Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Apoptosis is the mechanism by which complex multicellular organisms induce the programmed death of damaged cells, thus maintaining tissue homeostasis. One of the main hallmarks of cancer, apoptosis is tightly regulated by pro- and anti-apoptotic factors whose equilibrium will decide of the fate of the cell. Among these factors, the cellular inhibitor of apoptosis cIAP1 is a key regulator of nuclear factor-κB dependent signaling and of caspase-8 mediated apoptosis. cIAP1 expression is controlled primarily at the translational level through an internal ribosome entry site (IRES) that facilitates the recruitment of the ribosome to the translation initiation start independently of the 5’ cap. We have previously identified four putative IRES trans-acting factors (ITAFs) that bind specifically to the cIAP1 IRES, namely NF45, NF90, IGF2BP1 and RH1. My research project characterised NF45 as an ITAF that positively regulates the IRES-mediated translation of cIAP1 and of the Xlinked inhibitor of apoptosis, XIAP. This regulation is important for maintaining Survivin and Cyclin E protein levels and insuring proper cell division. Furthermore, I showed that IGF2BP1 is another ITAF that is overexpressed in rhabdomyosarcoma cancer (RMS) and positively regulates cIAP1 translation, thus leading to apoptotic resistance in these cells. Importantly, the use of Smac mimetics, chemical compounds that cause cIAP1 proteasomal degradation, induces TNFα-mediated apoptosis of RMS cells and leads to growth inhibition of RMS xenograft tumors as well as significantly improved survival. Finally, I show that certain modulators of innate immunity synergize with Smac mimetics to improve the killing of RMS cancer cells. Hence, cIAP1 translation regulation by NF45 and IGF2BP1 is highly important for maintaining proper functioning of the cell and dysregulation of these ITAFs can lead to carcinogenesis.

Macrophages can be polarized into M1 and M2 macrophages based on the composition of the milieu. Human macrophages have been poorly characterized. In this study, various macrophage subsets were generated by treating monocyte-derived macrophages (MDMs) with IFNγ (M1), IL-4 (M2a), LPS and IL-1β (M2b) or IL-10 (M2c) which were characterized with respect to their cell surface marker profile and functional profile in the context of cytokine production, susceptibility to HIV infection and apoptosis. Each polarization state demonstrated a unique cell surface marker profile and cytokine profile. In addition M1 macrophages were shown to produce IFNγ post TLR stimulation. Moreover, M1 macrophages were highly sensitive to apoptosis following Smac mimetic treatment. Furthermore, M2a and M2c macrophages were resistant to apoptosis, induced by PI3K blockage and IAPs degradation respectively, and at the same time supported productive HIV infection unlike the other macrophage subsets. These findings might lead to better understanding of HIV reservoir formation and be used to develop therapies to eradicate it.

A novel marine active polypeptide (PCF), isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether UVB-induced HaCaT cell apoptosis occurs via the mitochondrial pathways Apaf-1/caspase-9 and Smac/XIAP/caspase-3. We then investigated the molecular mechanisms controlling the anti-apoptotic effect of PCF. Pre-treatment with PCF and caspase-9 inhibitor significantly inhibited UVB-induced apoptosis in HaCaT cells based on a DNA fragmentation assay and Hoechst 33258 staining. The expression of Apaf-1 and the cleavage of procaspase-9 were dose-dependently reduced by 1.42-5.96 mmol/L PCF pretreatment in UVB-irradiated HaCaT cells. This was followed by inhibition of cleavage of procaspase-3, whose activation induced cell apoptosis. Meanwhile, PCF significantly and dose-dependently enhanced the activation of ATPase. Furthermore, we demonstrated that PCF strongly inhibited the release of Smac from the mitochondria to cytosol by reducing the degradation of XIAP dose-dependently. We conclude that the protective effect of PCF against UVB irradiation in HaCaT cells may be attributed to the inhibition of the Apaf-1/caspase-9 and Smac/XIAP/caspase-3 apoptotic signaling pathways.

