No description available.

No description available.

No description available.

Past studies of mammary tissue specific expression of transgenes using the murine whey acidic protein (WAP) promoter have shown widely variable, position-dependent and copy number-dependent expression. This study evaluates a series of three WAP transgenes containing the cDNA of human protein C (hPC) for the expression of human protein C in the milk of mice. In two of the transgenes studied, the cDNA of (hPC) was inserted at the translational start site of a 7.8 kbp mouse WAP genomic DNA Eco RI fragment containing 2.6 kbp of 5’ flanking, 3.9 kbp WAP coding (exons and introns), and 1.3 kbp 3’ untranslated region (UTR) and flanking sequences (designated WAPPC1 and WAPPC2). A third transgene consisted of only the 2.6 kbp of WAP 5’ UTR and flanking DNA, 1.4 kbp hPC cDNA, and 1.3 kbp of 3’ WAP UTR and flanking DNA with no linker sequences (designated WAPPC3). The WAPPC1 and WAPPC2 transgenes expressed up to about 10 μg/ml recombinant hPC in mouse milk while WAPPC3 expressed 30-300 (n=10, n=5, n=11, number of founder lines evaluated for each transgene, respectively). In contrast to past studies with WAP-cDNA fusion transgenes where the maximal expression was about 5% of endogenous WAP expression, the WAPPC3 transgene gave maximal expression which was about 30% of endogenous WAP expression. Thus, results from the combination in WAPPC3 of intact 5’ and 3’ WAP UTR with the cDNA of hPC suggests that introns are not necessary to enable high level expression in the mammary gland when using WAP regulatory elements. Relative specific transcript and protein levels in the transgenic animals studied suggest that the rates of translation initiation may be different for the mRNAs of each of the transgenes studied. / Ph. D.

Calcium (Ca²⁺) influx through Caᵥ1.2 channels mediates cardiac excitation-contraction coupling, tunes cardiac action potential duration and excitability, and regulates cardiomyocytes’ (CM) gene expression. Mechanisms regulating the sub-cellular localization, trafficking, and dynamics of surface Caᵥ1.2 in ventricular CMs are poorly understood though these are critical determinants of cardiac function. To gain new insights into Caᵥ1.2 organization, dynamics, and regulation at the CM surface we generated transgenic mice expressing an αMHC controlled cardiac-specific, dihydropyridine (DHP)- resistant α₁_ᴄ construct, tagged at the N-terminus with FLAG and HA epitopes, at the C- terminus with YFP, a 13-residue bungarotoxin binding site (BBS) inserted into in the third extracellular loop of domain II, and mutations that prevent cleavage of the C-terminus. We found robust inducible expression of DHP-resistant FLAG-HA-BBS-α₁_ᴄ-YFP in the heart that targeted to dyadic junctions, generated nisoldipine-resistant Ca²⁺ currents, supported cardiac excitation-contraction coupling, and was normally up-regulated by β-adrenergic activation with isoproterenol. Incubating transgenic CMs with AlexaFluor₆₄₇-conjugated α- bungarotoxin (BTX₆₄₇) enabled selective labeling of surface BBS-tagged Caᵥ1.2 channels. We used total internal fluorescence (TIRF) microscopy to investigate the spatiotemporal organization and dynamics of surface Caᵥ1.2 channels. Similar to endogenous Caᵥ1.2, transgenic α1C-YFP forms clusters with exponentially distributed sizes at the cell surface. A flow cytometry-based optical pulse-chase assay revealed surface Caᵥ1.2 channels in adult cardiomyocytes fully turn over within two hours. Application of angiotensin II (Ang II) decreased transgenic Caᵥ1.2 surface density and this effect was blocked by the selective Ang II receptor type I (AT1R) blocker losartan. Application of losartan by itself increased Caᵥ1.2 surface density, suggesting the potential presence of constitutively active Ang II receptors in adult CMs. Our results provide new insights into spatiotemporal organization, dynamics, and regulation of Caᵥ1.2 channels in adult CMs and introduce an approach that can be widely applied to elucidate spatiotemporal dynamics of cardiac ion channels and membrane proteins.

