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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: 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: Eleven Words That Sound Like "Orange"
Creator: Monica, Samelson
Date: 2012, 2012-04

Title: COMPARATIVE GENOMICS OF STREPTOCOCCUS SALIVARIUS ATCC 27945 AND 25975; FROM HELPFUL TO HARMFUL?
Creator: Soomer-james, Jahna T. A.
Date: 2015, 2015-07
Description: Streptococcus salivarius is a commensal bacterium that normally inhabits the oral mucosa. However, preliminary data indicated that the strain...
Show moreStreptococcus salivarius is a commensal bacterium that normally inhabits the oral mucosa. However, preliminary data indicated that the strain ATCC 25975 has acquired parasitic genes from Streptococcus pneumoniae. To investigate the nature of this genetic exchange, the genome of Streptococcus salivarius ATCC 27945 was first sequenced and served as a comparative model to provide insight into the possibility of development of pathogenicity within the salivarius group. Illumina and PacBio sequencing data were used complementarily to generate reliable genomes of the Streptococcus salivarius ATCC 27945 and ATCC 25975 strains. The reads were trimmed, filtered, assembled and annotated using custom Perl scripts and various software. The completed genomes of S. salivarius ATCC 27945 and ATCC 25975 are 2.11 Mbp and 2.20 Mbp long, respectively, with ATCC 25975 featuring an additional plasmid. Comparative genomics with other sequenced salivarius genomes revealed that strain ATCC 27945 was most closely related to strains JIM8777 and NCTC8618, while strain ATCC 25975 was more closely related to strains 57I and CCHSS3. The proteins that were common across the investigated salivarius genomes included housekeeping proteins involved in pathways such as DNA replication, metabolism and DNA repair but the unique protein types and their relative location to each other within the ATCC main chromosomes did not provide conclusive evidence to the identification of the parasitic cassette. However, the plasmid contained in S. salivarius ATCC 25975 showed promising signs of containing these genes of interest. Several genes typically found in S. pneumoniae such as capsular polysaccharide genes and two copies of integrative and conjugative genes were identified in close proximity. The phylogenetic analysis of the two S. salivarius ATCC strains suggested that the proposed genes acquired from S. pneumoniae might have been gained via horizontal gene transfer rather than through sporadic mutations. The plasmid had a 5.1% smaller GC content compared to the other salivarius chromosomes, further suggesting that it was acquired from a distinct organism. Overall, while these results provided useful insights into the nature and location of the parasitic cassette, further investigations will be required to assess the full extent of the genetic shuffling that occurred by horizontal gene transfer in these Streptococcus species.
M.S. in Biology, July 2015
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Title: BIODESULFURIZATION IMPROVEMENT OF A SYMBIOTIC PAENIBACILLUS CULTURE UTILIZING VITREOSCILLA HEMOGLOBIN
Creator: Liu, Benjamin Kwan
Date: 2015, 2015-05
Description: 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: 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: 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: THERMAL RESISTANCE OF SALMONELLA ENTERICA AND ESCHERICHIA COLI 0157:H7 IN PEANUT BUTTER
Creator: He, Yingshu
Date: 2014, 2014-05
Description: Salmonella enterica is a frequent food contaminant and the leading cause of foodborne bacterial illnesses in the United States. Our study...
