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Title: ENHANCEMENT OF BIODESULFURIZATION IN RHODOCOCCUS SPECIES (IGTS8) BY THE EXPRESSION OF VITREOSCILLA HEMOGLOBIN
Creator: Shivdas, Vrushali D.
Date: 2013, 2013-07
Description: The bacterium Rhodococcus sp. IGTS8 contains the dsz operon, which encodes a three enzyme pathway (the “4S pathway”) that is able to...
Show moreThe bacterium Rhodococcus sp. IGTS8 contains the dsz operon, which encodes a three enzyme pathway (the “4S pathway”) that is able to mineralize the sulfur contained in dibenzothiophene (DBT), an organic sulfur containing molecule found in petroleum. The gene vgb, which encodes Vitreoscilla hemoglobin (VHb), has shown wide usefulness in enhancing productivity and other useful properties when expressed in heterologous hosts. We engineered strain IGTS8 to express VHb and measured the effects on growth and desulfurization of DBT, using minimal medium containing DBT as the sole source of sulfur. VHb was clearly detected in the engineered strain using the standard COdifference spectral analysis, but its level (0.38-0.63 nmoles/gm wet weight of cells) was about 10-fold lower than commonly seen for expression of VHb in other heterologous bacterial hosts. The VHb-expressing strain was tested for growth at both low and high aeration in minimal medium containing DBT as sole sulfur source; growth was about 50% lower at low aeration compared with high aeration. Despite this, metabolism of DBT (as detected by accumulation of the end product of the 4S pathway, 2-Hydroxy biphenyl (2-HBP), in the growth medium) was about 30 % higher in the low aeration compared to the high aeration culture. A possible explanation for these results is direct enhancement of the first two (monooxygenase) steps in conversion of DBT to 2-HBP. It was thus concluded from the studies that the expression of vgb in Rhodococcus sp. IGTS8 enhances the process of biodesulfurization under conditions of low aeration
M.S. in Biology, July 2013
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Title: GENOME ANNOTATION AND PHYLOGENETIC ANALYSIS OF 27 SALMONELLA STRAINS BASED ON BIOINFORMATIC ANALYSIS OF RESPECTIVE GENOMES AND THREE GENES
Creator: Li, Xinyue
Date: 2013-04-15, 2013-05
Description: Salmonella is the most common food-borne bacterial infectious pathogen worldwide. Different serovars of Salmonella are capable of infecting...
Show moreSalmonella is the most common food-borne bacterial infectious pathogen worldwide. Different serovars of Salmonella are capable of infecting different kinds of hosts, such as humans, mice, pigs, chickens, and can also lead to different syndromes, such as enterica fever, enterocolitis and diarrhea, bacteremia and chronic asymptomatic carriage. Although Salmonella strains are quite diverse, strains within the same serovar usually infect the same host and cause similar symptoms. Thus, it is important, especially in food-borne disease outbreaks, to know which type of Salmonella is present. The current method of typing Salmonella is based on the Kaufmann-White scheme and MLEE, which are laborious and expensive. Although the reliability of this method has not been previously verified, the evolutionary relationship reflected by phylogenetic trees can be a possible alternative to the way of typing the Salmonella strains; this method would be less labor intensive and more economical. MLST is considered as a “gold standard” of typing for many species includes Salmonella. And genome sequence, which certainly reflects the evolutionary relationship of strains, is the most ideal data to construct a more reliable phylogenetic tree; however, genome sequencing is also a laborious and expensive process. Thus, conserved and ubiquitous gene data, which can be accessed with little effort, are generally used to minimize cost. Using16s rRNA is the most widely used method. In this study, 27 Salmonella genome sequences are annotated with RAST, and phylogenetic trees are constructed using three software, (phylip3.69, MEGA5.1, and CVTree). And MLST is also used to construct phylogenetic tree in this study, and the result is used to be compared with genome phylogenetic tree to find a more reliable reference tree. Although Neighbor-Joining method is the only algorithms x available in CVTree, phylip3.69 and MEGA5.1 are capable to use three separate algorithms(Maximum Parsimony, Maximum Likelihood, and Neighbor-Joining, respectively). Finally, these trees are compared in an effort to find a good alternative to replace the reference phylogenetic tree. In this study, it was determined that the groEL gene would be the best replacement.
M.S. Biological and Chemical Sciences, May 2013
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Title: ISOLATION OF MONOSPECIFIC ANTIBODY AGAINST SPLICED VARIANT OF DYSTROPHIN PROTEIN USING YEAST SURFACE DISPLAY TECHNIQUE
Creator: Saraswathi, Raj Prabu Vijayakumar
Date: 2011-05-04, 2011-05
Description: Dystrophin gene is the largest in the human genome with 79 exons covering greater than 0.1% of the total genome, located on the Xp21 locus of...
