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Age-related macular degeneration (AMD) is the leading cause of blindness in the United States of America. The pyridinium bis-retinoid A2-ethanolamine (A2E) has been implicated to play a role in AMD. We have observed novel pyridinium bis-retinoids through melanolipofuscin and human RPE extractions that may also play a role in the pathology of AMD. We have begun the construction of an amino-retinoid library in order to identify these ocular compounds. The compounds from the amino-retinoid library are also used in a targeted and triggered drug delivery system for treating cancer. Folic acid is coupled with the amino-retinoids to specifically target cancer cells. The first two amino-retinoids to be synthesized and characterized were A2-dopamine (A2D) and A2-cadaverine (A2C). Both pyridinium bis-retinoids were shown to generate cytotoxic oxidation products similar to A2E. Successful coupling of folic acid to A2C was achieved to form the folic acid-A2-cadaverine (FA-A2C) product. Preliminary irradiation results suggest that the FA-A2C product may be more photoreactive than initially anticipated. This could mean less drug and light exposure required to induce apoptosis and could eventually lead to a less invasive and toxic cancer treatment.

Cancer is not only difficult to treat but the patients also suffer from the pain associated with anticancer treatments. Targeted chemotherapeutics can reduce the adverse effects by reducing the dose required for tumor cell kill. Cancers of various origins often have characteristic marker molecules that distinguish them from the normal tissues. Folate receptors are such marker molecules present in ovarian and cervical cancers. The hypothesis for the current study is that amphiphiles constructed out of folic acid, the natural ligand for the folate receptor, can deliver paclitaxel, a chemotherapeutic compound, to folate receptor expressing cancer cells. To test this hypothesis, amphiphilic molecules were synthesized out of folic acid and fatty acids or long chain aliphatic amines. The gamma carboxylic group of folic acid was converted to an N-alkyl substituted amide. The alkyl group had various chain lengths varying from eleven methylene groups to seventeen methylene groups giving rise to a number of amphiphiles. The amphiphiles formed micelles in aqueous solutions. The critical micellization concentrations of the amphiphiles were measured by pyrene fluorescence and were found to be in the range of 10–70μM. HeLa and Caco-2 cells were taken as in vitro tumor models. Folate receptor expression was verified in HeLa and Caco-2 cells by western blot analysis. HeLa showed more than forty fold expression of the receptor when compared to Caco-2 and was chosen as receptor positive cell line while Caco-2 served as a negative control. Uptake of the folate labeled delivery system in the cell lines was tested by a fluorescent probe (aminocoumarin) labeled amphiphile. To test the specificity of the delivery system towards the receptor positive HeLa cells, the receptors were knocked down (70%) by folate receptor specific siRNA. Fluorescent amphiphile uptake in the knockdown cells was comparable to that of the negative control, Caco-2. Finally cytotoxicity studies were performed for paclitaxel formulated with the folate labeled amphiphiles and compared to free drug treatment in HeLa and Caco-2. IC50 values in HeLa for formulations with the folate labeled amphiphiles were ten folds less than those observed for free drug treatment whereas in Caco-2 no significant difference was noted.

This thesis is divided into two parts.Part I describes the organic extraction, separation, and liquid chromatographic-mass spectrometric analysis of chromophores from human and bovine retinal pigment epithelium. Flurorophores in the retinal pigment epithelium have been implicated in age related macular degeneration. In addition, the synthesis and characterization of a number of bis-retinoid type compounds that may potentially be found in such extracts, or that may be used for insight into pyridinium bis-retinoid reactivity, was accomplished.Part II describes a study of pyridinium bis-retinoid-folic acid coupling with respect to linker type, linker length, and nature of the linkage. Folic acid has been used as a targeting compound for a variety of cancer types. Development of HPLC and UV-Vis conditions suitable for the analysis of this new type of macromolecule was performed.