Show moreSalmonella enterica is a frequent food contaminant and the leading cause of foodborne bacterial illnesses in the United States. Our study demonstrated that a 5-strain S. enterica cocktail displayed increased heat resistance in peanut butter of low water activity (aw). Significant differences (P < 0.05) were found between the survival rates of Salmonella enterica and Escherichia coli O157:H7 in peanut butter with different formulations and water activity. High carbohydrate content in peanut butter and low incubation temperature resulted in higher levels of bacterial survival during storage but lower levels of bacterial resistance to heat treatment. Furthermore, we also compared the relative heat resistance of three individual strains of S. enterica representing serotypes Typhimurium, Enteritidis and Tennessee and the 3-strain cocktail treated at both 90oC and 126oC in two different peanut butter formulations with varied fat and carbohydrate contents and adjusted water activities (aw from 0.2 to 0.8). When treated at 90oC, increased water activity in peanut butter significantly (P < 0.05) reduced the heat resistance of desiccation-stressed S. enterica cells. Differences in heat resistance were also detected among the three S. enterica serotypes and between the two peanut butter formulations. When treated at 126oC, the differences in bacterial heat resistance among serotypes and adjusted water activities were less notable (P > 0.05). Based on the Weibull model, an average of 52 to 132 min was required to achieve a 5-log reduction of the 3-strain cocktail at 90oC in peanut butter with an aw of 0.2. When aw was increased to 0.6, to achieve the same 5-log reduction required only 23-27 min. At aw of 0.8, S. enterica could be completely killed in less than 10 min in peanut butter with a fat content of 48.49%. Using scanning electron microscopy, we observed minor morphological changes xiii of S. enterica cells during desiccation and rehydration processes in peanut oil, which was used as a surrogate for peanut putter. Results from this study collectively suggest that water activity plays a critical role in determining S. enterica heat resistance in peanut butter. The variability that exists among the heat resistance of different S. enterica serotypes in different peanut butter formulations should also be taken into consideration for developing and validating effective intervention and mitigation strategies in peanut butter production.
PH.D in Biology, May 2014
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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: 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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Title: ROLE OF EXTRACELLULAR MATRIX IN CELLULAR BEHAVIOR AND TISSUE FUNCTION
Creator: Sridharan, Indumathi
Date: 2012-04-22, 2012-05
Description: Matrix-dictated control of stem cell differentiation and tissue status are of considerable interest to cell biologists and tissue engineers....
Show moreMatrix-dictated control of stem cell differentiation and tissue status are of considerable interest to cell biologists and tissue engineers. To create suitable biological scaffolds for tissue engineering and cell therapeutics, it is essential to understand the matrix mediated specification of cell lineage. Our study examines the role of matrix properties on cellular behavior and tissue mechanics. To this end, we studied the effect of collagen type I on stem cell differentiation and its mechanical properties within a live tissue. We altered the properties of collagen type 1 by incorporating CNT. The collagen-carbon nanotube (collagen-CNT) composite material was stiffer with thicker fibers and longer D-period. We find that the enhanced mechanical and structural properties of collagen-CNT allow for rapid and efficient derivation of neural progenitors from human decidua parietalis placental stem cells (hdpPSC). Both structure and stiffness of the matrix are important determinants of neural differentiation rate. Strikingly, the collagen-CNT matrix, unlike collagen, imposes the neural fate by an alternate mechanism that is independent of beta-1 integrin and beta-catenin. The study demonstrates the sensitivity of stem cells to subtle changes in the matrix and the utilization of a novel biocomposite material for efficient and directed differentiation of stem cells. Investigation of connective tissue disorders has led to the understanding of the important role played by collagen. So far, native collagen fibers within an intact tissue have not been examined. In this study, we employed a unique approach- histochemical staining guided high-resolution elasticity mapping- to study collagen and smooth muscle in fresh vaginal wall connective tissue. The comparative study of tissues collected from healthy pre-menopausal (pre-M) and post-menopausal (post-M) women suggest that during menopause, collagen’s structure and elasticity are subtly altered. The systematic analysis enables detection of minute changes in collagen in non-fatal conditions such as pelvic organ prolapse and other genitor-urinary disorders, where the initial symptoms are subtle and multivariate and where early diagnosis will allow non-invasive interventions and reduce incidence of surgical correction for these common disorders.
Ph.D. in Molecular Biochemistry and Biophysics, May 2012
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Title: DIRECTED EVOLUTION TO IMPROVE BIODESULFURIZATION OF PETROLEUM
Creator: Khayyat, Naghmeh Hassanzadeh
Date: 2013, 2013-12
Description: Dibenzothiophene (DBT) is an organosulfur compound found in petroleum that is refractory to the current hydrodesulfurization (HDS) method in...
