Network decontamination is a well known mobile agent problem with many applications. We assume that all nodes of a network are contaminated (e.g., by a virus) and a set of agents is deployed to decontaminate them. An agent passing by a node decontaminates it, however a decontaminated node can be recontaminated if any of its neighbours is contaminated. In the vast literature a variety of models are considered and different assumptions are made on the power of the agents. In this thesis we study variation of the decontamination problem in mesh and tori topologies, under the assumption that when a node is decontaminated, it is immune to recontamination for a predefined amount of time t (called immunity time). After the immunity time is elapsed, recontamination can occur. We focus on three different models: mobile agents (MA), cellular automata (CA), and mobile cellular automata (MCA). The first two models are commonly studied and employed in several other contexts, the third model is introduced in this thesis for the first time. In each model we study the temporal decontamination problem (adapted to the particular setting) under a variety of assumptions on the capabilities of the decontaminating elements (agents for MA and MCA, decontaminating cells for CA). Some of the parameters we consider in this study are: visibility of the active elements, their ability to make copies of themselves, their ability to communicate, and the possibility to remember their past actions (memory). We describe several solutions in the various scenarios and we analyze their complexity. Efficiency is evaluated slightly differently in each model, but essentially the effort is in the minimization of the number of simultaneous decontaminating elements active in the system while performing the decontamination with a given immunity time.

Previous studies with conjugates containing the NH2-terminal and COOH-terminal antigenic determinants of oxidized ferredoxin from C. pasteurianum indicated a need for at least two determinants to stimulate DNA synthesis in sensitized lymphocytes. This suggested a mechanism involving cell cooperation, a possibility which has been investigated here by selectively inactivating cells binding one or the other of the determinants. Cells from immunized guinea pigs were tested in vitro for their capacity to bind antigen or to be stimulated by it before and after "antigen suicide" with radioiodinated conjugates containing the NH2-terminal or COOH-terminal determinants of oxidized ferredoxin. A microculture system for assessing antigen induced stimulation of 3H-thymidine uptake by lymphocytes was developed for this work. The data show that: 1) Lymphocytes from unimmunized guinea pigs bind both NH2-terminal and COOH-terminal determinants at a frequency of about 10-4. In immune animals the proportion of antigen binding cells increased about 4-6 fold. The frequency of cells binding the determinants depends markedly on the specific activity of antigens employed. 2) Both T and B lymphocytes bind the antigenic determinants from oxidized ferredoxin. 3) Specific inactivation of cells binding either determinant was achieved by antigen suicide with ¹²⁵I-NH₂-terminal or ¹²⁵I COOH-terminal s-BSA conjugates. Synergy occurs between the NH2-terminal binding cells and COOH-terminal binding cells in the proliferative response of sensitized lymph node cells challenged with oxidized ferredoxin in vitro. Evidence from B cell depletion studies indicates that this is a T cell-T cell interaction. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Outer membrane proteins (OMPs) were recovered from eleven strains (eight serogroups) of Legionella pneumophila by sequential treatment with Tris buffer (pH 8), citrate buffer(pH 2.75) and Tris buffer (pH 8). Transmission electron microscopy revealed clearly the separation of the outer membrane from the bacteria. The development of delayed hypersensitivity was also noted by measuring the area of arythema and induration produced by intradermal injections of the MPSs from Chicago 8 strain. The adjuvants enhanced greatly both active and cell-meditated immunity (CMI). Transient lymphocytopenia with a slight rise in neutrophils was noted in each of the immunized groups. Intraperitoneal challenge, seven days after the OMP booster, of one LD (1.5 x10^6) of legionellae resulted in lymphocytopenia with elevated neutrophils. All immunized rats survived the challenge, although those in the saline-OMP group were clearly the sickest. Post-challenge, legionella antibody titers rose greatly and CMI was heightened. Passive immunization (homologous and heterologous) was found to protect the rats from a challenge of on LD. Actively-immunized rats retained their immunity for at least six months as determined by their resistance to a second challenge.

