EXAMINATION OF THE EFFECTS OF VALPROIC ACID EXPOSURE OR P300 PROTEIN INHIBITION ON THE BALANCE OF APOPTOSIS AND PROLIFERATION IN P19 EMBRYONAL CARCINOMA CELLS

Valproic acid (VPA), a commonly-used anticonvulsant drug, is associated with increased risk of fetal malformations, including neural tube defects (NTDs). Previous in vivo studies determined that VPA-exposed embryos with a NTD had altered expression of several proteins regulated by p300, a histone acetyltransferase (HAT) protein. p300 is capable of acetylating histones and non-histone proteins through its HAT activity, allowing it to transcriptionally regulate genes as well as modulate the stability and activity of specific proteins. NFκB, Stat3 and Egr1, all of which function as transcription factors, are regulated by p300 through its HAT activity. Together, these proteins all play an important role in maintaining the balance of apoptosis, proliferation and differentiation, the regulation of which is extremely important for proper embryonic development. The studies in this thesis utilized P19 embryonal carcinoma (EC) cells in order to determine the effects of VPA exposure on the expression of p300 and the aforementioned transcription factors, as well as apoptosis and proliferation, in vitro. P19 EC cells were exposed to C646, a selective p300 inhibitor, in order to assess whether the effects observed as a result of VPA exposure were due to p300 protein degradation. It was found that VPA exposure for 24 hours in P19 EC cells in vitro resulted in a significant decrease in p300 protein expression. VPA exposure also significantly decreased NFκB protein expression, while resulting in increased Stat3 protein expression. However, Stat3 acetylation and phosphorylation, which both contribute to Stat3 activation, were significantly decreased as a result of VPA exposure. p300 inhibition resulted in a significant decrease in NFκB, similar to what was observed as a result of VPA exposure, which suggests that VPA-mediated degradation of p300 may play a role in reduced NFκB protein expression following VPA exposure. Conversely, Stat3 protein expression, acetylation and phosphorylation were not significantly changed as a result of p300 inhibition, suggesting that p300 degradation does not play a role in VPA’s effects on Stat3 protein expression and activation. VPA exposure also resulted in a significant increase in apoptosis, while p300 inhibition did not significantly increase apoptosis. These data suggest that p300 degradation plays a role in VPA-mediated teratogenicity, and that VPA may target other cellular mechanisms in order to exert its teratogenic effects.

EXAMINATION OF VALPROIC ACID-INDUCED PERTURBATIONS TO DNA REPAIR, NF-ΚB SIGNALING AND CBP/P300 TRANSCRIPTIONAL CO-ACTIVATORS

Exposure to the antiepileptic drug valproic acid (VPA) is associated with an increased risk of congenital malformations including heart, skeletal and most frequently neural tube defects. Although the mechanisms contributing to its teratogenesis are not well understood, VPA was previously shown to increase homologous recombination (HR)-mediated DNA repair and decrease protein expression of the transcription factor NF-κB/p65. The studies in this thesis utilized in vivo and in vitro models to evaluate the expression of HR mediators, investigate the implications of decreased p65 including DNA binding and transcriptional activation, and the expression and histone acetyltransferase activity of Cbp/p300 with an aim to provide mechanistic insight into VPA-mediated alterations. The first study demonstrated that following maternal administration of VPA, mouse embryonic mRNA expression of HR mediators Rad51, Brca1 and Brca2 exhibited temporal and tissue-specific alterations. Protein expression of Rad51 was similarly altered and preceded increased cleavage of caspase-3 and PARP; indicative of apoptosis. The second study confirms previous findings of decreased total cellular p65 protein using P19 cells, but is the first to demonstrate that nuclear p65 protein is unchanged. NF-κB DNA binding was decreased following VPA exposure and maybe mediated by decreased p50 protein, which dimerizes with p65 prior to DNA binding. Transcriptional activity of NF-κB was also increased with VPA exposure which was not due to increased p65 phosphorylation at Ser276. Furthermore, the transcriptional activation capacity was unaffected by VPA exposure as combined exposure to VPA and TNFα additively increased NF-κB activity. The third study demonstrated that VPA exposure in P19 cells decreased Cbp/p300 total cellular and nuclear protein attributed primarily to ubiquitin proteasome-mediated degradation. Histone acetyltransferase (HAT) activity of p300 was decreased proportionately to nuclear protein following VPA exposure. Inhibition of Cbp/p300 HAT activity decreased p65 total cellular protein, increased caspase-3 cleavage and ROS similar to VPA exposures. Furthermore, pre-treatment with the antioxidant enzyme catalase attenuated the increase in caspase-3 cleavage, but not p65 protein. Overall, this thesis demonstrates that VPA exposure impacts the expression and activity of the transcription factor NF-κB and transcriptional co-activators/HATs Cbp/p300, which has implications for downstream VPA targets including Rad51, Brca1 and Brca2.