Show moreDibenzothiophene (DBT) is an organosulfur compound found in petroleum that is refractory to the current hydrodesulfurization (HDS) method in refineries and raises the need for alternative methods such as biodesulfurization. Rhodococcus erythropolis strain IGTS8 naturally contains a dszABC operon, which encodes enzymes that can desulfurize DBT through the 4S pathway. Desulfurization-negative Rhodococcus opacus was transformed with plasmids pRESXdszABC and pRESXdszAS1BC, conferring the ability to desulfurize DBT. pRESXdszAS1BC had been modified from pRESXdszABC by placing a synthetic “sulpeptide gene” (S1) within the operon between dszA and dszB; S1 encodes a short polypeptide with high methionine and cysteine content, which puts additional sulfur demands on the 4S pathway when DBT is the only source of sulfur. Here we performed directed evolution on the two transformed R. opacus strains for 50 passages in a minimal (CDM) medium with DBT as the sole sulfur source in an attempt to drive evolution of greater DBT desulfurization activity. Desulfurization specific activity experiments were performed every 4 to 10 passages to compare the specific activities of these strains through the production of 2-HBP as measured by the Gibbs assay. Desulfurization positive strain Rhodococcus erythropolis IGTS8 was used as the control culture. R. opacus/pRESXdszABC demonstrated a maximum increase of 28-fold in desulfurization specific activity after 40 passages to a level as high as that of the control culture R. erythropolis IGTS8. Thereafter (passages 41-50), there was a 7% decrease from the maximum of desulfurization activity level (passage 40). R. opacus/pRESXdszAS1BC, however, showed only a maximum increase of 4-fold in specific activity after 37 passages. Moreover, there was a 61% decrease from the ix maximum of desulfurization activity level (passage 37) thereafter (passage 38-50). These could be due to the unexpected mutations and/or epigenetic changes in the pRESXdszAS1BC plasmid or host genomic sequences. Further DNA sequence analysis will be helpful in identification of these possible mutations.
M.S. in Biology, December 2013
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Title: Factors Affecting Sanitizer Efficacy on Preventing Cross-Contamination of E. Coli 0157:H7 During Postharvest Washing of Romaine Lettuce
Creator: Li, Yichen
Date: 2011-12-05, 2011-12
Description: Outbreaks of E. coli O157:H7 infections have been a continuing food safety challenge for the produce industry. Previous studies showed that...
Show moreOutbreaks of E. coli O157:H7 infections have been a continuing food safety challenge for the produce industry. Previous studies showed that contamination that originated in the farm can spread during postharvest processing. The objective of this study was to examine the spread of E. coli O157:H7 during postharvest washing of contaminated lettuce and to determine factors affecting the efficacy of sanitizer use in preventing cross-contamination. A bench-scale washing system was established to simulate industry operations. This system was equipped with a submersible pump and instruments to measure wash water properties including pH, temperature, chlorine level, oxidation reduction potential (ORP), turbidity and total organic carbon (TOC). Fresh-cut romaine lettuce (8 or 20 g) inoculated with approximately 8 log CFU/ml of E. coli O157:H7 with green fluorescence protein (GFP) were added into 40 L of tap water or industry water together with uninoculated lettuce (800 or 2000 g). The wash procedure lasted for 2 minutes. Washing operations were performed at two temperatures (3°C and 20°C) combined with different levels of chlorine treatments (0, 5, 20 and 30 ppm). Smaller-scale (50 – 100 mL) washing experiments were performed separately to determine the effects of organic contents and solid contents on the efficacy of sanitizer. Without chlorine treatment, the spread of E. coli O157:H7 occurred in both tap water and industry water at both 20°C and 3°C. With 20 ppm chlorine, no E. coli O157:H7 was detected in either wash water or uninoculated lettuce after washing in tap water. In industry water, chlorine level at 30 ppm or above could prevent crosscontamination of E. coli O157:H7. Neither the lettuce load nor the wash water temperature was proved to affect the efficacy of sanitizer. At 5 ppm of chlorine, increases in organic carbon (0% to 20%) led to the drop of free chlorine which resulted in a decrease in the microbial reduction from 2.51 to 0.01 log CFU/g. Increases in solid contents (0 g/L to 20 g/L) also caused a decrease in the microbial reduction from 2.52 to 1.17 log CFU/g but it did not change the free chlorine concentration. The utility of ORP as a measure of the antimicrobial efficiency of wash water was evaluated. ORP readings increased with increasing chlorine levels but reached a plateau and failed to correlate with the concentration of chlorine at chlorine levels > 20 ppm.
