Browsing by Subject "Gene expression"
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Item Acclimation of Holstein Calves to Transit Stress: The Integration of Endocrine, Immune, and Behavior Systems(2012-10-19) Adams, AmberLittle is known about the adaptation of livestock to repeated transport. This study determined how repeated transport affected calf feed intake, plasma cortisol (CORT), post-transport behavior, and the expression of immune-related genes. Thirty-six 4-month-old Holstein steer calves were housed in groups of six with each group randomly assigned to either transport (T) or control (C) treatments. The T calves were hauled for 6 h in a 7.3 m x 2.4 m goose-neck trailer, at an average density of 0.87 m2/calf, every 7 d for five consecutive weeks. Individual daily intake was determined using Calan gate feeders. Blood samples were obtained in the trailer or home pen via jugular venipuncture before loading, and after 2, 4, and 6 h of transport. Samples were analyzed for CORT, serotonin, tryptophan, and the gene expression of interleukin-4 (IL-4), interleukin-6 (IL-6), chemokine (C-X-C motif) receptor 2, interleukin-12, toll-like receptor-4, toll-like receptor-2, and 5-hydroxytryptamine receptor 2A in leukocytes. Behavior was recorded for transported calves at 5-min intervals for 1 h after return to their home pens. The C calves had a higher feed intake than T calves overall (P = 0.01), on the day of transport (P = 0.007), and the day after transport (P = 0.02). Pre-transport CORT concentrations did not differ by treatment (P = 0.77) or trial (P = 0.32). However, the T calves had higher response CORT concentrations than C calves during Transport 3 (P = 0.006), Transport 4 (P = 0.001) and Transport 5 (P = 0.02). The T calves had the highest response CORT concentrations after 2 h of transport and the lowest response CORT concentrations after 6 h of transport (P < 0.0001). Treatment did not affect gene expression in leukocytes, however, the expression of IL-4 (P = 0.01) and IL-6 (P = 0.05) was significantly lower after 2 h of transport than any other sampling times. These results suggest conflicting conclusions on whether the calves started to acclimate after being transported five times. However, CORT and gene expression differences occurred in response to the blood sampling regimen, which may provide insight to how calves acclimate during prolonged stress.Item Algorithms for the analysis of whole genomes(2004) Wyman, Stacia Kathleen; Kuipers, Benjamin; Jansen, Robert K.With the advent of whole genome sequencing, we now have an abundance of whole genomes which have been sequenced and we have entered an era when algorithms can address problems at the whole genome level. In the past, sequencing efforts often focused on a single gene, and therefore, existing algorithms are at the scale of a single gene. With whole genome sequencing, we have access to sequence data for the entire genome of an organism or an organelle and algorithms are needed for whole genome analysis. In this research, we have addressed new computational problems that have arisen out of the availability and abundance of whole genome data. In genome annotation, all of the genes of a genome are located and identified in preparation for publication of the complete genome sequence. We address the problem of genome annotation with a software package that allows researchers to locate and identify all the genes in a genome and prepare the genome for direct submission to GenBank. A difficult problem that arises in the annotation of organellar genomes is the identification of animal mitochondrial transfer RNA genes. We present an experimental evaluation a set of methods (including our own) for identifying tRNAs. The problem of reconstructing phylogenies from gene order data involves recreating the evolutionary history of a set of organisms based on the order and direction of the genes in the genomes. This can give insight into mechanisms of large-scale evolutionary events. We present a new method for gene order phylogeny reconstruction, as well as improvements to an existing method, and evaluate the results on both real and simulated datasets. Finally, we address the problem of identification of regulatory elements. Understanding gene expression is one of the most pressing unsolved problems in molecular biology today because gene expression controls all of the metabolic and developmental processes in an organism. We present a new method which uses a comparative genomics approach which is made possible now that we have access to the complete DNA sequences of many sets of related organisms.Item Analysis of sodium pump gene expression in microdissected nephrons using competitive RT-PCR and a novel HPLC technique(Texas Tech University, 1996-12) Hayward-Lester, Amanda LHypertension is determined by the interaction of multiple genetic and environmental factors, making it difficult to elucidate any single genetic determinant. Biochemical markers such as intraerythrocytic sodium concentration, erythrocytic ouabain binding-site density and passive sodium leak indicate that abnormal membrane cation flux segregates with some hypertension subtypes. The sodium pump, Na+, K+ ATP-ase (NKA) may therefore participate in the development of hypertension. It is a multi-subunit cell membrane protein which translocates sodium and potassium ions with hydrolysis of ATP. It is inhibited by ouabain and regulated by phosphorylation. The alpha subunit (of which there are four