SERS of meso-droplets supported on superhydrophobic wires allows exquisitely sensitive detection of dipicolinic acid, an anthrax biomarker, considerably below the infective dose

Surface-enhanced Raman measurements of <1 μL analyte/colloid meso-droplets on superhydrophobic wires with hydrophilic tips allowed dipicolinic acid, a spore biomarker for Bacillus anthracis (anthrax), to be detected at 10(-6) mol dm(-3). This is equivalent to 18 spores, significantly below the infective dose of 10(4) spores and 2 orders of magnitude better than previous measurements.

Cost-effectiveness of zoledronic acid and strontium-89 as bone protecting treatments in addition to chemotherapy in patients with metastatic castrate-refractory prostate cancer: results from the TRAPEZE trial (ISRCTN 12808747)

OBJECTIVE: To evaluate the cost-effectiveness of adding zoledronic acid or strontium-89 to standard docetaxel chemotherapy for patients with castrate-refractory prostate cancer (CRPC).

PATIENTS AND METHODS: Data on resource use and quality of life for 707 patients collected prospectively in the TRAPEZE 2 × 2 factorial randomised trial (ISRCTN 12808747) were used to assess the cost-effectiveness of i) zoledronic acid versus no zoledronic acid (ZA vs. no ZA), and ii) strontium-89 versus no strontium-89 (Sr89 vs. no Sr89). Costs were estimated from the perspective of the National Health Service in the UK and included expenditures for trial treatments, concomitant medications, and use of related hospital and primary care services. Quality-adjusted life-years (QALYs) were calculated according to patients' responses to the generic EuroQol EQ-5D-3L instrument, which evaluates health status. Results are expressed as incremental cost-effectiveness ratios (ICERs) and cost-effectiveness acceptability curves.

RESULTS: The per-patient cost for ZA was £12 667, £251 higher than the equivalent cost in the no ZA group. Patients in the ZA group had on average 0.03 QALYs more than their counterparts in no ZA group. The ICER for this comparison was £8 005. Sr89 was associated with a cost of £13 230, £1365 higher than no Sr89, and a gain of 0.08 QALYs compared to no Sr89. The ICER for Sr89 was £16 884. The probabilities of ZA and Sr89 being cost-effective were 0.64 and 0.60, respectively.

CONCLUSIONS: The addition of bone-targeting treatments to standard chemotherapy led to a small improvement in QALYs for a modest increase in cost (or cost-savings). ZA and Sr89 resulted in ICERs below conventional willingness-to-pay per QALY thresholds, suggesting that their addition to chemotherapy may represent a cost-effective use of resources.

Comparative study between the effects of hyaluronic acid and acid galactan purified from eggs of the mollusk Pomacea sp in wound healing

To compare the effect of hyaluronic acid (HA) and of AG on the healing of intestine wounds. Methods: The semi-purified extract of the eggs of the mollusc was obtained by fractionation with ammonium sulfate and purification for ion-exchange chromatography. The obtained galactans were eluted in water (neutral galactan) and in 0.1 and 0.2M NaCl (acidic galactans). The in vivo study was performed with 45 “Wistar” rats, separated in three groups (n=15). Solutions containing HA 1%, GA 1% or saline solution 0,9%, was placed topically on the sutures of wounds in the small intestine of the rats. After 05, 10 and 21 days the animals were sacrificed and biopsy of the healing tissue was done. Results: The hystologic grading was more significant for HA and AG groups when compared to the group C. AG stimulated the appearance of macrophages, giant cells and increase in the concentration of collagen in the area of the wound when compared to HA. Conclusion: The topical use of GA in intestinal wounds promoted the anticipation of events that are important in the wound healing

Erythrocyte Phospholipid Molecular Species and Fatty Acids of Down Syndrome Children Compared with Non-affected Siblings