The inflammatory and anti-inflammatory macrophages have been implicated in many diseases including rheumatoid arthritis, inflammatory bowel disease and chronic rhinosinusitis. Recent studies suggest targeting macrophage function and activation may represent a potential target to treat these diseases. Herein, I investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of normal human pro- and anti-inflammatory macrophage subsets. It has been shown that human monocytes are highly susceptible to the cytotoxic effects of SMs, however, differentiated macrophages (M0) develop resistance to the cytocidal abilities of SMs. Whether human macrophage subsets are also resistant to the cytotoxic effects of SM remains unknown. My results show that differentiation of M0 macrophages towards M1 state rendered them highly susceptible to SM-induced cell death, whereas M2a, M2b and M2c differentiated subsets were resistant, with M2c being the most resistant. SM-induced cell death in M1 macrophages was mediated by apoptosis as well as necroptosis and activated both extrinsic and intrinsic pathways of apoptosis. The susceptibility of M1 macrophages to SM-induced cell death was attributed to the IFN-𝛾-mediated polarization as JAK inhibitor reversed their susceptibility. In contrast, M2c and M0 macrophages experienced cell death through necroptosis pathway following simultaneous blockage of the IAPs pathways by SM-LCL161 and the caspase pathways by the pan-caspase inhibitors (zVAD.fmk). I investigated the molecular mechanism governing SM-induced cell death in M1 macrophages. My results show that in contrast to the cancer cell lines, SM-induced cell death in M1 macrophages is independent of endogenously produced TNF-⍺, the canonical and non- canonical NF-𝜅B pathways. The susceptibility of M1 macrophages to SM-induced cell death was found to be dependent on IFN-𝛾-mediated differentiation through the JAK-STAT pathway and subsequent activation of IRF-1. In addition, the selective cell death in SM-treated M1 macrophages is mediated by simultaneous degradation of cellular IAP-2 (cIAP-2) and RIPK-1/3 through the activation of mTORC signaling pathway. Overall, the results suggest that survival of human macrophages is critically linked to the activation of the IAPs pathways. Moreover, agents blocking cIAP-1/2, mTORC and IRF-1 can be exploited therapeutically to address inflammation-related diseases. These observations hold a promising therapeutic strategy to limit the activation of proinflammatory M1 macrophages and eventually controlling the M1-associated diseases.

Multiple myeloma is an aggressive plasma cell cancer that consistently acquires multi-drug resistance and relapses despite initial treatment successes. Patients may go through greater than 10-lines of therapy, highlighting the need for more effective treatment options. Immunotherapies are the latest evolution in targeted cancer treatments, and thus far have displayed impressive results in several hematological cancers, including multiple myeloma. T cells possess robust anti-tumor functions which can be harnessed and refined for the treatment of cancers. Genetic engineering of T cells to express a chimeric antigen receptor (CAR) confers antigen-specific tumor-targeting, and adoptive transfer of patient-derived CAR-engineered T (CAR T) cells has been efficacious in relapsed/refractory multiple myeloma. Despite the high efficacy, CAR T cell therapy for myeloma is associated with serious adverse events, which limits dose levels and patient eligibility. We have developed a novel synthetic antigen receptor platform, called the T cell antigen coupler (TAC) receptor, which has shown comparatively higher efficacy with a reduced pro-inflammatory profile compared with CAR T cells in pre-clinical models. The TAC receptor was purpose-built to co-opt the natural T cell activation machinery and lacks the costimulatory signaling typically incorporated in CAR designs. This thesis investigates strategies to augment TAC T cell function against for multiple myeloma through the evaluation of ancillary pharmacological and protein stimuli that would complement the anti-tumor functions of TAC T cells without modifying the TAC receptor design. In chapter 2, I investigated a strategy combining TAC T cells with the SMAC mimetic LCL161 to provide transient costimulatory effects. While