<p>Previous studies suggest that nicotine impairs pancreatic function, which may explain the increased risk of T2DM in smokers. We have previously shown that nicotine exposure results in decreased beta cell function, an effect which appears to be mediated via increased beta cell oxidative stress. The goal of this study is to determine whether folic acid, an antioxidant, can prevent nicotine-induced beta cell dysfunction in the beta cell.</p> <p>INS 1E cells, a rat pancreatic beta cell line, were treated with nicotine or vehicle ± 10µM folic acid for 48 hours. Nicotine treatment decreased both basal and glucose stimulated insulin secretion, but had no effect on insulin content, mitochondrial function or markers of apoptosis. Expression of oxidative stress/damage markers (HSP70 and 4-HNE), antioxidant enzymes (Cu/ZnSOD, MnSOD and CAT), insulin gene transcription factor PDX1 and K_ATPchannel subunit kir_6.2 were determined by western blot analysis. Expression of HSP70, 4-HNE and MnSOD were significantly increased with nicotine treatment (p=0.002, 0.05 and 0.03 respectively). Cu/ZnSOD and CAT expression remained unchanged with nicotine treatment. The addition of folic acid significantly reduced HSP70 expression, 4-HNE expression, CAT expression, but did not alter the expression of MnSOD. There was a significant (p6.2expression (p=0.019) which showed a trend toward reduced expression following treatment with folic acid (p=0.067).</p> <p>Nicotine treatment significantly increases markers of oxidative stress and oxidative damage in pancreatic beta cells; an effect which was reversed by folic acid administration. Nicotine and folic acid treatment increased insulin content, likely mediated through an increase in the insulin gene transcription factor, PDX1. Furthermore, nicotine treatment increased expression of kir_6.2,suggesting a defect in the insulin secretory mechanism. This effect was reversed with folic acid treatment.Although many studies suggest that Canadians are meeting or exceeding recommended folate levels, this is not true in smokers. Our data suggest that additional folate supplementation in smokers may prevent nicotine-induced damage to the pancreas and thus reduce the risk of type 2 diabetes.</p> / Master of Science (MSc)

Introduction : Les interventions d'exercice aérobie améliorent la fonction endothéliale chez les hommes âgés en bonne santé. Cependant, cet effet n'est pas toujours observé chez les femmes post-ménopausées en bonne santé et les mécanismes possibles sous-jacents à cette différence liée au sexe restent inconnus. Les objectifs de ce mémoire étaient de 1) Évaluer l’hypothèse que les femmes post-ménopausées démontrent une sensibilité réduite de l’endothélium à la contrainte de cisaillement et 2) Explorer si une consommation aiguë d’acide folique améliore la sensibilité de l’endothélium à la contrainte de cisaillement. Méthodes : Dix-huit femmes post-ménopausées en bonne santé (66 ± 8 ans) et 14 hommes du même âge (66 ± 6 ans) ont effectué des contractions avec leur main dominante pendant 4 minutes, à des intensités de 20%, 40%, 60% et 80% de leur force de préhension maximale. Tous les participants ont effectué ce protocole 2h après avoir consommé un placebo ou 5 mg d’acide folique selon un devis croisé à double aveugle. Le diamètre et la vélocité sanguine de l’artère brachiale ont été mesurés en continu à l'aide d'une échographie pour quantifier la sensibilité de l’endothélium à la contrainte de cisaillement. Résultats : Durant la visite placebo, la sensibilité de l’endothélium à la contrainte de cisaillement ne différait pas entre les hommes (0,036 ± 0,018 %/s-1) et les femmes (0,052 ± 0,027 %/s-1, p = 0,086). L'acide folique n'a pas amélioré la sensibilité de l’endothélium chez les hommes (placebo : 0,036 ± 0,018 %/s-1 vs. acide folique : 0,054 ± 0,030 %/s-1, p = 0,054) ni chez les femmes (placebo : 0,052 ± 0,027 %/s-1 vs. acide folique : 0,052 ± 0,030 %/s-1, p = 0,957). Conclusion : La sensibilité de l’endothélium à la contrainte de cisaillement induite par l'exercice ne diffère pas entre des femmes post-ménopausées en bonne santé et des hommes du même âge. Une consommation aiguë d’acide folique n’améliore pas la sensibilité de l’endothélium à la contrainte de cisaillement chez des hommes et des femmes âgées et en bonne santé. Ces résultats suggèrent qu’une sensibilité réduite