Show moreDystrophin gene is the largest in the human genome with 79 exons covering greater than 0.1% of the total genome, located on the Xp21 locus of the “X” chromosome resulting in a 427kDa protein, “Dystrophin”. Dystrophin is an important cytoskeletal protein which belongs to the β-Spectrin/α-actinin family of proteins. It comprises of an amino terminal domain, alpha helical coiled structure COOH domain, central rod region with 24 STRs and four proline rich hinge regions. It plays a vital role in localizing the Dystrophin glycoprotein complex (DGC) in the Sarcollema and is associated with the DGC in controlling the signaling events of certain proteins associated with DGC. The large size of the gene makes it more vulnerable to mutations resulting in partially functional or non-functional Dystrophin. The absence of Dystrophin results in disruption of sub sarcolemma-extracellular matrix linkage, loss of nitric oxide, progressive muscle weakening and muscle wasting leading to the death of patients typically before the end of their teenage. In certain cases alternatively spliced isoforms produce Dystrophin with reduced length yet stable and completely functionality. The main focus of this project was to select monospecific antibody against the more stable alternatively spliced variant D14 (15”16”) 17, which is functional and more stable compared to unspliced parent D14:17. The yeast surface display technique was used to effectively screen and select the yeast scFv clones containing the monospecific antibody against our target spliced variant D14 (15”16”) 17 protein and D2:3. The yeast scFv sub population was enriched by repeated MACS and FACS selection. Test colonies picked from the enriched scFv pool were confirmed via PCR and restriction digestion analysis. The scFv for the respective antigens were then sub cloned into pPnnl-9 secretion vector using YVH10 yeast cells via LiTRAFCO method. It was clear that by repeated MACS and FACS selection the scFv pool can be enriched and the yeast scFv clone sub population can be reduced to a significant level. The scFv sub cloned into Pnnl-9 secretion vector can be purified using affinity chromatography and the further affinity and avidity studies can be conducted.
M.S. in Biology, May 2011
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Title: BIOLOGICAL STRATEGIES FOR ENHANCEMENT OF LIQUID FUELS: SULFUR REMOVAL FROM PETROLEUM AND BIOETHANOL PRODUCTION
Creator: Wang, Jia
Date: 2013, 2013-12
Description: Rhodococcus baikonurensis CW25 was transformed with the Rhodococcus erythropolis strain IGTS8 desulfurization operon (dszABC, which encodes...
Show moreRhodococcus baikonurensis CW25 was transformed with the Rhodococcus erythropolis strain IGTS8 desulfurization operon (dszABC, which encodes the enzymes of the “4S” desulfurization pathway) or this operon modified to contain a synthetic cysteine-methionine rich “sulpeptide” gene (S1) (dszAS1BC). The two CW25 derivatives were subjected to directed evolution to select faster growing cells using the key 4S pathway substrate dibenzothiophene (DBT) as the sole source of sulfur. Data of cell doubling times verified the success of selection of cultures with increasingly rapid growth. The desulfurization activities of resting cells of early passages demonstrated improvements, and the highest activity of the dszAS1BC-bearing CW25 derivative was 115% higher than that of the CW25 derivative without S1. In addition, a trend of initial high activity was followed by a decrease in subsequent passages. Rates of DBT metabolism of growing cells demonstrated a different trend, probably because the activity of growing cells concurrently reflects the activity of DszABC enzymes and the growth rates of the recombinants. Dry cell weights fluctuated during the evolution process, probably because of variations in the efficiency of the conversion of the sulfur in DBT into sulfite, then into sulfate or biomass, or, for the S1-bearing cells, because the secretion of the S1 peptide from cells might have variable efficiency. A mixed culture of two Paenibacillus species (“W” and “Y”) was isolated that can metabolize DBT at temperatures up to 54 ºC. Strain Y is the only one of the two with desulfurization activity, while strain W enhances the desulfurization ability of Y. The W-Y culture may be a useful starting point for selection of desulfurization cultures with even greater thermal stability. xiii Ethanologenic Escherichia coli strain FBR5 was compared with Vitreoscilla hemoglobin (VHb)-expressing FBR5 (TS3) regarding the concentrations of ATP, NAD+, NADH, NAD+/NADH ratio; and growth and ethanol production at various points during growth. The significant finding was that the NAD+/NADH ratio for TS3 was lower in early growth, but higher in later growth compared to that for FBR5. This is probably because more NADH was required by TS3 for its enhanced ethanol production and VHb-related increased respiration under microaeration conditions.
PH.D in Biology, December 2013
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Title: PROBING THE PAN-GENOME OF LISTERIA MONOCYTOGENES
Creator: Deng, Xiangyu
Date: 2011-04-26, 2011-05
Description: Bacterial pathogens often show significant intra-species variations in ecological fitness, host preference and pathogenic potential to cause...
