No description available.

Bacterial infection is a leading cause of morbidity and mortality arising from platelet transfusions (1, 2). Storage of platelet products at room temperature (20 to 24ºC) provides ideal conditions for bacterial proliferation (1, 3-6). Furthermore, platelets are stored in plasma containing bioavailable iron that bacteria require to survive (7). Thus we hypothesize that the inclusion of iron chelators will bind and remove iron, thereby inhibiting bacterial growth in both culture medium and platelet concentrates. Additionally, we hypothesize that residual red blood cells (RBCs) in platelet units may contribute bioavailable iron that promotes bacterial growth. To test these hypotheses, we first assessed growth of Staphylococcus epidermidis in culture medium after treatment with the iron chelators deferoxamine (DFO) or phytic acid. DFO significantly inhibited bacterial growth in a dose dependent manner (p < 0.009). Conversely, phytate only inhibited bacterial growth at concentrations ≥ 100 mM (p < 0.001); at ≤ 5 mM, phytate supplied S. epidermidis with additional nutrients and significantly promoted growth (p < 0.001). Subsequently, we monitored the change in RBCs over time. Hemolysis, methemoglobin, and iron levels all significantly increased over the 7-day storage period (p < 0.001) releasing bioavailable iron. Indeed, we found that S. epidermidis growth in iron-poor medium drastically increased with the addition of RBCs, thus supporting our second hypothesis. Surprisingly, the inclusion of DFO in minimal medium did not demonstrate a bacteriostatic effect in the presence of RBCs. The inhibitory effect of DFO was likely overcome by iron released from the elevated methemoglobin levels arising from the direct interaction of DFO with hemoglobin. Previous studies demonstrate that methemoglobin releases iron more quickly than normal hemoglobin (8). Lastly, we evaluated the effect of DFO on microbial growth in platelet concentrates using the BacT/ALERT system. The presence of DFO significantly inhibited S. epidermidis growth in buffy coat platelets in a dose dependent manner (p < 0.001). With these findings, the inclusion of iron chelators is a promising approach to preventing transfusion-transmitted bacterial infection and providing patients with a safer platelet product.

Bacterial infection is a leading cause of morbidity and mortality arising from platelet transfusions (1, 2). Storage of platelet products at room temperature (20 to 24ºC) provides ideal conditions for bacterial proliferation (1, 3-6). Furthermore, platelets are stored in plasma containing bioavailable iron that bacteria require to survive (7). Thus we hypothesize that the inclusion of iron chelators will bind and remove iron, thereby inhibiting bacterial growth in both culture medium and platelet concentrates. Additionally, we hypothesize that residual red blood cells (RBCs) in platelet units may contribute bioavailable iron that promotes bacterial growth. To test these hypotheses, we first assessed growth of Staphylococcus epidermidis in culture medium after treatment with the iron chelators deferoxamine (DFO) or phytic acid. DFO significantly inhibited bacterial growth in a dose dependent manner (p < 0.009). Conversely, phytate only inhibited bacterial growth at concentrations ≥ 100 mM (p < 0.001); at ≤ 5 mM, phytate supplied S. epidermidis with additional nutrients and significantly promoted growth (p < 0.001). Subsequently, we monitored the change in RBCs over time. Hemolysis, methemoglobin, and iron levels all significantly increased over the 7-day storage period (p < 0.001) releasing bioavailable iron. Indeed, we found that S. epidermidis growth in iron-poor medium drastically increased with the addition of RBCs, thus supporting our second hypothesis. Surprisingly, the inclusion of DFO in minimal medium did not demonstrate a bacteriostatic effect in the presence of RBCs. The inhibitory effect of DFO was likely overcome by iron released from the elevated methemoglobin levels arising from the direct interaction of DFO with hemoglobin. Previous studies demonstrate that methemoglobin releases iron more quickly than normal hemoglobin (8). Lastly, we evaluated the effect of DFO on microbial growth in platelet concentrates using the BacT/ALERT system. The presence of DFO significantly inhibited S. epidermidis growth in buffy coat platelets in a dose dependent manner (p < 0.001). With these findings, the inclusion of iron chelators is a promising approach to preventing transfusion-transmitted bacterial infection and providing patients with a safer platelet product.

