Arsenic contamination of the environment is a worldwide health hazard. This research project focused on four areas: development and testing of low cost, field portable devices capable of measuring levels of arsenic at 10 μg L-1 or less; specific chemical techniques for such testing; creation of educational tools and techniques to allow operators who lack advanced chemistry training to perform accurate testing; and the determination and use of a biomarker in DNA as a cancer predictor in individuals exposed to environmental arsenic. The analytical techniques explored include: (1) the Gutzeit method of arsenic determination though arsine gas production, which was investigated in three experiments: measuring arsenic levels in soil samples, using Gutzeit-based kits using silver nitrate as a reactant for arsine gas, and sensitivity comparison of three commercial test kits over varying time periods up to twenty-four hours. (2) The molybdenum blue method, technologically quantified through three different experiments: digital photographic analysis, spectroscopic analysis, and flow injection. (3) Filtration of arsenic contaminated water with wood-ash, sand, ferric oxide, and commercially available steel wool; and the construction of a filtering device constructed of recyclable discarded soda bottles. Further, single nucleotide polymorphisms in the DNA of arsenic exposed individuals were studied to determine what immune response genes might be implicated in arsenic susceptibility. The major conclusions of this research were: digital image analysis used with the Gutzeit method improves precision and accuracy; silver nitrate proved to be a better measurement tool at low concentrations of arsenic than mercuric bromide; and the Gutzeit method can be applied to soils in the Hach kit.

Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDS-related Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxel-accumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa-4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium -mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.

Progesterone regulates female reproduction in many ways, yet it is still unclear how signals are conveyed through nuclear and extranuclear receptors. The traditional notion was that progesterone binds classical progesterone receptors to alter gene transcription. This view has been challenged by the discovery of additional progesterone signaling molecules important for progesterone actions in non-neural cells. In granulosa cells, the progesterone receptor membrane component 1 (Pgrmc1) mediates progesterone effects by forming a receptor complex with binding partner, Serpine mRNA binding protein 1, but it is unknown whether these molecules function similarly in the brain. To begin to address these issues, I investigated the neural role of Pgrmc1 in female mouse brain, rat brain and in neural cells. By examining the neuroanatomical localization, hormonal regulation, and colocalization of Pgrmc1 within key neurons in the neural control of ovulation, Pgrmc1 emerged as a candidate signaling molecule likely to mediate progesterone functions. Furthermore, Pgrmc1 levels regulate the expression of several diverse genes and signaling pathways in neural cells. Taken together, these results demonstrate that Pgrmc1 function is likely to impact diverse neural functions.

Transcription initiates when the enzyme binds to the promoter region and melts open an initial transcribing bubble that extends from position -4 to +4. The initiation phase continues until the enzyme synthesizes ∼8mer RNA, at which point T7 RNA polymerase begins its transition into the elongation phase. The initiation phase is characterized by a energetic instability, which leads to release of small RNA 2-8 bases in length, known as abortive cycling. Abortive cycling, which is the release of small RNA transcripts during synthesis of the first 8 bases of a transcript, has been well documented in most single and multisubunit RNA polymerases, and has been shown to occur in vivo. Structural studies have prompted the ‘scrunched intermediate’ mechanistic model (an elaboration of the earlier stressed intermediate model), which proposes that compaction of the upstream template DNA within the enzyme and/or expansion of the bubble during initiation leads to instability and the release of abortive RNAs. T7 polymerases represent one of the most well characterized transcription systems and despite having no structural similarities to other multi subunit polymerases, shares very similar fundamental mechanistic features. In the initially transcribing abortive phase, the bubble expands as the initial RNA:DNA hybrid grows and the hybrid pushes on components of the enzyme: both key features in the proposed scrunching mechanism. In this work, we directly test predictions of the scrunching model. The introduction of nicks or gaps into the template (scrunched) strand should reduce stress and therefore reduce abortive. Similarly, the introduction of extra bases in this region should increase the release of abortive RNAs or shift their profile to shorter lengths. For all of these modifications, our results show no systematic change in the abortive amounts or profile. An alternate model predicts that a critical source of stress during abortive initiation is the stress caused by the steric clash of the DNA-RNA hybrid against the N-terminal domain. It is already known that this steric clash leads to the transition of the enzyme into a stable elongation complex, however it is unclear as to whether it induces energetic instability within the system. By introducing bulk at the 5’ end of an initiating RNA primer we have increased the putative stress of the DNA-RNA primer against the N terminal domain and demonstrated a slight increase in the release of abortive products. The increase in abortive RNA was however not systematic and even with an increase in the steric push, the enzyme continues to transition into the elongation phase and make run off RNA. Our results suggest that abortive cycling is a kinetically controlled process as opposed to any structurally mediated mechanism.