isoforms encoded by different genes) is currently ascribed all catalytic function, while the beta and gamma subunits may have regulatory roles. We examined NBCA alpha and gamma subunit gene expression in spontaneously hypertensive rats (SHR) and Wistar Kyoto controls (WKY). Both prehypertensive and adult SHR exhibit abnormal renal sodium retention. Solution hybridization studies in adult SHR revealed a decrease in alpha 1 expression in kidney. Kidney is a heterogeneous tissue whose functional unit, the nephron, may be divided into 12 distinct segments. To examine NKA expression in individual segments requires an assay allowing quantitation of NKA alpha and gamma isoform RNA in microdissected tissue samples. We combined competitive RT-PCR with a novel ion-paired reversed phase HPLC to produce rapid, accurate and precise measurement of gene expression in a single-tube assay. The ability of HPLC to resolve heteroduplex molecules formed between native and competitor products proved essential. Assay validation confirmed absolute quantification is possible if competitors have identical reverse-transcription efficiency to the native RNA. We used the assay to examine qualitative and quantitative expression of NKA subunits in normotensive Sprague-Dawley, prehypertensive and adult SHR and WKY. Qualitative analysis revealed alpha 1 and gamma expression in all segments examined. Expression of the other alpha isoforms was not detected. Quantitative analysis in the prehypertensive SHR revealed that a selective alteration in alpha 1 expression in proximal convoluted tubule may explain the results obtained in whole kidney and suggest an attempted feedback response by SHR to reduce sodium reabsorption.Item Characterization and functional analysis of arabinogalactan protein 31 in Arabidopsis(2007-12) Liu, Chenggang, 1970-; Mehdy, Mona M.Arabinogalactan proteins (AGPs) are highly glycosylated cell wall proteins specific to plants. AGPs have been implicated in almost all aspects of plant development and defense responses, nevertheless, most of such studies are correlative. To define the specific functions of individual AGPs, direct evidence from analyses of genetic knockout mutants of individual AGPs is required. Up to now, only a few AGPs have been demonstrated to have defined functions by mutant analyses. This dissertation identified a non-classical AGP (AGP31), described its expression and characterized the null mutant of AGP31 in Arabidopsis. In agp31 mutant, microarray analyses revealed that the expressions of genes encoding a subset of seed storage proteins (SSP): CRU3, CRA1 and OLEOSIN2 were induced. Further analysis showed that induction by agp31 knockout was specific to these three SSP genes, indicating a novel pathway to regulate the SSP gene expression. Comprehensive characterizations of AGP31 were carried out. Yariv reagent staining and monosaccharide analysis of purified AGP31 showed that AGP31 was a bona fide galactose-rich AGP. The cell wall localization of AGP31 was confirmed by expression of an AGP31Item Characterization of the XTH gene family in cotton(Texas Tech University, 2006-08) Lee, Joohyun; Allen, Randy D.; Auld, Dick L.; Zhang, Hong; Shi, Huazhong; Wright, Robert J.Cotton is the single most important crop in Texas agriculture, reaching a value of 4 billion dollars per year. Cotton fiber is the major product in cotton cultivation which is converted into economic revenue. Therefore, most research groups have focused on fiber development and improvement of fiber quality. The xyloglucan endotransglucosylase/hydrolase gene family has a unique role in plant cell wall elongation and development during plant growth. It can cut and paste xyloglucan fragments into xyloglucan hemicellulose matrix in Type I plant cell wall, allowing cell wall extension and loosening while bearing tension for normal cell metabolism. XTH genes encode enzymes that may play a role in determining fiber length and other fiber qualities during cotton fiber development. Both the structures and functions of the XTH family have been well studied in Arabidopsis. In this research the cotton XTH genes were cloned by using the sequence information of Arabidopsis XTHs. A cotton boll cDNA library and a cotton genomic BAC library were screened and used for cotton XTH gene cloning. The clones of cotton XTHs were classified to three distinctive groups based on their 3ŒUTR sequences. The three groups were denoted as GhXTH-G1, G2, and G3. The sequences of abnormal transcripts of GhXTH-G1 showed transcription termination in the middle of introns, producing shortened forms of mRNAs. In addition, two GhXTH-G1 genes were analyzed and assigned to A and D sub genomes. Real time quantitative RT-PCR was used to study tissue and development specific patterns of XTH gene expression. GhXTH-G3 appears to be the most important cotton XTHs in cotton fiber development. Also, hormone treatment results indicated that brassinolide (BL), gibberellin (GA3), naphthalene acetic acid (NAA) induced GhXTH expression in developing cotton fiber.Item Characterization of the XTH gene family in cotton(2006-08) Lee, Joohyun; Allen, Randy D.; Auld, Dick L.; Wright, Robert J.; Shi, Huazhong; Zhang, HongCotton is the single most important crop in Texas agriculture, reaching a value of 4 billion dollars per year. Cotton fiber is the major product in cotton cultivation which is converted into economic revenue. Therefore, most research groups have focused on fiber development and improvement of fiber