Insufficient maternal folate during pregnancy increases the risk of the baby being small for gestational age (SGA). Studies in teenagers, a population vulnerable to folate deficiency and SGA birth, have shown that low maternal folate status is associated with impaired placental cell turnover and reduced transport suggesting placental dysfunction underlies SGA in maternal folate deficiency. Mechanisms through which folate-depletion compromises placental function are currently unknown. In non-placental cells, folate modulates microRNAs (miRs), post-transcriptional regulators of cellular functions. Expression of miRs is altered in placentas of SGA compared to normally grown babies but there are no data on differential miR expression or regulation in placentas from folate deficient women. This PhD investigated the hypothesis that placental dysfunction observed in folate deficient women is mediated by altered miR expression. Three placental preparations were compared (villous tissue in explant culture, BeWo choriocarcinoma cells and isolated cytotrophoblast cells) to determine the optimum in vitro system to study the direct effects of folate deficiency. In cytotrophoblast cells, folate deficiency significantly elevated apoptosis and reduced the activity of the system A amino acid transporter, consistent with observations in the placentas of folate-deficient teenagers. The reduction in system A activity by low folate was not associated with altered mRNA expression for the isoforms of system A, implicating an effect of low folate on post-translational regulation of the nutrient transporter. Targeted examination of villous tissue from teenagers with low folate status identified up-regulation of miR-222-3p a folate-sensitive miR. An unbiased miR array identified up-regulation of a further 16 miRs suggesting that maternal folate deficiency in vivo results in aberrant placental miR expression. Bioinformatic analysis of the folate sensitive miRs predicted gene targets known to be altered in placentas from SGA pregnancy that were likely to alter placental function. Two miRs altered in placentas from women with low folate status, miR-30e-3p and miR-34b-5p, were also significantly altered in folate deficient cytotrophoblasts confirming a direct effect of folate on trophoblast miR expression. Inhibition of these miRs in vitro had no effects on placental functions that are altered in vivo in folate-deficient women. Gene array and in silico analysis identified functional endpoints affected by these folate sensitive miRs, including cell signalling for proliferation and survival and oxidative stress, which might contribute to placental dysfunction in folate deplete women. Overall, this study has demonstrated for the first time that folate deficient conditions can directly alter trophoblast system A transport and cell survival and thus could contribute to the increased susceptibility to SGA births in folate deficient women. It has also contributed to the knowledge that miR expression is differentially altered in placentas exposed to folate-deficient versus sufficient conditions in vivo and that miRs are directly altered by folate depletion in vitro. These studies provide the foundation for future research to define the functional consequences of altered expression of folate-sensitive miRs and their target genes to explain how altered miRs could be affecting placental function resulting in development of SGA.

4 pp. / Before they may even know they are pregnant; women’s bodies and their level of folate play a critical role in preventing certain birth defects, specifically neural tube defects (NTDs). NTDs are birth defects in the brain, spinal cord, or spine. Considered ‘one of the most important public health discoveries of this century’ is that daily supplemental folic acid taken before becoming pregnant significantly reduces the risk of NTDs (1). In 1998, the United States made sweeping efforts that fortified cereal grains with folic acid to ensure all Americans consume adequate amounts of this vitamin. So what exactly is folate? What are the functions of this vitamin? What foods have high levels of folate and what is the recommended daily intake? This article will answer these questions and will go on to explain folic acid fortification and the impact fortification has had on the incidence of NTDs in Arizona.

Many Mexican women consume inadequate amounts of folic acid. Fortification of the corn tortilla could be an effective way to help increase the folic acid levels among the Mexican population. Previous studies have shown significant folic acid losses in fortified tortilla dough (masa) as it is held before baking. This loss in folic acid could be due to degradation by lactic acid bacteria naturally present in the masa. The microflora of traditionally made nixtamalized corn masa from six tortilla mills in Guadalajara, Mexico were isolated and characterized, and their effect on folic acid content was evaluated. Isolated bacteria were identified using whole cell fatty acid analysis via MIDI Inc.'s Microbial Identification System. Twenty-two unique bacterial species were identified, primarily belonging to the Streptococcus and Lactobacillus genera. Lactic acid bacteria were the predominant microorganisms, with counts ranging from 10^4 to 10^7 cfu/g. Aerobic mesophilic bacteria also ranged from 10^4 to 10^7 cfu/g. Coliforms and yeasts and mold were present at significantly lower levels. Masa samples, prepared from sterile fortified corn masa flour, were inoculated with a cocktail of bacteria isolated from the individual mills. Control samples were prepared using sterile media. Inoculated and uninoculated control samples were held at 56°C for 0, 3 and 6 hours, mimicking the elevated temperature of the masa as it is held before baking. The loss of folic acid in the sterile control was not different from the inoculated samples, indicating that the decline in folic acid is not due to bacteria present in the masa, but appears to be a chemical degradation related to time and temperature.

