In order to assess the benefit or toxicity of chromium in dietary supplements, trivalent chromium and hexavalent chromium must be measured and verified with mass balance (sum of both species equaling total chromium). This is necessary because dietary supplements report trivalent chromium, an essential trace element, as an ingredient, but hexavalent chromium, a toxic carcinogen, may also be present. Because trivalent chromium is stable in acidic conditions and hexavalent chromium in alkaline conditions, interconversions between species occur and increase the difficulty of quantification. Therefore, EPA Method 3060A was first performed to extract hexavalent chromium. Then, EPA Method 3052 was performed on the residue to digest the remaining trivalent chromium. Speciated Isotope Dilution Mass Spectrometry (SIDMS) with Ion-Exchange Chromatography-Inductively Coupled Plasma-Mass Spectrometry (IC-ICP-MS) was used to account for interconversions as well as determination of trivalent and hexavalent chromium concentrations in the studied samples. Mass balance indicated that the analyzed supplements contained hexavalent chromium ranging from 0 to 16% of the total chromium content. / Bayer School of Natural and Environmental Sciences; / Environmental Science and Management (ESM) / MS; / Thesis;

Dictyostelium discoideum serves as an ideal system to study social evolution because of the social stage of its lifecycle, where individuals aggregate to build a multicellular structure. However, much of its basic biology remains unknown and this limits its utility. I used three separate projects to fill these gaps. In my first project, I examined how speciation and genetic diversity affects kin discrimination using a related dictyostelid, Polysphondylium violaceum . I sequenced the ribosomal DNA of 90 clones of P. violaceum and found that P. violaceum is split into several morphologically identical groups. When allowed to cooperate in pairwise mixes, I found that some clones cooperated with others in their group, but in mixes between groups, clones did not cooperate. For my second project, I looked at whether D. discoideum has sex in natural populations. While sex has been observed in laboratory clones of D. discoideum , it is unclear whether sex occurs in natural populations, and sex can influence the evolution of traits. I used a dataset of microsatellites in 24 clones of D. discoideum to look for a decrease in linkage disequilibrium as a molecular sign of sex. Linkage disequilibrium is higher between physically close loci than between loci on different chromosomes. From this, I conclude that D. discoideum undergoes recombination in nature. Lastly, I used the genome sequence of D. discoideum to look at large scale patterns of evolution. Mutations tend to be biased towards A/T from G/C so, on average, mutations should lower the nucleotide content of sequences. The removal of these mutations, purifying selection, should preserve nucleotide content. I used the genomes of D. discoideum and Plasmodium falciparum identify classes of sequences that should be under different amounts of purifying selection and compared their nucleotide contents. In all cases, those sequences under more purifying selection had higher GC contents than sequences under less purifying selection. Looking at relative nucleotide content may thus serve as an indicator purifying selection. These three studies add insight on how cooperation works in dictyostelids as well as adding an understanding of how traits, social and otherwise, would evolve in this system.

Copper is one of the most important trace elements in ruminant nutrition. Its deficiency causes certain pathologies that can be cured by supplementation, by either five ("inorganic") or complexed ("chelated") forms. With the objective of being able to quantitatively compare the distribution of copper between the two forms of supplements, the speciation of copper in ruminant fluids was studied here. For this study, copper lysine supplement was used. It was necessary to first study the acid-base and complexation chemistry of lysine with copper (II). In addition, the complexation chemistry of glycine and histidine, were investigated. Mass balance equations were used to describe the distribution of copper (II) amongst different ligands. The results of the distribution of copper (II) ions in McDougall's solution (a simulated form of bovine saliva), indicate no significant differences in the distribution of copper using the different form of supplements. 1H NMR was used to validate the results from the computer model. Using a combination of the results from the saliva simulation model and the chemical shifts from the NMR studies, the chemical shift changes that would accompany the addition of copper (II) to McDougall's solution were predicted. Results from the models do not show any appreciable differences from experimental values. Rumen samples were collected. Important peaks in the 1H NMR spectrum were assigned. The spectrum indicated that acetic acid, resulting from the fermentation in the rumen, was a good probe for monitoring the speciation pattern. Speciation calculation indicated that the bulk of the copper would be bound to ammonia in the rumen. Changes in chemical shifts that result from the introduction of copper (II) to the rumen contents were predicted. Results were compared with experimental values. Agreement between the two sets of results was found to be satisfactory. The study shows that any advantages that result from the use of copper lysine supplement are not as a result of its remaining intact. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process. The methods developed here could be extended to other metal complexation problems in biological fluids. (Abstract shortened by UMI.)

