Totally 84 differentially expressed mRNA clones from infective L3 larvae of the parasite Haemonchus contortus, a blood sucking nematode, were analyzed with single strand hybridization assay (SSH). Altogether 79 clones were sequenced, edited, and compared with proteins found via BLAST in GeneBank. The aim was to investigate gene expression and potential protein expression following storage at 5 °C for 32 weeks. mRNA was extracted from fresh and stored L3. The SSH derived products were cloned into E. coli, purified and sequenced with Big Dye Terminator v3.1, and then compared with uploaded sequences in GeneBank. BLAST showed 59 (70 %) reliable protein results, where 39 (66 %) were of nematode origin.. Three sequences (4 %) were recognized as H. contortus-related metabolic proteins. Further research is necessary to elucidate the role of these proteins in relation to storage. Many of the sequences (36 %) are also present in other nematodes, such as Caenorhabditis briggsae and C. elegans, where whole-genome projects have been conducted. Bigger and more accurate databases need to be developed. Maybe the most significant future project is to sequence the whole genome of H. contortus.

<p>Current techniques for nucleic acid analysis often involve extensive sample preparation that requires skilled personnel and multiple purification steps. In this dissertation, we introduce an on-chip, isothermal, post-hybridization labeling and signal amplification technique that can directly interrogate unmodified DNA and RNA samples on a microarray format, eliminating the need for microarray sample pre-processing. </p><p>We name this technique Surface Initiated Enzymatic Polymerization (SIEP), where we exploit the ability of a template independent DNA polymerase called Terminal Deoxynucleotidyl Transferase (TdT) to catalyze the formation of long single-stranded DNA (ssDNA) chain from the 3'-end of a short DNA primer, which is tethered on the surface, and TdT's ability to incorporate unnatural reporter nucleotides, such as fluorescent nucleotides. We hypothesize that polymerization of a long ssDNA chain while incorporating multiple fluorescent nucleotides on target DNA or RNA hybridized to probe printed on a surface will provide a simple and powerful, isothermal method for on-chip labeling and signal amplification. </p><p>We developed the SIEP methodology by first characterizing TdT biochemical reaction to polymerize long homopolymer ssDNA (> 1000 bases) starting from the 3'-OH of ten bases oligonucleotides. We found that the preferred monomers (deoxynucleotide, dNTP) are dATP and dTTP, and that the length of the ssDNA extension is determined by the ratio of input monomer (dNTP) to initiator (short oligonucleotides). We also investigated TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the initiation efficiency, the degree of fluorophore incorporation, the length and the polydispersity of the polymerized DNA strand. These experiments allowed us to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using SIEP as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a up to ~45 fold increase in signal amplification compared to the incorporation of a single fluorophore.</p><p>SIEP was then employed to detect hybridization of DNA (25 bases), short miRNA (21 bases) and long mRNA (1400 bases) by the post-hybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of hybridized target strands. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection (LOD) and a 2-log linear dynamic range while the detection of short miRNA and fragmented mRNA targets resulted in ~2 pM and ~10 pM LOD, respectively with a 3-log linear dynamic range.</p><p>We further developed SIEP for colorimetric detection by exploiting the presence of negatively charged phosphate backbone on the surface as target DNA or RNA hybridizes on the immobilized probe. The net negative charge on the surface is further increased by TdT catalyzed polymerization of long ssDNA. We then used positively charged gold nanoparticles as reporters, which can be further amplified through electroless metallization, creating DNA spots that are visible by eye. We observed an increase of 100 fold in LOD due to SIEP amplification.</p><p>Overall, we demonstrated the use of SIEP methodology to label unmodified target DNA and RNA on chip, which can be detected through fluorescence signal or colorimetric signal of metallized DNA spots. This methodology is straightforward and versatile, is compatible with current microarray technology, and can be implemented using commercially available reagents.</p> / Dissertation

Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

Expansion of common bean (<i>Phaseolus vulgaris</i> L.) crops in the northern Great Planes has been hampered due to the lack of cultivars demonstrating sufficient vitality under low temperature conditions. <i>Phaseolus angustissimus</i> L., a wild bean species, has been previously shown to possess the ability to survive low temperatures in field trials. Freezing tolerance experiments under controlled conditions resulted in P. angustissimus demonstrating a greater capacity for freezing tolerance than <i>P. vulgaris</i>, as all P. vulgaris plants studied were dead at -2.5oC while most P. angustissimus plants treated to the same conditions survived. Exposure to chilling temperatures over five days resulted in stunted growth in both species, but the cultivated bean suffered more compared to the wild bean, as noted by a marked loss in tissue water content over the first three days of chilling. Interspecific macroarray hybridizations of a cDNA library from cold acclimated Medicago sativa L. using cDNAs derived from non-chilled and three-day chilled <i>P. vulgaris</i> and <i>P. angustissimus</i> plants showed that <i>P. vulgaris</i> showed more changes in gene expression after three days of chilling. Also, <i>P. vulgaris</i> showed a general trend towards down-regulation of the transcripts sampled on the third day of chilling compared to <i>P. angustissimus</i>. RT-PCR experiments were conducted using cDNAs from plant tissues exposed to various durations of chilling to confirm the results from the macroarray experiment. These time-course RT-PCR experiments revealed expression patterns across various chilling durations in genes identified from the macroarray. Data from these experiments suggest that <i>P. vulgaris</i> and <i>P. angustissimus </i> seedlings respond differently to low temperature exposure, and that some of the changes in <i>P. angustissimus</i> transcripts monitored here may be useful for researchers in better understanding how Phaseolus species can respond better to chilling temperatures.

The federally mandated 36 billion gallons a year production goal for "advanced biofuels" by 2022 has created a demand for lignocellulosic feedstocks that are inexpensive to produce. The current lack of market development for lignocellulosic feedstocks incentivizes the development of versatile biomass products with greater end-use possibilities, as in either a forage or bioenergy system. High-biomass, perennial grasses offer dual-use potential in either forage or biofuel systems. In 2009 and 2010 controlled pollinations were made to evaluate the efficiency of producing interspecific hybrids between homozygous recessive iap/iap and Iap/- Sorghum bicolor (L.) Moench, cultivated sorghum, and three S. halepense (L.) Pers., johnsongrass, genotypes. The iap/iap genotype removes reproductive barriers to alien pollen in S. bicolor and aids in wide hybridization. Total seed set, germinable seed set, and hybrid production were significantly higher using the iap/iap genotype. The iap/iap S. bicolor genotype is a valuable tool available to plant breeders for the creation of wide hybrids with S. halepense. In a related study a bulked segregant analysis was conducted using bulked samples of S. bicolor, typical flowering S. halepense, non-flowering S. halepense, and putative triploid hybrids of the two species to identify unique markers for each bulk and to evaluate S. bicolor genetic material introgression into the non-flowering S. halepense genome. Thirty-nine and 23 markers were found to be unique to the S. bicolor and typical flowering S. halepense bulks, respectively. These unique markers could be used in a breeding program to identify interspecific hybrids. Alleles at fifteen markers were found in both the S. bicolor and non-flowering S. halepense bulks but not in typical flowering S. halepense and may help explain the non-flowering phenotype. In 2010 and 2011 a study was conducted to investigate the rhizome composition of 11 genotypes of Sorghum species and its relationship to overwintering. Genotype, environment, and sampling date had significant effects on rhizome metabolite concentrations. Overwintering capacity was related to fructans and crude protein concentrations and NIRS (Near Infrared Spectroscopy) was effective at estimating these values. This information can be used to screen for stronger perennial parents to be used in future breeding programs.

