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Title: MOLECULAR MECHANISMS UNDERLYING SALMONELLA SURVIVAL ON SURFACE OF SELECTED NUTS AND FRUITS
Creator: Li, Ye
Date: 2017, 2017-07
Description: Salmonella enterica is the leading cause of bacterial foodborne illness in the United States. In recent years, S. enterica has been frequently...
Show moreSalmonella enterica is the leading cause of bacterial foodborne illness in the United States. In recent years, S. enterica has been frequently linked to foodborne outbreaks associated with nuts and fruits; however, the underlying mechanisms of such association have not been fully understood. In the first part of this study, we evaluated the impact of various environmental factors and food surface attributes on the attachment and survival of five S. enterica strains representing serotypes Typhimurium, Enteritidis, Montevideo, Mbandanka, and Braenderup on three different raw nuts (i.e. black peppers, almonds and hazelnuts) and two different S. enterica strains including serotypes Typhimurium and Enteritidis on two fresh fruits (i.e. grape tomatoes and cantaloupes) under storage conditions relevant to industrial practice. We observed significant inter-strain variations in S. enterica survival on nut and fruit surface. A direct correlation was found between the nut and fruit surface roughness and S. enterica attachment and survival. Lower relative humidity (20%) and higher storage temperature (25oC) resulted in significant S. enterica reduction on nut shells. Lower storage temperature at 4oC significantly reduced S. enterica population on grape tomatoes. In the second part of this study, we used a newly-developed transposon mutagenesis library in S. enterica serotype Enteritidis genome and highthroughput sequencing analysis to identify genes with potential roles in S. enterica attachment to and survival on almonds and grape tomatoes. A total of 336 and 210 S. enterica genes displayed significant selection on almonds and grape tomatoes over a 7-d storage period at 25oC (p<0.05), respectively. Our results suggest that various food attributes, environmental factors as well as bacterial determinants collectively contribute to the survival and persistence of S. enterica on nuts and fruits, providing new data for future development of knowledge-based intervention strategies.
Ph.D. in Biology, July 2017
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Title: STUDIES ON CONNECTIVE AND NEUROLOGICAL TISSUES IN RELATION TO DISEASE
Creator: Madhurapantula, Rama Sashank
Date: 2015, 2015-12
Description: The structure of connective tissue is of great importance for homeostasis of the cells present within it. Pathologies leading to changes in...
Show moreThe structure of connective tissue is of great importance for homeostasis of the cells present within it. Pathologies leading to changes in the structure of the extracellular matrix (ECM), in particular collagen have been shown to play a pivotal role in the progression of various diseases. Similarly, changes in the structure of specific elements in neurological tissues, such as myelin, have been shown to elicit adverse responses to injury. This thesis explores two main aspects: 1) the structural changes brought about by high sugar concentrations, much similar to that found in diabetic patients, to the structure of type I collagen and 2) possible effects of traumatic brain injury (TBI) to the structure of neurons in rat brains. Specific changes in the structure and packing of collagens in various tissues could be potential therapeutic targets to control the progression of related diseases. However, the information available on the nature, specificity and the relevance of these changes at a molecular level are largely unknown and have been explored only sparsely. The result of non-enzymatic glycosylation i.e. glycation, is the formation of sugar- mediated crosslinks within the native structure of type I collagen. The chemistry behind these crosslinks, also known as Advanced Glycation Endproducts (AGEs), has been known for decades. However, the exact locations or regions of high propensity for the formation of these crosslinks within the packing structure of collagen are largely unknown. The results presented in this thesis inform on the location of possible crosslinks using the principle of Multiple Isomorphic Replacement (MIR) to and correlate the effects of crosslinks to the structural and functional sites present on the D-periodic arrangement of collagen into fibrils. An extension to this is the study of the effects of povidone-iodine on the packing structure of collagen. Iodine is used as a common disinfectant in surgery and first aid. Prolonged treatment with iodine is detrimental to the structure of collagen underlying the wound site (surgical or otherwise). This is particularly important in large surface area wounds, as seen in open-heart, hip and joint replacement surgeries and amputations. Diabetic patients are more prone to injuries to limb extremities and a common procedure to stop infections from spreading to the rest of the body is amputation of the limb and constant treatment with low doses of iodine immediately following surgery for a certain length of time. The results presented in this thesis demonstrate specific disintegration of collagen fibrils in rat tail tendons, from a short iodine treatment. This is detrimental for cellular activity, more so in processes like wound healing. TBI results in the loss of neurological control and/or function of various parts of the body, governed by this region. The results presented herein, inform and support the finding that neuroplasticity, in the hemisphere opposite to that where injury was delivered, compensates for the functional deficits as a result of TBI. The data presented here can be used in developing rehabilitation regimens for TBI patients on case-to-case basis to restore most of the functional deficits observed thereof, and also as a factor of predicting the onset of secondary neurological disorders (for instance amyloid related pathologies) at a later stage in life.