quality. The xyloglucan endotransglucosylase/hydrolase gene family has a unique role in plant cell wall elongation and development during plant growth. It can cut and paste xyloglucan fragments into xyloglucan hemicellulose matrix in Type I plant cell wall, allowing cell wall extension and loosening while bearing tension for normal cell metabolism. XTH genes encode enzymes that may play a role in determining fiber length and other fiber qualities during cotton fiber development. Both the structures and functions of the XTH family have been well studied in Arabidopsis. In this research the cotton XTH genes were cloned by using the sequence information of Arabidopsis XTHs. A cotton boll cDNA library and a cotton genomic BAC library were screened and used for cotton XTH gene cloning. The clones of cotton XTHs were classified to three distinctive groups based on their 3ŒUTR sequences. The three groups were denoted as GhXTH-G1, G2, and G3. The sequences of abnormal transcripts of GhXTH-G1 showed transcription termination in the middle of introns, producing shortened forms of mRNAs. In addition, two GhXTH-G1 genes were analyzed and assigned to A and D sub genomes. Real time quantitative RT-PCR was used to study tissue and development specific patterns of XTH gene expression. GhXTH-G3 appears to be the most important cotton XTHs in cotton fiber development. Also, hormone treatment results indicated that brassinolide (BL), gibberellin (GA3), naphthalene acetic acid (NAA) induced GhXTH expression in developing cotton fiber.Item Characterizing perchlorate exposure and effects in mammals(Texas Tech University, 2006-05) Cheng, QiuqiongPerchlorate contamination in the U.S. appears to be more widespread than originally thought. Perchlorate is naturally occurring, in addition to its anthropogenic sources. Animals including humans have been exposed to perchlorate through drinking water and/or trophic transfer; perchlorate has been detected in dairy milk, some food items, human milk, and urine. Perchlorate exposure and effects in mammals were characterized in the current study. The ability to detect perchlorate in exposed animals is critical for effects studies and risk assessments; perchlorate residues in biological fluids such as plasma, urine, and milk can serve as biomarkers for perchlorate exposure. A well-developed method for perchlorate determination in these matrices would contribute greatly to perchlorate exposure assessment. In this study, analytical methods for perchlorate determination in these matrices were explored. Alumina-neutral cartridges combined with C18 cartridges and NAX combined with alumina-neutral provided the best cleanup with significantly reduced background signal of plasma and urine, respectively, and relatively high recovery of perchlorate using conventional ion chromatography with suppressed conductivity detection (IC-SCD). However, the cleanup method was not robust enough to be used on some urine matrices such as deer mouse urine collected in perchlorate excretion and elimination experiments. As an alternative to IC-SCD, IC-MS/MS provides an excellent method with high selectivity and sensitivity for part per trillion perchlorate determination in both aqueous and deer mice urine matrices. A study on perchlorate exposure and absorption in beef cattle showed that constant exposure to 25 ng/mL perchlorate in water over 14 weeks did not result in measurable residues in blood plasma or edible tissues of cattle at the first test site (McLennan County, Texas). However, perchlorate was detected in 4 of 33 and 17 of 26 cattle at the two Kansas farms with the highest plasma perchlorate concentrations of 43 and 32 ng/mL, respectively. Compared to perchlorate residues in urine, perchlorate residues in plasma may not be a proper biomarker for perchlorate exposure assessment. A study on perchlorate distribution, excretion, and depuration in rodents showed that urine was the major pathway for perchlorate fate in the body. Higher levels of perchlorate exposure corresponded well to high levels of perchlorate excreted in the urine. Perchlorate excretion via urine reached a steady state after one day in the 28-day exposure experiment. An average of 46, 46, and 61% of perchlorate was recovered in urine over the exposure period in high, medium, and low dose groups, respectively. Metabolism of perchlorate may occur in the body based in part on the 40% perchlorate unaccounted for in this study. Endogenous perchlorate may also exist. Perchlorate exposure through dosed drinking water for 28 days increased sodium-iodide symporter (NIS) gene expression in the kidney and stomach, and pendrin gene expression in the kidney, without significant difference. No significant difference was observed neither between the low and high dose groups in the depuration experiment regarding either gene expression in the kidney or stomach. No significant linear relationship was found between perchlorate urinary excretion and either gene expressions in the kidney. A partial sequence of deer mice NIS gene cDNA with 425 bps was discovered in the current study for the first time. Quantitative analysis of NIS mRNA expression in various tissues was also studied for the first time in the current study with expression levels from highest to lowest in deer mice tissues in the following order: stomach, testes, brain, large intestine, and barely expression in the lung, kidney, heart, and liver. The effect of perchlorate exposure on the fatty acid profile in