Show moreBacterial pathogens often show significant intra-species variations in ecological fitness, host preference and pathogenic potential to cause infectious disease. The species of Listeria monocytogenes, a facultative intracellular pathogen and the causative agent of human listeriosis, consists of at least three distinct genetic lineages. Two of these lineages predominantly cause human sporadic and epidemic infections, whereas the third lineage has never been implicated in human disease outbreaks despite its overall conservation of many known virulence factors. The genomes of 26 L. monocytogenes strains representing the three lineages are compared based on both in silico comparative genomic analysis and high-density, pan-genomic DNA microarray hybridizations. We uncover 86 genes and 8 small regulatory RNAs that likely make L. monocytogenes lineages differ in carbohydrate utilization and stress resistance during their residence in natural habitats and passage through the host gastrointestinal tract. We also identify 2,330 to 2,456 core genes that define this species along with an open pan-genome pool that contains more than 4,052 genes. Phylogenomic reconstructions based on 3,560 homologous groups allowed robust estimation of phylogenetic relatedness among L. monocytogenes strains. The pan-genome approach enables accurate co-analysis of DNA sequence and hybridization array data for both core gene estimation and phylogenomic reconstruction. Application of our method to the pan-genome of L. monocytogenes sheds new insights into the intra-species genomic diversification, niche expansion and evolution of this important foodborne pathogen.
Ph.D. in Biology, May 2011
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Title: INVOLVEMENT OF MIR-182 IN THE ACTION OF ATORVASTATIN IN PROSTATE CANCER CELLS
Creator: Li, Wenping
Date: 2013-04-10, 2013-05
Description: Atorvastatin (ATO), a widely used statin for lowering cholesterol was examined for its chemopreventive/therapeutic activities in prostate...
Show moreAtorvastatin (ATO), a widely used statin for lowering cholesterol was examined for its chemopreventive/therapeutic activities in prostate cancer cells. We found that ATO inhibited cell proliferation and induced autophagy in PC3 prostate cancer cells, as marked by significant induction of an autophagy marker LC3-II. Using Taqman RT-PCR technique, we also found that ATO treatment for 24h and 48h consistently up-regulated miR-182 in PC3 cells. However, adding geranylgeraniol (GGOH) to the culture media reversed the effect of ATO in regulating miR-182, suggesting that ATO up-regulates miR-182 through inhibition of geranylgeranyl biosynthesis. Overexpression of miR-182 in PC3 cells significantly decreased cell proliferation by about 36% (MTT assay), while knock-down of miR-182 stimulated cell proliferation by about 43% (MTT assay). In screening for miR-182 target genes, we found that Bcl2 and p21 are potential miR-182 target genes; Bcl2 was significantly down-regulated by ATO at both mRNA and protein levels and miR-182 knock-down up-regulated Bcl2 protein; p21 protein expression was positively correlated with alteration of miR-182 expression levels in PC3 cells. Through screening database of miR-182 target genes from TargetScanHuman 6.2 and PicTar, we found that p21 is not the direct target gene of miR-182, so it could be regulated by miR-182 indirectly. It has recently been established that miR-182 regulation is p53-dependent, since PC3 cells are p53 negative, it is clear that ATO regulates miR-182 in a p53-independent manner. These data demonstrate that miR-182 up-regulation and Bcl2 down-regulation by ATO could be two independent events and both could be involved in ATO mediated cell proliferation and autophagy.
M.S. in Biology, May 2013
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Title: CHANGES OF BACTERIAL SPECIES AND HEME PROTEIN OCCURRENCE IN ACTIVATED SLUDGE COMMUNITIES CULTURED IN THE LABORATORY
Creator: Wang, Xiaomeng
Date: 2016, 2016-05
Description: An activated sludge sample that had originally been collected from an aeration tank of the Stickney wastewater treatment plant in Chicago, and...
Show moreAn activated sludge sample that had originally been collected from an aeration tank of the Stickney wastewater treatment plant in Chicago, and had previously been cultured at low dissolved oxygen (DO) for 48 weekly passages was used as starting material for continuation of the low DO acclimation. The culture was continued at low dissolved oxygen in synthetic wastewater for 25 additional weekly passages to study what would happen to the activated sludge if the low DO continued. In order to do that, some important data were measured during the culture, including the specific oxygen uptake rates (SOUR) which could reflect the ability of oxygen utilization, 16S rDNA information which could tell the community diversity of sludge, and the dominant species genome data which suggested what really happened to the sludge and some reasons. The results showed that SOUR decreased modestly during the course of low DO adaptation, which was contrary to the results of the previous study. There were significant changes in community structure with respect to bacterial species during the first fifteen additional passages. Species known to produce both flavohemoglobins (FHbs) and truncated hemoglobins (trHbs) were common at all passages tested, although the dominant species were totally different from passage to passage. Specifically, during the course of the experiment, the frequency of cells encoding an FHb decreased substantially, from 84% to 50%, while the percentage of cells encoding a trHb decreased slightly from 84% to 78%. The overall content in the culture of heme b (the heme type found in bacterial hemoglobins) decreased, however, during continuation of the low DO conditions. So it is indicated that the oxygen utilization ability of the activated sludge does not increase all the time.
M.S. in Biology, May 2016
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Title: SITE-DIRECTED MUTAGENESIS STUDY OF VITREOSCILLA HEMOGLOBIN-THE ROLE OF TYROSINE (B10) AND PROLINE (E8) IN THE STRUCTURE OF THE LIGAND-BINDING SITE
Creator: Zhang, Yifan
Date: 2015, 2015-05
Description: Vitreoscilla is a genus of Gram-negative aerobic bacterium which has the capability to synthesis a soluble, homodimertic hemoglobin,...