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Anticancer therapeutics are often limited to suboptimal doses due to their lack of selectivity for tumor cells and resultant damage to healthy tissue. These limitations motivated researchers to develop tumor-specific delivery systems for improved therapeutic efficacy and reduced unintended cytotoxicity. Polyamidoamine dendrimers offer an ideal platform for designing targeted therapeutics with tunable characteristics that optimize pharmacokinetic behavior and targeting specificity. Ligand conjugation to dendrimer provides the biochemical interaction necessary to activate tumor-specific receptors for receptor-mediated endocytosis and effective internalization of polyplexes. Tumor-specific receptors overexpressed in carcinomas, like folate receptor-alpha (FOLRα), are targeted by ligand-conjugated dendrimer to allow enhanced internalization of dendrimer and its therapeutic cargo. We examined the cellular trafficking dynamics and potential of folate-conjugated dendrimer for nucleic acid delivery in vitro. Results show folate-conjugation to G4 PAMAM dendrimer (G4FA) confers enhanced uptake in FOLRα-positive tumor cells. Cells internalize G4FA in a receptor-dependent manner with specificity for FOLRα-positive tumor cells.

Le pronostic des cancers ovariens reste sombre, en particulier en raison du retard diagnostic. Les traitements actuels associent une chirurgie de cytoréduction complète à l'administration de chimiothérapie à base de sels de platine. Les métastases viscérales sont rares dans cette pathologie, et la maladie est longtemps localisée à la cavité péritonéale. Pour cette raison, une attention particulière est portée au traitement des métastases péritonéales. Il est admis en effet que le facteur principal de réduction des récidives est l'absence de résidu tumoral en fin d'intervention. Malgré les progrès et la standardisation des techniques chirurgicales, la chirurgie de cytoréduction macroscopiquement complète, associée à une chimiothérapie efficace, ne prévient pas la survenue des récidives qui concerneront 60% des femmes en rémission à l’issu de ce traitement.Parmi les hypothèses expliquant ce taux élevé de récidive, l’existence d’une maladie microscopique résiduelle à l’issu de la chirurgie est évoquée. Le traitement de cette maladie microscopique représente un nouveau défi à relever pour les oncologues médicaux et les chirurgiens, et de nouvelles stratégies sont à développer dans ce domaine.Notre objectif est de réaliser la destruction ciblée par thérapie photodynamique (PDT) des métastases péritonéales microscopiques qui sont ignorées lors de la chirurgie. Nous espérons ainsi diminuer l’incidence des récidives locales qui concernent la majorité des patientes. Pour apporter la preuve de l’efficacité de cette stratégie innovante, un ciblage thérapeutique est indispensable car le développement de la PDT dans cette indication est limité par la mauvaise tolérance des tissus sains.Nous présentons ici les résultats précliniques obtenus in vitro et in vivo pour l’évaluation de photosensibilisateurs couplés à l’acide folique (PS1 et PS2) et ainsi dirigés vers le récepteur au folate, récepteur membranaire spécifique des cancers épithéliaux de l’ovaire (CEO).Nous avons travaillé sur des lignées cellulaires murine (NuTu-19) et humaines (SKOV-3, OVCAR-3) de CEO et sur un modèle animal de carcinose péritonéale. Après validation du modèle animal pour l'évaluation de molécule couplée à l'acide folique, nous avons montré la bonne spécificité du PS1 pour sa cible tumorale, meilleure que celle rapportée pour les autres photosensibilisateurs utilisés dans cette indication. Les lignées cellulaires émettent une fluorescence détectable après mise en culture dans un milieu enrichi en PS ce qui indique leur capacité à incorporer la molécule d’intérêt. Cette fluorescence a été détectée par spectrofluorimétrie (PS1 et PS2) et en photodiagnostic (PS2) in vivo au niveau des métastases péritonéales. La PDT permet d'obtenir la mort cellulaire des cellules humaines in vitro avec une excellente efficacité. Les premières données précliniques obtenues in vitro sur lignées humaines indiquent que la PDT utilisant un photosensibilisateur couplé à l'acide folique pourrait avoir des applications en immunothérapie.Un photosensibilisateur spécifique pourrait autoriser le développement d'une technique de PDT sure et efficace et jouer ainsi un rôle dans le traitement et la prévention des récidives péritonéales des cancers épithéliaux de l'ovaire. / Ovarian cancer’s prognosis remains dire after primary therapy. The standard of care remains debulking surgery in combination with platinum-based chemotherapy. This consists of either primary debulking surgery and adjuvant chemotherapy or neoadjuvant chemotherapy followed by interval debulking surgery, depending on FIGO stage and predictive factors concerning residual macroscopic disease after surgery. Recurrence rate is disappointingly high as 60-80% of women with epithelial ovarian cancer (EOC) considered in remission will develop recurrent disease within five years. Special attention to undetected peritoneal metastases and residual tumorous cells during surgery is necessary as they are the main predictive factors of recurrences.An option to improve the completion of cytoreductive surgery is using photodynamic therapy (PDT) to induce necrosis of micrometastases. A limit of this technique is the toxicity induced by the low photosensitizer (PS) specificity for tumor tissue if the light cannot be specifically applied. This would be the case in advanced ovarian cancer. To solve this problem, a solution is the design of selective PS, that is to say PS coupled to a unit that target over-expressed receptors on tumor cells. Approximately, 72-100% of ovarian carcinoma overexpress Folate Receptor α (FRα) in particular the serous carcinoma. FRα is absent in most of the healthy tissues; thus, representing a promising target for EOC targeted therapy.We present preclinical results of in vitro and in vivo studies concerning properties of folic-acid targeted photosensitizers (PS1 and PS2). Those studies have been performed on murine and human cell lines of EOC and on a preclinical model of peritoneal carcinomatosis (Fisher F344 rat / NuTu-19 cell line). Results suggest that specificity for ovarian cancer metastases is better than previously reported with other photosensitizer. Fluorescence emission was higher in peritoneal metastases than in liver and healthy peritoneum. Tissue quantification of the PS showed specific incorporation of the folate-targeted PS within tumor tissue. Folic acid targeted PDT induced cellular death on EOC human cell lines.Specific PS may allow the development of efficient and safe intraperitoneal PDT procedure which could play a role in the prevention of EOC peritoneal recurrences.