milk was observed in lactating goats dosed with perchlorate from Monday to Friday each week for 4.5 weeks. Omega-6 fatty acids and total polyunsaturated fatty acids (PUFA) at the 1 mg/kg treatment were significantly reduced at day 10 (p = 0.0113 and 0.0053, respectively), day 17 (p < 0.001), and day 24 (p < 0.05), but not at day 2 and 31. Monounsaturated fatty acid (MUFA) was significantly reduced only at day 17 (p = 0.0130). Significant reductions in short- and long-chain fatty acids were observed at day 24 only (p = 0.0431 and 0.0097, respectively) in the high and low dose groups, respectively. Additionally, a weak negative correlation between milk perchlorate concentrations and total PUFA levels was found in human breast milk samples collected from Lubbock, TX or nearby counties. To our knowledge, this is the first report on the effect of perchlorate exposure on fatty acid profiles in milk. Further study is urged to investigate mechanistic aspects of the effect. This work will contribute to the human risk assessment of perchlorate, particularly for the development of infants with maternal exposure to perchlorate.Item Characterizing perchlorate exposure and effects in mammals(2006-05) Cheng, Qiuqiong; Anderson, Todd T.; Hooper, Michael J.; Jackson, W. Andrew; McMurry, Scott T.; Smith, Ernest E.Perchlorate contamination in the U.S. appears to be more widespread than originally thought. Perchlorate is naturally occurring, in addition to its anthropogenic sources. Animals including humans have been exposed to perchlorate through drinking water and/or trophic transfer; perchlorate has been detected in dairy milk, some food items, human milk, and urine. Perchlorate exposure and effects in mammals were characterized in the current study. The ability to detect perchlorate in exposed animals is critical for effects studies and risk assessments; perchlorate residues in biological fluids such as plasma, urine, and milk can serve as biomarkers for perchlorate exposure. A well-developed method for perchlorate determination in these matrices would contribute greatly to perchlorate exposure assessment. In this study, analytical methods for perchlorate determination in these matrices were explored. Alumina-neutral cartridges combined with C18 cartridges and NAX combined with alumina-neutral provided the best cleanup with significantly reduced background signal of plasma and urine, respectively, and relatively high recovery of perchlorate using conventional ion chromatography with suppressed conductivity detection (IC-SCD). However, the cleanup method was not robust enough to be used on some urine matrices such as deer mouse urine collected in perchlorate excretion and elimination experiments. As an alternative to IC-SCD, IC-MS/MS provides an excellent method with high selectivity and sensitivity for part per trillion perchlorate determination in both aqueous and deer mice urine matrices. A study on perchlorate exposure and absorption in beef cattle showed that constant exposure to 25 ng/mL perchlorate in water over 14 weeks did not result in measurable residues in blood plasma or edible tissues of cattle at the first test site (McLennan County, Texas). However, perchlorate was detected in 4 of 33 and 17 of 26 cattle at the two Kansas farms with the highest plasma perchlorate concentrations of 43 and 32 ng/mL, respectively. Compared to perchlorate residues in urine, perchlorate residues in plasma may not be a proper biomarker for perchlorate exposure assessment. A study on perchlorate distribution, excretion, and depuration in rodents showed that urine was the major pathway for perchlorate fate in the body. Higher levels of perchlorate exposure corresponded well to high levels of perchlorate excreted in the urine. Perchlorate excretion via urine reached a steady state after one day in the 28-day exposure experiment. An average of 46, 46, and 61% of perchlorate was recovered in urine over the exposure period in high, medium, and low dose groups, respectively. Metabolism of perchlorate may occur in the body based in part on the 40% perchlorate unaccounted for in this study. Endogenous perchlorate may also exist. Perchlorate exposure through dosed drinking water for 28 days increased sodium-iodide symporter (NIS) gene expression in the kidney and stomach, and pendrin gene expression in the kidney, without significant difference. No significant difference was observed neither between the low and high dose groups in the depuration experiment regarding either gene expression in the kidney or stomach. No significant linear relationship was found between perchlorate urinary excretion and either gene expressions in the kidney. A partial sequence of deer mice NIS gene cDNA with 425 bps was discovered in the current study for the first time. Quantitative analysis of NIS mRNA expression in various tissues was also studied for the first time in the current study with expression levels from highest to lowest in deer mice tissues in the following order: stomach, testes, brain, large intestine, and barely expression in the lung, kidney, heart, and liver. The effect of perchlorate exposure on the fatty acid profile in milk was observed in lactating goats dosed with perchlorate from Monday to Friday each week for 4.5 weeks. Omega-6 fatty acids and total polyunsaturated fatty acids (PUFA) at the 1 mg/kg treatment were significantly reduced at day 10 (p = 0.0113 and 0.0053, respectively), day 17 (p < 0.001), and day 24 (p < 0.05), but not at day 2 and 