Stroke is a pathological condition that causes extensive brain damage. During ischemic stroke, an excess of the excitatory neurotransmitter glutamate exerts many deleterious effects, which leads to cellular damage and cell death, a phenomenon appropriately termed excitotoxicity. Among the events triggered is the activation of the enzyme calpain, a protease whose action is dependent on the intracellular concentration of calcium, which is known to be elevated during excitotoxicity. In this thesis, I hypothesize that neuroprotective drugs can be better accumulated into stroke-affected regions by utilizing the actions of calpain. The extent of calpain activation was first investigated, and it was found to increase over time in both in vitro and in vivo models of stroke. Different amino acid sequences recognized and cleaved by calpain were then incorporated into the neuroprotective Tat-GluR2/3Y peptide. Although in vivo detection of modified Tat-GluR2/3Y peptides was unsuccessful due to technical difficulties, the accumulation of the therapeutic 3Y peptide fragments in neurons under excitotoxic conditions in vitro was found to increase with the CP-3 peptide, a peptide that is a modified version of the Tat-GluR2/3Y, with a sequence cleavable by calpain from the protein Collapsin Response Mediator Protein-3 (CRMP-3). These results suggest that it is possible to concentrate therapeutic agents into stroke-affected neurons, and this may translate into enhanced neuroprotective properties in both in vitro and in vivo animal stroke models. / Medicine, Faculty of / Graduate

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TAT peptide is one of the best-characterised cell penetrating peptides (CPPs) derived from the transactivator of transcription protein from the human immunodeficiency virus 1 (HIV-1). TAT peptide is able to cross the cell membrane and deliver various biomolecules into cells with low immunogenicity and no toxicity. However, the exact mechanism of internalization still remains a subject of controversy. Lamellar neutron scattering was used to determine the location of TAT peptide in the negativelycharged phospholipids bilayers. The results reveal two locations, one in the peripheral aqueous phase between the adjacent bilayers and the second one below the glycerol backbone region of the lipid bilayer. A concentrationindependent membrane thinning above a peptide concentration threshold (1mol%) and a contiguous transbilayer water channel at the largest peptide concentration (10mol%) were also found. This evidence led to the suggestion that the toroidal pore model might be involved in the transmembrane mechanism at high peptide concentration. Another set of neutron diffraction experiments examined the interaction between the TAT peptide and neutral phospholipids showed that TAT peptide preferentially intercalated into the hydrophobic core and the glycerol backbone region of the neutral lipid bilayer at the lowest peptide concentration investigated (0.1mol%), indicating that the insertion did not require negatively-charged phospholipids. There was also clear evidence for the concentration-dependent reorientation of TAT peptide. A plasmid containing the human copper-zinc SOD gene linked with the coding sequence for a 11-aa HIV-TAT peptide (pGEX-TAT-SOD, 513bp) was constructed and used to express a recombinant fusion protein in Escherichia coli strain BL21 (DE3). High-level expression of TAT-SOD soluble protein with a GST tag (44-kDa) was achieved under optimal expression conditions and a small-scale glutathione affinity column or large-scale ion-exchange chromatography used for its purification. The potential protective effect of TAT-SOD against UV-induced cell damage was studied on UVC-irradiated MDCK epithelial cells. Before any further clinical study, the UV full-length absorption of TAT-SOD protein was measured. The results showed the potential UV protective effect of TAT-SOD was not due to the physical absorption of UV irradiation. In a preclinical study with five healthy volunteers, the penetration of TAT-SOD through human stratum corneum on the inner upper arm was identified by the tape stripping and specific SOD activity analysis. Significant increases on SOD activity were found on the outer layers of stratum corneum in TAT-SOD treated group, compared to placebo treated control, indicating that the TAT peptide assisted SOD to penetrate into the human stratum corneum . In a clinical study with ten healthy volunteers, eight showed a significant increase of minimal erythema dose (MED) with TAT-SOD pre-treatment. The median blood flow value of ten subjects at the UVB-irradiated site decreased with TAT-SOD pretreatment. Taken together, this evidence showed that TATvi SOD did have a marked protective effect against UVB induced skin damage. In a second clinical study, five healthy volunteers were challenged with a series of UVB doses. Skin punch biopsies were taken from four test sites on the lower back for H&E and immunohistochemical staining analysis. UVB-induced apoptotic sunburn cell (SBC) formation, p53 up-regulation and thymine dimer formation in epidermis were not attenuated by pretreatment with TAT-SOD. These data suggest that transdermal superoxide scavenger TAT-SOD reduced the UVB-induced inflammation, but did not abrogate the direct DNA damage of UVB irradiation on the skin. However, the hope of TAT-SOD could reduce UVA indirect DNA damage remains.