GABA is the main inhibitory neurotransmitter of the central nervous system. GABA[subscript A]Rs are multimeric transmembrane receptors, which are composed of 5 subunits. It is known that there are 19 subunits that can make up the GABA[subscript A]Rs, allowing for a vast array of receptor subtypes. In addition to the GABA binding site GABA[subscript A]Rs have distinct allosteric binding sites for benzodiazepines, barbiturates, ethanol, certain general anaesthetics and neuroactive steroids. The molecular heterogeneity of the GABA[subscript A]R is accompanied by distinct pharmacological profiles of the different receptor subtypes. The advance of transgenic mouse models has allowed the functional significance of this heterogeneity to be studied in vivo. Therefore, this thesis utilises a variety of transgenic mouse models carrying either mutations or deletions of certain subunits to study the functional significance of the receptor heterogeneity. Mice lacking the α1 subunit (α1[superscript(-/-)]), carrying a point mutation of the α1 subunit (α1H101R), and mice lacking the δ subunit (δ[superscript(-/-)]) have been utilised to investigate the role of these subunits in the sedative actions of benzodiazepines and the GABA[subscript A]R agonist THIP. Although there are limitations to the interpretation of these results due genetic background of the α1[superscript(-/-)] and α1H101R, experiments suggest that the α1H101R mutation is not behaviourally silent as previously suggested and provide further evidence that the α1 subunit mediates the sedative properties of benzodiazepines. These experiments also reveal that the extrasynaptic δ containing receptors are responsible for mediating the sedative effects of THIP, and these findings combined with evidence from collaborators, implicates the thalamus as an anatomical mediator of these effects. An investigation of the putative cognitive enhancing effects of THIP using an attentional set-shifting task for mice suggested that pre-treatment with THIP reduces the number of errors to reach criterion. δ[superscript(-/-)] mice could not be trained to perform the task, therefore further behavioural investigation of these mice was performed, which suggested a heightened level of anxiety and reduced motivation for a food reward. This thesis has furthered our understanding of the functional role of GABA[subscript A]R subtypes. With the advance in genetic manipulations that allow for regionally selective mutations of the receptor the anatomical structures involved in these functions can be identified.

The Arctic mutation in the Amyloid-β (Aβ) domain of the Amyloid-β precursor protein (APP) causes Alzheimer’s disease (AD) and confers unique biochemical characteristics to Aβ peptides. The aims of this thesis were to evaluate a transgenic model with the Arctic mutation, and to use it to gain new insights into the mechanisms of early (pre-plaque) and late-stage Aβ pathogenesis in AD. The Arctic mutation made Aβ more prone to aggregate, to accumulate in intracellular compartments and to form extracellular plaques when the models tg-ArcSwe and tg-Swe were compared. By inhibiting APP processing genetically or pharmacologically, the intraneuronal granular immunoreactivity with antibodies binding the Aβ domain was shown to largely represent Aβ, and not APP or APP-fragments. At two months of age, the intracellularly accumulated Aβ decreased rapidly, likely because it was still accessible to intracellular clearance. Extracellular Aβ deposits emerged at 5-6 months of age and the amyloid fibril structure was more compact than in tg-Swe. Moreover, Aβ deposits in tg-ArcSwe were more resistant to chemical extraction than those of established models carrying the Swedish APP mutation only, e.g. tg-Swe mice. The stability of deposits better reflects the biochemistry of senile plaques in AD. Thus, the tg-ArcSwe model may better predict the outcome of clinical trials, particularly therapies designed to enhance clearance of Aβ aggregates and deposits. Postmortem brain of Arctic mutation carriers contained extensive parenchymal plaque pathology. Differential immunostaining patterns with C- and N-terminal Aβ antibodies revealed a subset of plaques that were unique to the brains of Arctic mutation carriers. Aβ deposits in the cerebral vessel walls were congophilic and mainly composed of full-length Aβ. In contrast, N-terminally truncated Aβ was more prominent in the parenchymal plaques, all of which essentially lacked amyloid cores. A heterogeneous assembly of mutant and wild-type Aβ was shown to favor the formation of diffuse deposits in bitransgenic mice, and such mechanisms may at least partly explain observations of plaques lacking amyloid cores in postmortem Arctic mutant brain. In the bitransgenic mice, a low level of Arctic Aβ was sufficient to facilitate aggregation of wild-type Aβ. This observation, but also our findings of differences in amyloid fibril structure in tg-ArcSwe and tg-Swe, further highlights similarities between AD and prion disorders in which PrPsc refolds PrPc and facilitates fibril formation. / (Faculty of medicine)

