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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
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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
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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
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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
- INVOLVEMENT OF MIR-182 IN THE ACTION OF ATORVASTATIN IN PROSTATE CANCER CELLS
- Creator
- Li, Wenping
- Date
- 2013-04-10, 2013-05
- Description
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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
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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
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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
- 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
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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
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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
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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
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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
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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
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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
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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
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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
- 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
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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 204C. At 232C, 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 232C 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
- INFLUENCE OF FOUR BACILLUS SP. STRAINS ON GROWTH AND DESULFURIZATION ABILITY OF MYCOBACTERIUM STRAIN U
- Creator
- Tian, Fangzhou
- Date
- 2016, 2016-05
- Description
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Desulfurization is an important step in crude oil processing and is commonly achieved through a chemical process known as hydrodesulfurization...
Show moreDesulfurization is an important step in crude oil processing and is commonly achieved through a chemical process known as hydrodesulfurization (HDS). Because this process is expensive and produces H2S as a by-product, the alternative of biodesulfurization (BDS) has been investigated for many years. The most potentially useful biodesulfurization process is the 4S pathway, which is found in a number of bacterial species, including Mycobacterium Strain U, which was isolated in our lab. To reach the requirement of BDS for use in an actual industrial-scale process, U has to survive at temperatures approaching 60 OC. In work in our lab, natural selection methods have been introduced for improving the U strain. During this natural selection, four contaminant strains, identified by 16S rDNA sequencing as Bacillus sp., were isolated from extraordinary U cultures which have BDS activity at 54 OC. Meanwhile the BDS activity of U on its own was found to have an upper temperature limitation of 53 OC. Additional experiments proved that all four Bacillus strains interact with U and improve its BDS ability.
M.S. in Biology, May 2016
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- Title
- BIODESULFURIZATION IMPROVEMENT OF A SYMBIOTIC PAENIBACILLUS CULTURE UTILIZING VITREOSCILLA HEMOGLOBIN
- Creator
- Liu, Benjamin Kwan
- Date
- 2015, 2015-05
- Description
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Biodesulfurization (BDS) of petroleum has been investigated as an alternative method to conventional chemical desulfurization for many years....
Show moreBiodesulfurization (BDS) of petroleum has been investigated as an alternative method to conventional chemical desulfurization for many years. Despite its potential to be an environmentally benign method, it has not been developed sufficiently to be useful in real world applications. This is due to its low efficiency and the necessity for it to work at temperatures high enough to lower the viscosity of petroleum so that mixing can be achieved. This study places the spotlight on two strains of Paenibacillus isolated in our laboratory that, together, possess biodesulfurization ability at moderately high temperatures and attempts to enhance biodesulfurization by expression of Vitreoscilla hemoglobin (VHb) in the Paenibacillus strains. The effects of expression of the VHb gene (vgb) on growth and desulfurizing activity was examined in a symbiotic system between the Paenibacillus strains 32O-Y and 32O-W. Of the two, 32O-Y is the one with the ability to metabolize dibenzothiophene (DBT), a common compound in petroleum that contains organic sulfur, while 32O-W enhances this ability, forming a symbiotic relationship between the two. The transformant of 32O-Y bearing vgb cloned into the shuttle vector pNW33N had been previously constructed in our laboratory. Presence of pNW33N-vgb was verified in one strain of 32O-Y through isolation of DNA, PCR, and gel electrophoresis. Mixtures of 32O-Y/32O-W or 32O-Y[pNW33N-vgb]/32O-W were cultured in minimal medium (CDM) with DBT as the sole sulfur source and subjected to multiple trials of growth and assay of DBT metabolism at varying temperatures. At 45 ˚C there was a substantial increase in both growth and DBT metabolizing coincident with VHb expression, whereas at lower (37 ˚C) and higher (50 ˚C) temperatures, VHb expression had little to no effect on either parameter. For both growth and DBT metabolism tested at 37 ˚C, 45 ˚C and 50 ˚C the highest absolute levels were seen at 37 ˚C, and the lowest at 45 ˚C.
M.S. in Biology, May 2015
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- Title
- A PHAGE-DISPLAY SELECTION FOR AN AFFINITY REAGENT OF ASXL2 PROTEIN
- Creator
- Balyan, Arjun
- Date
- 2013-04-07, 2013-05
- Description
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Asxl2 is a chromatin factor that regulates histone H3 methylation and H2A deubiquitination. Previous research in our lab has shown that Asxl2...
