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Antisense RNA (asRNA) strategies are identified as an effective and specific method for gene down-regulation at the post-transcriptional level. In this study, the major purpose is to find a correlation between the expression level and minimum free energy to enable the design of specific asRNA fragments. The thermodynamics of asRNA and mRNA hybridization were computed based on the fluorescent protein reporter genes. Three different fluorescent proteins (i) green fluorescent protein (GFP), (ii) cyan fluorescent protein (CFP) and (iii) yellow fluorescent protein (YFP) were used as reporters. Each fluorescent protein was cloned into the common pUC19 vector. The asRNA fragments were randomly amplified and the resulted antisense DNA fragments were inserted into the constructed plasmid under the control of an additional inducible plac promoter and terminator. The expression levels of fluorescent reporter protein were determined in real time by plate reader. Different results have been observed according to the fluorescent protein and the antisense fragment sequence. The CFP expression level was decreased by 50 to 78% compared to the control. However, with the GFP, the down-regulation did not exceed 30% for the different constructs used. For certain constructs, the effect was the opposite of expected and the expression level was increased. In addition, the YFP showed a weak signal compared to growth media, therefore the expression level was hard to be defined. Based on these results, a thermodynamic model to describe the relationship between the particular asRNA used and the observed expression level of the fluorescent reporter was developed. The minimum free energy and binding percentage of asRNA-mRNA complex were computed by NUPACK software. The expression level was drawn as a function of the minimum free energy. The results showed a weak correlation, but linear trends were observed for low energy values and low expression levels the CFP gene. The linear aspect is not verified for higher energy values. These findings suggest that the lower the energy is, the more stable is the complex asRNA-mRNA and therefore more reduction of the expression is obtained. Meanwhile, the non-linearity involves that there are other parameters to be investigated to improve the mathematical correlation. This model is expected to offer the chance to "fine-tune" asRNA effectiveness and subsequently modulate gene expression and redirect metabolic pathways toward the desired component. In addition, the investigation of the localization of antisense binding indicates that there are some regions that favors the hybridization and promote hence the down-regulation mechanisms.

Arsenic (As) is a well established cause of cancer in humans, and increasing evidence indicates that As has deleterious effects on the immune system that are not directly related to carcinogenesis. However, the mechanisms of As toxicity remain poorly understood. Our laboratory previously reported decreased urinary levels of human beta-defensin-1 (HBD1) peptides in As-exposed individuals from two cross-sectional studies based in Nevada and Chile, and confirmed in vitro that As exposure suppressed HBD1 mRNA expression which is encoded by the DEFB1 gene. DEFB1 is constitutively expressed in epithelial tissues, plays a role in both the innate and adaptive branches of the immune system, and is implicated in anti-tumor immunity. Therefore, the objectives of this dissertation are to review the immunotoxicological effects of As, characterize the effects of As on DEFB1 gene and protein expression in relevant in vitro model systems, investigate the molecular mechanisms mediating these effects, and explore the influence of other metals on HBD1 levels. A comprehensive review of the literature on the immune-related effects associated with As exposure in humans, animals and in vitro models reveals that chronic exposure to As can severely impair various aspects of immune function and consequently result in elevated risk of infections and chronic diseases. However, further investigation is needed to better understand the relationship between As exposure and the development of disease, and several recommendations are discussed to help bridge the gaps in knowledge.The current research investigated the effects of As exposure on DEFB1 in cells derived from target tissues of toxicity using immortalized non-tumorigenic human HOK-16B keratinocytes and HK-2 kidney epithelial cells. DEFB1 mRNA levels were more abundant in HK-2 cells than in HOK-16B cells, and were suppressed by exposure to arsenite (AsIII) or monomethylarsonous acid (MMAIII), the postulated more toxic metabolite. The suppressive effect of AsIII and MMAIII treatments on DEFB1 transcript levels continued for several passages after removal of As. HBD1 peptide levels were significantly reduced following exposure to AsIII, but were not affected by treatment with lead, cadmium or chromium, suggesting that decreased HBD1 may be a specific response to As. Finally, AsIII treatment was found to suppress DEFB1 promoter activity, indicating that the inhibition of DEFB1 mRNA by As is likely due to transcriptional down-regulation. Taken together, the research presented here provides evidence that our previous findings of decreased urinary HBD1 levels are likely due to a direct effect of As on the kidney, and suggest a novel mechanism by which As exposure may promote cancer development.This dissertation summarizes the known in vivo and in vitro effects of As on the immune system, characterizes the effects of As on DEFB1 using relevant cell culture models, and establishes DEFB1 as a potentially relevant biomarker of response to As. Future studies should address the role of DEFB1 inhibition in As immunotoxicity and carcinogenicity.

A number of epidemiological studies have correlated arsenic exposurwith cancer, skin diseases, cardiovascular diseases, and adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, which would indicate that arsenic has the ability to act as a developmental toxicant. Myoblast cells were exposed to 20nM sodium arsenite and allowed to differentiate into myotubes and expression of the muscle-specific transcription factor myogenin, along with the expression of myosin light chain 2, and tropomyosin were investigated using real time PCR and immunofluorescence. Exposing C2C12 cells to 20nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes. Additionally, arsenic exposure caused a time-dependant decrease in myogenin mRNA expression, as compared to the control cells, starting on day two of the differentiation process. Arsenic reduced myogenin mRNA levels by 1.4-fold on day two, 2.7-fold on day three, and 5.1-fold on day four of differentiation. This reduction in transcript number was confirmed by immunofluorescence, which also showed a decrease in the total number of nuclei expressing myogenin protein. Interestingly, myosin light chain 2 mRNA was significantly upregulated in the arsenic-exposed cells, although this did not translate into altered protein expression. This study demonstrated that low concentrations of arsenic are able to disturb the differentiation process of myoblasts without causing overt toxicity.