Ph.D. in Biology, December 2015
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Title: STUDY OF THE STRUCTURE OF THE INDIRECT FLIGHT MUSCLE OF LETHOCERUS INDICUS BY LABELING WITH HEAVY ATOMS
Creator: Xie, Luping
Date: 2012-04-24, 2012-05
Description: Insect flight muscle (IFM) from Lethocerus indicus is an asynchronous muscle which can keep on oscillating after a neural stimulation, as long...
Show moreInsect flight muscle (IFM) from Lethocerus indicus is an asynchronous muscle which can keep on oscillating after a neural stimulation, as long as the load is mechanically-resonant. It has high degree of structural order as well. These characteristics make it an ideal material to study the structure of IFM in vitro. In this research, the structure of IFM from Lethocerus indicus was studied using X-ray diffraction. Multiple isomorphous replacement (MIR) using heavy atoms to alter the structure of biological macromolecules was used in an attempt to solve the well-known phase problem of crystallography. MIR is less commonly used in non-crystalline systems. Here we showed that, by labeling with two heavy atoms, potassium tetrachloroaurate (III) (KAuCl4) and p-Chloromercuribenzoic acid (PCMB), the diffraction patterns from IFM samples changed, in particular the intensities of reflections on the meridian. The positions and intensities of every layer line on the meridian before and after labeling were compared, and the best conditions for the two heavy atoms to use for labeling were discussed. These results indicate that this approach may be a feasible way of determining the electron density in this material with further development.
M.S. in Biology, May 2012
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Title: BIOPHYSICAL CHARACTERIZATION OF TYROSINE (B10) AND PROLINE (E8) MUTANT FORMS OF VITREOSCILLA HEMOGLOBIN
Creator: Pei, Yumeng
Date: 2016, 2016-05
Description: Vitreoscilla spp. is an aerobic, Gram-negative bacterium that produces a dimeric hemoglobin, designated as Vitreoscilla hemoglobin(VHb). The...
Show moreVitreoscilla spp. is an aerobic, Gram-negative bacterium that produces a dimeric hemoglobin, designated as Vitreoscilla hemoglobin(VHb). The distal site of VHb has a unique structure, which has becomes quite a hot spot for study. The first crystallographic structure of wild type VHb indicated that Tyr29 might play an important role in the ligand-binding properties. In an earlier study of a Tyr29Ala mutant, the oxygen-binding properties were shown to be unaffected relative to the wild-type, possibly because the space occupied by the aromatic ring of Tyr 29 in the wild-type is occupied by the aliphatic ring of Pro 54. However, there is no crystal structure that can validate this hypothesis. This project aims to determine the structural changes of VHb when both Tyr29 and Pro54 are mutated to Ala, and the crystal structure of VHb when Tyr29 is mutated to Ala. We added a hexahistidine tag on the C terminus of both the mutants in order to simplify the process of protein purification. The protein was obtained by precipitation and then purified over a HisPur Spin Column. For further purification, the protein was purified by ion exchange and hydrophobic interaction chromatography. The circular dichroism spectrum data of both mutants showed that the basic structure of VHb remains similar, and the thermal denaturation ability is also similar to that of wild-type VHb.
M.S. in Biology, May 2016
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Title: Structural Survey of Select Spectrin Type Repeats within the Rod Region of Dystrophin Using Hydrophobic Moment Analysis and Molecular Dynamics Supplementing Crystallographic Data
Creator: Reinfelds, Pauls Edvins
Date: 2012-07-18, 2012-07
Description: Dystrophin is a rod shaped protein consisting of amino- and carboxy-terminal binding domains linked by a large central rod composed of 24...