Show moreVitreoscilla is a genus of Gram-negative aerobic bacterium which has the capability to synthesis a soluble, homodimertic hemoglobin, Vitreoscilla hemoglobin (VHb). The Vitreoscilla hemoglobin was the first bacterial hemoglobin discovered, and has a wide range of biological and biotechnological applications. The distal site is one of the hot spots in VHb studies because of its unique structure. The Tyrosine residue at B10 and its hydrogen bonded Proline at E8 were considered as the ligand binding functional sites in distal space of VHb according to the previous study. In this study, two single mutated and one double mutated Vitreoscilla hemoglobin at position B10 and E8 were constructed and purified. In the two single mutants, the Tyr at B10 and the Pro at E8 were mutated to Ala. In the double mutant, both of the sites were mutated to Ala. The CO di↵erence spectrum data of the mutants indicate that the ligand binding ability of the Vitreoscilla hemoglobin was not neutralized by the mutations at ProE8 and TyrB10. Circular dichroism spectrum data of the mutants is similar to the wild type Vitreoscilla hemoglobin, which means the globin secondary structure is conserved. However the micro-environment in the distal sites is changed: the IR spectrum of the carbonyl stretch bond red-shifted in the CO bound VHb double mutant. A molecular dynamic simulation was introduced in the study to o↵er some guidance for future research plans. The simulation results showed that the B10 and E8 residue mutated to Ala might reduce the flexibility of the D-region, because of the more completed C and E helix. The volume of cavity where the heme group inserts changes significantly in various mutant models, which may provide a rough explanation of the change in carbonyl stretch bond IR spectrum. Additionally, an interesting conformation of Gln E7 was found in the simulation of double mutant model.
M.S. in Biology, May 2015
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Title: BIOPHYSICAL AND BIOCHEMICAL STUDY OF NATIVE AND EDITED DYSTROPHIN ROD REGION
Creator: Mangat, Khushdeep
Date: 2014, 2014-12
Description: Duchenne Muscular Dystrophy (DMD) is a severe X-linked recessive disease affecting 1 in 3500 boys that is characterized by the degeneration of...
Show moreDuchenne Muscular Dystrophy (DMD) is a severe X-linked recessive disease affecting 1 in 3500 boys that is characterized by the degeneration of muscle function and strength. The cause of this disease lies in gene defects that eliminate expression of the protein dystrophin. Becker Muscular Dystrophy, BMD is a milder form of disease that has a later onset and much longer survival (up to the 7th decade of life, compared to median survival of 25 years for DMD patients) because of the presence of low levels of modified dystrophin protein. BMD is very heterogeneous, however, and many cases are nearly as severe as DMD. A major therapy for DMD involves exon skipping, which produces modified forms of dystrophin that are very similar to BMD. However, how these edits impact the function of dystrophin, and how they are linked to the severity of BMD or the BMD-like state produced in DMD exon skip therapy is unknown. We investigated this in two specific cases involving a specific panel of BMD defects linked to a major cause of death, dilated cardiomyopathy (DCM). We also investigated the contribution of various exons to interaction with a signaling partner of dystrophin, neuronal nitric oxide synthetase (nNOS).
Ph.D. in Biological and Chemical Sciences, December 2014
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Title: INTEGRATION OF TANGENTIAL FLOW FILTRATION AND IMMUNOMAGNETIC SEPARATION FOR RAPID DETECTION OF ESCHERICHIA COLI 0157:H7 IN PRODUCE WASH WATER
Creator: Ren, Yan
Date: 2011-12-12, 2011-12
Description: Escherichia coli O157:H7 is one of the most commonly reported pathogens associated with microbial contamination of leafy greens. Since washing...
Show moreEscherichia coli O157:H7 is one of the most commonly reported pathogens associated with microbial contamination of leafy greens. Since washing is a major postharvest processing step, microbial testing of spent wash water has been suggested as a good marker to determine the contamination status of the products. In this study, the efficiency of four commercial rapid methods (BAX®, IQ-Check, Reveal® and mini-VIDAS®) for detection of E.coli O157:H7 in lettuce wash water was evaluated in comparison with the FDA BAM method. The improvement of the detection sensitivity of these tests by immunomagnetic separation (IMS) technology and sample pre-concentration by Tangential Flow Filtration (TFF) was determined. Twenty-five ml of lab prepared lettuce wash water samples were spiked with 0, 1, 10, 100 CFU of E.coli O157:H7, and subjected to enrichment protocols recommended by each of the methods. The presence of E.coli O157:H7 in the enriched samples were then assayed by the test kits, either directly or after IMS (IMS-Pathatrix ™ or IMS-Dynabeads™) treatments. All four test kits and BAM were able to detect E.coli O157:H7 at levels as low as 1 CFU/25ml of wash water. IMS treatments did not lead to further improvement in detection sensitivity. Experiments were also performed to determine the feasibility of incorporating IMS and sample pre-concentration to achieve culture-free detection. Fifty ml of wash water samples were inoculated with E.coli O157:H7 at levels of 0 to 107 CFU and analyzed by the test kits either directly or after IMS-Pathatrix™ treatment. Additionally, 10 L of wash water either prepared in the lab or collected from a commercial fresh-cut processing facility were inoculated with 0 – 106 CFU of the pathogen and subjected to TFF concentration prior to IMS or test kit analyses. IQ-Check showed the highest sensitivity with a detection limit as low as 103 CFU/50ml, and, with IMS, achieved a sensitivity of 100 CFU/50ml. Combining TFF concentration and IMS, 10 L of lab prepared wash water can be tested with IQ-Check and achieve a detection limit of approx. 100 CFU/10 L within 6 hours. For 10L of industry spent wash water, IQ-check also showed the highest sensitivity but the results lacked consistency and required additional evaluations.