Skeletal muscle is dynamic and actively regenerates following damage or altered functional demand. Regeneration is essential for the maintenance of muscle mass and, when dysregulated as a result of disease or aging, can lead to losses in functional capacity and increased mortality. Limited data exist on the molecular mechanisms that govern skeletal muscle regeneration in humans. Therefore, the overall objective of this dissertation was to characterize early molecular alterations in human skeletal muscle to strenuous exercise known to induce a muscle regenerative response. Thirty-five subjects completed 100 eccentric (muscle lengthening) contractions (EC) of the knee extensors with one leg and muscle biopsies were taken from both legs 3 h post-EC. The sample from the non-EC leg served as the control. A well-powered transcriptomic screen and network analysis using Ingenuity Pathway software was first conducted on mRNA from the biopsy samples. Network analysis identified the transcription factor NF-kappaB (NF-kB) as a key molecular element affected by EC. Conformational qRT-PCR confirmed alterations in genes associated with NF-kappaB. A transcription factor ELISA, using nuclear extracts from EC and control muscle samples showed a 1.6 fold increase in NF-kB DNA binding activity following EC. Immunohistochemical experiments then localized the majority of NF-kB positive nuclei to cells in the interstitium, which stained positive for markers of pericyte cells and not satellite cells. To ascertain the mechanistic significance of NF-kB activation following muscle damage, in vitro analyses were carried out using a novel primary pericyte/myoblast co-culture model. Primary pericyte/myoblast co-culture experiments demonstrated that pericytes, transfected with a DNA vector designed to drive NF-kB activation, enhanced proliferation and inhibited myogenic differentiation of co-cultured skeletal muscle myoblasts. Furthermore, reduced NF-kB activation led to enhanced myogenic potential of primary pericytes. Taken together, the data in this dissertation suggest that NF-kB dependent signaling in pericytes regulates myogenic differentiation in a cell- and non-cell autonomous manner and may affect the early regenerative response following muscle damage by inhibiting differentiation and promoting proliferation of muscle satellite cells.

Hsp70 molecular chaperones protect proteins from aggregation, assist in their native structure formation, and regulate stress responses in the cell. A mechanistic understanding of Hsp70 function will be necessary to explain its physiological roles and guide the therapeutic modulation of various disease states. To this end, several fundamental features of the Hsp70 structure-function relationship are investigated. The central component of Hsp70 chaperone function is its capacity for allosteric signaling between structural domains and tunable binding of misfolded protein substrates. In order to identify a cooperative network of sites that mediates interdomain allostery within Hsp70, a mutational correlation analysis is performed using genetic data. Evolutionarily correlations that describe an allosteric network are validated by examining roles for implicated sites in cellular fitness and molecular function. In a second component of the Hsp70 molecular mechanism, a novel function is discovered for the disordered C-terminal tail. This region of the protein enhances the refolding efficiency of substrate proteins independently of interdomain allostery and is required in the cell upon depletion of compensatory chaperones, suggesting a previously undescribed mode of chaperone action. Finally, experiments are initiated to assess the dynamic assembly of Hsp70 domains in various allosteric states and how domain orientations may be guided through interaction with partner co-chaperone proteins.