Show moreDystrophin is a rod shaped protein consisting of amino- and carboxy-terminal binding domains linked by a large central rod composed of 24 homologous copies of the STR motif and 5 non-homologous regions termed hinges. There are no high-resolution crystallographic structures available for the STR repeats. This work yielded protein crystals and data sets of two STR fragments (d5 & d16-17). Though unable to produce a high resolution structure, this work examines the physical properties of the rod region utilizing hydrophobic moment analysis and molecular dynamics to suggest flexibility among the helices of the STR fragments. Helical shifting appears to possibly occur among the more stable STR tandems to increase the cooperation among certain adjacent fragments. A full understanding of how these properties vary along the length of the rod has implications for the engineering of these rods regions in future dystrophin therapies.
Ph.D. in Biology, July 2012
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Title: REGULATION OF THE CELL DEATH SIGNALING PATHWAY IN ANDROGEN-INDEPENDENT PROSTATE CANCER CELLS
Creator: Lin, Yuting
Date: 2012-04-16, 2012-05
Description: Prostate cancer is the 2nd leading cause of cancer death in American men, mainly due to therapy-resistance in the advanced stage, androgen...
Show moreProstate cancer is the 2nd leading cause of cancer death in American men, mainly due to therapy-resistance in the advanced stage, androgen-independent prostate cancer (AIPCa). One major defect is that the cancer cells are insensitive to apoptosis induced by androgen ablation, chemotherapy or radiation therapy. However, the underline molecular mechanism still remains unclear. In this thesis, we focused on cell death signaling regulation in the development of AIPCa cells. We first show that up-regulation of Bcl-2, an anti-apoptotic oncogene, is required for the transition of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. Knockdown Bcl-2 impairs the transition process and blocks androgen-independent prostate tumor formation in vivo. Second, we show that Androgen-receptor (AR), which is generally considered as a survival factor in prostate cancer, promotes stress-induced apoptosis in AIPCa cells. AR promotes apoptosis through augmenting the mitochondrial translocation of Bax, a pro-death family member of Bcl-2. Finally, we show that AR can execute both pro-death and pro-survival events in same AIPCa cells. The AR pro-survival role is transcription-dependent, while its pro-death activity is transcription-independent. Interestingly, the AR exerts both functions through regulating p21 and JNK signaling pathways. These findings will help us to understand the dynamic survival signaling process in the development and progression of AIPCa. The key molecules identified here also provides potential therapeutic targets for the treatment of prostate cancer.
Ph.D. in Biology, May 2012
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Title: Development of Genetically Engineered Aromatase Overexpressing and Aromatase Inhibitior Resistant Breast Cancer Cell Lines to Study Possible Therapeutic Implications of a Novel Antiprogestin
Creator: Gupta, Akash
Date: 2012-05-07, 2012-05
Description: Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI....
Show moreAromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the antiprogestin CDB-4124 (Proellex) in aromatase overexpressing and Letrozole resistant human breast cancer cell lines. Mechanism of antiproliferative action of Proellex was also explored. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom cells for 75 weeks in the presence of 10μM Letrozole. Cell proliferation was determined by MTT or Crystal violet assays. Gene expressions were quantified by quantitative RTPCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student’s ‘t’ test or ANOVA followed by Bonferroni’s post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50%to 60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex or other AIs. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PRB / EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients. Mouse mammary organ culture (MMOC) has been classically employed for evaluating the efficacy of chemopreventive agents against development of carcinogeninduced preneoplastic lesions. Efficacy of chemopreventive agents observed in MMOC correlates well with that observed in in-vivo carcinogenesis models. In the present study, we developed a new ex-vivo Human in Mouse organ culture model which mimics in-vivo orthotopic breast cancer model. Since we introduced human breast cancer cells in mouse mammary gland and cultured in vitro, this model is termed as human Breast cancer (BCA) in Mouse Mammary Organ Culture (BCa-in-MMOC). Three to four week old female Balb/c mice were sensitized with estradiol (1mg) + progesterone (1mg) for 9 days. On the 10 th day animals were sacrificed and 2.5x10 4 T47D parental or T47D aromatase overexpressing cells were injected into the fourth pair of mammary glands. The glands were excised then cultured at 37C under 95% O2 / 5% CO2 in hMEM medium containing 10% charcoal stripped FBS supplemented with Testosterone (1nM) and progesterone (1mM) and growth promoting hormones (5 μg insulin, 5 μg prolactin per ml medium). At the end of the experiment, the glands were fixed in formalin. The paraffin embedded sections (longitudinal) of entire glands were processed for histopathological examination (H and E stain) and immnohistochemical staining of various proteins. Mammary glands were evaluated for the presence of T47D cells, their growth pattern and their molecular responsiveness to estradiol. T47D cells (both types) injected into mammary glands were easily identified against mouse cells by intense human specific CK18 immunofluorescence staining. Histopathological observation of mammary gland sections showed that growth pattern of injected cancer cells was identical to that observed of breast cancer cells injected in vivo in athymic mice. Interestingly, clusters of cancer cells in the mammary gland stroma appear similar to those observed in breast tumors in women. Cancer cells injected into glands survived and continued to grow (as evident from Ki-67 immunostaining) after 15 days in culture. Cancer cells maintained their original characteristics (ER+, PR+, EGFR+, and aromatase). T47D cells with enhanced aromatase expression growing in the MMOC could metabolize testosterone to estrogen, which resulted in enhanced cell proliferation and induction of estrogen target genes such as ER and PR-B. Mouse mammary glands with T47D aromatase overexpressing cells also showed changes typical to estrogenic milieu. In summary this model provides a novel, inexpensive ex vivo model, which could be used to study effects of therapeutic agents on the cancer cells growing in orthotopic micromilieu.
Ph.D. in Biology, May 2012
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Title: The Effect of 25-Hydroxyvitamn D3 on TIMP-2 Expression and Function in Colon Cancer
Creator: Roy, Sarbani
Date: 2011-12-19, 2011-12
Description: The efficacy of the vitamin D metabolite, 25-hydroxyvitamin D3 as a chemopreventive agent has already been implicated in colon cancer. In a...
Show moreThe efficacy of the vitamin D metabolite, 25-hydroxyvitamin D3 as a chemopreventive agent has already been implicated in colon cancer. In a microarray analysis data from our laboratory, 25D3 treatment upregulated TIMP-2 mRNA expression in human colon cancer cells. TIMP-2 is a multifunctional protein affecting cell migration, cell growth and differentiation, anti-angiogenesis, pro and anti – apoptosis, and synaptic plasticity of cells. In this study we characterized the TIMP-2 expression with the treatment of 25-Hydroxyvitamin D3, in colon cancer cells. We found that TIMP-2 was significantly upregulated at transcriptional, translational and post translational level when treated with 500nM 25D3, in several human colon cancer cell lines HT29, Caco2, SW620, but was not regulated in SW480. Concomitantly, the cell proliferation was significantly inhibited by different concentrations (250nM, 500nM and 100nM) of 25D3 treatment for 7days in Caco2 and HT29, but not in SW480. Similarly, cell invasion and cell migration were also significantly inhibited in HT29 cells, but not in SW480 when conditioned media generated from treatment of HT29 and SW480 cells with 500nM 25D3 for 72 , were used as chemoattractant. These results suggested that TIMP-2 present in the conditioned media from 25D3 treated HT29 cells inhibited the invasive and metastatic abilities of the human colon cancer cell line. This data was also supported in the in vivo mice experiment, in which the lung metastasis of mice colon adenocarcinoma cells, CT26 was inhibited by 25% (p=0.01), when pre-treated with 25D3 (500mg/kg). At the same time, the TIMP-2 levels were also higher in the 25D3 treated groups.
M.S. in Biology, December 2011
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Title: EFFECT OF METABOLIC INHIBITORS ON GROWTH AND BIOFILM DEVELOPMENT OF ENTEROBACTER AEROGENES AND KLEBSIELLA PNEUMONIAE
Creator: Sanjana, Krithica
Date: 2017, 2017-05
Description: Enterobacter aerogenes and Klebsiella pneumoniae are the leading cause of severe hospital-acquired infections across the globe, particularly...
