Thesis submitted in fulfillment of the requirements for the Degree MSc

Masters of ScienceThe Coronaviridae family consists of RNA viruses within the order Nidovirales. The family is classified into two genera, namely the corona- and toroviruses. Coronaviruses are enveloped, single stranded, positive sense RNA viruses with genomes ranging between 27-32kb in size. The 5’ two-thirds of the genome encodes for the 1a/b polyprotein, while the 3’ one-third of the genome encodes for the structural proteins that mediate viral entry into the host cell. These structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The nucleocapsid protein is expressed at high levels within an infected cell. Studies have shown that this protein plays a key regulatory role in different cellular pathways, including the inhibition of interferon production and the up-regulation of the AP1 signal transduction pathway, amongst others. Also, the N protein is vital in the formation of the ribonucleocapsid core by binding to the viral RNA during virion assembly. The focus of this study is the immune response in whole blood cultures to the presence of human coronavirus (HCoV) NL63 N protein. To characterise the stimulation of the immune activity against HCoV-NL63 N in blood cultures, the HCoV-NL63 N gene was expressed in a bacterial system. In this pilot study, GSTtagged N constructs were then purified and used to treat whole blood cultures from three volunteers. ELISAs were used to measure the cytokine response in these treated whole blood cultures. Results showed that the nucleocapsid protein has an inflammatory response on whole blood cultures. These results have generated vital information in the potential function of the HCoV-NL63 N protein on the immune system. It is suffice to say that the HCoV-NL63 N protein is able to elicit an effective inflammatory response within the host cell. Future studies into the cellular pathways affected by the HCoV-NL63 N protein will clarify its exact role in stimulating the host immune system

Capacity of Asynchronous Random-Access Scheduling in Wireless Networks

Abstract—We study the throughput capacity of wireless networks which employ (asynchronous) random-access scheduling as opposed to deterministic scheduling. The central question we answer is: how should we set the channel-access probability for each link in the network so that the network operates close to its optimal throughput capacity? We design simple and distributed channel-access strategies for random-access networks which are provably competitive with respect to the optimal scheduling strategy, which is deterministic, centralized, and computationally infeasible. We show that the competitiveness of our strategies are nearly the best achievable via random-access scheduling, thus establishing fundamental limits on the performance of randomaccess. A notable outcome of our work is that random access compares well with deterministic scheduling when link transmission durations differ by small factors, and much worse otherwise. The distinguishing aspects of our work include modeling and rigorous analysis of asynchronous communication, asymmetry in link transmission durations, and hidden terminals under arbitrary link-conflict based wireless interference models. I

EXAFS study of intermetallics of the type RGe<SUB>2</SUB> (R=La, Ce, Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er and Y) Part I: determination of Ge-Ge distances

A study of the EXAFS associated with the K x-ray absorption discontinuity of germanium in pure germanium and in the rare-earth germanides RGe2 (where R=La, Ce, Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er and Y) has been carried out. The Ge-Ge distances have been obtained in these compounds. Considering the phase to the RGe2 system, the bond lengths in these compounds have been determined. The values obtained by us for the RGe2 compounds (R=La, Ce, Pr, Nd, Sm, Gd, Dy and Y) agree with those obtained earlier by crystallographic methods. The bond lengths for the compounds TbGe2, HoGe2 and ErGe2 are also being reported

Branched KLVFF tetramers strongly potentiate inhibition of beta-amyloid aggregation

The key pathogenic event in the onset of Alzheimer's disease (AD) is the aggregation of beta-amyloid (Abeta) peptides into toxic aggregates. Molecules that interfere with this process might act as therapeutic agents for the treatment of AD. The amino acid residues 16-20 (KLVFF) are known to be essential for the aggregation of Abeta. In this study, we have used a first-generation dendrimer as a scaffold for the multivalent display of the KLVFF peptide. The effect of four KLVFF peptides attached to the dendrimer (K(4)) on Abeta aggregation was compared to the effect of monomeric KLVFF (K(1)). Our data show that K(4) very effectively inhibits the aggregation of low-molecular-weight and protofibrillar Abeta(1-42) into fibrils, in a concentration-dependent manner, and much more potently than K(1). Moreover, we show that K(4) can lead to the disassembly of existing aggregates. Our data lead us to propose that conjugates that bear multiple copies of KLVFF might be useful as therapeutic agents for the treatment of Alzheimer's disease

Amitriptyline-Mediated Cognitive Enhancement in Aged 3×Tg Alzheimer's Disease Mice Is Associated with Neurogenesis and Neurotrophic Activity

Approximately 35 million people worldwide suffer from Alzheimer's disease (AD). Existing therapeutics, while moderately effective, are currently unable to stem the widespread rise in AD prevalence. AD is associated with an increase in amyloid beta (Aβ) oligomers and hyperphosphorylated tau, along with cognitive impairment and neurodegeneration. Several antidepressants have shown promise in improving cognition and alleviating oxidative stress in AD but have failed as long-term therapeutics. In this study, amitriptyline, an FDA-approved tricyclic antidepressant, was administered orally to aged and cognitively impaired transgenic AD mice (3×TgAD). After amitriptyline treatment, cognitive behavior testing demonstrated that there was a significant improvement in both long- and short-term memory retention. Amitriptyline treatment also caused a significant potentiation of non-toxic Aβ monomer with a concomitant decrease in cytotoxic dimer Aβ load, compared to vehicle-treated 3×TgAD controls. In addition, amitriptyline administration caused a significant increase in dentate gyrus neurogenesis as well as increases in expression of neurosynaptic marker proteins. Amitriptyline treatment resulted in increases in hippocampal brain-derived neurotrophic factor protein as well as increased tyrosine phosphorylation of its cognate receptor (TrkB). These results indicate that amitriptyline has significant beneficial actions in aged and damaged AD brains and that it shows promise as a tolerable novel therapeutic for the treatment of AD

Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

<p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p