4-Aminobiphenyl is an aromatic amine compound that is present in cigarette smoke, diesel exhaust, cooking oil fumes and dye intermediates. It is a well-known human bladder carcinogen and liver carcinogen in experimental animals that is metabolically activated by liver CYP1A1/2. We have used the “Accelerated Cytotoxicity Mechanism Screening” (ACMS) techniques to analyze the molecular cytotoxic mechanisms of 4-aminobiphenyl. Hepatocyte exposure to an inflammatory system significantly increased hepatocyte susceptibility to 4-aminobiphenyl. 4-Aminobiphenyl- induced cytotoxicity and lipid peroxidation were both prevented by altering cellular redox status and with the addition of antioxidants. Toxicity was increased with the depletion of hepatocyte GSH levels and by inhibiting N-acetyltransferase. These results will provide more insight into the cytotoxic and genotoxic mechanisms of 4-aminobiphenyl and also suggest that inflammation may be responsible for an increase in arylamine carcinogenesis.

4-Aminobiphenyl is an aromatic amine compound that is present in cigarette smoke, diesel exhaust, cooking oil fumes and dye intermediates. It is a well-known human bladder carcinogen and liver carcinogen in experimental animals that is metabolically activated by liver CYP1A1/2. We have used the “Accelerated Cytotoxicity Mechanism Screening” (ACMS) techniques to analyze the molecular cytotoxic mechanisms of 4-aminobiphenyl. Hepatocyte exposure to an inflammatory system significantly increased hepatocyte susceptibility to 4-aminobiphenyl. 4-Aminobiphenyl- induced cytotoxicity and lipid peroxidation were both prevented by altering cellular redox status and with the addition of antioxidants. Toxicity was increased with the depletion of hepatocyte GSH levels and by inhibiting N-acetyltransferase. These results will provide more insight into the cytotoxic and genotoxic mechanisms of 4-aminobiphenyl and also suggest that inflammation may be responsible for an increase in arylamine carcinogenesis.

Aptamers are single-stranded oligonucleotides, DNA or RNA, which can bind to a myriad of targets such as ions, peptides, proteins, drugs, organic and inorganic molecules with high affinity and specificity. Aptamers are derived using combinatorial libraries comprised of a variable region flanked by two primer regions used for a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The central theme of my thesis was to use this technology to develop aptamers able to bind to validated therapeutic targets, specifically the Tumour Necrosis Factor alpha (TNFα) and Carcinoembryonic Antigen (CEA), and block their biological functions. As well, I investigated the use of CEA and MUC1 binding aptamers as targeting agents to guide and detect the delivery of contrast agent-loaded liposomes in tumour-bearing mice using computed tomography (CT) imaging. Aptamer selections successfully identified a 25-base aptamer (VR11) that can bind with high affinity and specificity to TNFα. VR11 blocked TNFα signaling, prevented apoptosis, reduced nitric oxide (NO) production in cultured cells and was non-immunogenic when injected into C57BL/6 mice. As well, aptamers were derived to the IgV-like N-domain of CEA. Two DNA aptamers were isolated containing a 40-base variable region, N54 and N56, bearing anti- CEA homotypic adhesive properties. These aptamers are not cytotoxic or immunogenic and III are able to prevent CEA-mediated homotypic and heterotypic cell adhesion events. In addition, the pretreatment of murine cancer cells expressing CEA with these aptamers prior to their intraperitoneal injection into C57BL/6 mice resulted in the prevention of tumour foci formation. Finally, the in vivo targeting of nanoparticles such as pegylated liposomes to tumour cells was enhanced by introducing tumour marker-specific DNA aptamers on their surface. The CEA-specific aptamer N54 and a 40-base second generation aptamer MUC1-VR1 that recognizes the tumour-associated mucin MUC1 were incorporated into liposomes containing the CT contrast agent Omnipaque350™ and Cy5 to characterize their binding to CEA and MUC1-expressing cancer cells in vitro. Pharmacokinetic studies also revealed that the incorporation of these aptamers into pegylated liposomes significantly lenghthened their circulation half-lives to values that parrallel that of untargeted pegylated liposomes.

