Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

As multidrug resistance in Gram-negative bacilli increases, the old antibiotic colistin has rapidly gained attention as one of few last line treatment options in the form of colistin methanesulfonate (CMS), which is hydrolyzed to colistin both in vitro and in vivo. There is a dearth of knowledge on fundamental aspects of colistin, including pharmacokinetics and optimal dosing regimens. The aim of this thesis was to improve the basis for optimal colistin therapy. To be able to study colistin, an LC-MS/MS assay method was developed which is sensitive, specific and useful in both in vivo and in vitro studies. Using this method we detected a significant loss of colistin during standard laboratory procedures. This loss was characterized and quantified, the hypothesis being that the loss is mainly caused by adsorption to labware. The pharmacokinetics of colistin was studied in two populations of critically ill patients, one with normal renal function and one with renal replacement therapy. Plasma concentrations were assayed with the method above, and population modeling was employed to describe the data. The results include a previously unseen, long elimination half-life of colistin. The data from the population on renal replacement therapy was described without modeling, and showed that both CMS and colistin are cleared by hemodiafiltration. Combination therapy is an approach that is often used when treating patients infected with multidrug-resistant pathogens. The thesis discusses how the joint effect of antibiotics can be measured using colistin and meropenem as a model, and proposes a method for testing antibiotic combinations. Furthermore, a PKPD model was adapted to describe the pharmacodynamics of the combination. In conclusion, a specific and sensitive method for analysis of colistin was developed and the adsorption of colistin to materials was described. The assay method has been well accepted internationally. The pharmacokinetics of colistin and CMS was described in two important patient populations, partly with surprising results that have influenced dosages of colistin worldwide. The pharmacodynamics of combination therapy was investigated and quantified, and the methods applied could be further developed into clinically useful tools for selection of antibiotic combinations.

Malaria is a disease of major public health importance, killing approximately one million people and causing about 250 million cases of fever annually. It mostly affects children under the age of five and pregnant women in many developing countries, making it a prominent issue in international health and maternal and child health. The most aggressive form of malaria is caused by the parasite Plasmodium falciparum which is responsible for 80% of infections and 90% of deaths from malaria, and is most prevalent in sub-Saharan Africa. Public Health interventions include the implementation of prevention programs, health education, and chemotherapy. The latter has experienced multiple problems in the past years whereby resistance of the parasite to the available drugs has emerged, rendering the majority of them ineffective. Furthermore, the high cost of those drugs represents a major obstacle to their dispensation in areas of the world where the affected people are often the less fortunate. The enzyme Cytochrome b5 Reductase (cb5r) and its electron acceptor Cytochrome b5 (cb5) play a role in fatty acid elongation, cholesterol biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. Therefore, these proteins are suitable as potential novel drug targets for malaria. These two proteins have been thoroughly studied in mammals but have to be characterized in microorganisms such as fungi and parasites, including Plasmodium falciparum. It is important to note that plant cb5r has been identified as a novel herbicidal target. Considering the close phylogenetic relationship between plant cb5r and Plasmodium falciparum cb5r, we conclude that these plant inhibitors may also serve as promising candidates for a new class of antimalarial drugs against the parasite. In this project, we want to obtain the biochemical and enzymatic characterization of cb5r and cb5 in order to establish whether these two proteins represent potential novel drug targets in Plasmodium falciparum malaria. This initial work may lead to the development of novel drugs which will consequently affect the field of public health with respect to drug delivery, drug resistance, and drug chemotherapy.

Nowell (1976) first proposed that unless cytotoxic cancer therapy eradicates all tumor cells, genetic or heritable variation within heterogeneous tumors will inevitably lead to the evolution of chemotherapeutic resistance through clonal selection. This evolutionary hypothesis was formalized by Goldie and Coldman (1979), who developed one of the earliest mathematical kinetic models of resistance evolution in neoplasms. Their model predicted that the likelihood of response and cure would be increased in combination vs single agent cytotoxic therapies. In a later study, Gardner (2002) developed a computational kinetic model to predict chemotherapeutic combinations, doses, and schedules most likely to result in patient response and prolonged life. This model predicts that combination therapy involving both cytotoxic and cytostatic drugs will be more effective than combination therapy involving only cytotoxic drugs. Thus far, no systematic evaluation of the Goldie and Coldman and Gardner hypotheses have been conducted in the metastatic clinical trial setting. Here I test these hypotheses using the results of over 700 phase II, III and II/III clinical trials. I show that, as predicted by Goldie and Coldman, both overall response rate and overall survival were greater in combination arms. Moreover, median duration of response – the key indicator of the rate of resistance evolution - was also greater in combination vs single agent arms. These results suggest that generally combination chemotherapy is more effective than single agent therapy for advanced solid tumors as predicted by Goldie and Coldman (1979) hypothesis and that, at least in the metastatic setting, the potential disadvantages of combination therapy with respect to accelerated resistance evolution are outweighed by the greater waiting times for resistance mutations to arise. By contrast, although combination cytotoxic and cytostatic therapy is associated with a greater average overall response rate than multi agent cytotoxic therapy, this is not the case for both median duration of response and overall survival. Hence, there is no evidence that, in contrast to the predictions of the Gardner (2002) model, combination cytotoxic and cytostatic therapy decreases the rate of resistance evolution relative to that obtaining under combination cytotoxic therapy.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

