungarotoxin binding, are activity-dependent and regulated by a calcium/calmodulin-dependent protein kinase pathway. The results presented in this thesis provide insights on how neurons are influenced in their extension of dendrites and how this extension affects neurotransmitter receptor expression.

Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death.

Candidate virulence genes were sought through the genetic analyses of two strains of Mycobacterium tuberculosis, one virulent, H37Rv, one attenuated, H37Ra. Derived from the same parent, H37, genomic differences between strains were first examined via two-dimensional DNA technologies: two-dimensional bacterial genome display, and bacterial comparative genomic hybridisation. The two-dimensional technologies were optimised for mycobacterial use, but failed to yield reproducible genomic differences between the two strains. Expression differences between strains during their infection of murine bone-marrow-derived macrophages were then assessed using Bacterial Artificial Chromosome Fingerprint Arrays. This technique successfully identified expression differences between intracellular M. tuberculosis H37Ra and H37Rv, and six candidate genes were confirmed via quantitative real-time PCR for their differential expression at 168 hours post-infection. Genes identified to be upregulated in the attenuated H37Ra were frdB, frdC, and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE, and Rv1571. Further qPCR analysis of these genes at 4 and 96h post-infection revealed that the frd operon (encoding for the fumarate reductase enzyme complex or FRD) was expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 was higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of culture when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Furthermore, inhibition of the fumarate reductase complex in intracellular bacteria resulted in a significant reduction of intracellular growth. Microarray technology was also applied in the expression analysis of intracellular bacteria at 168h post-infection. Forty-eight genes were revealed to be differentially expressed between the H37Ra and H37Rv strains, and a subset were further analysed via qPCR to confirm and validate the microarray data. phoP was expressed at a lower level in the attenuated M. tuberculosis H37Ra, whereas members of the phoPR regulon were up-regulated in the virulent H37Rv. Additionally, a group of genes (Rv3616c-Rv3613c) that may associate with the region of difference 1 were also up-regulated in the virulent H37Rv.

The family Adenoviridae consists of five genera, including the genus Aviadenovirus, which infects avian species. The genus Aviadenovirus currently comprises five fowl (Fowl adenovirus A-E), one falcon (Falcon adenovirus A), and one goose (Goose adenovirus) adenovirus species. Fowl adenoviruses (FAdVs) have a worldwide distribution. Some are associated with diseases such as inclusion body hepatitis (IBH), while FAdV species C serotype 4 (FAdV-4) has been associated with hydropericardium-hepatitis syndrome (HHS). In this study, the complete nucleotide sequence of fowl adenovirus serotype 8 (FAdV-8) was determined. The full genome was 44,055 nucleotides (nt) in length, with an organization similar to that of the FAdV-1 and FAdV-9 genomes. No regions homologous to early regions E1, E3, and E4 of mastadenoviruses were recognized Pathogenicity of FAdV-8 and FAdV-4 were studied in specific-pathogen-free chickens following oral and intramuscular inoculations. Pathogenicity was determined on the basis of clinical signs and gross and histological lesions. Additionally, virus shedding and viral genome copy numbers in liver, cecal tonsil, and bursa of Fabricius were determined. The role of interleukins (IL) in the pathogenicity of and immune response to FAdVs is unknown. Therefore, in a chicken experiment, interferon-γ, IL-10, IL-18, and IL-8 gene expression was evaluated following FAdV-8 and FAdV-4 infection. Cytokine gene expression was examined in the liver, spleen, and cecal tonsils. This study explored the ability of fowl adenoviruses to subvert the host cell’s secretion of cytokines in response to infection as an important viral mechanism for immune evasion during infection. Variations in virulence of FAdVs are likely to be determined by the fiber alone as shown by Pallister et al. (1996). Therefore, we compared and analyzed the nt and amino acid (aa) sequences of the fiber gene of pathogenic and non-pathogenic FAdVs representing species groups D (FAdV-11) and E (FAdV-8). According to our data, virulence might not be associated only with sequence of the fiber gene. This work is a continuation of our efforts towards better understanding of the molecular biology of FAdVs and the pathogenesis of the disease, with an emphasis on the role of interleukins, an unknown area.