M.S. in Food Safety and Technology, December 2011
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Title: Investigation of Fresh Produce Washing on E. Coli O157:H7 and Murine Norovirus with Peroxyacetic Acid and High Power Ultrasound
Creator: Yuan, Wen
Date: 2011-12-05, 2011-12
Description: E. coli O157:H7 and norovirus have each been responsible for outbreaks of foodborne illness involving fresh leafy green vegetables....
Show moreE. coli O157:H7 and norovirus have each been responsible for outbreaks of foodborne illness involving fresh leafy green vegetables. Peroxyacetic acid (POAA) has the potential to be an effective sanitizer during commercial fresh-cut produce washing. The addition of high power ultrasound (HPU) to the washing system may enhance the POAA efficacy. The purpose of this study was to reduce E. coli O157:H7 and norovirus contamination during commercial fresh-cut lettuce washing using POAA and HPU. Fresh-cut romaine lettuce leaves were inoculated with E. coli O157:H7 or murine norovirus (MNV, a surrogate for the human norovirus) and washed by POAA and HPU. Cross-contamination was tested by washing clean leaves in contaminated processing water where pre-inoculated leaves were washed previously. A high level of cross-contamination (4.5-log CFU/g E. coli) occurred after uninoculated leaves were rinsed in contaminated wash water for 2 min. A subsequent 2 min wash in POAA alone or in combination with HPU reduced counts by approximately 1.2-log and 2.3-log, respectively. More than 2-log norovirus was removed from washing 2 min in either DI water or POAA. However infectious MNV washed from leaves was not detected in wash water containing POAA. Those results implied a high possibility of cross-contamination during freshproduce washing, and indicate that adding POAA and HPU in addition to washing water in wash water can reduce cross-contamination rate.
M.S. in Food Safety and Technology, December 2011
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Title: STUDY OF VITREOSCILLA HEMOGLOBIN VARIANTS PRODUCED BY RANDOM MUTAGENESIS
Creator: Lin, Xiaodan
Date: 2015, 2015-05
Description: This study is focused on comparing the wild type and mutated versions of the Vitreoscilla hemoglobin gene (vgb). The purpose of this focus is...
Show moreThis study is focused on comparing the wild type and mutated versions of the Vitreoscilla hemoglobin gene (vgb). The purpose of this focus is to find out whether any of the vgb mutations provides an advantage regarding cell growth rate, as well as on the expression level of Vitreoscilla hemoglobin protein (VHb). A negative control Escherichia coli DH5α (E. coli DH5α) bearing no pUC plasmid, as well as seven E. coli DH5α strains bearing different pUC-based plasmids were tested in the experiments. Among these were one vector-only negative control (pUC18), one wild type positive control (pUC8:16, which carries wild type vgb) and five different types of pUC-bearing vgb mutants (pUC-vgb M1, M2, M3, M4 and pUC18-vgb M3). In order to compare cell growth rate among these strains, the growth rate assay was carried out under three different conditions: (1) Luria-Bertani (LB) medium, aerobic conditions; (2) Terrific Broth (TB) medium, low oxygen conditions; and (3) TB medium, microaerobic conditions. In addition, the carbon monoxide (CO) difference spectra assay was conducted to measure functioning VHb protein expression levels for the strains grown under aerobic conditions. In contrast to the results obtained by our Australian collaborators, our growth rate assay and CO difference spectra assay showed no growth advantage or higher expression level of functioning VHb protein due to any of the vgb mutations. For the further study of the vgb mutants, four different recombinant plasmids were constructed by cloning three types of mutated vgb (vgb M1, M3 and M4) as well as wild type vgb into the prokaryotic expression vector pUC8 with ampicillin (Amp) resistance. After being transformed into competent E. coli DH5α cell, these resulting xii strains, as well as the plasmid-free negative control (E. coli DH5α) and vector-only negative control (E. coli DH5α bearing plasmid pUC8) were tested by the CO difference spectra assay. Except strain E. coli DH5α [pUC8-vgb M3], which showed a slight increase in the VHb expression level, the strain bearing other mutated vgbs did not demonstrate any elevation in VHb protein expression level, compared to the positive control containing wild type vgb.