Copper is one of the most vital trace elements in ruminant nutrition. It is required for several metabolic activities and it is also an essential component of several physiologically important metalloenzymes. Thus copper deficiency in ruminants results in distinctive pathologies, and hence in significant economic losses to farmers. Copper deficiency results from very low copper in diet (primary copper deficiency) and interference with Cu absorption in the animal due to Mo and S in food or water (secondary copper deficiency). The molybdenum-induced copper deficiency that affects ruminants can be attributed to the formation of thiomolybdates (TMs)from molybdate and sulfide in the rumen. The TMs formed then react irreversibly with copper to form insoluble Cu-TM complex which ultimately end up being excreted, thus reducing copper bioavailability to the ruminant. <p>In this study, an attempt has been made to use computer simulations to model speciation of copper in rumen fluid in the presence of TMs with the aim of understanding the extent to which TMs affects the levels of copper in the rumen. <p>This was done by initially refining the computer model of copper speciation with respect to low molecular mass (LMM) ligands in bovine rumen with the aim of correcting the discrepancy that was observed during experimental validation of the computer model in a previous study. To this end, mass balance equations which describes the distribution of Cu(II) amongst the different ligands were encoded into a spreadsheet to calculate equilibrium concentration of all species. Formation constants obtained from literature as well as those obtained from studies in our group were used as input values in the spreadsheet. Results show that at average ruminal pH, the metal would be present mostly as carbonate and phosphate complexes. The results obtained from the computer model in the present study were validated using 1H NMR experiments on simulated rumen fluid as well as actual rumen fluid containing Cu(II); using acetic acid chemical shift as the probe for monitoring the speciation pattern. Excellent agreement was observed between the computer model and experimental results. Discrepancy was however observed upon introduction of copper lysine as copper source into the model. Incorporation of a mixed ligand complex of Cu(II), acetate and lysine into the computer model gave an excellent agreement between the computer model and experimental results. <p>The study was extended to include glycine, histidine, methionine and EDTA complexes as the copper source in both rumen saliva (McDougalls solution) and rumen fluid. Results show that only the histidine and EDTA complexes persist to any significant extent, in spite of the large number of competing ligands present in these matrices.<p>In this study, success has also been achieved in the integration of the slow (kinetically controlled) formation of TMs and copper-tetrathiomolybdate (TM4) complexation into the previously developed model for the rapidly equilibrating copper-ligand speciation. To simulate the formation of the TMs and Cu-TM4 complex with respect to time, the differential equations representing rate expressions for each chemical species were solved to obtain an analytical solution using the Laplace transform method. The analytical solutions obtained were encoded in a spreadsheet and calculated as function of time to obtain time dependent concentrations of TMs and Cu-TM4 complex. This was then integrated with previously developed model for the rapidly equilibrating copper-ligand speciation in the rumen. The kinetic data used in the simulation of the formation thiomolybdates was obtained fron literature wheras that for Cu-TM4 complexation was obtained from our lab using Cu(II) - Ion Selective Electrode. The results show that that in the presence of TM4 the, Cu(II) bound to low molecular ligands in the rumen is drastically reduced confirming the effect TM4 on Cu(II) observed in several in vitro studies.<p>The study shows that in thiomolybdate contaminated rumen environment, the bioavailability of copper is considerably reduced. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process.