M.S. in Food Safety and Technology, December 2011
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Title: COMPLETE PLASTID GENOME OF THE HEMIPARASITIC PLANT PEDICULARIS REX
Creator: Yang, Jingyi
Date: 2015, 2015-07
Description: Pedicularis rex is a hemiparasitic plant from the Orobanchaceae family. Hemiparasites, also called semiparasites, are partly dependent on...
Show morePedicularis rex is a hemiparasitic plant from the Orobanchaceae family. Hemiparasites, also called semiparasites, are partly dependent on their host and, while they steal nutrients and other metabolites like other parasites, they have retained their photosynthesis ability. To better understand the hemiparasitic lifestyle of Pedicularis rex and what remains of its photosynthetic capability, we have determined the complete sequence of its plastid chromosome. The chloroplast genome of Pedicularis rex is 153,650 base pairs-long and exhibits a typical quadripartite structure with a large single copy (LSC) region and a small single copy (SSC) region separated by two inverted repeats (IR). A total of 79 unique protein-coding genes, including 9 pseudogenes, 30 tRNA- and 4 rRNA-encoding genes were retained in the plastid genome. Compared to the plastome of its close non-parasitic relative Lindenbergia philippensis, only one protein-coding gene and one intron are missing from P. rex but many genes show signs of pseudogenization. Pseudogenization in the P. rex plastid genome was found to be mainly caused by single site insertions, deletions or substitutions. The overall high level of homology between the P. rex and L. philippensis plastomes may explain why P. rex shows weak host dependence. However, how P. rex maintains its photosynthetic capability despite featuring a number of potentially dysfunctional pseudogenes involved in photosynthesis is unclear, and the minimal set of genes that is required for hemiparasitic plants to keep their autotrophic lifestyle is still unknown.
M.S. in Biology, July 2015
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Title: P21 IS A BIOMARKER OF CELL FATE AFTER UV IRRADIATION
Creator: Lu, Ziyan
Date: 2011-04-19, 2011-05
Description: UV irradiation can cause DNA damage, which leads to either cell cycle arrest or apoptosis. As a major transcriptional target of p53 protein in...
Show moreUV irradiation can cause DNA damage, which leads to either cell cycle arrest or apoptosis. As a major transcriptional target of p53 protein in response to DNA damage, p21 protein plays critical roles in both cell cycle arrest and apoptosis. However, the specific range of UV doses and functions of p21 protein leading to the determination of cell fate in human androgen-independent prostate cancer cells has not been completely elucidated. Here, we show that low doses of UV irradiation (< 40 J/m2) induced cell cycle arrest with up-regulation of p21 protein. However, high doses of UV irradiation (> 60 J/m2) can induce apoptotic cell death as indicated by caspase-3 activation, which is also consistent with apoptotic morphological changes. Interestingly, p21 protein is degraded at early time course of high-dose UV irradiation-induced apoptosis, pretreatment of proteasome inhibitor MG132 which can block p21 degradation but partially inhibits apoptotic cell death. Consistently, similar results were obtained in both 104-R1 cells and 104-IS cells. Taken together, the results suggest that there is a narrow window of UV doses range that serves as a “switch” point, in which cells make a decision: either cell cycle arrest or cell death. p21 protein serves as a good indicator for both cell cycle arrest and apoptotic cell death post-UV irradiation in human androgen-independent prostate cancer cells. Therefore, p21 may be a potential therapeutic target in prostate cancer.
M.S. in Biology, May 2011
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Title: CHARACTERIZATION OF A NOVEL TUMOR SUPPRESSOR BAX
Creator: Wang, Xin
Date: 2013-04-16, 2013-05
Description: Bax is a pro-death tumor suppressor. The loss of functional Bax expression can enhance the tumor growth. Recently we discovered a new family...