La protéine Tat (Trans-activator of transcription) est certainement l’une des cibles présentant le plus d’intérêts dans la lutte contre le VIH-1. En effet, synthétisée précocement, elle joue un rôle central dans le cycle viral et protège les cellules infectées. Secrétée dans le milieu extracellulaire, elle participe à l’immunodéficience en inhibant certaines fonctions ou en induisant l’apoptose des cellules du système immunitaire. Elle est également impliquée directement dans de nombreuses pathologies associées au VIH-1. Dans une première étude, nous avons voulu savoir si la trithérapie était capable d’inhiber la synthèse et la sécrétion de la protéine Tat. Nous avons proposé à des patients infectés, sous trithérapie, ayant une charge virale indétectable, de nous permettre de faire cinq prélèvements sanguins (un tous les trois mois pendant un an) afin de vérifier la présence d’anticorps anti-Tat. Nous avons pu constater que 86% des patients avaient des anticorps anti-Tat mais que ces anticorps pouvaient disparaître ou apparaître chez une majorité de patients, démontrant que la protéine Tat continue d’être sécrétée malgré les antiviraux. Une deuxième étude, sur des sérums de patients, a été effectuée afin de déterminer si la protéine Tat était structurée dans le sang des patients. Il existait une polémique, dans la littérature, sur le fait que la protéine Tat soit naturellement non structurée. Nous avons démontré que la protéine Tat est structurée dans le sérum de patients. De plus, l’activité biologique de la protéine Tat est étroitement liée à l’acquisition de sa conformation. Dans le cadre du développement clinique d’un vaccin anti-Tat dans le laboratoire, nous avons effectué des vaccinations sur des souris afin de déterminer la dose, l’adjuvant, la voie d’administration et le nombre de rappels à effectuer ainsi que la vérification de la tolérance et de la toxicité du vaccin. Des essais cliniques sont en préparation dans le cadre d’un protocole thérapeutique. Le laboratoire développe également une autre approche thérapeutique avec un anticorps monoclonal de souris capable de reconnaitre et de neutraliser les variants Tat représentatifs des cinq principaux sous-types du VIH-1. Cet anticorps qui sera humanisé, servirait à une future immunothérapie en combinaison à la trithérapie pour des patients en phase précoce ou tardive ou encore pour des nouveaux nés dont le système immunitaire est peu fonctionnel. / The protein Tat (Trans-activator of transcription) is definitely one of the most interesting targets in the fight against HIV-1. Synthesized early, it plays a central role in the viral life cycle and protects infected cells. Secreted into the extracellular medium, it participates in the immunodeficiency by inhibiting some functions or inducing apoptosis of immune cells. It is also directly involved in many diseases associated with HIV-1. In a first study, we examined whether HAART was able to inhibit the synthesis and secretion of the Tat protein. We proposed to HIV-1 infected patients under HAART, with undetectable viral load, to allow us to do five blood samplings (one every three months for one year) to verify the presence of antibodies against Tat. We found that 86% of patients had antibodies against Tat but these antibodies could disappear or appear in a majority of patients showing that Tat protein continues to be secreted in spite of antiviral treatment. A second study of patient sera was carried out to determine if Tat was structured in the blood of patients. There was a controversy in the literature about the fact that Tat could be naturally unstructured. We showed that Tat is structured in the serum of patients. In addition, the biological activity of Tat is closely related to the acquisition of its conformation. As part of a clinical development of a Tat vaccine in the laboratory, we carried out vaccinations in mice to determine the dose, the adjuvant, the route of administration, the number of boosts, tolerance and toxicity of the vaccine. Clinical trials are planed with a therapeutic protocol. The laboratory is also developing another therapeutic approach with a mouse monoclonal antibody able to recognize and neutralize Tat variants representative of the five major subtypes of HIV-1. This antibody will be humanized and could be used for future immunotherapy, in combination with HAART for patients in early or late stage of the pathology or to newborn babies who have a weak immune system.

Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

Impaired cell mediated immunity is the clinical hallmark of HIV infection yet the manner in which CD8 T-cells are disabled is not yet fully understood. IL-7 signalling is essential for normal CD8 T-cell development and function. Our lab has previously shown decreased expression of the IL-7 receptor a-chain (CD127) on circulating CD8 T-cells in HIV+ patients is mediated by the HIV Tat protein which results in poor CD8 T-cell function. Soluble Tat protein is secreted by infected CD4 T-cells and taken up by neighbouring uninfected CD8 T-cells through endocytosis. Once in the cytoplasm, Tat translocates to the inner leaflet of the cell membrane where it binds directly to the cytoplasmic tail of CD127 inducing receptor aggregation, internalization, and degradation by the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is able to reduce IL-7 signaling and impair CD8 T-cell proliferation and function. To determine which domain(s) in the cytoplamic tail of CD127 are required for interaction with Tat, a series of plasmids encoding for CD127 deletion mutants were successfully created. These series of mutant CD127 coding sequences were transfected into a eukaryotic expression system, the Jurakt cell line, where CD127 mutants were successfully expressed. Before determine which region on CD127 is required for Tat binding, an optimized Ni-NTA column system was used to successfully isolate histidine-tagged HIV-1 Tat at a high yield and purity from E. coli. This HIV Tat protein was used to treat the lysates of the Jurakt cells transfected with the panel of CD127 mutants. CD127 was then immunoprecipitated, followed by Western analysis of the immune complexes to detect Tat protein. Tat was immunoprecipitated with all CD127 mutants suggests neither tyrosine 449, box 1, the acidic region, serine region nor C-tail are specifically required for Tat binding to CD127.