Aptamers are single-stranded oligonucleotides, DNA or RNA, which can bind to a myriad of targets such as ions, peptides, proteins, drugs, organic and inorganic molecules with high affinity and specificity. Aptamers are derived using combinatorial libraries comprised of a variable region flanked by two primer regions used for a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The central theme of my thesis was to use this technology to develop aptamers able to bind to validated therapeutic targets, specifically the Tumour Necrosis Factor alpha (TNFα) and Carcinoembryonic Antigen (CEA), and block their biological functions. As well, I investigated the use of CEA and MUC1 binding aptamers as targeting agents to guide and detect the delivery of contrast agent-loaded liposomes in tumour-bearing mice using computed tomography (CT) imaging. Aptamer selections successfully identified a 25-base aptamer (VR11) that can bind with high affinity and specificity to TNFα. VR11 blocked TNFα signaling, prevented apoptosis, reduced nitric oxide (NO) production in cultured cells and was non-immunogenic when injected into C57BL/6 mice. As well, aptamers were derived to the IgV-like N-domain of CEA. Two DNA aptamers were isolated containing a 40-base variable region, N54 and N56, bearing anti- CEA homotypic adhesive properties. These aptamers are not cytotoxic or immunogenic and III are able to prevent CEA-mediated homotypic and heterotypic cell adhesion events. In addition, the pretreatment of murine cancer cells expressing CEA with these aptamers prior to their intraperitoneal injection into C57BL/6 mice resulted in the prevention of tumour foci formation. Finally, the in vivo targeting of nanoparticles such as pegylated liposomes to tumour cells was enhanced by introducing tumour marker-specific DNA aptamers on their surface. The CEA-specific aptamer N54 and a 40-base second generation aptamer MUC1-VR1 that recognizes the tumour-associated mucin MUC1 were incorporated into liposomes containing the CT contrast agent Omnipaque350™ and Cy5 to characterize their binding to CEA and MUC1-expressing cancer cells in vitro. Pharmacokinetic studies also revealed that the incorporation of these aptamers into pegylated liposomes significantly lenghthened their circulation half-lives to values that parrallel that of untargeted pegylated liposomes.

The delivery of biomolecules by cell penetrating peptides (CPPs) is an innovative therapeutic strategy. However delivery efficiency is hindered by the entrapment of CPPs in vesicles, degradation, or recycling out of cells, which limits their delivery into the cell cytoplasm and nucleus. To overcome these barriers, we investigated a bacterial protein domain derived from Pseudomonas aeruginosa, Exotoxin A (ETA, residues 253-412) that is able to exit vesicular compartments. A series of CPP-ETA(253-412) fusion proteins were constructed, expressed, and purified. Confocal microscopy and flow cytometry confirmed the internalization at 37oC of constructs containing CPPs (poly-arginine or TAT). In addition, constructs containing CPP-ETA(253-412)-eGFP were shown to relocate from endosomes to the cytosol. CPP-ETA(253-412) constructs were also able to act as carriers of DNA cargos facilitating their delivery to the cytosol. The ETA(253-412) translocation domain may prove useful for the intracellular delivery of drugs, protein therapeutics, siRNA delivery, and vaccine formulations.

The delivery of biomolecules by cell penetrating peptides (CPPs) is an innovative therapeutic strategy. However delivery efficiency is hindered by the entrapment of CPPs in vesicles, degradation, or recycling out of cells, which limits their delivery into the cell cytoplasm and nucleus. To overcome these barriers, we investigated a bacterial protein domain derived from Pseudomonas aeruginosa, Exotoxin A (ETA, residues 253-412) that is able to exit vesicular compartments. A series of CPP-ETA(253-412) fusion proteins were constructed, expressed, and purified. Confocal microscopy and flow cytometry confirmed the internalization at 37oC of constructs containing CPPs (poly-arginine or TAT). In addition, constructs containing CPP-ETA(253-412)-eGFP were shown to relocate from endosomes to the cytosol. CPP-ETA(253-412) constructs were also able to act as carriers of DNA cargos facilitating their delivery to the cytosol. The ETA(253-412) translocation domain may prove useful for the intracellular delivery of drugs, protein therapeutics, siRNA delivery, and vaccine formulations.