Bacterial infection is a leading cause of morbidity and mortality arising from platelet transfusions (1, 2). Storage of platelet products at room temperature (20 to 24ºC) provides ideal conditions for bacterial proliferation (1, 3-6). Furthermore, platelets are stored in plasma containing bioavailable iron that bacteria require to survive (7). Thus we hypothesize that the inclusion of iron chelators will bind and remove iron, thereby inhibiting bacterial growth in both culture medium and platelet concentrates. Additionally, we hypothesize that residual red blood cells (RBCs) in platelet units may contribute bioavailable iron that promotes bacterial growth. To test these hypotheses, we first assessed growth of Staphylococcus epidermidis in culture medium after treatment with the iron chelators deferoxamine (DFO) or phytic acid. DFO significantly inhibited bacterial growth in a dose dependent manner (p < 0.009). Conversely, phytate only inhibited bacterial growth at concentrations ≥ 100 mM (p < 0.001); at ≤ 5 mM, phytate supplied S. epidermidis with additional nutrients and significantly promoted growth (p < 0.001). Subsequently, we monitored the change in RBCs over time. Hemolysis, methemoglobin, and iron levels all significantly increased over the 7-day storage period (p < 0.001) releasing bioavailable iron. Indeed, we found that S. epidermidis growth in iron-poor medium drastically increased with the addition of RBCs, thus supporting our second hypothesis. Surprisingly, the inclusion of DFO in minimal medium did not demonstrate a bacteriostatic effect in the presence of RBCs. The inhibitory effect of DFO was likely overcome by iron released from the elevated methemoglobin levels arising from the direct interaction of DFO with hemoglobin. Previous studies demonstrate that methemoglobin releases iron more quickly than normal hemoglobin (8). Lastly, we evaluated the effect of DFO on microbial growth in platelet concentrates using the BacT/ALERT system. The presence of DFO significantly inhibited S. epidermidis growth in buffy coat platelets in a dose dependent manner (p < 0.001). With these findings, the inclusion of iron chelators is a promising approach to preventing transfusion-transmitted bacterial infection and providing patients with a safer platelet product. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Iron is the most abundant trace mineral in the body and an essential element in all living systems. In humans, iron is found at the active site of a number of key proteins involved in oxygen transport, metabolism, respiration and DNA synthesis. The development of agents that inhibit the iron dependent enzyme ribonucleotide reductase (RR) is an established strategy in cancer therapy. Hydroxyurea is the first agent to target RR by inactivating the nonheme iron centre, but its efficacy appears to be limited to myeloproliferative disorders. Currently, Triapine™ (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) is a successful iron chelator, which inhibits enzyme activity and cell growth in vitro at 100 to 1000 fold lower concentrations than hydroxyurea. Preclinical studies suggested that Triapine™ may be an attractive agent to move into clinical development in patients with cancer. Therefore, heterocyclic thiosemicarbazones as well as their iron complexes are an important series of compounds, which may show promising anticancer properties. Thiosemicarbazone derivatives of the HDpT (di-2-pyridyl ketone-3-thiosemicarbazone), HBpT (2-benzoylpyridine-3-thiosemicarbazone), HNBpT (2-(3΄-nitrobenzoyl)pyridine-3-thiosemicarbazone) and HApT (2-acetylpyridine-3-thiosemicarbazone) series and their FeII and FeIII complexes were synthesised and characterised by elemental analyses, UV-vis spectroscopy, NMR spectroscopy, IR (ATR, attenuated total reflectance), EPR spectroscopy, electrochemistry, and X-ray crystallography. To further understand their physical and chemical properties, other transition metal Mn, Co, Ni, Cu and Zn complexes of the HDpT and HBpT analogues were synthesised and characterised. The X-ray crystal structure of trivalent Fe complexes and divalent (Mn, Ni, Cu and Zn) complexes were determined. The HDpT, HBpT, HNBpT and HApT analogues of thiosemicarbazone class of iron chelators have the capability to bind metal ions as tridentate (N,N,S) ligands, forming 2:1 ligand:Fe complexes with an N4S2 octahedral coordination sphere. During the course of investigation, the formation constants of MnII, NiII, CuII and ZnII complexes of these series were determined by spectrophotometric competition titration with nitrilotriacetic acid (NTA). Several interesting aspects of the coordination chemistry of these iron chelators have also been discovered To better understand the biological activities of these ligands, the chemical and the physical properties of these ligands and their iron and other transition metal complexes have been studied. Chiefly, we are concerned with their ability to cross the cell membrane (lipophilicity) as well as the redox chemistry of their metal complexes. The lipophilicity of the ligands was determined by direct partitioning between 1-octanol and water at pH 7.4. The Fe complexes of the HApT series exhibit lower redox potentials than their corresponding HDpT, HBpT and HNBpT complexes and higher anticancer activity indicating a link between their Fe redox reactivity and their biological properties.