Through their motion, proteins perform essential functions in the living cell. Although we cannot observe protein motion directly, over 68,000 crystal structures are freely available from the Protein Data Bank. Computational protein rigidity analysis systems leverage this data, building a mechanical model from atoms and pairwise interactions determined from a static 3D structure. The rigid and flexible components of the model are then calculated with a pebble game algorithm, predicting a protein's flexibility with much more computational efficiency than physical simulation. In prior work with rigidity analysis systems, the available modeling options were hard-coded, and evaluation was limited to case studies. The focus of this thesis is improving accuracy and robustness of rigidity analysis systems. The first contribution is in new approaches to mechanical modeling of noncovalent interactions, namely hydrogen bonds and hydrophobic interactions. Unlike covalent bonds, the behavior of these interactions varies with their energies. I systematically investigate energy-refined modeling of these interactions. Included in this is a method to assign a score to a predicted cluster decomposition, adapted from the B-cubed score from information retrieval. Another contribution of this thesis is in new approaches to measuring the robustness of rigidity analysis results. The protein's fold is held in place by weak, noncovalent interactions, known to break and form during natural fluctuations. Rigidity analysis has been conventionally performed on only a single snapshot, rather than on an entire trajectory, and no information was made available on the sensitivity of the clusters to variations in the interaction network. I propose an approach to measure the robustness of rigidity results, by studying how detrimental the loss of a single interaction may be to a cluster's rigidity. The accompanying study shows that, when present, highly critical interactions are concentrated around the active site, indicating that nature has designed a very versatile system for transitioning between unique conformations. Over the course of this thesis, we develop the KINARI library for experimenting with extensions to rigidity analysis. The modular design of the software allows for easy extensions and tool development. A specific feature is the inclusion of several modeling options, allowing more freedom in exploring biological hypotheses and future benchmarking experiments.

Removal of protein activity by genetic mutation or pharmacological inhibition has been used extensively to understand the normal function of a protein. However, null mutations eliminate gene function in all cells and pharmacological agents can diffuse through tissues to have similar global effects that can obscure the physiological function of a protein. This is a particular problem when studying proteins that function in many cell types or that have different cell-specific activities. The most direct strategy to study the function of a protein is to reduce or eliminate its activity only in specific cell types, rather than in all cells of an organism. The idea of targeting gene knockdown to specific cell types or to individual cells is not new and many strategies aim to do just this. However, these strategies result in variable knockdown efficiencies and can have silencing effects in neighboring cells and therefore knockdown is never cell-specific. We developed a novel method to knock down the expression of any gene and to restrict this knockdown to specific cell types in C. elegans. In this method we replaced endogenous genes with single copy integrated transgenes containing an engineered sequence tag that introduces premature stop codons (PTCs) into transgene mRNA. This tag causes the natural stop codon to be recognized as a PTC by the host's nonsense-mediated decay (NMD) machinery and does not disrupt gene function. In NMD-competent animals, a PTC-containing transgene is degraded and in NMD-defective animals, a PTC-containing transgene is expressed. Therefore, the expression of PTC-containing transgenes can be controlled by cell-specific activation of NMD. Using this technique, we replaced two endogenous genes with PTC-containing transgenes and directed degradation of their mRNA to specific cell types by restoring NMD activity in these cells. The single copy transgenes were expressed at levels comparable to the endogenous genes and were knocked down to ∼10% of endogenous by NMD, resulting in both global and cell-specific null-like phenotypes. This knockdown strategy can be used to cell-specifically knock down essentially any gene in the C. elegans genome and should provide new insights into understanding protein function.