LCL161 boosted TAC T cells survival and proliferation, the drug also enhanced susceptibility of TAC T cells to apoptosis and offered no advantage to the TAC T cells when challenged with myeloma. In chapter 3, I engineered TAC T cells to secrete IL-27 in an attempt to modulate the myeloma microenvironment and support T cell cytolytic function. IL-27 did not enhance the anti-tumor activity of TAC T cells but forced expression of IL-27 led to a reduction in the production of pro-inflammatory cytokines without altering cytotoxicity. In appendix I, I describe the process of optimizing CRISPR/Cas9 editing of primary TAC T cells. This methodology was required for much of the work in chapter 2. Ph.D. Thesis – Arya Afsahi McMaster University – Biochemistry and Biomedical Sciences v In appendix II, I describe an assessment of mRNA-engineering as a method to produce TAC T cells. This approach proved to be therapeutically futile and was not pursued beyond the work described herein. The work presented here highlights methods of combining TAC T cells with a clinically relevant SMAC mimetic, or the cytokine IL-27, and provides insights into the biological mechanisms that are affected by these approaches. / Thesis / Doctor of Philosophy (PhD)

Made available in DSpace on 2014-12-17T14:55:35Z (GMT). No. of bitstreams: 1 FelipeDMO.pdf: 2614728 bytes, checksum: fd69e303891800912ab5260562a5545d (MD5) Previous issue date: 2009-02-13 / This dissertation aims to develop a software applied to a communication system for a wireless sensor network (WSN) for tracking analog and digital variables and control valve of the gas flow in artificial oil s elevation units, Plunger Lift type. The reason for this implementation is due to the fact that, in the studied plant configuration, the sensors communicate with the PLC (Programmable and Logic Controller) by the cables and pipelines, making any changes in that system, such as changing the layout of it, as well as inconveniences that arise from the nature of the site, such as the vicinity s animals presence that tend to destroy the cables for interconnection of sensors to the PLC. For software development, was used communication polling method via SMAC protocol (Simple Medium Access ControlIEEE 802.15.4 standard) in the CodeWarrior environment to which generated a firmware, loaded into the WSN s transceivers, present in the kit MC13193-EVK, (all items described above are owners of Freescale Semiconductors Inc.). The network monitoring and parameterization used in its application, was developed in LabVIEW software from National Instruments. The results were obtained through the observation of the network s behavior of sensors proposal, focusing on aspects such as: indoor and outdoor quantity of packages received and lost, general aspects of reliability in data transmission, coexistence with other types of wireless networks and power consumption under different operating conditions. The results were considered satisfactory, which showed the software efficiency in this communication system / Este trabalho tem por finalidade desenvolver um software aplicado a um sistema de comunica??o de uma rede de sensores sem fio (RSSF), para monitoramento de vari?veis anal?gicas, digitais e comando de v?lvulas de passagem do fluxo de g?s em unidades de eleva??o artificial de petr?leo e g?s natural do tipo Plunger Lift. A raz?o desta implementa??o deve-se ao fato que, na configura??o da planta estudada, os sensores comunicam-se com o CLP (Controlador L?gico Program?vel) atrav?s de cabos e dutos, dificultando eventuais modifica??es nesse sistema, tais como mudan?a de layout do mesmo, al?m de inconveni?ncias que venham a surgir da pr?pria natureza do local, como a presen?a de animais nas redondezas que tendem a destruir os cabos de interconex?o dos sensores ao CLP. Para o desenvolvimento do software, foi utilizado o m?todo de comunica??o polling, atrav?s do protocolo SMAC (Simple Medium Access Control - padr?o IEEE 802.15.4), no ambiente CodeWarrior, ao qual gerou um firmware, carregado nas placas de monitoramento da RSSF, presentes no kit MC13193-EVK, (todos os itens descritos acima s?o propriet?rios da Freescale Semiconductors Inc.). O monitoramento e parametriza??o da rede utilizou uma aplica??o, desenvolvida no software LabVIEW, da National Instruments. Os resultados foram obtidos atrav?s da observa??o do comportamento da rede de sensores proposta, focando aspectos, tais como: quantidade de pacotes recebidos e perdidos em ambientes externos (Outdoor) e internos (Indoor), aspectos gerais de confiabilidade na transmiss?o dos dados, coexist?ncia entre outros tipos de redes sem fio e consumo de energia sob diferentes condi??es de opera??o. Os resultados obtidos foram considerados satisfat?rios, o que comprovou a efici?ncia do software neste sistema de comunica??o