31. Monounsaturated fatty acid (MUFA) was significantly reduced only at day 17 (p = 0.0130). Significant reductions in short- and long-chain fatty acids were observed at day 24 only (p = 0.0431 and 0.0097, respectively) in the high and low dose groups, respectively. Additionally, a weak negative correlation between milk perchlorate concentrations and total PUFA levels was found in human breast milk samples collected from Lubbock, TX or nearby counties. To our knowledge, this is the first report on the effect of perchlorate exposure on fatty acid profiles in milk. Further study is urged to investigate mechanistic aspects of the effect. This work will contribute to the human risk assessment of perchlorate, particularly for the development of infants with maternal exposure to perchlorate.Item Cloning and functional characterization of the WdSTUA and WdPACC genes of Wangiella dermatitidis(2007) Wang, Qin, 1970 Nov. 15-; Szaniszlo, Paul J.To study the function of WdStuAp and WdPacCp in Wangiella dermatitidis, a black, polymorphic fungal pathogen of humans with yeast phase predominance, WdSTUA and WdPACC were cloned, sequenced, disrupted and expressed. WdStuAp was most similar to the APSES proteins of Aspergillus species and its APSES DNA-binding domain was located in its N-terminal half. Deletion of WdSTUA in W. dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich, yeast maintenance agar medium, YPDA, at 37°C. Additionally, deletion of WdSTUA repressed aerial hyphal growth, conidiation and invasive hyphal growth on the nitrogen poor, hyphae-inducing agar medium, PDA, at 25°C. Ectopic expression of WdSTU Arepressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°C. Expression of WdSTUA in S. cerevisiae induced pseudohyphal growth on the nitrogen poor medium. WdPacCp was also most similar to the PacCp proteins of Aspergillus species. Three zinc finger DNA-binding motifs were at the N-terminus, and the C-terminus had the signaling protease cleavage site. WdPACC was more expressed at neutral-alkaline pH than at acidic pH. Truncation of the coding sequence for about 40 residues upstream of the conserved processing protease cleavage site of WdPacCp affected growth on YPDA, increased sensitivity to Na⁺ stress, decreased growth level at neutral-alkaline pH, and repressed hyphal growth on PDA at 25°C. Truncation of the coding sequence for the conserved signaling protease box of WdPacCp impaired growth and reduced RNA expression of class II chitin synthase gene WdCHS1 at acidic pH, and activated hyphal growth on PDA. My results suggested that WdStuAp and WdPacCp play important roles in yeast-hyphal transitions in W. dermatitidis, and that WdPacCp is particularly important for W. dermatitidis to adapt to different ambient pH conditions.Item Determine the spatial and temporal expression patterns of Drosophila miRNAs(2007-12) Ao, Xue, 1977-; Macdonald, Paul M.microRNAs (miRNAs), are a class of small (21-23nt), endogenous, non-coding RNAs which regulate gene expression. In animals, most miRNAs recognize their mRNA targets though imperfect base paring with complementary sites at the 3’UTR of the mRNAs, resulting in translational repression of the target genes. The specific functions of miRNAs in Drosophila are generally unknown. To determine the spatial and temporal expression patterns of miRNAs, it is necessary to identify both the sites and time of miRNA action and further elucidate miRNA functions. I made a series of GFP reporter transgenes (miRNA sensors) in UAS vector containing multiple copies of synthesized miRNA targets in the 3’UTR. The sensors were expressed in a range of tissues driven by different GAL4 drivers. We expected that our sensors would reveal when and where miRNAs are actively regulate gene expression. However, the sensor strategy failed to detect clear expression patterns of the miRNAs we tried.Item Development and evaluation of an imidazole-modified chitosan for nucleic acid and contrast agent delivery(2009-05) Ghosn, Bilal; Roy, KrishnenduOver the past several decades, gene therapy technologies have been developed for a diverse number of applications ranging from DNA-based vaccines to gene silencing with RNAi. While all are powerful tools, a common limitation for these technologies is the need for effective and safe delivery to target sites within the body. Such delivery vectors are necessary for retention of bioactivity and stability, while also providing a method of cellular and tissue uptake and distribution, which may require endosomal escape. Although, viral and lipid-based technologies have shown promise as nucleic acid delivery vectors, both have inherent issues such as cytoxicity, oncogenicity, and immunogenicity. Thus, the development of polymer-based non-viral vectors has been an area of great focus over the past decade. While many polymeric vectors have been developed for plasmid DNA (pDNA) delivery, very few have shown effective delivery of short interfering RNA (siRNA), a powerful tool for gene silencing via the RNA interference mechanism. Furthermore, very few prospective delivery vectors have shown versatility for the administration of siRNA through multiple routes of administration. The overall goal of this research was to develop a biocompatible non-viral delivery system for the delivery of plasmid DNA, siRNA, and contrast agents through the modification of the natural biopolymer chitosan. We have synthesized an imidazole modified chitosan (chitosan-IAA) by conjugation of imidazole acetic