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Medicine, Department of Medicine. Title from pdf file main screen (viewed on January 17, 2010). Includes bibliographical references.

No description available.

Amyotrophic lateral sclerosis (ALS) is a disease in which the motor neurons die in a progressive manner, leading to paralysis and muscle wasting. ALS is always fatal, usually through respiratory failure when the disease reaches muscles needed for breathing. Most cases are sporadic, but approximately 5–10% are familial. The first gene to be linked to familial ALS encodes the antioxidant enzyme superoxide dismutase-1 (SOD1). Today, more than 160 different mutations in SOD1 have been found in ALS patients.  The mutant SOD1 proteins cause ALS by gain of a toxic property that should be common to all. Aggregates of SOD1 in motor neurons are hallmarks of ALS patients and transgenic models carrying mutant SOD1s, suggesting that misfolding, oligomerization, and aggregation of the protein may be involved in the pathogenesis. SOD1 is normally a very stable enzyme, but the structure has several components that make SOD1 sensitive to misfolding. The aim of the work in this thesis was to study misfolded SOD1 in vivo. Small amounts of soluble misfolded SOD1 were identified as a common denominator in transgenic ALS models expressing widely different forms of mutant SOD1, as well as wild-type SOD1. The highest levels of misfolded SOD1 were found in the vulnerable spinal cord. The amounts of misfolded SOD1 were similar in all the different models and showed a broad correlation with the lifespan of the different mouse strains. The misfolded SOD1 lacked the C57-C146 intrasubunit disulfide bond and the stabilizing zinc and copper ions, and was prinsipally monomeric. Forms with higher apparent molecular weights were also found, some of which might be oligomers. Misfolding-prone monomeric SOD1 appeared to be the principal source of misfolded SOD1 in the CNS. Misfolded SOD1 in the spinal cord was found to interact mainly with chaperones, with Hsc70 being the most important. Only a minor proportion of the Hsc70 was sequestered by SOD1, however, suggesting that chaperone depletion is not involved in ALS.  SOD1 is normally found in the cytoplasm but can be secreted. Extracellular mutant SOD1 has been found to be toxic to motor neurons and glial cells. Misfolded SOD1 in the extracellular space could be involved in the spread of the disease between different areas of the CNS and activate glial cells known to be important in ALS. The best way to study the interstitium of the CNS is through the cerebrospinal fluid (CSF), 30% of which is derived from the interstitial fluid. Antibodies specific for misfolded SOD1 were used to probe CSF from ALS patients and controls for misfolded SOD1. We did find misfolded SOD1 in CSF, but at very low levels, and there was no difference between ALS patients and controls. This argues against there being a direct toxic effect of extracellular SOD1 in ALS pathogenesis. In conclusion, soluble misfolded SOD1 is a common denominator for transgenic ALS model mice expressing widely different mutant SOD1 proteins. The misfolded SOD1 is mainly monomeric, but also bound to chaperones, and possibly exists in oligomeric forms also. Misfolded SOD1 in the interstitium might promote spread of aggregation and activate glial cells, but it is too scarce to directly cause cytotoxicity.