de l’endothélium à la contrainte de cisaillement ne sous-tend pas l’effet mitigé des interventions d’exercice aérobie sur la fonction endothéliale des femmes post-ménopausées en bonne santé. Les résultats suggèrent également que la consommation d’acide folique n’est peut-être pas une stratégie qui pourrait restaurer les effets de l’exercice aérobie sur la fonction endothéliale des femmes post-ménopausées en bonne santé. / Purpose: Aerobic exercise interventions improve peripheral endothelial function in healthy older males. However, this effect is not always observed in healthy postmenopausal females and possible mechanisms underlying this sex-related difference remain unknown. The objectives of this thesis were to 1) Test the hypothesis that postmenopausal females have reduced endothelial sensitivity to exercise-induced shear rate compared to age-matched males and 2) Explore the effect of folic acid consumption on endothelial sensitivity to exercise-induced shear rate. Methods: Eighteen healthy postmenopausal females (66 ± 8 years) and 14 males of similar age (66 ± 6 years) performed handgrip exercise for 4 minutes at 20%, 40%, 60%, and 80% of their maximal voluntary contraction. All participants performed this protocol 2h after consuming either a placebo pill or 5 mg of folic acid in a randomized, double-blind and crossover design. Brachial artery diameter and blood velocity were continuously measured using high-resolution ultrasound. Results: During the placebo visit, endothelial sensitivity to exercise-induced shear rate did not differ between males (0.036 ± 0.018%/s-1 ) and females (0.052 ± 0.027%/s-1 , p=0.086). Folic acid consumption did not change endothelial sensitivity to exercise-induced shear rate in males (placebo: 0.036 ± 0.018%/s1 vs. folic acid: 0.054 ± 0.030 %/s-1 , p=0.054) or females (placebo: 0.052 ± 0.027%/s-1 vs. folic acid: 0.052 ± 0.030%/s-1 , p=0.957. Conclusion: Endothelial sensitivity to exercise-induced shear rate does not differ between healthy postmenopausal females and age-matched males. Acute folic acid consumption does not improve endothelial sensitivity to exercise-induced shear rate in healthy older adults. These results suggest that a reduced endothelial sensitivity to exercise-induced shear rate may not underlie the inconsistent effects of aerobic exercise interventions on peripheral endothelial function in healthy postmenopausal females. The results also suggest that folic acid consumption may not restore the endothelial benefits of aerobic exercise in healthy postmenopausal females.

Submitted in fulfillment of the requirements of the degree of Doctor of Technology: Health Sciences, Durban University of Technology, Durban, South Africa, 2015. / Background: In a vulnerable low-income group with a confirmed high risk of cardiovascular disease, like the elderly in the Sharpeville care centre, an acute intervention is needed in order to improve their health profile. Previous studies suggested homocysteine lowering by vitamin B12, B6 and folate supplementation. The effect of vitamin B12, B6 and folate supplementation on the inflammatory response, thrombotic risk, lipid profile, hypertension, risk of metabolic syndrome and homocysteine metabolism in an elderly, black South African population has never been reported. Objectives: The main aim of this interventional study was to assess the effect of vitamins B12, B6 and folate supplementation at 200% RDA for six months on cardiovascular risk markers of an elderly semi-urbanised black South African community. Design: This study was an experimental intervention non-equivalent control group study design in 104 purposively selected samples of all the elderly attending the day-care centre. Setting and participants: A homogeneous group of respondents was included in the study. All subjects were equivalent in age (>60 years), race (black), unemployed/pensioners (socio-demographic) and 60 years and older attending a day care centre in Sharpeville, situated in the Vaal region, Gauteng, SA. Measurements: The distinctiveness of this study lies in the broad panel of parameters evaluating the CVR in correlation with the increased nutritional intake of vitamin B6, B12 and folate. These included: weight, height, waist, serum cholesterol, high density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, blood pressure, fibrinogen, high-sensitivity