Show moreBax is a pro-death tumor suppressor. The loss of functional Bax expression can enhance the tumor growth. Recently we discovered a new family of functional Bax isoforms generated specifically in certain tumors; one of them is Bax 2 . In this thesis, I characterized the properties of Bax 2 protein by comparing with the parental Bax 2. Bax 2 was cloned into a GFP mammalian expression vector and transfected into the bax knockout cells. I examined Bax 2 expression, cellular distribution, and ability to induce cell death. The results show that the Bax 2 protein aggregated as granules around the nucleus in cytoplasm and induced more cell death than that from previously studied Bax 2. The results implicate that cancer patients with Bax 2 isoform might have better prognosis or response to treatment.
M.S. in Biology, May 2013
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Title: MOLECULAR AND GENETIC ENGINEERING STUDIES OF VITREOSCILLA HEMOGLOBIN
Creator: Chen, Yang
Date: 2014, 2014-05
Description: Biodesulfurization is a promising field that can be applied to the processing of crude oil for removing dibenzothiophene (DBT) and its...
Show moreBiodesulfurization is a promising field that can be applied to the processing of crude oil for removing dibenzothiophene (DBT) and its derivatives. A thermophilic bacterial strain Paenibacillus naphthalenovorans (32O-Y) was found to metabolize DBT as sole sulfur source. Another thermophilic strain Paenibacillus apiaries (32O-W) which cannot utilize DBT, however, was found to increase the desulfurization activity of 32O-Y in mixed 32O-Y+W culture in minimal-DBT medium at temperatures between 45 and 50 °C. In order to increase the desulfurization activity of these strains, we genetically engineered strain 32O-Y to express Vitreoscilla hemoglobin (VHb). VHb is the first hemoglobin found in bacteria and has been used to increase the growth and product yields various cells. The VHb gene (vgb) was successfully cloned into shuttle vector pNW33N. 32O-Y was successfully transformed with pNW33N-vgb while 32O-W was not. Compared to untransformed 32O-Y, 32O-Y[pNW33N-vgb] grew slower and reached a lower maximum OD600 when cultured in minimal-DBT medium at 45 °C. However, the Gibbs assay showed that VHb expression in 32O-Y increased its desulfurization activity by 18%. Thus, VHb can help 32O-Y metabolizing DBT while it might not promote growth. A series of high temperatures cultures need to be conducted to select an even more thermophilic strain which can fulfill the 60 °C requirement of industrial process. The VHb mutant (vgbM3) was also studied, which was previously found correlated with increased growth of E. coli DH5α, compared with the effect of wild type VHb. The effect that the mutant amino acids in VHbM3 was determined. Plasmid pUC- vgbM3 was obtained and successfully transformed to DH5α. After sequencing, two copies of vgbM3 were found in the plasmid. In order to study only one copy of vgbM3, x primers were designed for amplification. Fragment vgbM3 was successfully inserted into vector pUC18, followed by successful transformation into DH5α. Cell free extract was obtained from strains DH5α, DH5α[pUC8:16] and DH5α[pUC18-vgbM3] for CO-difference assay. As expected, DH5α expressed no hemoglobin. DH5α[pUC8:16] expressed wild type hemoglobin at 20 nmol/gm wet weight, while DH5α[pUC18-vgbM3] expressed mutant hemoglobin at 4 nmol/gm wet weight. Oxygen dissociation constant determination of VHbM3 will be the next step.
M.S. in Biology, May 2014
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Title: DIFFERENTIAL SENSITIVITY OF BAXΔ2 POSITIVE AND NEGATIVE CELLS TO ANTI-TUMOR AGENT BORTEZOMIB IN COLON CANCER
Creator: Chen, Wenjing
Date: 2015, 2015-05
Description: Tumor suppressor BaxΔ2 is a functional Bax isoform that is found in microsatellite unstable (MSI) colon cancer. However, BaxΔ2 proteins are...