En nanomédecine, une nouvelle approche consiste à développer des vecteurs synthétiques pour co-délivrer au sein d’une cellule tumorale, un anticancéreux ainsi qu’un siARN, capable de supprimer l’expression d’une protéine impliquée dans les mécanismes de résistance. Les travaux décrits dans ce manuscrit ont été consacrés à la synthèse de nano-vecteurs micellaires pour la délivrance simultanée de ces deux agents thérapeutiques. Une première partie décrit la synthèse et la formulation de micelles nanométriques diacétyléniques photopolymérisables conçues pour délivrer efficacement un siARN. Les propriétés d’encapsulation et de délivrance de ces micelles ont ensuite été étudiées in vitro et in vivo pour une application en thérapie combinatoire. Enfin, une dernière partie présente la fonctionnalisation par interaction électrostatique de ces vecteurs cationiques avec des anticorps préalablement modifiés par des oligonucléotides anioniques pour réaliser un ciblage actif des cellules tumorales. / In the nanomedecine field, a new approach consists in developing synthetic vectors able to co-deliver into a cancer cell, an antitumoral drug and siRNAs that target protein(s) involved in MDR. The work described in this manuscript was dedicated to the development of micellar nanovectors for the intracellular co-delivery of these two therapeutic agents. The first part details the synthesis and the formulation of nanometric photopolymerized diacetylenic micelles adapted for the delivery and intracellular release of the siRNA. Then, the encapsulation and delivery properties of these micelles, bearing histidine polar heads have been investigated in vitro and in vivo for the application of combination therapy. Finally, the last part presents the functionalization by electrostatic interaction of these cationic vectors with antibodies, priorly modified by anionic oligonucleotides. This original and versatile system allowed achieving an active targeting of tumoral cells.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

Breast cancer is the most common cancer among Canadian women and 14-20% will develop lethal metastases within 5 years. A potential novel therapeutic target is Focal Adhesion Kinase (FAK), a cytoplasmic tyrosine kinase. FAK’s expression is inversely correlated with survival and is known to regulate cell migration, proliferation and invasion. While tyrosine kinase inhibitors are historically ineffective as single agents, they are commonly used as part of combination therapies. Therefore, given its central role in tumor cell biology and cell signaling, we hypothesized that inhibiting FAK in combination with pharmacological agents commonly used to treat metastatic breast cancer patients will result in enhanced anti-tumor activity. We combined a commercial FAK inhibitor (PF-562271) with a range of chemotherapeutic agents commonly used to treat metastatic breast cancer and searched for synergistic partners. Only DNA topoisomerase inhibitors showed potential to synergistically reduce cell viability when paired with low doses of the FAK inhibitor. However, the combination does not induce an increase in cell death or apoptosis. It was then discovered that both agents in isolation and in combination produce increased levels of ROS, a toxic metabolite. This, along with other more preliminary data, provides clues for a novel proposed mechanism of action for this interaction.