During the course of normal DNA replication, replication forks are constantly encountering "housekeeping" types of routine damage to the DNA template that may cause the forks to stall or collapse. One product of this fork collapse is the induction of the SOS response, a coordinated global response to help pause the growth and replication of a cell while DNA damage is addressed and repaired. In E. coli, this response is activated by the formation of ssDNA, to which the RecA protein binds and forms a nucleoprotein filament, which acts as the activator for autocleavage of the LexA transcriptional repressor, which normally represses expression of SOS genes. Damage responses are crucial to maintaining genomic integrity, and are therefore essential to all forms of life, and this type of regulatory system is highly conserved. However, cells have mechanisms for tightly regulating induction of these responses, and can often repair routine damage to their chromosomes without the need to induce SOS. This is chiefly evidenced by the observation that more than 20% of cells in a population have RecA filaments, but less than 1% are induced for SOS. How cells make this decision to induce SOS is the subject of this work. This dissertation describes three projects aimed at examining molecular mechanisms by which cells regulate RecA filaments, and therefore the decision to induce the SOS response. The first examines the disparity between the formation of RecA filaments, as evidenced by RecA-GFP foci, and the induction of SOS in the absence of damage, using a psulA-gfp reporter system. It is shown that there are three independent factors that repress SOS expression in undamaged E. coli cells. These are radA, the amount of recA in the cell, and in some circumstances recX. The first two limit SOS in wild type cells in the absence of external damage, while the third is an additional factor required in xthA mutants, likely due to the fact there are more RecA loading events in these mutants. These factors are thought to change the character and reduce the half-life and persistence of RecA filaments in the cell. The second project shows that suppression of SOS through the use of recA4162 and uvrD303 mutants is substrate and situation-specific. This specificity is demonstrated by the fact that, while both recA4162 and uvrD303 can suppress SOS in the SOS constitutive mutant recA730, recA4162 can only suppress SOS when the signal occurs at replication forks and not at any other place on the chromosome, while uvrD303 appears to suppress SOS with less specificity, and can suppress after UV (shown previously), at induced DSBs, and other places not directly at the replication fork. Here mutants of different replication factors are used that uncouple the replisome and induce SOS to a high degree. The third project determines the factors necessary for loading RecA filaments at the replication fork versus other locations on the chromosome when SOS is induced in the absence of damage, and helps elucidate further mechanisms for induction of SOS at these substrates. It is shown that the sbcB and recJ exonucleases assist in inappropriate RecA filament formation by substrate processing exclusively at replication forks, but not other substrates, likely through mechanisms that are reliant on the activities of the RecA loading factors RecBCD and RecFOR.

Lysosomal storage diseases are disorders caused by deficiencies of enzymes responsible for the degradation of substances present in lysosomes. The loss of activity of a lysosomal enzyme leads to the accumulation of substrates within the lysosome, which is the initial step in the process leading to a lysosomal storage disease. Over fifty lysosomal storage diseases are known and have a collective incidence of approximately 1 in 7700 live births. One treatment for these diseases is enzyme replacement therapy, where the defective enzyme is replaced by a recombinant enzyme. Pharmacological chaperone therapy is an alternative treatment which uses small molecule inhibitors to stabilize the defective enzymes. The purpose of this project is to extend the study of lysosomal storage diseases into the field of molecular medicine by exploring the structure and function of human lysosomal enzymes, specifically those responsible for three variants of the mucopolysaccharidosis (MPS) family of diseases. These three enzymes fall into two categories: N-acetylgalactosamine-6-sulfatase (GALNS) and N-sulfoglucosamine sulfohydrolase (SGSH) are human lysosomal sulfatases while β-galactosidase (GLB1) is a glycosidase. In order to accomplish this goal we used a biochemical and structural biology approach. We solved the structures of SGSH and GALNS using X-ray crystallography and analyzed the mutations that lead to their respective diseases MPS III A and MPS IV A. This analysis revealed that these diseases could reflect protein misfolding since the majority of the mutations is located in the hydrophobic core of the proteins. The active site pockets of these enzymes have features such as charged amino acids and hydrophobic amino acids suitable for drug design. We determined that the small molecules galactose and 1-deoxygalactonojirimycin (DGJ) and 4-epi-isofagomine are competitive inhibitors of GLB1. Using the published crystal structure of GLB1, we present an explanation for the inhibitory effects of 4-epi-isofagomine.Through the structural analysis of the disease causing mutations and the identification of novel inhibitors; we hope to gain insight into the enzymatic mechanisms of these enzymes and the potential candidates for pharmacological chaperone therapy as treatments for the MPS family of diseases.