published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.

Autosomal-dominant mutations in the amyloid precursor protein (APP) gene increase the production and aggregation of toxic amyloid-β (Aβ) peptides and cause early-onset Alzheimer’s disease (AD). Noradrenergic cell loss is well documented in AD and has been posited to play a role in cognitive symptoms as well as disease progression. We investigated memory and affect, tissue levels of catecholamines, brain-derived neurotrophic factor (BDNF) mRNA and bioenergetic homeostasis in TgCRND8 mice that express a double mutant (K670N/M671L + V717F) human APP695 transgene. We found that TgCRND8 mice develop object memory impairment and behavioural despair, as well as reductions in noradrenaline and BDNF expression in the hippocampus and cortex, before the appearance of Aβ plaques. Animals with more advanced Aβ pathology exhibit disruptions in energetic status, along with diminished complex I+III activity in the electron transport chain. To test whether the AD-like phenotypes of TgCRND8 mice might be due to altered noradrenergic tone, pre-plaque mice were treated with dexefaroxan, an antagonist of presynaptic inhibitory α2-adrenoceptors that are highly expressed on both noradrenergic and cholinergic terminals. Effects of dexefaroxan were compared to those of rivastigmine, a cholinesterase inhibitor. Both dexefaroxan and rivastigmine improved behavioural phenotypes and BDNF expression without affecting tissue Aβ load. Drug treatments also restored complex I+III mitochondrial activity and increased ATP levels. Reductions in noradrenergic tone appear to underlie Aβ-induced functional impairment in TgCRND8 mice, in addition to BDNF deficits and bioenergetic stress. These studies suggest that α2-adrenoceptor targeting may warrant consideration as a therapeutic strategy in AD.

Autosomal-dominant mutations in the amyloid precursor protein (APP) gene increase the production and aggregation of toxic amyloid-β (Aβ) peptides and cause early-onset Alzheimer’s disease (AD). Noradrenergic cell loss is well documented in AD and has been posited to play a role in cognitive symptoms as well as disease progression. We investigated memory and affect, tissue levels of catecholamines, brain-derived neurotrophic factor (BDNF) mRNA and bioenergetic homeostasis in TgCRND8 mice that express a double mutant (K670N/M671L + V717F) human APP695 transgene. We found that TgCRND8 mice develop object memory impairment and behavioural despair, as well as reductions in noradrenaline and BDNF expression in the hippocampus and cortex, before the appearance of Aβ plaques. Animals with more advanced Aβ pathology exhibit disruptions in energetic status, along with diminished complex I+III activity in the electron transport chain. To test whether the AD-like phenotypes of TgCRND8 mice might be due to altered noradrenergic tone, pre-plaque mice were treated with dexefaroxan, an antagonist of presynaptic inhibitory α2-adrenoceptors that are highly expressed on both noradrenergic and cholinergic terminals. Effects of dexefaroxan were compared to those of rivastigmine, a cholinesterase inhibitor. Both dexefaroxan and rivastigmine improved behavioural phenotypes and BDNF expression without affecting tissue Aβ load. Drug treatments also restored complex I+III mitochondrial activity and increased ATP levels. Reductions in noradrenergic tone appear to underlie Aβ-induced functional impairment in TgCRND8 mice, in addition to BDNF deficits and bioenergetic stress. These studies suggest that α2-adrenoceptor targeting may warrant consideration as a therapeutic strategy in AD.

Olfaction is vitally important to animals in all environments and is used to identify food, habitat, conspecifics and predators. Some odors, like pheromones or the pungent smell of spoiled foods, can trigger pre-existing behavioral responses that appear to require no learning. Most odors, however, are only attended to as a result of prior experience. It is believed that different types of odors are processed in different olfactory pathways in the forebrain. This thesis examines the relationship between innate and learned olfactory behaviors and the anatomy of the neural pathways that underlie them, using the zebrafish olfactory system as a model. I first characterized an appetitive olfactory behavior, which is displayed promptly by zebrafish when they encounter amino acid odors. A similar appetitive behavior can also be learned by the fish for another, initially neutral odorant, if it is repeatedly paired with food rewards. Zebrafish can therefore respond to, and learn to respond to certain odors. I then conducted an in-depth anatomical analysis of the structure and distribution of glomeruli in the zebrafish olfactory system. Glomeruli are spheroidal synaptic aggregates that organize and shape olfactory information that arrives in the brain. Throughout the development of zebrafish, I identified two distinct populations of glomeruli. One population consisted of 25 individually identifiable, anatomically stereotypic glomeruli that closely resembled specialized glomeruli in mammals and insects. These glomeruli were already formed during embryonic development and persisted in remarkably stable configurations throughout later developmental stages. I hypothesize that the 25 individually identifiable glomeruli constitute stable olfactory pathways (i.e., for innate olfactory behaviors). Most glomeruli, however, were anatomically variable and displayed different distributions within coarsely circumscribed regions in the zebrafish olfactory bulbs. The development of these glomeruli could be modified by sensory experience, suggesting that they may comprise plastic olfactory pathways that subserve the establishment of learned olfactory behaviors. Collectively my results show that innate and learned olfactory behaviors may indeed be represented in different olfactory pathways, and that these types of pathways may be located in both main and accessory olfactory systems.

In this work we studied various aspects of DNA repair in plants, focusing mainly on point mutation repair and its interconnection with double-strand break repair. We were using transgenic point mutation and recombination substrates as a primary tool in our experiments. We have compared two transgenic homologous recombination assays (B-glucuronidase- and luciferase-based), analyzed the sensitivity of DNA repair machinery to ultraviolet radiation and assessed the involvement of AtKu80, Atm and AtXpd repair genes in point mutation repair. Ours study revealed the following: the luciferase-based recombination assay is more sensitive then B-glucuronidase-based; double-stand break repair machinery is sensitive to ultraviolet radiation, which results in increased pint mutation formation; chosen DNA repair genes might be impaired in point mutation repair, however further experimentations are needed to confirm this. / xi, 132 leaves : ill. ; 29 cm.

No description available.

Human protein C (HPC) is a vitamin-K dependent plasma glycoprotein which is one of the major components regulating anticoagulation. HPC injection is a promising therapy for several diseases but a heterologous production system would be preferred over purifying HPC from human plasma because of its low concentration (4-5 $ mu$g/ml). A cDNA clone coding for HPC was inserted downstream of the CaMV 35S promoter and of a dimer of the CaMV 35S promoter. Tobacco plants were transformed using Agrobacterium and a binary vector strategy. Kanamycin resistant plants were regenerated and enzyme linked immunosorbent assay determined that HPC, in crude plant extracts, accounted for up to 0.03% of plant soluble proteins. HPC was found to be expressed by R$ sb1$ seedlings suggesting successful integration of the T-DNA into plant genome. A partial protein purification system was developed in order to enrich the protein mixture for HPC. HPC was found to bind tightly at pH 6.0 to Fast Flow Q Sepharose resin.