Iron is the most abundant trace mineral in the body and an essential element in all living systems. In humans, iron is found at the active site of a number of key proteins involved in oxygen transport, metabolism, respiration and DNA synthesis. The development of agents that inhibit the iron dependent enzyme ribonucleotide reductase (RR) is an established strategy in cancer therapy. Hydroxyurea is the first agent to target RR by inactivating the nonheme iron centre, but its efficacy appears to be limited to myeloproliferative disorders. Currently, Triapine™ (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) is a successful iron chelator, which inhibits enzyme activity and cell growth in vitro at 100 to 1000 fold lower concentrations than hydroxyurea. Preclinical studies suggested that Triapine™ may be an attractive agent to move into clinical development in patients with cancer. Therefore, heterocyclic thiosemicarbazones as well as their iron complexes are an important series of compounds, which may show promising anticancer properties. Thiosemicarbazone derivatives of the HDpT (di-2-pyridyl ketone-3-thiosemicarbazone), HBpT (2-benzoylpyridine-3-thiosemicarbazone), HNBpT (2-(3΄-nitrobenzoyl)pyridine-3-thiosemicarbazone) and HApT (2-acetylpyridine-3-thiosemicarbazone) series and their FeII and FeIII complexes were synthesised and characterised by elemental analyses, UV-vis spectroscopy, NMR spectroscopy, IR (ATR, attenuated total reflectance), EPR spectroscopy, electrochemistry, and X-ray crystallography. To further understand their physical and chemical properties, other transition metal Mn, Co, Ni, Cu and Zn complexes of the HDpT and HBpT analogues were synthesised and characterised. The X-ray crystal structure of trivalent Fe complexes and divalent (Mn, Ni, Cu and Zn) complexes were determined. The HDpT, HBpT, HNBpT and HApT analogues of thiosemicarbazone class of iron chelators have the capability to bind metal ions as tridentate (N,N,S) ligands, forming 2:1 ligand:Fe complexes with an N4S2 octahedral coordination sphere. During the course of investigation, the formation constants of MnII, NiII, CuII and ZnII complexes of these series were determined by spectrophotometric competition titration with nitrilotriacetic acid (NTA). Several interesting aspects of the coordination chemistry of these iron chelators have also been discovered To better understand the biological activities of these ligands, the chemical and the physical properties of these ligands and their iron and other transition metal complexes have been studied. Chiefly, we are concerned with their ability to cross the cell membrane (lipophilicity) as well as the redox chemistry of their metal complexes. The lipophilicity of the ligands was determined by direct partitioning between 1-octanol and water at pH 7.4. The Fe complexes of the HApT series exhibit lower redox potentials than their corresponding HDpT, HBpT and HNBpT complexes and higher anticancer activity indicating a link between their Fe redox reactivity and their biological properties.