acid to chitosan. Extensive evaluation and characterization of the modified polymer demonstrates enhanced solubility and buffering capacity within the physiological and endosomal pHs, thus providing enhanced endosomal escape by exploiting the "proton sponge" effect. We have demonstrated effective in vitro gene expression and gene silencing with chitosan-IAA mediated delivery of pDNA and siRNA, respectively. Furthermore, we have demonstrated in vivo gene silencing by delivery of siRNA through both intranasal and intravenous routes of delivery with chitosan-IAA/siRNA nanocomplexes. We have also demonstrated delivery of contrast agents up to 45 nm in size through mucosal tissue following treatment with chitosan and no contrast agent modification in both human and animal tissue. In conclusion, we have successfully developed a versatile and highly effective delivery vector for both nucleic acids and contrast agents.Item Development of imaging-based high-throughput genetic assays and genomic evaluation of yeast gene function in cell cycle progression(2007-12) Niu, Wei; Marcotte, Edward M.Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.Item Effects of experience and novelty on sexual behavior and associated neuronal activity in male Japanese quail(2008-08) Can, Adem, 1977-; Domjan, Michael, 1947-; Delville, YvonIn many behavioral paradigms, repeated exposures to a particular stimulus or event results in lower immediate early gene (IEG) expression. First, it was investigated if a similar reduction in IEG expression in the brain areas controlling male sexual behaviors would be observed after repeated copulation experiences in male Japanese quail. The results showed that IEG expression, as assessed by egr-1 immunoreactivity, did not increase in the POM, the BST, or the PAG after a copulation episode in highly sexually experienced subjects. One possibility was that the pattern of initial elevation of neuronal activity during the early trials of sexual interactions and the lack of increase in IEG expression later was associated with the novelty of sexual stimuli. While early exposures to certain stimuli constitute a new learning experience, the significance of such exposures would be lower as the level of experience increases. It was hypothesized that the introduction of a novel stimulus would increase the IEG expression in the POM, the BST and the PAG of experienced subjects. To evaluate this prediction, subjects were tested to see if they learn to respond to females decorated with distinct novel artificial cues after repeated exposures. The results showed that control subjects that did not have sexual experiences with decorated females discriminate against such females and directed their responses to normal females. Trained subjects did not show such preferences and responded to both types of females. In the next experiment, contrary to the prediction, no increase in IEG expression was observed after the introduction of the novel stimulus. This might be due to lower sexual motivation in subjects exposed to novel females. Effects of sexual experience were also tested in the catecholaminergic system. It was hypothesized that TH innervation in the POM and the BST would increase as a result of sexual experience. IEG expression in the catecholaminergic areas was predicted to be lower after repeated sexual experiences. The results showed no effect of experience in either tyrosine hydroxylase (TH) innervation, nor TH-egr-1 colocalization. These findings suggest that experience-related changes in male sexual behavior may be mediated by a different neurotransmitter system.Item Exploring the eukaryotic gene expression machinery using a single-cell yeast gene expression reporter(2014-08) Sorenson, Matthew Richard; Stevens, Scott W.; Ellington, Andrew D; Iyer, Vishwanath R; Johnson, Arlen W; Marcotte, Edward MIt has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex, independent macromolecular machines. To query these processes and to explore the potential relationships between them in the budding yeast Saccharomyces cerevisiae, we designed a versatile reporter employing multicolor high-throughput flow cytometry. Due to its design, this single reporter exhibits a distinctive signature for many defects in gene expression including transcription, histone modification, pre-mRNA splicing, mRNA export, nonsense-mediated decay, and mRNA degradation. Analysis of the reporter in 4967 non-essential yeast genes not only revealed striking phenotypic overlaps between similar functions, but also provided us a dataset in which to compare additional genetic or chemical perturbations. Utilizing a binning and clustering algorithm we developed we are able to compare reporter expression data from thousands of mutants in a semi-automated fashion, essentially grouping mutants or conditions based on the levels of reporter signal. I further utilized our reporter to screen a vast library of in vivo produced cyclic peptides using fluorescence-activated cell sorting (FACS), identifying a cyclic peptide that resulted in mild gene-specific pre-mRNA splicing inhibition. Additionally, I adapted our reporter assay to perform a high-throughput small molecule screen to identify inhibitors or modulators of specific gene expression processes. Our efforts led to the identification of a small molecule that inhibits pre-mRNA splicing in a dose-dependent manner. Moreover, I utilized our reporter system to quickly identify loss-of-function mutants in the poorly