The majority of children with Down syndrome (DS) develop Alzheimer's disease (AD) at an early age. Although long-chain n-3 fatty acids (FA) are protective of neurodegeneration, little is known about the FA status in DS. In the present study, we aimed to investigate whether children with DS presented altered plasma and erythrocyte membrane phospholipids (PL) FA composition, when compared with their non-affected siblings. Venous blood samples were analysed for plasma and erythrocyte membrane FA composition by TLC followed by GC techniques. Lipid molecular species were determined by electrospray ionisation/tandem MS (ESI-MS/MS). FA analysis measured by standard GC showed an increased concentration of MUFA and a decreased concentration of plasmalogens in major PL fractions, but there were no differences in the concentrations of arachidonic acid or DHA. However, as identified by ESI-MS/MS, children with DS had increased levels of the following erythrocyte PL molecular species: 16 : 0–16 : 0, 16 : 0–18 : 1 and 16 : 0–18 : 2n-6, with reduced levels of 16 : 0–20 : 4n-6 species. Children with DS presented significantly higher levels of MUFA in both plasma and erythrocyte membrane, as well as higher levels of saturated and monounsaturated molecular species. Of interest was the almost double proportion of 16 : 0–18 : 2n-6 and nearly half the proportion of 16 : 0–20 : 4n-6 of choline phosphoacylglycerol species in children with DS compared with their non-affected siblings. These significant differences were only revealed by ESI-MS/MS and were not observed in the GC analysis. Further investigations are needed to explore molecular mechanisms and to test the association between the pathophysiology of DS and the risk of AD.

Catalytic decomposition of formic acid using supported metal nanoparticles

Upgrade of hydrogen to valuable fuel is a central topic in modern research due to its high availability and low price. For the difficulties in hydrogen storage, different pathways are still under investigation. A promising way is in the liquid-phase chemical hydrogen storage materials, because they can lead to greener transformation processes with the on line development of hydrogen for fuel cells. The aim of my work was the optimization of catalysts for the decomposition of formic acid made by sol immobilisation method (a typical colloidal method). Formic acid was selected because of the following features: it is a versatile renewable reagent for green synthesis studies. The first aim of my research was the synthesis and optimisation of Pd nanoparticles by sol-immobilisation to achieve better catalytic performances and investigate the effect of particle size, oxidation state, role of stabiliser and nature of the support. Palladium was chosen because it is a well-known active metal for the catalytic decomposition of formic acid. Noble metal nanoparticles of palladium were immobilized on carbon charcoal and on titania. In the second part the catalytic performance of the “homemade” catalyst Pd/C to a commercial Pd/C and the effect of different monometallic and bimetallic systems (AuxPdy) in the catalytic formic acid decomposition was investigated. The training period for the production of this work was carried out at the University of Cardiff (Group of Dr. N. Dimitratos).

Encapsulation of ascorbic acid promotes the reduction of Maillard reaction products in UHT milk

The presence of amino groups and carbonyls renders fortified milk with ascorbic acid particularly susceptible to the reduction of available lysine and to the formation of Maillard reaction products (MRPs), as Nε-(Carboxyethyl)-L-lysine (CEL), Nε-(Carboxymethyl)-L-lysine (CML), Amadori products (APs) and off-flavors. A novel approach was proposed to control the Maillard reaction (MR) in fortified milk: ascorbic acid was encapsulated in a lipid coating and the effects were tested after a lab scale UHT treatment. Encapsulation promoted a delayed release of ascorbic acid and a reduction in the formation of MRPs. Total lysine increased up to 45% in milk with encapsulated ascorbic acid, while reductions in CML, CEL and furosine ranged from 10% to 53% compared with control samples. The effects were also investigated towards the formation of amide-AGEs (advanced glycation end products) by high resolution mass spectrometry (HRMS) revealing that several mechanisms coincide with the MR in the presence of ascorbic acid (AA).

Anti-MDR and antitumoral action of acetylsalicylic acid on leukaemic cells

ASA (acetylsalicylic acid) is an NSAID (non-steroidal anti-inflammatory drug). ASA has gained attention as a potential chemopreventive and chemotherapeutic agent for several neoplasms. The aim of this study was to analyse the possible antitumoural effects of ASA in two erythroleukaemic cell lines, with or without the MDR (multidrug resistance) phenotype. The mechanism of action of different concentrations of ASA were compared in K562 (non-MDR) and Lucena (MDR) cells by analysing cell viability, apoptosis and necrosis, intracellular ROS (reactive oxygen species) formation and bcl-2, p53 and cox-2 gene expression. ASA inhibited the cellular proliferation or induced toxicity in K562 and Lucena cell lines, irrespective of the MDR phenotype. The ASA treatment provoked death by apoptosis and necrosis in K562 cells and only by necrosis in Lucena cells. ASA also showed antioxidant activity in both cell lines. The bcl-2, p53 and cox-2 genes in both cell lines treated with ASA seem to exhibit different patterns of expression. However, normal lymphocytes treated with the same ASA concentrations were more resistant than tumoral cells. The results of this work show that both cell lines responded to treatment with ASA, demonstrating a possible antitumoral and anti-MDR role for this drug.