Brown algae have been the focus of metal ecotoxicology research for over 60 years, mainly because of their high metal accumulation capacity and reputed resistance. Now that Ectocarpus siliculosus has been positioned as a model for the study of brown algae, and that the genome has been recently sequenced and annotated, new lines of research have been made possible on these ecologically and economically important organisms, including the field of ecotoxicology. Several strains of E. siliculosus have been collected and isolated from locations around the world, thus providing the opportunity to study inter-population differences in their responses to environmental stress. This investigation can be split into three main sections. In the first part Cu exposure experiments were carried out under laboratory conditions using three strains of E. siliculosus: Es524 from a Cu polluted location in Chile, REP10-11 from a metal polluted (including Cu) location in England and LIA4A from a pristine site in Scotland. Strains were exposed for 10 d to concentrations ranging between 0 and 2.4 μM Cu. We measured different parameters: relative growth rates; metal accumulation (extracellular and intracellular); phytochelatins and the expression of related enzymes; oxidative stress responses as manifested in lipid peroxidation and levels of H2O2, and levels of pigments; levels of antioxidants glutathione and ascorbate (in reduced and oxidised forms), and phenolic compounds; and the activity of the antioxidant enzymes superoxide dismutase, catalase, and ascorbate peroxidise. Strain Es524 was the most efficient in counteracting the effects of Cu stress as manifested by a combination of Cu exclusion production of metal chelators, upregulation of oxidative enzymes, and strong antioxidant metabolism. REP10-11 also showed effective Cu defences, especially related to glutathione-ascorbate interactions. LIA4A was the least tolerant strain, with metabolic defences significantly less effective against Cu exposure. In part two a novel transplantation experiment was developed to compare responses in the field with those obtained in the laboratory. The study was carried out at a metal polluted and a low-impacted site in central Chile using strain Es524 (as in the laboratory experiments) and Es147, isolated from a low metal-polluted site in Chile. From the biomass, we conducted similar measurements of Reactive Oxygen Metabolism (ROM) as for the laboratory experiments described in the first part. In agreement with the laboratory experiments, strain Es524 displayed a higher resistance to metal stress. Because they behaved similarly between strains, the best suggested biomarker candidates for future assessments are metal accumulation, glutathione and ascorbate in reduced and oxidised forms, phenolic compounds, and the activity of superoxide dismutase. The method is simple, widely applicable in temperate environments, cost-effective, and provides a reliable representation of metal bioavailability in the environment. In the final part of the study a novel technique for the co-extraction of RNA and DNA, using a high pH Tris-HCl buffer, from small amounts of biomass of different strains of E. siliculosus was successfully developed. The extraction of nucleic acids from brown algae is considered to be difficult and the product is of poor quality due to the high concentrations of interfering secondary metabolites such as phenolics and polysaccharides. The protocol devised here provided high yields of pure RNA and DNA that are suitable for molecular analyses. This investigation provides new insights on metal stress metabolism in brown algae, and demonstrates that metal resistance is dependent on inherited defences developed over a long history of exposure. Furthermore, the good agreement between the results obtained in the laboratory with those from the field study confirms that the responses expressed under controlled laboratory conditions are representative of stress metabolism of E. siliculosus under natural conditions.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lucie Sedlářová Supervisor: Dr. Přemysl Mladěnka, Ph.D. Title of thesis: Standardization of method for in vitro assessment of copper chelation. Cooper is a trace element, which is essential for many biochemical processes in the human body. Disruption of copper homeostasis can cause a variety of diseases. There are primarily two hereditary diseases associated with copper dyshomeostasis: Wilson's disease, which is linked with the excess of copper in the liver and other tissues, and Menkes disease, which is associated with a lack of systemic copper. In addition, local disturbances of copper play a role also in other diseases (neurodegenerative diseases, tumors, myocardial infarction). The main objective of this experimental work was the standardization of a simple but accurate method for rapid screening of copper chelators, which could be applied for the therapy of systemic or local copper excess in the future. The methodology is based on the spectrophotometric determination of copper in microplates using appropriate indicators, bathocuproindisulfonic acid disodium salt (BCS) and hematoxylin. This thesis has shown that the BCS methodology is able to determine chelation of cupric as...