Copper (Cu) is an essential trace element in ruminants such as the cow. Its deficiency leads to a number of debilitating symptoms and can eventually cause death. Secondary Cu deficiency is caused by the presence of chemical antagonists such as the thiomolybdates (TMs) (MoOxS4-x2-; x = 0 3). TMs form in the rumen and then form tightly bound insoluble complexes with Cu. These complexes are then excreted and the Cu is unavailable to the animal. The TMs and their effects on ruminant Cu are the focus of this thesis.<p>This study includes three main bodies of work. First, a method for quantifying the composition of TM mixtures in solution was developed. Second, the rate constants of TM interconversions were determined. Third, reactions between Cu and the TMs were studied. This work is part of an ongoing study in the Reid group to model Cu-ligand speciation in the rumen. <p>Thiomolybdates form by successive replacement of molybdate (MoO42-) O ligands with S. This makes synthesis of the uncontaminated compounds difficult. Mixtures of TMs are best analyzed by UV-visible spectroscopy despite the considerable spectral overlap. The combination of these two issues makes the characterization of the spectra difficult. In this study, a method was developed to establish the spectra of the pure TMs despite cross-contamination. Multivariate curve fitting methods were used to establish the composition of synthesized samples simultaneously with the molar absorptivities of the pure TMs. This was done using a Beers Law model. The composition was determined from a fit using assumed molar absorptivities. The absorptivities were then refined using the composition results. These processes were successively reiterated until both were optimized. The optimized absorptivities could then be used to determine the composition of any TM solution mixture. <p>The kinetics of TM formation under biologically relevant conditions were followed by UV-visible spectroscopy. The spectra were then analyzed, using the method developed above, to determine all TM concentrations. Curve fitting methods were used to simultaneously determine the rate constants of all processes that occurred. Integrated rate equations used in the fitting process were developed using the Laplace Transform method. Rate constants were determined at varying pH, ionic strengths and temperatures. From this information it was shown that TM formation and hydrolysis occurs via an associative mechanism. It was also determined that H2S and not HS- was the sulfide nucleophile in TM formation reactions.<p>This study was then extended to include reactions involving Cu and the TMs. First, the UV-visible spectra of the reaction products of Cu with each TM were characterized. This allowed the kinetics of reactions between Cu and the TMs to be followed. This reaction has been found to proceed via an intermediate. Molar absorptivities for this intermediate were optimized along with the appropriate rate constants. This was done using a combination of mathematical simulations and the curve fitting methods used for the TM kinetics. The resulting rate constants can be compared with those obtained previously in the group using a Cu ion selective electrode. <p>These rate constants were used in conjunction with the TM formation rate constants to perform simulations. The results of these simulations provide a picture of what is expected to occur in the rumen. Reactions were also performed in which TM formation occurred in the presence of Cu. This was then extended to mimic bovine feeding habits. Here, portions of the reactions mixture was removed at various time intervals and replaced with fresh reagents. These experiments were used to gain a qualitative picture of Cu speciation in the presence of the TMs over time. The work presented in this thesis provides a crucial step toward understanding the problem of bovine copper deficiency.