C-reactive protein (HS–CRP), homocysteine, vitamin B12, folate, glucose, insulin, adiponectin and fibronectin. Results: A very high incidence (66.36%) of hyperhomocysteinaemia is present in the sample. The mean serum homocysteine level in hyperhomocysteinaemic individuals decreased statistically significantly from 25.00±8.00 umol/l to 18.80±12.00 umol/l after the intervention. The number of respondents with an increased homocysteine level decreased from 100% (baseline) to 67% (follow-up). The supplementation was beneficial (statistically significant changes) to the glucose levels, fibrinolytic status, vitamin B6 serum levels, fibronectin levels and haemopoeiesis (decreased macrocytosis) of all the individuals (regardless of their homocysteine status). Conclusion: It is concluded that supplementation of vitamins B6, B12 and folate at 200% RDA for six months is an effective homocysteine-lowering approach as a strategy to reduce hyperhomocysteinaemia in an elderly population and thereby reduce cardiovascular risk (CVR). The supplementation intervention mentioned is not an effective multifactorial strategy to decrease CVR although beneficial effects were found with other CVR markers independent of homocysteine status.

En dépit des progrès réalisés ces dernières décennies, le cancer demeure un problème majeur de santé publique. La plupart des chimiothérapies conventionnelles exploitent les propriétés cytotoxiques d'agents non sélectifs qui affectent les cellules à division rapide, qu'elles soient cancéreuses ou non, ce qui peut aboutir à une toxicité importante et de sévères effets secondaires. Pour pallier à ce problème, l'utilisation de ligands de ciblage pour délivrer spécifiquement l'agent cytotoxique aux cellules malignes tout en épargnant les tissus sains est une approche prometteuse. Parmi les différents ligands exploités, l'acide folique fait l'objet d'une attention particulière due à la surexpression préférentielle de son récepteur par de nombreux types de cellules cancéreuses. Cependant, les essais cliniques menés sur des molécules reposant sur cette stratégie indiquent que leur efficacité dépend surtout du niveau d'expression du récepteur ciblé. Dans ce contexte, j'ai développé deux approches permettant d'augmenter l'efficacité du ciblage des récepteurs à l'acide folique. D'une part, j'ai réalisé la validation biologique de nouveaux conjugués conçus pour délivrer conjointement deux agents anticancéreux. D'autre part, j'ai mis au point un traitement capable d'augmenter, in vitro et in vivo, l'expression des récepteurs à l'acide folique à la surface des cellules tumorales. Cela aboutit à l'internalisation d'une plus grande quantité de vecteur, qui se traduit in vivo, par une inhibition plus importante de la croissance tumorale, sans toxicité sur les cellules saines. / Despite significant advances obtained during the last decades, cancer is still lack of effective treatments. Indeed, most conventional chemotherapies relies on non-selective agents that kill indifferently healthy and tumor cells with high rate of division. This can lead to elevated toxicity and severe side effects. In this context, therapeutic approaches that exploit targeting ligands to selectively deliver cytotoxic drugs to malignant cells are currently promising. Among these different ligands, folate conjugates are subject to special attention due to the preferential overexpression of the folate receptor on several human cancer cell types that can mediate specific attachment and internalization of folate-derived imaging and therapeutic agents. However, clinical trials carried on such molecules revealed that their efficiency is mainly governed by folate receptor expression level on tumor cells. In this respect, I developed two approaches to increase efficacy of folate receptor targeting. On one hand, I realized biological validation of new targeting devices designed for the simultaneous delivery of two therapeutic agents. On the other hand, I developed a treatment to enhance in vitro and in vivo folate receptor expression at the surface of malignant cells. This treatment allows the internalization of a larger amount of a folic acid conjugate, previously synthesized and validated in our laboratory. That results in a drastically improvement of the in vivo tumor growth inhibition, without toxicity toward healthy cells.