Nucleic acid analysis is one of the most important disease diagnostic approaches in medical practice, and has been commonly used in cancer biomarker detection, bacterial speciation and many other fields in laboratory. Currently, the application of powerful research methods for genetic analysis, including the polymerase chain reaction (PCR), DNA sequencing, and gene expression profiling using fluorescence microarrays, are not widely used in hospitals and extended-care units due to high-cost, long detection times, and extensive sample preparation. Bioassays, especially chip-based electrochemical sensors, may be suitable for the next generation of rapid, sensitive, and multiplexed detection tools. Herein, we report three different microelectrode platforms with capabilities enabled by nano- and microtechnology: nanoelectrode ensembles (NEEs), nanostructured microelectrodes (NMEs), and hierarchical nanostructured microelectrodes (HNMEs), all of which are able to directly detect unpurified RNA in clinical samples without enzymatic amplification. Biomarkers that are cancer and infectious disease relevant to clinical medicine were chosen to be the targets. Markers were successfully detected with clinically-relevant sensitivity. Using peptide nucleic acids (PNAs) as probes and an electrocatalytic reporter system, NEEs were able to detect prostate cancer-related gene fusions in tumor tissue samples with 100 ng of RNA. The development of NMEs improved the sensitivity of the assay further to 10 aM of DNA target, and multiplexed detection of RNA sequences of different prostate cancer-related gene fusion types was achieved on the chip-based NMEs platform. An HNMEs chip integrated with a bacterial lysis device was able to detect as few as 25 cfu bacteria in 30 minutes and monitor the detection in real time. Bacterial detection could also be performed in neat urine samples. The development of these versatile clinical diagnostic tools could be extended to the detection of various cancers, genetic, and infectious diseases.

Background: Economic evaluations of generalized anxiety disorder (GAD) have been limited to ≤18 months. A decision model was developed; quantifying the lifetime cost-of-illness (COI) of GAD. Methods: An incidence-based Markov-model was developed using TreeAge® software, reflecting 9 health-states (HS): physician-assessed patients (3HS), maintenance therapies(4HS), discontinuation(1HS) and death(1HS). Onset probability (ages 18-80) determined model entry. Canadian Psychiatric Association (CPA) guidelines determined pharmaco-therapy, with revisions/validation by an expert panel. Response, remission based on pooled-analysis of CPA-cited evidence. Remaining clinical rates, absenteeism and hospitalization retrieved from literature. Direct (clinician, pharmacotherapy, hospitalization) and indirect costs (wage rate) retrieved from government publications. Results discounted at 5%. Results: The mean COI was 2008 Canadian $31,213(SD=$9,100)/patient; 96% attributed to absenteeism. Mean age=31years, discontinued treatment=85% by 2nd year, treatment discontinuation duration, 14(SD=9) years. CONCLUSION: GAD is a costly disease with a lifetime COI<$32k/patient; absenteeism exerts a significant impact. Limited prospective data contributes to uncertainty of estimate.

BACKGROUND: Alcohol and tobacco misuse and dependence are highly comorbid disorders. Varenicline alleviates symptoms of cigarette craving while preventing nicotine from binding to nicotinic acetylcholine receptors, thereby reducing nicotine’s reinforcing effects. Recent studies have shown that varenicline decreased alcohol self-administration in animal models and in one human study of heavy-drinking smokers. AIMS: To assess the effect of two-week varenicline (0.5-2mg) vs. placebo administration on cue-induced craving for tobacco and alcohol in smokers with heavy alcohol use (n = 24). METHODS: Subjects participated in two study visits where nicotine and alcohol craving and withdrawal were assessed with self-report questionnaires under four conditions (abstinence/one cigarette/neutral cues/tobacco-alcohol cues). RESULTS: Two-week administration of varenicline reduced tobacco-alcohol cue-induced cigarette cravings and reduced emotionality aspects of alcohol craving after smoking a cigarette compared to abstinence in heavy-drinking smokers. CONCLUSION: It is possible that varenicline may have particular advantages as a smoking cessation aid in heavy drinkers.

Purpose Two new scales, CORECTS and PUQE-24 are introduced and validated, and the safety and effectiveness of Proctofoam-HC® in pregnancy is demonstrated. Method 315 of Motherisk NVP patients provided information on five clinical parameters as well as PUQE scores. 28 patients visiting a proctologist were graded for the severity of anal conditions by a proctologist before administering CORECTS. Pre and postnatal interviews were conducted with 204 pregnant women prescribed Proctofoam-HC®. Results Strong correlations were found between the following: PUQE-24 scores and parameters of well-being, hospitalization, and multivitamin intake; bleeding and pain components of CORECTS and the proctologist’s grade.There was no significant difference between mean birth weight of Proctofoam-HC® treated and comparison groups. There was a significant reduction in all symptoms of hemorrhoids. Conclusion PUQE-24 and CORECTS are the first validated scales used to assess the severity of NVP and hemorrhoids. Proctofoam-HC® is safe and effective for use in pregnancy.