Magister Public Health - MPH / In Zambia, there had been a large scaling up of new interventions to control malaria since 2003, which included the distribution of rapid diagnostic tests (RDTs), used to immediately determine if someone with symptoms suggestive of malaria actually has malaria; training of health workers in the use of the RDTs; and the prescription of artemisinin-based combination therapy (ACT) to which the malaria parasite is sensitive, rather than the old treatment regime of chloroquine to which the malaria parasite had become resistant. The use of RDTs to confirm the presence of malaria before treating for it with ACT became known as the „test and treat‟ policy. Previously, since the 1960s, in malaria endemic areas such as Zambia, children presenting with fever (the commonest symptom of malaria) without any obvious other cause for the fever, were assumed to have malaria and were hence treated for it with chloroquine. This was known as "presumptive treatment" of malaria. The combination of "presumptive treatment" and the use of a single medication led to the development of high levels of resistance to chloroquine, to the extent that it is now no longer an effective treatment for malaria. Years after the introduction of the "test and treat" policy, it was still unclear to what extent it was being implemented, as there was initial reluctance by health workers to test all children presenting with fever for malaria and if they did test they may not have followed the management guidelines of treating those who test positive with ACT and further investigating those who test negative for the cause of the fever. It seemed that staff had gotten used to the "presumptive treatment" approach to malaria over almost 4 decades and hence were quite reluctant to abandon it. The conflicting guidelines for malaria treatment in children between IMCI and "test and treat‟ has promoted a paradox between presumptive treatment for malaria and "test and treat" approach as IMCI teaches health workers to treat febrile children presumptively for malaria whereas the "test and treat" approach requires them to first make a definitive diagnosis before treating. Hence although the "test and treat" approach was instituted to overcome the problems with presumptive treatment approach it now had to contend with the competing and contradictory influence of the IMCI approach. This study therefore aimed to assess what proportion of children aged five years and younger who presented with fever were managed via the "test and treat" guidelines and which factors were associated with this, in Lusaka District, Zambia. Methodology: A cross sectional analytical study design was used based on a review of medical records. A sample size of 800 medical records of children presenting with fever was selected from 10 out of the 23 health care facilities in Lusaka, using a multistage stratified random sampling technique. Four hundred records were sampled from 2008 records (five years after commencement of the "test and treat" policy) and 400 from 2011 records (eight years after commencement of the "test and treat" policy). Trained data collectors used a data extraction tool to transcribe demographic and clinical data from the medical records in a standardized manner. Data Analysis: Univariate descriptive statistics analysis was performed using measures of central tendency and measures of dispersion to analyze numerical (continuous) variables such as age, weight and body temperature; and using frequencies for categorical variables such as gender, area of residence, RDTs/microscopy malaria tests conducted, received ACT if RDT positive, presence of an ACT treatment chart on the health centre wall and availability of a weighing scale. To determine the relationship between variables, bivariate analysis via the prevalence ratio was conducted. Results: Just over half (55%) of all children with fever were tested for malaria in 2008 and this gratifyingly increased to (73%) in 2011. Overall, the proportion of children correctly and appropriately treated with ACT, which means that those who tested positive for malaria were given ACT, was 85% in 2008 but regrettably dropped to 72% in 2011. Although "presumptive treatment" decreased from 24% in 2008 to 11% in 2011, the proportion of children with fever not tested for malaria, and although not treated for malaria, but left without a definitive diagnosis of their fever being made, remained high but dropping (22% in 2008 and 16% in 2011). Similarly the proportion of children who tested negative for malaria but then did not undergo any further investigation also unfortunately remained very high and rising (57% in 2008 and 89% in 2011). A combination of the above poor clinical management practises resulted in only 38% of children with fever in 2008 and unfortunately dropping to only 33% in 2011 being correctly managed (tested for malaria via RDT or microscopy and treated with ACT if positive, while further investigated for the cause of fever if negative). On preparedness of the health facility to implement the "test and treat" policy, it was noted that only 4 out of 10 health facilities were at least minimally prepared to do so, but paradoxically on bivariate analysis those minimally prepared were less likely (PR 0.62; 95% CI 0.41-0.94) to correctly manage the patients in 2011 than those who were unprepared. A similar paradox occurred for those correctly treated with ACT after testing positive, with facilities which were minimally prepared being less likely to do so (PR 0.28; 95% CI 0.14-0.58) in 2011 than those facilities which were unprepared to implement the "test and treat" policy. However these associations were inconsistent over time, as the associations were not present in 2008. Similarly all other factors such as staff category (doctor, nurse, clinical officer) and type of presenting symptoms besides fever (anorexia, lethargy, pallor) assessed, were not consistently associated with testing for malaria in both 2008 and 2011. The same applied for the other two main outcome variables of 'treated with ACT after test positive for malaria' and 'correctly managed child with fever', in that there were no factors that showed a consistent association with them in both 2008 and 2011. Conclusion: Testing of children with fever for malaria is at a low level but rose between 2008 and 2011. Paradoxically the proportion of those diagnosed with malaria who were correctly treated with ACT dropped between 2008 and 2011, as did the proportion of children with fever who were correctly managed. No factors assessed in this study were found to be consistently associated in both 2008 and 2011 with either testing for malaria, or treating confirmed malaria cases with ACT, or managing patients with fever correctly. Recommendations: In order for health workers to correctly implement the "test and treat" policy, which involves a series of complex steps, they ought to be formally trained to do so, mentored and constructively supervised. Additionally health facilities should be adequately equipped to enable health workers to fully implement the policy. Further studies to assess factors associated with the correct management of malaria via the "test and treat" policy are warranted.

Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu((R))) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.