The NF-κB family of transcription factors can be activated through canonical (classical) or non-canonical (alternative) signalling pathways, which are regulated by the redundant ubiquitin ligases, cellular inhibitor of apoptosis 1 and 2 (cIAP1 and cIAP2). While the canonical NF-κB pathway is needed for myoblast proliferation, it is inactivated during myoblast differentiation. However, the non-canonical NF-κB pathway is a major factor in promoting myoblast fusion, which is crucial to the processes of myogenesis and muscle repair. Ablation of cIAP1 levels through a chemical antagonist such as a SMAC- mimetic compound (SMC) activates non-canonical signalling to enhance myogenesis. The cytokine TNF-like weak inducer of apoptosis (TWEAK) has also been shown to activate primarily the alternative NF-κB pathway when signalling through its receptor Fn14. Here I show that alternative NF-κB signalling activity, stimulated by the addition of TWEAK or loss of cIAP1, can promote myogenesis. I also demonstrate that TWEAK is an endogenous myokine produced by myoblasts to promote their own differentiation, and suggest that targeting the alternative NF-κB pathway, with SMAC-mimetics or recombinant TWEAK for example, would be of therapeutic value in the repair and regeneration of muscle for various myopathies.

Contexte. Les carcinomes des voies aérodigestives supérieures (VADS) arrivent en 6ème position parmi les cancers les plus fréquents au niveau mondial. La fonction du récepteur TLR3 dans les cellules de carcinomes des VADS est encore très mal comprise. Objectifs et méthodes. 1) Déterminer le niveau d’expression du récepteur TLR3 dans les lignées et les biopsies de carcinomes des VADS par western blot et par immunohistochimie. 2) Etudier le rôle de TLR3 dans la croissance tumorale de ces tumeurs, en utilisant notamment des lignées invalidées de façon conditionnelle pour TLR3. 3) Evaluer in vitro les effets cytotoxiques de ligands artificiels de TLR3 soit seuls, soit utilisés en combinaisons avec un inhibiteur d’IAP (inhibitor of apoptosis protein).Résultats. La protéine TLR3 est détectée à un niveau élevé en western blot dans les lignées de carcinomes des VADS étudiées, comparativement à un panel d’autres tumeurs épithéliales humaines. TLR3 est également constamment détecté en immunohistochimie dans les biopsies. TLR3 semble jouer un rôle dans la croissance tumorale des carcinomes des VADS : dans certaines conditions de culture (culture en hypoxie ou en milieu pauvre en SVF et en nutriments), la stimulation de TLR3 par un ligand exogène, le poly(A:U), favorise la croissance des cellules tumorales. Nous avons étudié l’effet de la stimulation de TLR3 sur le métabolisme glucidique dans ces mêmes cellules en utilisant un appareil de type Seahorse® qui mesure la consommation d’oxygène et la production de protons à partir de cellules cultivées en microplaques. Ces expériences montrent que la stimulation de TLR3 fait augmenter l’activité des voies du métabolisme cellulaire anaérobie (glycolyse extra-mitochondriale). Une étude métabolomique a mis en évidence des différences significatives dans le profil métabolique des cellules tumorales stimulées par le poly(A:U) comparativement aux cellules non traitées. Par ailleurs, nous avons montré que la stimulation de TLR3 permettait de détecter le facteur de transcription HIF1 en Western blot, même en conditions normoxiques. Sachant que des ARN libérés par des cellules en état de nécrose peuvent stimuler TLR3, il est tentant de penser que ce récepteur pourrait favoriser la survie des cellules malignes en zone hypoxique au voisinage de cellules nécrotiques. Néanmoins, l’expression de TLR3 représente aussi un facteur de vulnérabilité pour les cellules de carcinome des VADS : en effet les ligands artificiels de TLR3 utilisés en combinaison avec un inhibiteur d’IAP (Inhibitor of Apoptosis Protein) produisent des effets cytotoxiques sur les lignées de carcinomes des VADS étudiées. / Background. Head and Neck (HN) carcinomas are the 6th most frequent type of cancer worldwide. The role of the TLR3 receptor in HN carcinomas remains poorly understood.Objectives and Methods. 