Anxiety disorders are one of the most prevalent mental disorders in the world. While normal anxiety serves as an important protective mechanism, pathological anxiety characteristic of an anxiety disorder is both maladaptive and disruptive. The majority of studies have focused on the neurotransmitter systems associated with the actions of known anxiety drugs. This focus may likely limit the exploration of mechanisms underlying anxiety disorders. This project aims to examine changes in gene expression that may underlie higher or lower levels of inherent anxiety. Using a well-established behavior test for anxiety, the elevated plus maze, we identified male Wistar rats exhibiting inherently high- or low-anxiety levels. Brain regions known to mediate anxiety, the amygdala, hippocampus and nucleus accumbens, were dissected and total mRNA isolated. The mRNA was converted to cDNA via reverse transcription-polymerase chain reaction (RT-PCR). Then, the cDNA was used in suppression subtractive hybridization, a technique used to compare two complete populations of cDNAs and identify cDNAs that are upregulated in one population in relation to the other. In this project suppression subtractive hybridization was used to compare high- and low-anxiety cDNA populations. The upregulated cDNAs were amplified in a PCR reaction that enables rare transcripts to be identified. The PCR products from the suppression subtractive hybridization were cloned and used to create two cDNA libraries for high- and low-anxiety related genes. These clones were sequenced to show over 1000 genes upregulated in high- and low-anxiety. The gene list was then subjected to bioinformatic analysis to identify one candidate to be studied in further detail. <p>The prion protein was identified as a potential candidate. Examination of the literature sparked an interest in studying other prion-like proteins, more specifically the cytoplasmic polyadenylation element binding protein (CPEB). The CPEB protein is a potent regulator of mRNA translation in both mature oocytes and the adult brain. While unphosphorylated the CPEB protein keeps specific mRNAs dormant in the cytoplasm. In its phosphorylated form CPEB catalyzes polyadenylation of the mRNA, leading to protein synthesis. p*PCR was used to show the presence of CPEB mRNA transcripts in the rat hippocampus. CPEB protein expression was examined in the brain samples isolated from control, high- and low-anxiety rats. It was found that CPEB was significantly upregulated in high- and low-anxiety rats compared to control. The protein expression of an upstream kinase, Aurora A kinase, and a downstream target, Calcium/Calmodulin Dependent Kinase II (CaMKII), was also investigated. The results from Aurora A kinase were inconclusive. CaMKII, on the other hand, was significantly upregulated in high-anxiety over both control and low-anxiety. These results suggest that CPEB may catalyze increased translation of mRNAs in high-anxiety while acting as a repressor of those same mRNAs in low-anxiety. <p>Recent studies have suggested that CPEB protein plays an important role in synaptic plasticity. The regulation of synaptic plasticity, and its impact on learning and memory, is believed to be a key mechanism behind the maintenance of anxiety disorders. Therefore the results of this study suggest a new molecular mechanism in the development of anxiety disorders.

Expansion of common bean (<i>Phaseolus vulgaris</i> L.) crops in the northern Great Planes has been hampered due to the lack of cultivars demonstrating sufficient vitality under low temperature conditions. <i>Phaseolus angustissimus</i> L., a wild bean species, has been previously shown to possess the ability to survive low temperatures in field trials. Freezing tolerance experiments under controlled conditions resulted in P. angustissimus demonstrating a greater capacity for freezing tolerance than <i>P. vulgaris</i>, as all P. vulgaris plants studied were dead at -2.5oC while most P. angustissimus plants treated to the same conditions survived. Exposure to chilling temperatures over five days resulted in stunted growth in both species, but the cultivated bean suffered more compared to the wild bean, as noted by a marked loss in tissue water content over the first three days of chilling. Interspecific macroarray hybridizations of a cDNA library from cold acclimated Medicago sativa L. using cDNAs derived from non-chilled and three-day chilled <i>P. vulgaris</i> and <i>P. angustissimus</i> plants showed that <i>P. vulgaris</i> showed more changes in gene expression after three days of chilling. Also, <i>P. vulgaris</i> showed a general trend towards down-regulation of the transcripts sampled on the third day of chilling compared to <i>P. angustissimus</i>. RT-PCR experiments were conducted using cDNAs from plant tissues exposed to various durations of chilling to confirm the results from the macroarray experiment. These time-course RT-PCR experiments revealed expression patterns across various chilling durations in genes identified from the macroarray. Data from these experiments suggest that <i>P. vulgaris</i> and <i>P. angustissimus </i> seedlings respond differently to low temperature exposure, and that some of the changes in <i>P. angustissimus</i> transcripts monitored here may be useful for researchers in better understanding how Phaseolus species can respond better to chilling temperatures.