Show moreTumor suppressor BaxΔ2 is a functional Bax isoform that is found in microsatellite unstable (MSI) colon cancer. However, BaxΔ2 proteins are not stable and are prone to be degraded by proteasomes in tumor cells. Bortezomib, a proteasome inhibitor, is an FDA approved anti-cancer drug mainly used for the treatment of myeloma and lymphoma. We tested if Bortezomib can block BaxΔ2 degradation and potentially be beneficial for the treatment of BaxΔ2 positive colon cancer. In this project, we compared the efficacy of Bortezomib-induced cell death in BaxΔ2-positive and BaxΔ2-negative colon cancer cells. We found that BaxΔ2-positive cells were highly sensitive to Bortezomib-induced cell death in comparison with that in BaxΔ2-negative cells. The half maximal inhibitory concentration (IC50) of cell viability for BaxΔ2-positive cells is 11.1 nM, while it is 453.8 nM for BaxΔ2-negative cells. The results indicate that Bortezomib has a selectivity towards BaxΔ2-expressive cells and could be a drug candidate for the treatment of BaxΔ2-positive colon cancer.
M.S. in Biology, May 2015
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Title: Thermal Resistance of Salmonella in Desiccation and Rehydration
Creator: Ahuja, Rameet
Date: 2011-12-06, 2011-12
Description: Transfer of salmonellae from a desiccated existence in dry food ingredients or processing environments to food products having higher water...
Show moreTransfer of salmonellae from a desiccated existence in dry food ingredients or processing environments to food products having higher water activities (e.g., peanut butter used in pie, chocolate in cake) leads to partial or full re-hydration of the bacteria. This study characterized the thermal behavior of Salmonella in response to desiccation and the subsequent rehydration. The thermal resistance of the desiccated S. enterica ser Tennessee was inversely correlated to aw: for example, desiccation at 11 to 97% equilibrated relative humidity (ERH) resulted in 0.5 to 3.3 log reduction, respectively, after 60ºC treatment for 10 min. Cells stored at lower ERH showed a lower survival rate, but higher thermal resistance. Once cells established their initial physiological response to desiccation, continual storage at 11% ERH up to three weeks did not further change the thermal resistance of Salmonella. Rehydration of the desiccated cells (11% ERH) to higher ERH conditions (84 to 97%) led to greater than 5 log reduction after heating cells at 60ºC for 10 min, in contrast, the same heat treatment resulted in approximate 3 log reduction for cells stored constantly at 84-97% ERH. There was no significant difference in regard to thermal sensitivity between cells rehydrated from 11% ERH to 33-55% ERH and that stored constantly at each ERH, only about 0.3-0.5 log CFU reduction in both cases. The study showed that rehydration moderately reduced cell viability, but greatly increased thermal sensitivity when a drastic aw shift occurred.
M.S. in Food Safety and Technology, December 2011
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Title: EFFECT OF SUCROSE AND HIGH PRESSURE PROCESSING ON FUNCTIONAL AND PHYCISOCHEMICAL PROPERTIES OF EGG WHITE PROTEINS
Creator: Liu, Hui
Date: 2013-04-25, 2013-05
Description: Egg white is widely used in food applications because of its nutritional benefits and versatile functionalities. Traditional treatment like...
Show moreEgg white is widely used in food applications because of its nutritional benefits and versatile functionalities. Traditional treatment like thermal pasteurization utilized for safety concern is disadvantageous to the egg white proteins due to the functionality impairment. High pressure processing (HPP) technology is known as a non-thermal food preserving method that can be operated at ambient temperatures. Sucrose is an essential ingredient that affects the functional behavior of egg white in numerous baking and pastry applications. The current study investigated the effect of sucrose and HPP on the functional and physicochemical properties of egg white proteins and verified the efficacy of HPP on microbial inactivation in egg white. Sucrose addition and HPP treatment were combined to study the foaming and physicochemical properties. Homogenized egg white samples were treated at selected HPP conditions (300-500 MPa) for 5-15 min, with an initial temperature of 4°C. Sucrose was added at 0-48% (w/v) to samples before or after HPP treatment. Foaming properties including foaming capacity and foam stability were measured immediately after whipping process. Turbidity and viscosity analyses were conducted within 24 hr after HPP treatment. The efficacy of HPP (500 MPa for 5 min) on microbial inactivation was verified using a pathogenic strain Salmonella enterica serotype Enteritidis (H7037) isolated from egg yolk implicated in a salmonellosis outbreak and a nonpathogenic Escherichia coli K12 strain (ATCC23716). All trials were completed in triplicate. Both addition of sucrose and HPP treatment decreased the foaming capacity but increased the foam stability of egg white proteins. Adding sucrose before HPP protected protein from turbidity and maintained foaming capacity, while adding sucrose after HPP greatly improved foam stability (p<0.05). Increasing HPP x holding time from 5 to 15 min did not affect the foaming properties significantly (p>0.05). A 5-log reduction of both strains was achieved after HPP treatment at 500 MPa for 5 min. These results exhibited that HPP can be used as a processing technology for the egg white safety control and that HPP could modify egg white foaming quality in food baking applications.