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<p>Lysosomal storage diseases (LSDs) are devastating neurological disorders caused by mutations in lysosomal hydrolases that result in accumulations of hydrolase substrates. Tay-Sachs disease (TSD) is an LSD that specifically results in the accumulation of GM2 gangliosides causing the activation of inflammatory signaling pathways, and leading to microglial activation and apoptotic cell death. The detailed mechanisms through which cell death occurs have not been completely elucidated, however, excitotoxicity is thought to play a major role. Here, we investigated the role of hypoxia-inducible factor-1α (HIF- 1α) and its effector microRNA, miR-210, and the impact they have on the expression of important molecules involved in excitotoxicity, namely neuronal pentraxin 1 (NPTX1) and potassium channel KCNK2 (KCNK2). We discovered that TSD neuroglia are inefficient at stabilizing HIF-1α in hypoxic conditions. Furthermore, miR-210 expression is significantly higher in TSD neuroglia compared to normal neuroglia at baseline and during hypoxia. In addition, TSD neuroglia expressed <em>NPTX1</em>, <em>NPTX2 </em>and <em>KCNK2 </em>at higher levels, and neuronal pentraxin receptor at lower levels than normal neuroglia, implicating excitotoxicity in disease pathogenesis. We also confirmed that miR-210 binds to the 3’ UTR of <em>NPTX1 </em>to repress its expression in TSD neuroglia. The presence of reverse hypoxia response elements in the promoter of KCNK2 and the repression of <em>KCNK2 </em>expression by HIF-1α stabilization suggest that KCNK2 is directly regulated by HIF-1α. Moreover, the glucosylceramide synthase inhibitor, NBDNJ, which is used to reduce ganglioside synthesis, caused expression of <em>NPTX1 </em>to decrease but <em>KCNK2 </em>expression to increase, indicating this drug can modify multiple parameters of disease. This study identifies major gene expression changes between normal and TSD neuroglia that affect the excitability and therefore the viability of TSD cells. This information provides new insight into the mechanisms of neurodegeneration experienced by TSD neuroglia.</p> / Master of Science (MSc)

<p>Telomeres are protein-DNA structures at the ends of eukaryotic chromosomes. The DNA portion is comprised of double-stranded and single-stranded G-rich repetitive DNA. The protein portion is anchored by the "shelterin complex" composed of six proteins. Inappropriate DNA repair and telomere length dysregulation result in cell cycle arrest, genome instability, and carcinogenesis. Thus, this DNA/protein structure protects telomere ends and regulates telomere length.</p><p>The shelterin component TRF1, a double-stranded telomeric DNA binding protein, was found to bind accessory protein PinX1 at mitosis. Given this, I investigated the effect of reducing PinX1 level on cell cycle progression and apoptosis. I found that reducing PinX1 expression with shRNA, as assessed by immunoblot, led to delayed entry into mitosis and elevated levels of apoptosis in human cells. These results indicated that PinX1 plays an important role in mitosis progression and cell viability.</p><p>Intriguingly, binding of PinX1 to TRF1 at mitosis increased the stability of the latter. Moreover, PinX1 binds to the same site on TRF1 as the protein TIN2, which can suppress degradation of TRF1 by inhibiting poly ADP-ribosylation of TRF1 by the enzyme tankyrase. Collectively, these results suggested that TIN2 might be released from TRF1 to promote the binding of PinX1 on TRF1 at mitosis. Given that proteins are often regulated in the cell cycle by phosphorylation, I investigated whether TIN2 was phosphorylated at mitosis. To this end, I performed phospho-proteomic analysis of human TIN2, which revealed two phosphorylated residues, serines 295 and 330. Both sites were phosphorylated specifically during mitosis, as detected by two independent approaches, namely Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2 in vitro and in vivo. The identification of these specifically timed post-translational events during the cell cycle demonstrates the mitotic regulation of TIN2 by phosphorylation. However, as expressing non-phosphorylatable mutants of TIN2 failed to reveal any overt phenotypes, the consequences of these phosphorylation events remain to be determined.</p><p>Lastly, the TRF1-related double-stranded telomeric DNA binding protein, TRF2, was shown to associate with another shelterin component, POT1. POT1 forms heterodimer with TPP1 to bind single-stranded telomeric DNA. Previous research found that mutations of POT1 with reduced binding affinity to either TRF2 or to TPP1 cause distinct phenotypes. To determine whether similar separation-of-function mutants could be generated to dissect the function of POT1s in mice, which are encoded by two genes, Pot1a and Pot1b, I screened a panel of substitution mutants of mPOT1a for loss of binding to mTRF2 and mTPP1. These studies revealed that mPOT1a does not bind mTRF2, but the association with mTPP1 could be disrupted.</p><p>In summary, the described studies have shed insight into the complexity of shelterin regulation, and in particular, highlighted protein-protein interactions and post-translational modifications.</p> / Dissertation

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy