Regions along the Mediterranean and Southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of vitamin E (tocopherols and tocotrienols). This study examines the potential of gamma tocopherol (GT) and gamma tocotrienol (GT3) to reduce prostate cancer proliferation by examining their effects on the NFκB pathway. NFκB is known to inhibit apoptosis in cancer cells. Our data shows that both GT and GT3 are capable of down regulation of NFκB precursors and up regulation of Caspase 8, indicating an induction of apoptosis.

Colorectal cancer is the third most prominent cancer world-wide and it is the second leading cause of cancer deaths in the United States. Many dietary components affect the risk of developing colorectal cancer, such as Vitamin E. Of the eight isomers of Vitamin E, four have a tocotrienol structure. Tocotrienols are found at highest concentrations in palm oil, which is ingested more in areas of Asia where the incidence of colorectal cancer is the lowest, suggesting a role of tocotrienols in the prevention of colorectal cancer. The metabolism of Arachidonic acid pathway produces a host of pro-inflammatory metabolites either by the Cyclooxygenase (Cox) pathway or the Lipoxygenase (Lox) pathway. The expression of Lox is increased in various human cancer lines; this over expression has been associated with tumor cell proliferation, resistance to apoptosis, and angiogenesis. Another important pathway related to cancer involves mTOR (mammalian target of rapamycin), which is involved in cell growth and human tumorigenesis. The focus of this study included treatment of the HCT-116 colon cancer cell line with gamma tocotrienol to examine potential pathways involved in the induction of apoptosis. Also, whether the Vitamin E-mediated signaling through Arachidonic acid metabolism is necessary for the down regulation of protein translation in the mTOR pathway by using chemical inhibitors specific to Arachidonic acid in the presence and absence of vitamin E treatment was explored. The colon cancer cell line, HCT-116, was treated with gamma tocotrienol isomer and then isolated at 18 and 24 hours. Cells lysates were analyzed by Western Blotting. Our data shows that the mTOR pathway is downregulated by treatment with gamma tocotrienol at 18 hours and 14 hours for 5 µM demonstrating that protein translation is abrogated. Phospho S6 ribosomal protein and phospho-p70 S6 kinase are both downregulated, and phospho-4EBP1 is up regulated upon treatment of gamma tocotrienol. Furthermore, at the same concentrations, Caspase 12 and Caspase 8 are cleaved indicating apoptosis. In addition, parallel up-regulation of 15-LOX-1 and down regulation of Cox-2 at 5 µM at 18 hours is observed. Upon treatment with Caffeic acid, 15-lox-1 is over expressed causing mTOR trend to reverse and down regulation of ERK. Thus, mTOR regulation is dependent on a delicate balance of fatty acid metabolites.

The zinc finger protein, zBED6, is a transcriptional regulator of IGF2 along with hundreds of other genes relating to development and growth. Studies on the growth of commercially bred pigs discovered a single nucleotide substitution in the third intron of IGF2 which disrupts the binding of zBED6 and is responsible for the three-fold upregulation of IGF2 in skeletal muscle. The mutation is linked to decreased subcutaneous fat deposition, larger organ size, and increased skeletal muscle mass. Three different constructs of the zBED6 protein made by Björklund 2018 were expressed and purified to characterize their binding affinity, where one contained both zinc finger domains and two of the constructs contained only one zinc finger domain each. Electrophoretic mobility shift assay protocol was optimized to determine the apparent Kd (= 210 ± 31nM) for the full-length construct C13 and to determine which zinc finger domain was sensitive to the mutation in the IGF2 gene. The first zinc finger domain seems to be more specific in its binding target. Preliminary microscale thermophoresis results were highly variable, needing further optimization of the protocol in order to obtain a full binding curve. The next steps involve site directed mutagenesis of residues binding DNA to determine which interactions are the most significant and possibly crystallization studies as well.

More conservation programs directed towards insects are needed in order to prevent the continue decline in which many insect species in the world are heading to. New ways to use the remaining microhabitats are needed to mitigate the fragmentation and degradation that urbanization and modern agriculture have contributed to in the southern Swedish landscapes. This study focusses on how microhabitats like road-verges can help the conservation projects of pollinating insects. To increase the knowledge about three groups of pollinators (Apidae, Rhopalocera and Syrphidae) and their distribution between two different microhabitats, a malaise trap was set up in road verges and in sample locations further away from the road-verges. The result from this study shows that road-verges are a microhabitat that could work as mitigation to the decline of some pollinating species, if right actions are taken. This study shows that Syrphidae was most acceptant to the hostile environment close to the roads. If these actions are going to help the most treated species is still unknown.

The purpose of this study was to identify the site of interaction of antimycin with the ubiquinone-cytochrome b-c1 oxidoreductase in the photosynthetic bacteria, Rhodopseudomonas sphaeroides. To accomplish this goal, three areas of research were undertaken: the synthesis of a radiolabeled, photoaffinity analog of antimycin, identification of the inhibitory characteristics of this analog, and the photoaffinity labeling of the antimycin binding site. All three areas were accomplished. The major finding of this study was the identification of an 11,000 dalton polypeptide as the predominantly labeled protein. Although this polypeptide was not exclusively labeled, it was consistently labeled and showed competition with antimycin. These results are consistent with a similar study performed by das Gupta and Rieske (1973) with a mitochondrial preparation. These results are not conclusive, but do show several interesting points. First, cytochrome b is not the only site of interaction of antimycin with the ubiquinone~cytochrome b-c1 region of the electron transport chain. Secondly, an 11,000 dalton polypeptide is an important component of this protein complex. The function of this polypeptide is unknown, but should provide interesting research for future studies.

Multi-drug resistance in bacterial pathogens has become a global health crisis. Each year, millions of people worldwide are infected with bacterial strains that are resistant to currently available antibiotics. Diseases such as tuberculosis, pneumonia, and gonorrhea have become increasingly more difficult to treat. It is essential that novel drugs and cellular targets be identified in order to treat this resistance. Bacterial topoisomerase IA is a novel drug target that is essential for cellular growth. As it has never been targeted by existing antibiotics, it is an attractive target. Topoisomerase IA is responsible for relieving torsional strain on DNA by relaxing supercoiled DNA following processes such as replication and transcription. The aim of this study is to find novel compounds that can be developed as leads for antibiotics targeting bacterial type IA topoisomerase. Various approaches were used in order to screen thousands of compounds against bacterial type IA topoisomerases, including mixture-based screening and virtual screening. In the mixture-based screen, scaffold mixtures were tested against the M. tuberculosis topoisomerase I enzyme and subsequently optimized for maximum potency and selectivity. The optimized compounds were effective at inhibiting the enzyme at low micromolar concentrations, as well as killing the tuberculosis bacteria. In a virtual screen, libraries with hundreds of thousands of compounds were screened against the E. coli and M. tuberculosis topoisomerase I crystal structures in order to find new classes of drugs. The top hits were effective at inhibiting the enzymes, as well as preventing the growth of M. smegmatis cells in the presence of efflux pump inhibitors. Organometallic complexes containing Cu(II) or Co(III) were tested as well against various topoisomerases in order to determine their selectivity. We discovered a poison for human type II topoisomerase that has utility as an anticancer agent, as it killed even very resistant cell lines of breast and colon cancer. The Co(III) complexes were found to inhibit the bacterial topoisomerase I very selectively over other topoisomerases. The various methods of drug discovery utilized here have been successful at identifying new classes of compounds that may be further developed into antibiotic drugs that specifically target bacterial type IA topoisomerases.

Melanoma incidence is increasing faster than any other cancer worldwide.1 Early detection is often curative, but metastatic melanoma is lethal (5-year survival <20%) due to the development of resistance to all approved drugs.1 However, emerging evidence suggests that differences in melanoma metabolism relative to non-malignant cells may provide a target to improve treatment.2-14 Specifically, melanoma cells have increased mitochondrial electron transport chain (ETC) activity, elevated levels of reactive oxygen species, and a simultaneous hyperpolarized mitochondrial membrane potential relative to non-malignant cells.4, 8, 11, 15-17 Furthermore, melanoma cells have upregulated glucose consumption and concurrent increased levels of glucose transporters (GLUTs) relative to non-malignant cells; the products of glycolysis (pyruvate and NADPH) aid in the detoxification reactive oxygen species (ROS), while the intermediates are utilized in energy production via increased oxidative metabolism.15, 18 Collectively, melanoma cells exhibit alterations in metabolic, mitochondrial, and cell-surface targets that can be potentially exploited for therapeutic strategies for selective cancer cell killing relative to non-malignant cells. The research presented here demonstrates the therapeutic potential for a new class of mitochondrial-targeted fluorescent lipophilic-cations: pyridinium derivatives (UIRF 17023.186PV1 U.S. Provisional Patent Application No. 62/268,980 Patent Pending). Importantly, the pyridinium derivatives presented in this study have not been previously investigated as a mitochondrial-targeted therapy.19-21 Furthermore, the research presented outlines the feasibility of improving melanoma cellular accumulation of these pyridinium derivatives by including a GLUT targeting moiety in the form of a hexosamine. The addition of a hexosamine molecule to pyridinium derivatives has the potential to increase melanoma cell accumulation by targeting upregulation of GLUT expression in melanoma cells relative to normal cells. Thus, the results of this study identified: (1) a triphenylvinylpyridine (TPVP) lipophilic cation derivative that increased melanoma oxidative metabolism and decreased melanoma cell viability; and (2) the targeting potential for GLUT-mediated melanoma cell specific delivery of glucosamine-modified TPVP derivatives. These findings support the hypothesis that TPVP-based therapies can be developed to exploit fundamental differences in glucose and mitochondrial metabolism to selectively kill melanoma cells relative to non-malignant cells.

Nitrogenase is the enzyme that catalyzes the reduction of nitrogen to ammonia in a reaction requiring MgATP hydrolysis. Two component proteins of nitrogenase are the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein). Nitrogenase contains two nucleotide binding sites. During catalysis, the Fe protein binds two MgATP first. The confonnational changes induced upon MgA TP binding allow the Fe protein to associate with the MoFe protein. After the formation of the Fe protein-MoFe protein complex, a single electron is transferred from the Fe protein to the MoFe protein, an event that is coupled to MgATP hydrolysis in the Fe protein. The wild-type Fe protein and all the altered Fe proteins studied so far are homodimeric. In order to assess the contribution of each nucleotide binding site in the Fe protein to the events occurring during nitrogenase catalysis, a heterodimeric Fe protein was constructed that has Asp 39 substituted by Asn in one subunit and the other subunit the same as the wild-type Fe protein. Characterization of this heterodimeric Fe protein showed that alterations in the properties of the [4Fe-4S] cluster that occur upon nucleotide binding to the Fe protein are due to the additive effect of each nucleotide binding to the Fe protein. The rates of MgATP hydrolysis and MgATP-dependent primary electron transfer of this heterodimeric Fe protein are intermediate between those of the homodimeric wild-type Fe protein and D39N Fe protein. These observations suggested that each ATP binding site contributes to the rate acceleration of primary electron transfer. After electron transfer, this heterodimeric Fe protein forms a tight complex with the MoFe protein, demonstrating that alteration in one subunit is enough for the formation of a tight nitrogenase complex. When this heterodimeric Fe protein was combined with the MoFe protein, no substrate reduction was detected. Therefore, two functional subunits of the Fe protein are necessary for reduction of substrates. The mechanism of ATP hydrolysis in the Fe protein was also investigated. Using site-directed mutagenesis, the role of lysine 10 of the Azotobacter vinelandii nitrogenase Fe protein in MgATP hydrolysis was examined. Changing Lys 10 of the protein to Arg resulted in an Fe protein that hydrolyzed MgATP at a rate 3% that of the wild-type Fe protein. The affinities of the K10R Fe protein for nucleotides and the changes in the properties of the [4Fe-4S] cluster of the K10R Fe protein upon nucleotide binding were compared with those of the wild-type Fe protein. These results indicated that in the absence of the MoFe protein, the interactions of the Kl OR Fe protein with nucleotides are similar to the wild-type Fe protein. After the Fe protein-MoFe protein complex formation, the dramatic decrease in the rate of MgATP hydrolysis of the K10R Fe protein indicated a role of Lys 10 in ATP hydrolysis. This conclusion is consistent with the X-ray crystal structure of the nitrogenase complex stabilized by the AlF4-•ADP, where Lys 10 is proposed to facilitate product formation in ATP hydrolysis.

Phosphorus has long been recognized by agriculturists as being one of the elements essential for plant growth. Although the total phosphorus content of a soil may be high, the amount of phosphate actually available for plant growth is often low. Many agencies such as inherent low solubilities of phosphate compounds, adsorption on Ca C03, adsorption in the diffuse double layer of clay micelles, and chemical precipitation of phosphate which refers to the removal of phosphate ions from solution and their chemical bonding to the solid phase have been studied to explain the lack of phosphate availability.

Asbestos related research began approximately 60 years ago, yet, the mechanism(s) by which asbestos exerts its biological effects is not well understood. The hypothesis upon which this dissertation is based is that mobilization of iron from asbestos enhances the iron-dependent biochemical reactivity of asbestos in vitro and contributes to asbestos-dependent cytotoxicity. The specific aims for this hypothesis were, 1) to determine whether iron was responsible for the biochemical reactivity of asbestos in vitro and asbestos-induced cytotoxicity in cultured cells, and 2) to determine whether mobilization of iron from asbestos enhanced the reactions catalyzed by asbestos in vitro and contributes to asbestos-induced cytotoxicity. It was shown that a chelator (e.g., citrate) had to be present to mobilize iron from asbestos in vitro at pH 7.5. Factors that affected iron mobilization from asbestos (e.g., chelator, pH, or surface area) were investigated. Iron on crocidolite reacted with reducing agents and o2, catalyzed the formation of hydroxyl radical, and induced the formation of DNA single-strand breaks in vitro. However, mobilization of iron from crocidolite by a chelator greatly enhanced crocidolite-dependent o2 consumption, hydroxyl radical formation, and DNA damage in vitro. Crocidolite was more cytotoxic, as measured by cloning efficiency, to cultured Syrian hamster embryo cells than crocidolite that had been pretreated to reduce the amount of iron associated with the fiber, suggesting that iron was responsible for the cytotoxicity. Crocidolite-dependent transformation of these cells was not detected. Crocidolite-dependent cytotoxicity to the human lung carcinoma cell line, A549, was directly dependent upon dose. Intracellular mobilization of iron (55 Fe) from crocidolite was determined using neutron-activated crocidolite and A549 cells. A time- and dose-dependent increase in the amount of 55 Fe mobilized intracellularly from crocidolite into a soluble, 10,000 x g supernatant fraction of lysed cells was observed for cultured cells treated up to 72 h. All of the results presented here support the hypothesis that iron and/or iron mobilization from asbestos may contribute to the some of the biological effects of asbestos in vivo.