characterized gene SWM2. The three mutants of interest have reduced interaction with Tgs1p, the conserved trimethylguanosine synthase, which we believe leads to decreased hypermethylation of the 5’ caps of spliceosomal snRNAs. Altogether this work describes the development, validation and utility of the versatile gene expression reporter system we developed, providing our lab and others a valuable tool to interrogate a wide-range of gene expression processes in yeast.Item Exploring the global gene expression programs and regulation in the response of quiescent human fibroblasts to distinct proliferative stimuli(2005) Gu, Jian; Iyer, Vishwanath R.Serum treatment of quiescent human dermal fibroblasts induces proliferation coupled with a complex physiological response that is indicative of their normal role in wound-healing. However, it is not known to what extent such complex transcriptional events are specific to a given cell type and signal, and to what extent these changes are innate programmed responses that are activated in a range of related cell types in response to a variety of stimuli. We have profiled the global transcriptional program of human fibroblasts from two distinct tissue sources to four different growth stimuli and identified a striking conservation in their gene expression signatures. However, there were specific differences among different stimuli with regard to signaling pathways that mediate these transcriptional programs. The use of a specific PI3-kinase pathway inhibitor suggested that this pathway is differentially involved in mediating the responses of cells to serum as compared to individual peptide growth factors. By applying siRNA knockdown technique, we demonstrated that putative targets of two important immediate early transcription factors, Myc and SRF, served functions related to cell cycle progression/cell survival and wound healing, indicating that these two transcription factors may serve as master transcription controllers during the transition of fibroblasts from quiescence to proliferation. In addition, different Myc targets were identified either between different cell types (Hela vs. foreskin fibroblasts) or between different cell states (unsynchronized vs. synchronized), while SRF targets included a group of genes only induced at certain time points during cell cycle progression, which was not observed in the Myc data. MicroRNA (miRNA) expression profiling indicated that let7 and other miRNAs with similar expression profiles may be involved in regulating the transcriptional program in response to proliferative signals. Our results indicate that conservation of transcriptional programs and their regulation among different cell types may be much broader than previously appreciated.Item Functional characterization of m-Bop, a transcriptional repressor essential for heart development(2002) Sims, Robert Joseph; Gottlieb, Paul D.Item Functional mapping and characterization of the responsive region required for polyunsaturated fatty acid regulation in the rat fatty acid synthase gene(2001-08) Teran-Garcia, Margarita de Lourdes; Clarke, Steven DonaldType and amount of dietary fats modify the onset of nutritionally related pathophysiologies such as diabetes mellitus, obesity and coronary heart disease. Among fats, arachidonic acid, 20:4(n-6) and docosahexaenoic acid, 22:6(n-3) are highly unsaturated long chain fatty acids (HUFA) that participate in abundant physiological events. The unique ability of HUFA to modulate lipid metabolism, and consequently energy homeostasis, depends on their coordinated effect on gene expression. HUFA inhibit the expression of genes responsible for fatty acid biosynthesis and at the same time stimulate the expression of genes involved in oxidative pathways and thermogenesis (Baillie et al., 1999; Jump and Clarke, 1999; Power and Newsholme, 1997). As a consequence, dietary HUFA improve insulin sensitivity and glucose utilization in skeletal muscle and adipose tissue (Baur et al., 1998; Pan et al., 1995; Storlien et al., 2000). This dissertation presents evidence of: a) the mapping and characterization of two candidate responsive regions required for HUFA regulation in the 5í flanking region of the rat fatty acid synthase (FAS), b) that the NF-Y and Sp1 sites in the proximal promoter of the FAS gene are essential for HUFA inhibition, c) that dietary HUFA inhibit the DNA binding of hepatic NF-Y and Sp1, and d) that there is a posttranslational mechanism implicated in this regulation. Identification of this two HUFA-DNA target sites open the possibilities to development of nutritional and pharmacological interventions in diabetes and obesity. Furthermore, it might allow us in the future to screen genetic mutations in humans and incorporate the appropriate preventive dietary changes to avoid the early onset of a metabolic disease.Item Gene expression in Autographa californica nuclear polyhedrosis virus infections exhibiting host specific modulation of occlusion body formation(Texas Tech University, 1989-08) Reinisch, Alesia JNuclear polyhedrosis viruses (NPVs) are enveloped, rod-shaped insect viruses containing a circular DNA genome (family: Baculoviridae). These viruses, which are being developed as biopesticides, are transmitted via polyhedral inclusion bodies (PIBs) in which viruses are embedded. In order to genetically engineer safe viral pesticides, it is necessary to understand the molecular basis of NPV host specificity and virulence. This research deals with