Show moreEnterobacter aerogenes and Klebsiella pneumoniae are the leading cause of severe hospital-acquired infections across the globe, particularly in the US and Europe. These bacteria contribute heavily to the alarming threat of emergence of multiple drug resistant strains, enabling them to survive a wide spectrum of antibiotics. One of the key factors involved in drug resistance is the production of biofilms, a complex exopolysaccharide matrix that acts as a protective component and increase their resistance to external factors including antibiotics. In this study, we described that targeting the bacterial respiratory metabolism entirely disrupts pathways used for energy synthesis and substantially inhibits bacterial growth. Moreover, the metabolic inhibitors decreased the production of biofilms by these bacteria. Two key factors being the bacterial growth and biofilm development were analyzed in this research study. The data indicates that HQNO has the highest inhibition effect which targets essential enzyme complex Na+-NQR in bacterial growth as well as biofilm formation. Thus, the structure of HQNO can potentially be suited for new antibiotic development to combat the problem of multidrug resistant bacteria.
M.S. in Biology, May 2017
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Title: PRFA-LIKE TRANSCRIPTION FACTOR, LMO0753, CONTRIBUTES TO VIRULENCE, L-RHAMNOSE UTILIZATION, AND PERSISTENCE OF LISTERIA MONOCYTOGENES HUMAN FOODBORNE OUTBREAK LINEAGES
Creator: Salazar, Joelle Krieger
Date: 2013, 2013-12
Description: Listeria monocytogenes is a foodborne bacterial pathogen and the causative agent of a human and animal disease, listeriosis. Among the three...
Show moreListeria monocytogenes is a foodborne bacterial pathogen and the causative agent of a human and animal disease, listeriosis. Among the three major genetic lineages of L. monocytogenes (i.e. LI, LII, and LIII), LI and LII are predominantly associated with foodborne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor lmo0753 that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains including a DNA-binding domain with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, lmo0753 deletion and complementation mutants were constructed in two fully sequenced L. monocytogenes LII strains 10403S and EGDe, and compared virulence-associated mechanisms of flagellar motility, phospholipase C production, hemolysis, and intracellular growth of the mutants and their respective wild types. Persistence-associated mechanisms of growth, biofilm production, attachment and soil survival were also assayed. Results suggested that lmo0753 plays a role in some virulence- and persistence-associated mechanisms in both EGDe and 10403S. More importantly, it was found that deletion of lmo0753 led to the loss of L-rhamnose utilization in EGDe but not in 10403S. Transcriptomic comparison of the EGDe lmo0753 deletion mutant and the wild type incubated in Phenol-red medium containing L-rhamnose as the sole carbon source revealed 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome that were up- and down-regulated for more than 2-fold, respectively. Genes involved in biotin biosynthesis, general stress response and rhamnose metabolism were shown to be differentially regulated by Lmo0753. Findings from this study may partially explain why xvi LIII of L. monocytogenes is underrepresented in the environment and rarely associated with human listeriosis outbreaks due to the inability of rhamnose utilization.
PH.D in Biology, December 2013
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Title: BAX EXON 2 CONTAINS A CRITICAL ACCEPTOR SITE FOR ALTERNATIVE SPLICING OF THE BAXΔ2 ISOFORM BY SAMUEL KISSINGER Submitted in partial fulfillment of the requirements for
Creator: Kissinger, Samuel
Date: 2013-04-17, 2013-05
Description: Previous studies have revealed that BAXΔ2, an alternatively spliced form of BAX with a microsatellite mutation (G8 to G7) on BAX exon 3, is a...
Show morePrevious studies have revealed that BAXΔ2, an alternatively spliced form of BAX with a microsatellite mutation (G8 to G7) on BAX exon 3, is a more potent inducer of cell death and a potential means of treatment for cancers with microsatellite instability (MSI). To define the critical splicing points for the splicing of the BAXΔ2 isoform, BAX mini-gene constructs with GFP reporter genes were used for site directed mutagenesis based disruption of predicted critical splicing points. The mutant constructs were transfected into mammalian cells, and expression of BAX-GFP fusion proteins were examined by both fluorescence microscopy and Western blot analysis. Our results indicate that the BAX exon 2 A667T mutation could completely abolish generation of the BAXΔ2 isoform. These results suggest that the acceptor site AG in BAX exon 2 position 667 is critical for alternative splicing to the BAXΔ2 isoform in microsatellite unstable BAX G7 tumor cells.