M.S. in Biology, May 2015
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Title: FUNCTIONAL ANALYSIS OF POTENTIAL PHOSPHORYLATION SITES IN BAXΔ2 UNIQUE OLIGOPEPTIDE
Creator: Tsai, Yu-tseng
Date: 2014, 2014-07
Description: The tumor suppressor gene, Bax, plays a critical role in tumor progression through regulating cell apoptosis. Mutations on the BAX gene often...
Show moreThe tumor suppressor gene, Bax, plays a critical role in tumor progression through regulating cell apoptosis. Mutations on the BAX gene often result in silencing its expression and the loss of pro-death ability. However, there is a unique Bax isoform, BaxΔ2, recently discovered in these Bax mutated cancer cells. BaxΔ2 isoform shows higher pro-apoptotic activity than Baxα. Unlike the parental Baxα, BaxΔ2 does not target mitochondria and forms aggregates in cytosol. There is a unique 10-amino-acid peptide in the N-terminus of BaxΔ2 protein possible function as a special signal. Two serines in this region are predicted as potential phosphorylation sites for regulation of the protein activity. To test this hypothesis, we mutated both serines (SS) into non-phosphorylatable alanines (AA) by site-directed mutagenesis approach. Both BaxΔ2 wild type (BaxΔ2-SS) and mutants (BaxΔ2-AA) were tagged with GFP, which allows us to monitor the protein expression and cellular localization in live cells. Here, we found that the distribution patterns of BaxΔ2-AA and BaxΔ2-SS were similar and appeared as aggregates in cytosol. BaxΔ2-AA mutant also possessed the similar pro-apoptotic activity with BaxΔ2-SS wild type. These results suggested that the two serines in BaxΔ2 unique oligopeptide might not play a critical role in BaxΔ2 localization and pro-death activity under the current ectopic expression conditions. Further study is needed to have better understanding of phosphorylation in contribution to unique behavior of BaxΔ2.
M.S. in Biology, July 2014
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Title: INVESTIGATION OF NOROVIRUS CROSS CONTAMINATION DURING FOOD SERVICE PROCEDURES USED IN PREPARATION OF FRESH PRODUCE
Creator: Suriyanarayanan, Annamalai
Date: 2011-11, 2011-12
Description: Human norovirus (HuNoV) is considered an important cause of foodborne outbreaks, often attributed to the preparation of fresh produce by...
Show moreHuman norovirus (HuNoV) is considered an important cause of foodborne outbreaks, often attributed to the preparation of fresh produce by infected food handlers. In this investigation, methods for recovery of murine norovirus (MNV-1), a surrogate for HuNoV, from food preparatory surfaces were optimized, and MNV-1 crosscontamination between various surfaces common in a food service setting were studied. Fifty microliters of MNV-1 was inoculated onto demarcated 1 x 1 inch squares of polypropylene cutting board, stainless steel knife and spigots. After drying, MNV-1 was recovered from each surface using either a cotton swab, composite tissue or sterile sponge in combination with different eluents such as tissue culture growth medium, 3% beef extract, glycine buffer (50mM glycine, 1% beef extract), stripping solution (0.04% K2HPO4, 1.01% Na2HPO4, 0.1% Triton X-100), and Earle’s Balanced Salt Solution (EBSS). The eluent/recovery tool combinations that recovered the highest percentage of MNV-1 from cutting board were stripping solution/sponge (20%) and growth medium/swab (20%). The greatest recovery from the knife blade was achieved with the growth medium/composite tissue combination (43%), while recovery from spigots was greatest using the stripping solution/sponge (28%) and the growth medium/sponge combinations (27%). In the second phase of this investigation, human volunteers were asked to perform various tasks in order to quantify the amount of MNV-1 cross contamination between various surfaces, including bare hands, fresh-cut lettuce, and spigots. The percentage of MNV-1 transfer from hands to spigots varied from 0.06% to 3.59%, spigots to hands varied from 10% to 90.4% and lettuce to hands varied from 0.30% to 4.33%. x The results of this investigation can be used in developing a model describing the transfer pattern of HuNoV between surfaces common in retail food service, and used in developing educational materials for food service workers.
M.S. in Science, Food Safety, and Technology, December 2011
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Title: LENGTH DEPENDENT ACTIVATION IN MANDUCA SEXTA FLIGHT MUSCLE
Creator: Kumar, Mohit
Date: 2011-08-09, 2011-07
Description: The flight muscle of Manduca sexta has some interesting properties. It is synchronous, like mammalian skeletal and cardiac striated muscle,...