<p>Biodiversity is generated by the process of speciation. Because biological species are defined as populations that are unable to exchange genes with one another, the study of the evolution of reproductive isolation occupies the center of speciation research. A key to deciphering how reproductive isolation evolves is to understand the genetic changes that underlie these barriers to gene flow. Intrinsic postzygotic barriers, such as hybrid sterility or inviability, are known to impede gene flow and especially lend themselves to genetic analysis because of their ease of study in a laboratory setting. Because hybrid sterility likely evolves before hybrid inviability, it potentially plays an important role in the cessation of gene flow. Yet, while their X-linked counterparts have been precisely localized, we remain ignorant of the numbers of and interactions among dominant autosomal loci that are predicted to contribute to F1 hybrid male sterility. </p><p> To address this conceptual void, I examine the genetic architecture of hybrid male sterility between the allopatric sister species Drosophila persimilis and D. pseudoobscura bogotana. First, using a large-scale backcross analysis, I fine-map autosomal QTL from D. persimilis that confer sterility in male hybrids. This fine-mapping shows that loci contributing to hybrid male sterility reside outside chromosomal rearrangements (i.e., regions of reduced recombination) in this allopatric species pairs. In contrast, these QTL do not contribute to hybrid male sterility in the comparable sympatric hybridizing species D. persimilis and D. pseudoobscura, as predicted by models that suggest that hybridizing species persist because of broad regions of reduced recombination. Next, I use a serial backcross design to introgress these sterility-conferring QTL from D. persimilis into a D. p. bogatana genetic background devoid of other alleles from D. persimilis. This introgression study tested a prediction of the dominance theory proposed to explain Haldane's rule: dominant-acting autosomal loci should interact with recessive-acting X-linked loci to produce sterile hybrid males. Surprisingly, the results demonstrated that the "composite" dominance of the autosomal QTL is more important than the dominance of individual QTL for producing Haldane's rule: epistasis among loci elevated their dominant effects on sterility such that individually-recessive-acting autosomal QTL can contribute to F1 male infertility. Finally, using recombination to generate independent lines bearing only small segments of the identified QTL regions, I examine whether single or multiple loci within these regions contribute to the overall effect of hybrid sterility. While the effect of one QTL depends on epistasis between several loci within that small region, the effect of the other QTL appears to derive from a single genetic factor. These results suggest that estimates of the number of genes that contribute to reproductive isolation are at best, likely too low and, at worst, unattainable with the mapping resolution attainable by standard backcross and introgression approaches.</p><p> This dissertation addresses both evolutionary and genetic hypotheses of intrinsic postzygotic isolation. Hybrid male sterility between D. persimilis and D. p. bogotana clearly involves highly specific and complex interactions between homoospecific loci. The mapping results presented here also lay the foundation for the identification and cloning of multiple autosomal sterility-conferring "speciation genes."</p> / Dissertation

<p>Recombination occurs during meiosis to produce new allelic combinations in natural populations, and thus strongly affects evolutionary processes. The model system Drosophila has been crucial for understanding the mechanics underlying recombination and assessing the association between recombination rate and several evolutionary parameters. Drosophila was the first system in which genetic maps were developed using recombination frequencies between genes. Further, Drosophila has been used to determine genetic and environmental conditions that cause variation in recombination rate. Finally, Drosophila has been instrumental in elucidating associations between local recombination rate and nucleotide diversity, divergence and codon bias, as well as helping determine the causes of these associations.</p><p>Here I present a fine-scale map of recombination rates across two major chromosomes in Drosophila persimilis using 181 SNP markers spanning two of five major chromosome arms. Using this map, I report significant fine-scale heterogeneity of local recombination rates. However, I also observed "recombinational neighborhoods", where adjacent intervals had similar recombination rates after excluding regions near the centromere and telomere. I further found significant positive associations of fine-scale recombination rate with repetitive element abundance and a 13-bp sequence motif known to associate with human recombination rates. I noted strong crossover interference extending 5-7 Mb from the initial crossover event. Further, I observed that fine-scale recombination rates in D. persimilis are strongly correlated with those obtained from a comparable study of its sister species, D. pseudoobscura. I documented a significant relationship between recombination rates and intron nucleotide sequence diversity within species, but no relationship between recombination rate and intron divergence between species. These results are consistent with selection models (hitchhiking and background selection) rather than mutagenic recombination models for explaining the relationship of recombination with nucleotide diversity within species. Finally, I found significant correlations between recombination rate and