a host range model (System III) in which Autographa californica multicapsid NPV (AcMNPV) replicates permissively in Spodoptera frugiperda (SF) cells but semipermissively in Bombyx mori (BM) cells. Early in infection, both cell lines displayed similar nuclear hypertrophy, but subsequent stages of cytopathic effect (CPE) were very different. PIBs were produced in SF but not BM cells. Instead, BM cells exhibited a unique CPE characterized by large sac-like bodies at the cell periphery. Virus growth kinetics experiments showed that infectious progeny virus was not produced in BM cells. Because PIBs were not found in the BM infection, gel electrophoresis was performed to determine whether polyhedrin, the major constituent protein of the PIB, was synthesized. Polyhedrin was not produced. The synthesis of another major late viral protein known as plO was also blocked. Thirteen other infected cell-specific polypeptides (ICSPs) were not detected in the semipermissive infection. Most of these appeared to be late ICSPs. Six ICSPs were found only in infected BM cells. Eight ICSPs were produced in both SF and BM infections but at different rates in each cell line. Inhibition of host proteins synthesis occurred in SF cells, but there was no clear evidence for host inhibition in BM cells. System III demonstrates a strong host range restriction which is manifested late in the BM infection. Additional research on the detailed mechanisms of the restriction should lead to the identification of viral DNA sequences responsible for host specificity and pave the way for the engineering of safe pesticides.Item Gene expression in cancer(Texas Tech University, 1992-05) Mittanck, Donald WayneCancer is a collection of diseases that affects the lives of many. One of every four Americans alive today will develop cancer and at least one in every five will die from cancer (Prescot et al., 1986). Thus it is very important to learn as much as possible about the modes of action of these diseases. In this work, we applied molecular biological techniques to study some of the genetic consequences of rat hepatoma cells and human endometrial carcinoma cells. In the first study the transcript levels for hexokinase, a major regulatory enzyme in glycolysis, were measured and compared in tumor and normal samples using Northern and slot blot analysis. This study was performed in response to previous observations by Nakashima and co-workers that there were significant increases in hexokinase activity in AS- 30D hepatoma cells. Our studies indicated that a significant increase of Type I hexokinase transcript did appear in AS-30D rat hepatoma cells. The second study was an investigation of the alpha-tubulin gene in human normal and tumor endometrial samples by restriction fragment length polymorphism (RFLP) analysis. The goal was to locate a tumor specific biomarker which could serve as a diagnostic marker for endometrial cancer. Previous reports had indicated that an alpha-tubulin polymorphism existed; however, no relationship was observed between the presence of this polymorphism and endometrial cancer. Our work indicated that there was an apparent, yet not fully explainable relationship between the presence of the observed polymorphism and endometrial cancer.Item Gene expression profiling and modeling of cervical cancer(2006) Carlson, Mark Wallace; Marcotte, EdwardCervical cancer is the number one cancer killer in women worldwide, but we still do not know how well our cell line models compare to the actual disease. Microarrays were used to create gene expression profiles of normal cervix and cervical cancer and were compared to nine different cervical cell lines typically used in research. Normal cervical expression was compared to cervical cancer expression and 140 genes were identified as differentially expressed. These genes were subjected to strict statistical testing and were further validated by a literature search. This validation increased our confidence in the analysis methods and enabled us to compare the data to cell lines. The Pearson correlation was used to quantitatively assess the expression similarity between cervical tissue and cell lines. The Primary normal cell line was the best global model of cervical tissue, with C4-I and C4-II the next best models. Cell lines were cultured in different types of media as well as in 3-dimensions (raft) that mimics the in vivo environment. These different culture conditions had varying changes in correlation to tissue; for instance simply using a different type of media could increase the correlation. Cell lines cultured as rafts had the largest change in correlation and were the best models of cervical tissue. The correlation of specific pathways were calculated to move beyond simple global comparisons. There were many cases where the correlation of a cell line in a specific pathway increased when cultured in a different environment. The cell adhesion pathway had a much higher correlation when cells were cultured as a raft instead of monolayer. In conclusion, new biomarkers of cervical cancer were discovered and current ones reconfirmed. A quantitative analysis of how well cell lines model cervical cancer was performed and revealed that small changes to the culture environment can improve their correlation to cervical tissue. This research improved our understanding of our ability to model cancer and will help translate in vitro results into in vivo care.
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