This dissertation comprises an introduction, background and current research progress in the area of classical antifolates as the targeted anticancer therapies.<br>In this study, twelve series of classical 5-, 6- and 7-substituted pyrrolo[2,3-d]pyrimidines were designed and synthesized. Extensive structure modifications of the pyrrolo[2,3-d] pyrimidine scaffold were investigated to determine selective transport via FR or/and PCFT and tumor targeted antifolates with GARFTase or multiple folate metabolizing enzyme inhibition.<br>The design strategies employed include: variation of the side chain substitution position (5-,6- and 7-substituted); variation of the side chain length (n=1-6); isosteric replacement of the 1,4-disubstituted phenyl ring with 1,2- and 1,3- disubstituted phenyl ring and 2,5- disubstituted thiophenyl ring; replacement the L-glutamate with variation at the á and ã carboxylic acids.<br>As a part of this study, a total of one hundred and fifty six new compounds (including new intermediates) were synthesized and separated. Of these, twelve series consisting of forty two classical antifolate final compounds were submitted for biological evaluation. In addition, bulk synthesis of some potent final compounds (2, 2.0 g; 161, 500 mg; 175, 1.0 g; 166, 500 mg; 194, 500 mg) was carried out to facilitate in vivo evaluation.<br>More importantly, a new Heck coupling of the thiophene iodide 301 and allyl alcohols to synthesize aldehydes in one step was discovered. Due to its potential use in analog synthesis of clinically used antifolates such as RTX and PMX, this mild conditioned and easy to handle Heck coupling reaction is highly attractive.<br>During this study, the SAR of folate transporters (RFC, FR and PCFT) and GARFTase inhibitors were extensively explored. The 6-substituted straight chain compound 166 (n=7) was extremely potent against KB tumor cells (IC50=1.3 nM, about 80-fold more potent than clinically used PMX) without any RFC activity. The 5- substituted phenyl compound 175 (n=4) showed AICARFTase as the primary target with potent KB tumor cell inhibition (IC50=7.9 nM, about 8-fold more potent than PMX) and also indirectly inhibited the mTOR pathway leading to tumor cell apoptosis. Due to their potent antitumor activities, these two compounds serve as leads for future structural optimization. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Medicinal Chemistry / PhD; / Dissertation;