GC content, supporting both GC-biased gene conversion (BGC) models and selection-driven codon bias models. </p><p>Next, I looked at the role of chromosomal inversions in species maintenance by examining the impact of inversions distinguishing species to disrupt recombination rates within inverted regions, at inversion boundaries and throughout the remainder of the genome. By screening nearly 10,000 offspring from females heterozygous for 3 major inversions, I observed recombination rates within an inverted region in hybrids between Drosophila pseudoobscura and D. persimilis to be ~10-4 (similar to rates of exchange for inversion heterozygotes within species). However, despite the apparent potential for exchange, I do not find empirical evidence of ongoing gene exchange within the largest of 3 major inversions in DNA sequence analyses of strains isolated from natural populations. Finally, I observe a strong 'interchromosomal effect' with up to 9-fold higher (>800% different) recombination rates along collinear segments of chromosome 2 in hybrids, revealing a significantly negative association between interchromosomal effect and recombination rate in homokaryotypes, and I show that interspecies nucleotide divergence is lower in regions with larger changes in recombination rates in hybrids, potentially resulting from greater interspecies exchange. This last result suggests an effect of chromosomal inversions on interspecies gene exchange not considered previously.</p><p>Finally, I experimentally tested for a novel male-mediated effect on female recombination rates by crossing males that differed by either induced treatment variation or standing genetic variation to genetically identical females. After assaying recombination frequency in the offspring of these genetic crosses, I fitted these data to a statistical model where I showed no effect of male temperature treatment or male genetic background on offspring recombination rate. However, I did observe a difference of recombination rates of offspring laid 5-8 days post-mating between males treated with Juvenile Hormone relative to control males. Environmental variation in male ability to affect recombination rate in their mates suggests the potential for sexual conflict on optimal proportion of recombinant offspring, perhaps leading to changes in population-level recombination rates with varying levels of sexual selection.</p><p>Overall, my map of fine-scale recombination rates allowed me to confirm findings of broader-scale studies and identify multiple novel features that merit further investigation. Furthermore, I have identified several similarities and differences between inversions segregating within vs. between species in their effects on recombination and divergence, and I have identified possible effects of inversions on interspecies gene exchange that had not been considered previously. Finally, I have provided some evidence that males may impact female recombination rates, although future work should attempt to explore the range of male differences that impact this trait and the mechanism through which males impact the outcome of female meiosis.</p> / Dissertation

Water samples were collected along salinity gradients during different seasons from three estuaries (Tseng-Wen, Gao-Ping, Er-Ren) and coasts in/off southwestern Taiwan. In order to assess the partitioning of trace metals between solution and particle, the concentrations of dissolve and particulate trace metals (Cd, Cu, Ni, Pb) along with their chemical affinity fractions, were determined. This study investigated the variations in distribution and partitioning of the different metals in estuarine waters, and examined the effects of oxides and particulate organic matter on the partitioning of trace metals in waters from different estuaries. Fractionation of dissolved trace metal species was based on ion exchange (Chelex-100 and AG MP-1 resins) separation techniques. The fractions obtained were operationally defined as labile (Chelex), organic (AG MP-1) and inert. Particles were extracted to three phases (surface adsorbed phases, Fe¡VMn oxide/organic phases and refractory phases) using sequential extraction techniques. Seasonally variable distributions of dissolved trace metals were found in the Tseng-Wen estuary. The behavior of trace metals was mainly influenced by anthropogenic input during the dry season in the upper Tseng-Wen estuary, while mixing processes controlled the distribution of trace metals during the wet season. The dilution effect was a major factor in the metal distribution in the Gao-Ping estuary due to high river discharge. The higher concentration of metals in the Er-Ren estuary, in contrast to other estuaries indicated that the Er-Ren estuary has serious pollution concerns. According to the results of particulate metal fractions obtained, Cd and Pb existed predominantly in the surface adsorbed phase. The speciation and spatial distribution of Cd were similar to those of Mn, indicating that the formation of authigenic Mn oxides affected the distribution of Cd in estuaries. The percentage of oxide /organic phase for Cu accounted for 25% of total particulate Cu, but dose not correlate well with particulate organic carbon, implying that organic carbon is not the only factor controlling particulate Cu distribution. Ni was present mainly in lattice phase, except in the Er-Ren estuary where anthropogenic Ni loading was high. In the Tseng-Wen and Gao-Ping estuaries, the percentages of lattice phase of all metals determined in this study during the wet season were higher than those during the dry season. These seasonal variations are probably resulted from different flushing times in dry/wet seasons, which control the extent of geochemical processes for trace elements.