Sponge are primitive multi-cellular organisms. They are important sources of secondary metabolites. In the previous studies indicated that the sponges harbor stable symbiotic microbial consortia. The mechanisms for maintenance and transmission of microbial consortia to the next generations are still not fully understood. The sponge, Cinachyrella australinesis, was chosen to further investigate relationship of the symbiotic bacteria within to the host. Fluorescent in situ hybridization ¡]FISH¡^was employed with non-specific ¡]EUB338¡^and specific oligonucleotide probes for bacteria. The sponge was cryo-sectioned¡]1£gm¡^and hybridized with fluorescent probes. The distribution and ratios of the bacteria in the sponge agreed with those of previous studies indicating that the symbiotic bacteria of C. australiensis are stable and endosymbiotic in nature.

This thesis explores, through fictional storytelling, the cultural duality of individuals inhabiting Singapore prior to World War II. The primary locale in many of these stories-an actual residence known as Eu Villa-interconnects each narrative and helps to uncover the hybridization of a Chinese family (and servants) living in a British colony. Many of the stories are imparted from different perspectives: wives, children and amahs, each of them pieced together to bridge the space between Chinese heritage overlaid and intermixed with British culture. In this way, the stories of this thesis reflect on the history that preceded the distinct multiculturalism of contemporary Singapore.

The morphological characters, pollen viability, and molecular markers are used in this study to assess the inter-species differentiation in Taiwan Trema( T. orientalis, T. tomentosa, T. cannabina, and the hybrid ). The hybrid was proposed to have been originated from T. tomentosa and T. cannabina natural hybridization with morphological, pollen viability and molecular marker evidences. The four taxa are variable in the following morphological characters: growth form, terminal bud color, leaf size, leaf apex, leaf base, leaf vestiture, leaf texture, leaf nerves, petiole, stipule size, inflorescence sex, male and female inflorescence length, flower number, mature fruit color, perianth vestiture, male flower perianth size, pistillode size, pistillode shape, filament length, and female flower size. The author also found differences in leaf shape, leaf size, leaf base, and leaf vestiture between adult and juvenile individuals of T. orientalis are ontogenetical variations. The hybrid is morphological intermediate between T. tomentosa and T. cannabina, possessing species-specific morphological characters of either species. Leaf trichome morphology was observed under scanning electronic microscope, and a unicellular trichome type with bulbous base, smooth surface, and creeping looking, is specific to T. orientalis leaf abaxial surface. In pollen viability tests, the hybrid had the lowest average pollen stainbility among tour taxa, but varied widely from 48.5 % to 81.6 %. In additivity test of molecular markers, for all 8 species-specific molecular markers of T. tomentosa, 6 were detected in hybrid; for all 14 species-specific molecular markers of T. cannabina, 11 were detected in hybrid; and none of 14 species-specific molecular markers of T. orientalis were detected in hybrid. Additionally, there were some recessive homozygote alleles detected in hybrid molecular marker, and even missing in T. tomentosa molecular markers. According to this evidence, there was a possible introgression between the hybrid and parental species-T. cannabina. In similarity dendrogram derived from molecular markers, all samples were clustered into four taxa-corresponded groups, the hybrid was placed between T. tomentosa and T. cannabina, and closely related to T. cannabina.