Show moreAsxl2 is a chromatin factor that regulates histone H3 methylation and H2A deubiquitination. Previous research in our lab has shown that Asxl2 is an important regulator of mammalian heart development and function. To facilitate the characterization of Asxl2, we proposed to generate an affinity reagent for the detection of Asxl2 proteins in vivo and in vitro. A recombinant GST-Asxl2 fusion protein in E. coli was produced that contained GST and the N-terminal 390 amino acids of Asxl2, separated by a Precision protease cleavage site. Asxl2 N1-390 was purified from GSTAsxl2 N1-390 and the purified polypeptide was biotinylated and used to select a phagedisplay antibody library. Finally, six antibodies were produced that exhibit various degrees of affinity with the recombinant Asxl2 N1-390 in ELISA assay. In the future the antibodies will be tested for their ability to detect endogenous Asxl2 proteins in western blot assay. This would facilitate the study of the role of Asxl2 in heart development and function.
M.S. in Biology, May 2013
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- Title
- ISOLATION OF TRANSPOSON MEDIATED TRANSGLUTAMINASE MUTANTS OF DROSOPHILA MELANOGASTER
- Creator
- Yang, Hua
- Date
- 2016, 2016-05
- Description
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Transglutaminase, Tg, catalyzes the formation of isopeptide bonds crosslinking and stabilizing protein complexes. The best-known factor XIII...
Show moreTransglutaminase, Tg, catalyzes the formation of isopeptide bonds crosslinking and stabilizing protein complexes. The best-known factor XIII crosslinks fibrin in clots. Paradoxically, there is an unexpected and counterintuitive correlation between factor XIII levels and heart attack mortality. Since factor XIII strengthens fibrin and resists fibrinolysis, higher XIII activity would be expected to lead to more stable fibrin clots and so more fatal atherosclerotic plaques; but it actually results in reduced mortality. One hypothesis to explain this is that Tg activity may also stabilize cardiac muscle tissue. Studies have shown that Tg is expressed in human embryonic myoblasts. Therefore, we suggest that muscular attachments may be strengthened by Tg, and play a vital unrecognized role in the muscular attachment. Humans have 9 Tg genes, complicating studies. However Drosophila melanogaster, D.m., has only one Tg gene, and is an attractive model system. We isolated 39 mutant D.m. lines gene produced by transposon mobilization in locus in a "dirty" fashion expected to create random deletions within the Tg locus. We then characterized them by PCR and sequencing, as well as functional assays, in an attempt to develop a Tg null line to test this hypothesis. The aim is to identify a line with all of Tg removed, but with neighboring genes unperturbed. Once the approximate break points were identified by PCR, we did DNA sequencing to fully characterize the genomic breakpoint. According to the DNA sequencing results, one line is specific for Tg exon1, and eliminates one of the two known Tg transcripts. Another line eliminated almost the entire catalytic domain. We also did RT-qPCR for two lines that were sequenced to determine their genomic characteristics. Both of these lines are viable but with subtle developmental defects, showing that Tg deficiency is not lethal in D.m.
M.S. in Biology, May 2016
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- Title
- DIFFERENT EXPLORATION ACTIVITY AND DETECTION THRESHOLD TOWARDS ODOR SETS BETWEEN CONNEXIN 36 KNOCKOUT MICE AND WILD-TYPE MICE
- Creator
- Chai, Xiaomeng
- Date
- 2012-04-16, 2012-05
- Description
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The sense of smell allows an organism to be aware of the chemical information of the external world. In most land animals, the olfactory...
Show moreThe sense of smell allows an organism to be aware of the chemical information of the external world. In most land animals, the olfactory system enables the perception of both volatile chemicals, as well as pheromones--chemicals released by animals that regulate their social activities. Among several factors that mediate olfaction, connexin 36 (Cx36), a subunit of gap junctions, has been suggested to play a crucial role in the olfactory system. In the research presented herein, I performed habituation/dishabituation studies on both Cx36 knockout (Cx36 KO) and wild-type mice to determine their exploration patterns towards four odorants. The results indicate that Cx36 KO and wild-type mice behaved differently when they were given defined odorants. In general, Cx36 KO mice spent more time exploring odorants. Cx36 KO and wild-type mice also exhibited different detection threshold towards given odorants. Moreover, cross-habituation between benzaldehyde and octaldehyde was established in Cx36 KO mice at the concentration of 10-4%, which was different from that in wild-type mice. These results may suggest important functions of Cx36 gap junctions in odor detection and integration in mammals. Keywords: Connexin 36, gap junction, olfactory system, habituation/dishabituation
M.S. in Biology, May 2012
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