Applesauce prepared for the US military is processed as MRE (meals-ready-to-eat) in several forms including Type VI - Applesauce with raspberry puree, and Type VII - Carbohydrate enriched applesauce. Production of MRE applesauce starts with commercially prepared and thermally processed applesauce that is further processed by a military contractor. The further processing includes adjusting pH, ºBrix, and ascorbic acid level, packaging into pouches, and again thermally processing. Both types of the MRE applesauce are very much liked by troops, but under stress storage applesauce darkens and its consumption is drastically reduced. The overall goal of this project was to identify additives to be used during further processing in order to slow darkening when exposed to elevated temperatures during shipping and storage. The specific objective was to determine whether different types of ascorbic acid, calcium salts, or addition of chelators can reduce deterioration under stress storage. Applesauce (AS), applesauce with raspberry puree (RPAS), MRE AS (Type VII) and MRE RPAS (Type VI) for all experiments were provided by Sopakco, Bennettsville S.C. The research was carried out in three phases. From the Phase 1, we learned that Type VII and Type VI darkened at faster rates at the beginning of the storage, but the effects of storage at 50°C for more than 2 weeks overcame any differences caused by further processing. Phase 2 helped us determine the formulations for the processing on the industrial scale. The formulations were: 0.15% L-ascorbic-acid (AA), and 0.15% AA with 300 ppm EDTA for both AS and RPAS, with 0.83% calcium lactate gluconate (CLG) for AS, and 0.15% ascorbyl-palmitate for RPAS. The results from the Phase 3 indicated that AS with addition of CLG and RPAS with total of 0.18% AA had the least total color change. In all samples, accumulation of HMF was related to amount of ascorbic acid with exception of samples with Pal which had the lowest HMF content. Our results indicate that current MRE Types VI and VII may have better stability at stress storage if the level of AA is limited to 0.18% and 0.83% CLG is added to the AS formulation.

<p>Fluorescent sensors provide a powerful analytical tool for the intracellular detection of metal cations. In some cases, these fluorescent metal-chelating sensors have helped elucidate the function of metal cations within complicated cellular systems. However, most measure or sense changes in the bulk concentration of a metal species and do not respond to those involved in a specific cellular event. For instance, misregulated copper and iron are implicated in neurodegenerative disease and cancer because of their ability to catalytically propagate the formation of the hydroxyl radical through reaction with hydrogen peroxide. A fluorescent sensor that is unresponsive to metal binding until activation by intracellular hydrogen peroxide could potentially pinpoint the location of this oxidative reaction and provide an understanding of the relationship between copper/iron and hydrogen peroxide. </p><p> Described here is the development of two fluorescent prochelators that show a selective fluorescence response to iron or copper only in the presence of hydrogen peroxide. A boronic ester masked spirolactam-based prochelator displays a copper-selective turn-on response after oxidation with hydrogen peroxide in organic solvents as determined by absorbance and fluorescence spectroscopy. However, a competing mechanism occurs in aqueous solution due to hydrolytic instability of the masked prochelator and results in a separate copper-dependent turn-on response as verified by liquid chromatography-mass spectroscopy. A second fluorescent prochelator design relies on metal-dependent fluorescence quenching after oxidation of a self-immolative boronic ester in both organic and aqueous solvents. Cellular microscopy studies show that the sensor's fluorescence intensity is unchanged until incubation with exogenous hydrogen peroxide, which resulted in a decreased fluorescent signal that is restored upon competitive chelation. Both of these prochelators provide a template for future applications and designs with improved properties.</p><p> Two additional chapters describe the development of a UV-activated iron prochelator and a new fluorescently tagged metal chelator. The UV-activated prochelator is protected with two nitrophenyl groups that are photolyzed with 350 nm light within 10 minutes to reveal a high affinity iron triazole-base chelator. A chelator of this nature may provide protection from UV-induced iron liberation and oxidative stress. A second triazole-based chelator with an embedded coumarin fluorophore was prepared as a potential metal sensor. However, this design showed off-target fluorescence responses, thus it cannot be utilized in its current form for metal detection.</p> / Dissertation