M.S. in Food Safety and Technology, May 2013
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Title: THE ELASTICITY AND PURIFICATION OF THE FLIGHT MUSCLE PROTEINS OF MENDUCA SEXTA
Creator: Dong, Sicong
Date: 2014, 2014-05
Description: Contraction of the DLM1 flight muscles of the Hawmoth, Manduca sexta are synchronous with the nervous impulse stimulation like mammalian...
Show moreContraction of the DLM1 flight muscles of the Hawmoth, Manduca sexta are synchronous with the nervous impulse stimulation like mammalian skeletal muscle. With cardiac-like behavior, DLM1 muscle of Manduca sexta can be a useful model to resemble mammalian cardiac muscle. Asynchronous flight muscles, like Lethocerus and Drosophila flight muscle, can only extend a few percent. But Manduca flight muscle has the ability to extend at least 100% in vitro and 9% in vivo. Very little is known about the protein composition and physiological behavior of Manduca flight muscle. Hence the length-tension behaviors of the DLM1 muscle of Manduca sexta are of considerable interest. In this experiment, we used PPi to remove thick filament in the sarcomere and focus on the passive tension generated by elastic proteins (projectin, kettin and sls proteins) in the sarcomere. The results showed elastic proteins (projectin, kettin and sls proteins)-based passive tension play a major role on muscle passive force in DLM1 muscles of Manduca sexta. We also tested a new way to prepare chemically skinned muscle samples where the dorsal and ventral muscle samples are pinned still in their shell on plates during the skinning step. In addition, preliminary experiments attempting to purify the DLM1 muscle proteins of Manduca sexta, fast protein liquid chromatography was used. Then 1% vertical SDS-agarose gel electrophoresis (VAGE) and 4-20% SDSPAGE gel are used to detect the samples after purification. We successfully purified 900kDa projectin and 700kDa kettin, which are major elastic proteins and useful for further research.
M.S. in Biology, May 2014
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Title: VITREOSCILLA HEMOGLOBIN: STRUCTURE-FUNCTION AND GENETIC ENGINEERING STUDIES
Creator: Li, Xiaodong
Date: 2014, 2014-05
Description: Paenibacillus strains 32O-W and 32O-Y were attempted to be transformed by electroporation with constructed plasmid pNW33N-vgb, modified to...
Show morePaenibacillus strains 32O-W and 32O-Y were attempted to be transformed by electroporation with constructed plasmid pNW33N-vgb, modified to contain the vgb gene which can be expressed as Vitreoscilla hemoglobin. Only attempts with 32O-Y were successful. Transformed 32O-Y/pNW33N-vgb was grown in CDM medium with dibenzothiophene (DBT) as the sole source of sulfur at different temperatures. Dramatic variability was observed in culture at different temperatures, so only the data at 45 °C was analyzed. The growth assay showed that the 32O-Y/pNW33N-vgb strain grew slower than untransformed 32O-Y, although Gibbs assay showed improvements in utilizing ability of DBT of 32O-Y/pNW33N-vgb compared to untransformed 32O-Y. This finding indicated that genetic engineering of introducing vgb into 32O-Y may cause deterioration in cell growth rate but improvement in desulfurization activities. Plasmid pUC-vgb-M2 was transformed into E. coli DH5α. The transformed DH5α/M2-vgb was cultured along with DH5α/pUC8:16, bearing plasmid pUC8:16 that was previously constructed in our lab and can be expressed to produce wild type VHb, and untransformed DH5α. CO-difference spectra were performed with the lysed cultures for the detection of VHb expression. As a result, DH5α/M2-vgb was confirmed to lack the ability to express functional VHb.
M.S. in Biology, May 2014
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