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The RET receptor tyrosine kinase is important during development of neural crest-derived tissues, particularly the enteric and sympathetic nervous systems, and in kidney morphogenesis. Activation of RET requires complex formation between a member of the Glial cell line-Derived Neurotrophic Factor family of ligands and a member of the GDNF-Family Receptor α co-receptors. Upon complex formation, RET becomes phosphorylated and subsequently activates multiple downstream signaling pathways, including those for cell-survival, differentiation, and migration. Mutations in RET can either interfere with or enhance normal RET signaling. Inhibiting RET mutations are associated with development of Hirschsprung disease, which is characterized by a lack of mature ganglia in the gut. Conversely, activating RET mutations are associated with several thyroid cancers. Papillary thyroid carcinoma is frequently associated with sporadic translocations between RET and other genes, known collectively as RET/PTC. A variety of heritable RET missense mutations lead to Multiple Endocrine Neoplasia type 2, which is associated with development of medullary thyroid carcinoma. Two cellular processes disrupted downstream of RET in these diseases are gene-expression and cell-migration. In order to clarify the effects of oncogenic mutations on gene-expression downstream of RET, we analyzed expression microarrays in a model using single mutant and isoform RET expression. We also examined the molecular mechanisms of cell-migration, using both functional cell-based assays and examination of integrins, cell-adhesion molecules important for cell-migration. Finally, we used a large cohort of thyroid tissues to examine RET and integrin expression. We showed that different forms of oncogenic RET do not affect transcription of different target genes, but rather target-gene transcription is proportional to phosphorylatability of mutant RET. We were also able to show that RET leads to activation of at least two integrin subunits (ITGB1 and ITGB3), and that they have unique activation patterns downstream of RET that correlate with cell-adhesion and migration. Finally, we showed that co-expression between RET, ITGB1, and ITGB3 is more frequent in malignant subtypes of thyroid tissues and that their co-expression is correlated to more aggressive thyroid cancer subtypes. Together, we have clarified how RET is able to mediate two important processes, gene-expression and cell-migration. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-11-08 13:53:32.474

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No description available.

No description available.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant surface antigens, encoded by the var gene family, play a crucial role in malaria pathogenesis through mediating immunomodulation and host cell adhesion. Var genes can be sub-grouped according to genetic or functional features. This thesis examined var gene transcription of conserved groups of var genes in the context of clinical malaria disease manifestation in African field isolates. Analysis of var gene transcription in 26 P. falciparum field isolates from Malian children revealed that field isolates from children with cerebral malaria show significantly higher transcription of group A var genes than the field isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia). These results suggest that group A var genes are important determinants of parasite virulence and strengthen the growing body of evidence associating group A var expression with severe disease in children. Analysis of var gene transcription in six P. falciparum placental malaria field isolates showed that var2csa was transcribed in all placental malaria field isolates, but not in 10 childhood isolates examined. This finding, also reported in other recent and subsequent studies, suggests that var2csa expression is a critical factor in the onset of clinical malaria disease in pregnant women. Examination of type 3 var gene transcription in laboratory and field isolates established that these var genes were commonly transcribed in blood-stage parasites, and sequence analysis of the transcribed domains confirmed a very high level of conservation across this var gene sub-family. Finally, rosetting is a property of some group A PfEMP1 and is associated with disease severity in African childhood malaria. Certain glycoconjugate compounds can disrupt rosetting, possibly due to the functional similarities of interactions between rosetting PfEMP1 and host rosetting ligands. A non-toxic compound (curdlan sulfate) was found to be effective at disrupting rosettes in all 18 rosetting field isolates examined, showing potential for use in treatment of severe malaria due to rosetting P. falciparum isolates. The findings presented in this thesis expand current knowledge of the role and significance of var genes/PfEMP1 in P. falciparum malaria disease pathogenesis. The work demonstrates the importance of continued research on var genes/PfEMP1 in further understanding this complex parasite, and ultimately in combating this severe disease.