Water samples were collected from coastal region off southwestern Taiwan during two cruises in different seasons (October, 2008 and March, 2010). In order to provide information for trace elements in this region, this study investigated the distribution and partitioning of dissolved trace elements (Cd, Cu, Ni, Zn), and to relate the influences of complicated hydrological conditions to trace element distributions. Fractionation of dissolved trace elements was applied by a one-step preconcentration technique which uses cationic and anionic exchange columns. Trace element fractions were separated to operationally defined reactive (Chelex-labile), organically complexed (anionic-organic), and stable (inert) species. Distributions of trace elements from near-shore surface waters off southwestern Taiwan were mainly affected by temporally variable terrigenous inputs and hydrological conditions. The most significant sources of trace elements were from the Gao-Ping, Er-Ren, and Tseng-Wen Rivers. Some near-shore vertical profiles of trace elements and nutrients showed abnormal distributions that could be attributed to complicated currents in this region. Trace elements in waters at offshore stations in this study showed nutrient-type distributions, and that is similar to other open ocean vertical profiles, except for the upper layers, where terrestrial influences were pronounced in this study. The major proportions of trace elements determined in this study were of the Chelex-labile fractions, indicating that they were reactive and bioavailable. There is a small part of inert fraction, and the proportions are different between inshore and offshore waters, with pronounced inert fractions in near-shore waters.

Arsenic (As) is a problem with rice production systems throughout the world as high As concentrations are reported in rice grains originating from several parts of the world. This characteristic is mainly due to the flooded conditions utilized in rice culture. We hypothesized that the soluble As concentrations in the rice rhizosphere can be decreased by growing rice more aerobically through intermittent flooding. Intermittent water management practices might also change microbial populations in the rice rhizosphere that might potentially impact As chemistry and bioavailability. Two field-scale experiments were conducted over two years to study the impact of intermittent and continuous flooding on As speciation and microbial populations in the rice rhizosphere. As levels and speciation in the rhizosphere soil, root-plaque and pore-water were determined using a high performance liquid chromatography-inductively coupled plasmamass spectroscopy (HPLC-ICP-MS). The microbial populations were assessed from the rhizosphere soil and root-plaque samples using quantitative polymerase chain reaction (qPCR) and 16S rRNA sequencing. Pore-water and root-plaque total-As concentrations significantly decreased in the intermittent compared to the continuous flood plots. Inorganic arsenite (iAsIII) was predominant in pore-water and inorganic arsenate (iAsV) in root-plaque and soil. Rootplaque sequestered significantly higher levels of As (almost tenfold higher) than the adjacent rhizosphere soil. Grain As concentrations also decreased by 35 to 45 percent in the intermittent compared to the continuously flooded plots. Organic As species, monomethyl and dimethyl arsenate were detected in the rhizosphere with relative increases and decreases among the treatments. Bacteria were the predominant group (91 to 94 percent and 48 to 78 percent of total community in root-plaque and rhizosphere soils, respectively). Archaea were also a major component of rhizosphere soil with their populations being higher under continuous flooding. The relative abundance of iron-reducing bacteria was around 3 to 6 percent of the total community in root-plaque and around 6 to 6 percent in soil, with significantly lower abundance in the intermittent compared to the continuously flooded plots. Results of these studies demonstrated that intermittent flooding could be a potential management option to reduce grain As in rice cultivated on fields with moderate to high As concentrations.