Characterization of rice starch- ι-carrageenan biodegradable edible film. Effect of stearic acid on the film properties

The main aim of this study was to develop rice starch (RS), ι-carrageenan (ι-car) based film. Different formulations of RS (1-4%, w/w), ι-car (0.5-2%, w/w) was blended with stearic acid (SA; 0.3-0.9%, w/w) and glycerol (1%, w/w) as a plasticizer. The effect of film ingredients on the thickness, water vapour permeability (WVP), film solubility (FS), moisture content (MC), colour, film opacity (FO), tensile strength (TS), elongation-at-break (EAB) of film was examined. Interactions and miscibility of partaking components was studied by using Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Hydrocolloid suspension solution of mix polysaccharides imparted a significant impact (p<0.05) on the important attributes of resulting edible film. TS and EAB of film were improved significantly (p<0.05) when ι-car was increased in the film matrix. Formulation F1 comprising 2% ι-car, 2% 33 RS, 0.3% SA, Gly 30% w/w and 0.2% surfactant (tween®20) provided film with good 34 physical, mechanical and barrier properties. FT-IR and XRD results reveal that molecular interactions between RS-ι-car have a great impact on the film properties confining the compatibility and miscibility of mixed polysaccharide. Results of the study offers new biodegradable formulation for application on fruit and vegetables.

ANTICANCER MECHANISM OF TOLFENAMIC ACID IN COLORECTAL CANCER

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States. Chemopreventive therapies could be effective way to treat CRC. Tolfenamic acid, one of the NSAIDs, shows anti-cancer activities in several types of cancer. Aberrant Wnt/β-catenin regulation pathway is a major mechanism of colon tumorigenesis. Here, we sought to better define the mechanism by which tolfenamic acid suppresses colorectal tumorigenesis focusing on regulation of β-catenin pathway. Treatment of tolfenamic acid led to a down-regulation of β-catenin expression in dose dependent manner in human colon cancer cell lines without changing mRNA. MG132 inhibited tolfenamic acid-induced downregulation of β-catenin and exogenously overexpression β-catenin was stabilized in the presence of tolfenamic acid. Tolfenamic acid induced an ubiquitin-mediated proteasomal degradation of β-catenin. In addition, tolfenamic acid treatment decreased transcriptional activity of β-catenin and expression of Smad2 and Smad3 while overexpression of Smad 2 inhibited tolfenamic acid-stimulated transcriptional activity of β-catenin. Moreover, tolfenamic acid decreased β-catenin target gene such as vascular endothelial growth factor (VEGF) and cyclin D1. In summary, tolfenamic acid is a promising therapeutic drug targeting Smad 2-mediated downregulation of β-catenin in CRC.

Analgesic Effects of Fatty Acid Amide Hydrolase Inhibition in a Rat Model of Neuropathic Pain

Cannabinoid-based medicines have therapeutic potential for the treatment of pain. Augmentation of levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is analgesic in models of acute and inflammatory pain states. The aim of this study was to determine whether local inhibition of FAAH alters nociceptive responses of spinal neurons in the spinal nerve ligation model of neuropathic pain. Electrophysiological studies were performed 14-18 days after spinal nerve ligation or sham surgery, and the effects of the FAAHinhibitor cyclohexylcarbamic acid 3-carbamoyl biphenyl-3-yl ester (URB597) on mechanically evoked responses of spinal neurons and levels of endocannabinoids were determined. Intraplantar URB597 (25 _g in 50 _l) significantly ( p _ 0.01) attenuated mechanically evoked responses of spinal neurons in sham-operated rats. Effects of URB597 were blocked by the cannabinoid 1 receptor (CB1 ) antagonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (30_g in50_l) and the opioid receptor antagonist naloxone. URB597 treatment increased levels of anandamide, 2-arachidonyl glycerol, and oleoyl ethanolamide in the ipsilateral hindpaw of shamoperated rats. Intraplantar URB597 (25 _g in 50 _l) did not, however, alter mechanically evoked responses of spinal neurons in spinal nerve ligated (SNL) rats or hindpaw levels of endocannabinoids. Intraplantar injection of a higher dose of URB597 (100 _g in 50 _l) significantly ( p_0.05) attenuated evoked responses of spinal neurons in SNL rats but did not alter hindpaw levels of endocannabinoids. Spinal administration of URB597 attenuated evoked responses of spinal neurons and elevated levels of endocannabinoids in shamoperated and SNL rats. These data suggest that peripheral FAAH activity may be altered or that alternative pathways of metabolism have greater importance in SNL rats.