1) To assess the expression level of TLR3 in HN carcinoma cell lines and biopsies by Western blot and immunohistochemistry, respectively. 2) To study the role of TLR3 in tumour growth using specific cell lines with conditional knock-down of TLR3. 3). To assess in vitro the cytotoxic effects of artificial ligands of TLR3 used either alone or in combination with an IAP (inhibitor of apoptosis protein) inhibitor.Results. TLR3 protein was detected at a high level by Western blot analysis in HN carcinoma cell lines, by comparison with a panel of other human epithelial cancer cell lines. TLR3 was also consistently detected by immunohistochemistry in tumour biopsies. TLR3 seem to play a role in HN carcinoma cell growth: under certain culture conditions (hypoxic or low fetal calf serum/low nutrient culture conditions), TLR3 stimulation by a synthetic ligand, the poly(A:U), favours tumour cell growth. We investigated the effects of TLR3 stimulation on glucose metabolism using a Seahorse® analyzer, which measures the oxygen consumption and the proton production in living cells. Our results indicate that TLR3 stimulation induces an increase in anaerobic metabolism (extra-mitochondrial glycolysis). A metabolomic study revealed significant changes in the metabolic profile of cancer cells treated by poly(A:U) by comparison with untreated cells. We also showed that under TLR3 stimulation, HIF1 became detectable by Western blot analysis, even in normoxia. Given the fact that RNA fragments released by dying cells are able to trigger TLR3, one can assume that TLR3 might favour cancer cell survival in hypoxic areas located near the necrotic core of the tumour. However, TLR3 expression is also a factor of vulnerability for HN carcinoma cells: indeed, the combination of TLR3 artificial ligands with an IAP inhibitor has a strong cytotoxic effect on HN carcinoma cells in vitro.

Made available in DSpace on 2014-12-17T14:56:21Z (GMT). No. of bitstreams: 1 CarloFPSC.pdf: 941846 bytes, checksum: 5fe2e2c9851039018a0653de693d001c (MD5) Previous issue date: 2006-11-27 / Wireless sensor networks are reality nowadays. The growing necessity of connectivity between existing industrial plant equipments pushes the research and development of several technologies. The IEEE 802.15.4 LR-WPAN comes as a low-cost and powersaving viable solution, which are important concerns while making decisions on remote sensoring projects. This study intends to propose a wireless communication system which makes possible the monitoring of analogic and/or digital variables (i. e., the pressure studied) involved on the artificial methods for oil and gas lifting. The main issues are: To develop a software based on SMAC Standard in order to create a wireless network to monitoring analogic and/or digital variables; To evaluate the communication link based on the number of lost packets tested in different environments (indoor and outdoor) and To propose an instrumentation system consisting of wireless devices / As redes de sensores sem fio (RSSS) j? s?o uma realidade hoje em dia, pois a grande necessidade de conectividade entre os equipamentos existentes numa planta industrial impulsiona a pesquisa e o desenvolvimento de diversas tecnologias. O padr?o LR-WPAN IEE 802.15.4 apresenta-se como uma solu??o vi?vel, com baixo custo e baixo consumo de energia, requisitos importantes para a implanta??o de um sistema de sensoriamento remoto. A contribui??o deste trabalho consiste em propor um sistema de comunica??o sem fio, que possa monitorar vari?veis anal?gicas e/ou digitais (no caso do estudo em quest?o, a press?o) nos m?todos de eleva??o artificial de petr?leo e g?s natural. Os pontos principais deste estudo s?o: Desenvolver um software baseado no Padr?o SMAC com intuito de criar uma rede sem fio com fins de monitoramento de vari?veis anal?gicas e/ou digitais; Avaliar o enlace de comunica??o com rela??o a quantidade de pacotes perdidos em diferentes ambientes (ambientes internos (Indoor) e externos (Outdoor)) e, Propor um sistema de instrumenta??o utilizando tecnologia sem fio