Show moreThe flight muscle of Manduca sexta has some interesting properties. It is synchronous, like mammalian skeletal and cardiac striated muscle, but it is structurally similar to the more widely studied asynchronous insect flight muscles of Drosophila and Lethocerus. One of the main goals of the thesis is to generate a useful skinned preparation for mechanical studies in vitro. A number of different skinning protocols were tried and evaluated for preservation of structural integrity by using light and X-ray diffraction. In all muscles studied to date, isometric force is a function of the [Ca2+] of the bathing solution and also a function of the sarcomere length whereby more force is generated at a longer sarcomere length than at a shorter for the same [Ca2+]. This phenomenon is termed “length dependent activation” (LDA). To date no real studies on the force-pCa relationship has been done on Manduca sexta flight muscle. This force-pCa analysis would give us some insight into the length dependent activation (LDA) in this novel insect flight muscle system. The present studies were undertaken to characterize and analyze the force-pCa relationships in Manduca sexta. Conditions were found that allowed skinning muscles while maintaining good structural order. We found that both DLMs dorsal and ventral show length-dependent activation at longer SL. Our study also shows that ventral muscles are more cooperative than the dorsal muscles which may be related to their different functions in vivo.
M.S. in Biological, Chemical, and Physical Sciences, July 2011
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Title: CONSTRUCTION OF ZEBRAFISH OLFACTORY RECEPTORS INTO A MAMMALIAN EXPRESSION VECTOR FOR FUTURE LIGAND-BINDING STUDIES
Creator: Tian, Chen
Date: 2011-07, 2011-07
Description: The olfactory system is an important chemosensory system. Our identification of odorants is occurred through binding of odorant molecules with...
Show moreThe olfactory system is an important chemosensory system. Our identification of odorants is occurred through binding of odorant molecules with olfactory receptors in the olfactory epithelium. In zebrafish, there are four classes of olfactory receptors: trace amine-associated receptor (TAAR), odorant receptor (OR), vomeronasal type 1-like receptor (V1R-like) and vomeronasal type 2-like receptor (V2R-like). In this study, I amplified four olfactory receptors taar1a, or1016, ora5 and olfcc1, which are respectively belong to the four olfactory receptor classes, from cDNA of the zebrafish olfactory epithelium and cloned genes into a mammalian expression vector pCI. The gene sequences of these receptors were not identical as the sequences obtained from GenBank database. The possible reasons for the inconsistency of gene sequence are discussed. I further tranfected these four cloned olfactory receptors in a mammalian cell line Hana3A. Expression of all four olfactory receptors in Hana3A cells was confirmed by immunocytochemistry. In summary, these four olfactory receptors taar1a, or1016, ora5 and olfcc1 were successfully cloned into the pCI vector and can be expressed on cell membrane surface of Hana3A cells.
M.S. in Biology, July 2011
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Title: CHARACTERIZATION OF VERY LARGE YET VERY MILD BMD TYPE EDITS OF DYSTROPHIN
Creator: Lin, Yi-hsiu
Date: 2014, 2014-12
Description: Duchenne muscular dystrophy, DMD, is a severe X-linked recessive disease that kills boys in their second or third decade of life. A related...
Show moreDuchenne muscular dystrophy, DMD, is a severe X-linked recessive disease that kills boys in their second or third decade of life. A related condition Becker Muscular Dystrophy, BMD, is very heterogeneous with clinical severity ranging from nearly as bad as DMD, to nearly benign. Both of these are caused by defects in the dystrophin gene, which encodes a 427 kDa cytoskeletal protein, dystrophin, which can provide a mechanical link between the cytoskeleton and the muscle membrane and stables muscle tissue. In general, DMD results from defects that eliminate all dystrophin expression, whereas BMD patients carry mutated products: modified and malfunctioned dystrophin proteins. Since DMD is invariably fatal, monogenic, has a high incidence (1:3500 male births) and afflicts a charismatic patient profile, it has been an attractive target for gene therapy. However, the huge size of the dystrophin gene (2.4 Mbp) and more importantly its processed mRNA (14.2 kb) poses a problem for viral gene therapy which is limited to payloads of about 8 kb. This has prompted efforts to reduce the size of the dystrophin protein while still maintaining functionality. A major inspiration for such efforts was the identification of a very unusual, very large deletion serendipitously found in an extremely mild BMD case. The patient identified with this defect was ambulatory at 65 year of ages. This edit removes more than half the central rod region, from exon 17 to 48, Δe17-48, and, fortunately, the size of this BMD type edit protein is available for viral vector loading. In this project, edited dystrophin with central rod region deletion (D2:22 Δe17-48) and other two related edits, D2:22 Δe17-47 (exon 48 is added in D2:22 Δe17-48) and D2:22 Δe17-49 (exon 49 is deleted from D2:22 Δe17-48), were studied. According to these results, D2:22 Δe17-47 demonstrated the best biophysical and biochemical properties, but D2:22 Δe17-48 displayed higher to protease sensitivity, and D2:22 Δe17-49 had the worst thermal stability. This work will help us to understand the factors that result in benign large scale edits, and facilitate the development of effective viral gene therapy vectors.
M.S. in Biology, December 2014
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Title: FUNCTIONAL ANALYSIS OF UBIQUITIN-LIKE PROTEIN 4A
Creator: Zhao, Yu
Date: 2015, 2015-12
Description: Ubiquitin-like protein 4A (Ubl4A) was identified as a housekeeping gene at X chromosome. It involves in the guided entry of tail-anchored (GET...