Alcohol abuse during pregnancy has been associated with Fetal Alcohol Spectrum Disorder (FASD). Research to date has focused on the role played by ethanol in the development of this disorder. In addition to ethanol, alcoholic drinks also contain methanol. Hence, consumption of alcohol can also lead to methanol accumulation. Methanol is metabolized to formaldehyde, which is then rapidly metabolized to formate, a toxic metabolite. Folate, a B-vitamin and antoxidant, is a cofactor in the metabolism of formate. This study assessed the relationship between formate and folate, formate kinetics in folate deficiency and, changes in antioxidant capacity during formate insult in folate deficiency. The findings of this study would lead to a better understanding of the role of formate in the development of the etiology of FASD and form the basis of future research. The relationship between formate and folate was investigated in intoxicated human female subjects, sober drug rehabilitating females and, pregnant women. A negative (inverse) relationship was observed between plasma formate and folate in pregnant sober women (correlation coefficient = -0.4989). Such a relationship, however, was not observed in whole blood in alcohol intoxicated (correlation coefficient = 0.0899) and detox women (correlation coefficient = 0.2382). Because of the health promoting ingredients in grain and fruit based alcoholic drinks, antioxidant B-vitamins were higher during intoxication while homocysteine levels were lower.<p>Formate kinetics during folate deficiency and changes in the body antioxidant capacity was investigated in folate deficient young swine. Folate deficiency altered formate kinetics leading to decreased systemic clearance (by approximately 2.3 fold), increased half-life (by 2.5 fold) and, consequently increased exposure (by 2.7 fold). Folate deficiency alone compromised antioxidant capacity. However, the combination of folate deficiency and formate insult further compromised antioxidant capacity.<p>In conclusion, methanol accumulates after alcohol intoxication, which can lead to formate build up in the body. During folate deficiency formate kinetics is altered leading to reduced formate clearance and increased exposure. Exposure to formate coupled to folate deficiency compromises antioxidant capacity, which can have deleterious effects on the fetus.

Alcohol abuse during pregnancy has been associated with Fetal Alcohol Spectrum Disorder (FASD). Research to date has focused on the role played by ethanol in the development of this disorder. In addition to ethanol, alcoholic drinks also contain methanol. Hence, consumption of alcohol can also lead to methanol accumulation. Methanol is metabolized to formaldehyde, which is then rapidly metabolized to formate, a toxic metabolite. Folate, a B-vitamin and antoxidant, is a cofactor in the metabolism of formate. This study assessed the relationship between formate and folate, formate kinetics in folate deficiency and, changes in antioxidant capacity during formate insult in folate deficiency. The findings of this study would lead to a better understanding of the role of formate in the development of the etiology of FASD and form the basis of future research. The relationship between formate and folate was investigated in intoxicated human female subjects, sober drug rehabilitating females and, pregnant women. A negative (inverse) relationship was observed between plasma formate and folate in pregnant sober women (correlation coefficient = -0.4989). Such a relationship, however, was not observed in whole blood in alcohol intoxicated (correlation coefficient = 0.0899) and detox women (correlation coefficient = 0.2382). Because of the health promoting ingredients in grain and fruit based alcoholic drinks, antioxidant B-vitamins were higher during intoxication while homocysteine levels were lower.<p>Formate kinetics during folate deficiency and changes in the body antioxidant capacity was investigated in folate deficient young swine. Folate deficiency altered formate kinetics leading to decreased systemic clearance (by approximately 2.3 fold), increased half-life (by 2.5 fold) and, consequently increased exposure (by 2.7 fold). Folate deficiency alone compromised antioxidant capacity. However, the combination of folate deficiency and formate insult further compromised antioxidant capacity.<p>In conclusion, methanol accumulates after alcohol intoxication, which can lead to formate build up in the body. During folate deficiency formate kinetics is altered leading to reduced formate clearance and increased exposure. Exposure to formate coupled to folate deficiency compromises antioxidant capacity, which can have deleterious effects on the fetus.