Endothelial dysfunction and/or injury is pivotal to the development of cardiovascular and inflammatory pathology. Endothelial dysfunction and/or injury has been described in Human Immunodeficiency Virus (HIV) infection. Elaboration of circulating markers of endothelial activation, such as soluble adhesion molecules and procoagulant proteins, occurs in HIV infection. Certain endothelial cells, such as those lining liver sinusoids, human umbilical vein endothelial cells, bone marrow stromal endothelial cells or brain microvascular endothelial cells, have been shown to be variably permissive for HIV infection. Entry of virus into endothelial cells may occur via CD4 antigen or galactosyl-ceramide receptors. Other mechanisms of entry including chemokine receptors have been proposed. Nevertheless, endothelial activation may also occur in HIV infection either by cytokines secreted in response to mononuclear or adventitial cell activation by virus or else by the effects of the secreted HIV-associated proteins, gp 120 (envelope glycoprotein) and Tat (transactivator of viral replication) on endothelium. Enhanced adhesiveness of endothelial cells, endothelial cell proliferation and apoptosis as well as activation of cytokine secretion have all been demonstrated. Synergy between select inflammatory cytokines and viral proteins in inducing endothelial injury has been shown. In HIV infection, dysfunctional or injured endothelial cells potentiate tissue injury, inflammation and remodeling, and accelerate the development of cardiovascular disease.

Unlike infrastructure-based networks, mobile ad hoc networks consist of nodes independent of any infrastructure. Cooperation among these nodes is essential for the sustenance of multi hop communication. However, battery and bandwidth constraints may lead nodes in an ad hoc network to adopt energy- and bandwidth-conserving strategies. As routing and packet forwarding are end results of cooperation, network performance is affected when nodes in the network behave selfishly to conserve their resources. Our work involves characterizing selfish behavior by nodes in ad hoc networks and assessing the effectiveness of adopting tit for tat based strategies, which are meant to discourage selfish behavior in the network. We show that in an ad hoc network where other nodes act selfishly to conserve their resources, a node can benefit by adopting a generous tit for tat strategy. We also show that a node can gain benefit by avoiding selfish nodes in an ad hoc network, adopting a strategy that we call generous tit for tat with selfish avoidance (GTFT-SA) To analyze the effectiveness of cooperation strategies in selfish ad hoc networks we create an emulation environment based on virtualization. Such an emulation environment is more flexible to changes and is simpler to replicate than real life testbeds, while providing higher fidelity than simulations. / Master of Science

Le peptide Tat est un des "cell penetrating peptides" (CPP) les plus utilisés pour l'internalisation cellulaire de diverses molécules cargos. La molécule chimérique (Tat-cargo) induit une réponse biologique plus efficace comparée au cargo seul. Cependant, en marquant le peptide Tat à l'iode 125, il a été déterminé que seulement moins de 1 % de la quantité initiale de peptide est internalisé. Il y a donc une opportunité d'augmenter l'efficacité de cette internalisation. L'étape cruciale du processus d'internalisation est le passage transmembranaire. Cette thèse présente l'évaluation de l'impact d'un groupement hydrophobe intégré en différentes positions au peptide Tat, afin de favoriser son interaction avec la membrane. Un acide aminé modifié chimiquement comportant un groupement cholestéryle a été développé dans ce sens. Cet aminoacide peut être intégré en toute position du peptide Tat. Différentes positions au sein du peptide Tat ont été cholestérylées et l'effet sur le taux d'internalisation a été étudié par cytométrie en flux et par comptage suite au radiomarquage des peptides à l'iode 125.L'ajout de cholestérol en position centrale du peptide Tat induit une efficacité d'internalisation supérieure d'un facteur 30 alors qu'une augmentation moindre est observée suite à l'ajout du groupement hydrophobe en positions latérales, N- ou C-terminale. / The Tat peptide is one of the most used cell penetrating peptides for internalizing various cargo molecules into cells. The chimaeric molecule thus triggers an efficient cellular biological response when compared with the cargo molecule alone. However, following labeling of the Tat peptide with radiolabeled iodine, less than 1% of the external peptide was internalized. Therefore, there is an opportunity to improve the level of CPP internalization. The ultimate step is the crossing through the plasma membrane. This thesis presents an evaluation of the impact of a hydrophobic group incorporated at different positions to Tat peptide, to promote its interaction with the membrane.A chemically modified amino acid comprising a cholesteryl group was developed in this direction. This amino acid can be inserted at any position within the Tat peptide. Different positions within the Tat peptide were cholesterylated and the effect on the internalization rate of Tat CPP was investigated by flow cytometry and by counting following the radiolabeling of peptides with iodine 125.The addition of cholesterol in the central position of the peptide Tat induces internalization efficiency than a factor of 30 while a smaller increase was observed after the addition of hydrophobic group in lateral positions, N-or C-terminus.