Show moreUbiquitin-like protein 4A (Ubl4A) was identified as a housekeeping gene at X chromosome. It involves in the guided entry of tail-anchored (GET) protein pathway in which tail-anchored (TA) proteins are transported to endoplasmic reticulum (ER). However, Ubl4A also involves other functions not related to GET pathway, such as tumor suppression and DNA damage-mediated apoptosis. Up to date, the function of Ubl4A in mammals is still largely unknown. We found that either overexpression or knockdown of Ubl4A promoted cell death in cell culture system. Using an in vivo genetic knockout system, we found that Ubl4A knockout mice displayed a high neonatal mortality and had a defect in glycogen synthesis, which is mainly controlled by a key protein kinase Akt. Loss of Ubl4A resulted in the impairment of insulin-induced Akt translocation to the plasma membrane, an essential step for Akt activation. We demonstrated that Ubl4A directly interacted with actin-related protein 2/3 (Arp2/3) complex, further accelerated Arp2/3 complex-dependent actin branching, thereby bringing Akt to proximity into the plasma membrane for activation. Furthermore, we showed that Ubl4A-mediated actin branching also played important roles in other cellular activities, such as formations oflamellipodia and filopodia, macrophage phagocytosis, wound healing, and neutrophil chemotaxis. These findings provide us a new insight into understanding the roles of Ubl4A in cellular function and a molecular basis for treatment of related human diseases.
Ph.D. in Biology, December 2015
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Title: IMPACT OF THERMAL PROCESSING ON THE STRUCTURAL AND IMMUNOLOGICAL PROPERTIES OF MAJOR EGG AND MILK ALLERGENS
Creator: Chandra, Srinivasa Rao
Date: 2011-05-03, 2011-05
Description: The underlying mechanism of food allergy is not well understood. Research has increasingly focused on the characterization of food allergens....
Show moreThe underlying mechanism of food allergy is not well understood. Research has increasingly focused on the characterization of food allergens. Since most foods are cooked prior to consumption, information relating to the impact of thermal processing on the properties of allergenic proteins is critical for allergen risk assessment. This study examined the impact of thermal processing on the structure and the antigenic potential of the major egg and milk allergens, ovomucoid (OVO) and -lactoglobulin (BLG) both A and B variants respectively. OVO and BLG were subjected to thermal processing under moist and dry heat conditions for 10 min. No significant changes in the solubility of both proteins were observed after boiling, autoclaving or dry heating up to 204C. At 232C, a significant protein loss was observed. Inhibition ELISA was used to determine the effect of heat treatment on the capacity of these proteins to bind rabbit derived IgG antibodies. While boiling and autoclaving caused a decrease in IgG binding of OVO, an increase in IgG binding of BLG was observed under the same experimental conditions. A similar pattern that a decrease in antigen binding potentials by OVO and an increase in antigen binding potentials by BLG A and B variants was noticed during dry heat treatment at temperatures 232C and above. Structural analyses were performed using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). Both proteins showed variations in the secondary structure when subjected to heating in water and PBS. In the presence of water, variable temperature scan with CD resulted in transition temperatures of OVO and BLG variants in the range of 70-75oC and 80-85oC respectively. There is no significant change in the secondary structure of BLG variants prepared in PBS. DSC study showed the transition temperatures of 84oC, 129oC for OVO and at 80oC, 195oC for BLG (A & B) variants under moist and dry heat conditions respectively. Overall, both proteins were highly resistant to thermal denaturation and retained their antigenic potential at typical cooking temperatures.
M.S. in Food Safety and Technology, May 2011
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Title: STRUCTURE AND FUNCTION OF NATIVE AND EDITED DYSTROPHIN RODS
Creator: Sahni, Neha
Date: 2011-05-10, 2011-05
Description: The purpose of this study is to examine the biophysical properties of the rod region of the dystrophin protein. This is important due to the...
Show moreThe purpose of this study is to examine the biophysical properties of the rod region of the dystrophin protein. This is important due to the severity of the disease Duchenne Muscular Dystrophy, (DMD), which is associated with the malfunction of this protein. DMD is one of the most serious single gene genetic defects of man. This rod region consists of a number of repeat motifs called spectrin type repeats or STRs. The thermodynamical and biochemical stability analysis shows, which single motifs are unstable on their own and which ones become more stable when linked to their appropriate tandem neighbors. This knowledge will impact strategies to produce modified mini dystrophins for gene therapy. Exon skipping therapy is an emerging approach to treat such genetic diseases. This is done by the administration of modified antisense oligonucleotides, AONs, which can interfere with exon splicing process and eliminate certain exons from the mature transcript. Furthermore, the rod region has a number of ancillary functions, such as providing secondary binding sites for actin, neuronal NO synthetase and phospholipids, which may be adversely perturbed by the edits.
Ph.D. in Biological, Chemical, and Physical Sciences, May 2011
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