M.S. in Biology, May 2013
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Title: ANALYSIS OF MITOCHONDRIAL RE-LOCALIZATION IN CHLAMYDIA TRACHOMATIS INFECTION
Creator: Patel, Dhwani B.
Date: 2017, 2017-05
Description: Chlamydia trachomatis is an obligate intracellular parasite that causes eye and genital tract infection in humans. C. trachomatis genital...
Show moreChlamydia trachomatis is an obligate intracellular parasite that causes eye and genital tract infection in humans. C. trachomatis genital tract infection is one of the most prevalent sexually transmitted diseases in United States. This pathogenic bacterium depends on the host cell for essential metabolites for energy generation and ATP as readily available energy source. Several previous studies have established interactions between the chlamydial inclusion and various host cell organelles, including the Golgi complex, nutrient rich exocytic vesicles and the cytoskeleton for survival in the host cell. However, the data available for chlamydial interactions with mitochondria is limited. In order to gain proper understanding of the host- pathogen relationship, the chlamydial inclusion interaction with mitochondria were evaluated, using HeLa cells as host. Immunostaining technique was used to stain uninfected and C. trachomatis (serovar L2b) infected HeLa cells at 7 different time points post infection- 8, 10, 12, 16, 18, 24, 36 hour post infection (hpi) and analyzed by fluorescence microscopy. The data showed that the chlamydial infection disrupts the well-organized mitochondrial network found in uninfected HeLa cells. C. trachomatis infected HeLa cells showed re-localization of cellular mitochondria toward the bacterial inclusion. The mitochondria were observed to begin re-localization at 10 hpi with gradual increase in per cent re-localization with subsequent time points. Highest per cent mitochondrial re-localization was observed at 36 hpi (81.1%), displaying all most all mitochondria accumulated around Chlamydial inclusion. The results clearly indicated the close association between host mitochondria and Chlamydial inclusion. Moreover, re-localization of mitochondria in close proximity of inclusion also indicated potential acquisition of essential metabolites and energy in form of ATP by bacterial inclusion from host mitochondria.
M.S. in Biology, May 2017
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Title: CHARACTERIZATION OF CELL SURFACE PROPERTIES, CHLORINE RESISTANCE AND SURVIVAL OF ESCHERICHIA COLI O157:H7 STRAINS
Creator: Depa, Keshava Reddy
Date: 2011-11-28, 2011-12
Description: Escherichia coli O157:H7 outbreaks linked to tainted fresh produce have increased during recent years. The aim of this study is to...
Show moreEscherichia coli O157:H7 outbreaks linked to tainted fresh produce have increased during recent years. The aim of this study is to characterize the cell surface properties, chlorine resistance and attachment to leafy greens of several E. coli O157:H7 strains belonging to different phylogenetic clades, in order to reveal if there is any correlation between their survival and bacterial cell surface properties. Six strains chosen among the nine E. coli O157:H7 clades included the fresh produce outbreak associated strains Sakai (1996 sprout outbreak in Japan) and TW14359 (2006 spinach outbreak in the US). The growth kinetics of these strains was compared in BHI broth containing 0.81 ppm free chlorine. Cell surface hydrophobicity, auto-aggregation and curli production were determined by in vitro assays and compared to the generic E. coli strain BW25113. The results showed that these E. coli O157:H7 strains exhibited different chlorine resistances regardless of their phylogenetic distance. They were approximately 20% more hydrophilic (p=0.0072) and showed 77% less auto-aggregated cells (p=0.049) after overnight incubation in broth than the generic E. coli strain. However, no distinguishable hydrophobic or aggregation properties were observed among the six E. coli O157:H7 strains despite their different resistances toward chlorine treatment. Curli production measured by a Congo-Red binding assay in suspension varied among the strains and exhibited no correlation with chlorine resistance. In preliminary studies of chlorine exposure (0.81 ppm) of cells being attached to spinach leaf surface, the spinach outbreak strain TW14359 showed less inactivation (2.6-fold) than the sprout outbreak strain Sakai (15-fold). The information generated from this study will help to develop more effective interventions used in the produce industry.
M.S. in Food Safety and Technology, December 2011
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Title: DEVELOPMENT OF AN INDUCIBLE SYSTEM FOR EXPRESSION OF PROTEINS IN DIFFUSE LARGE B CELL LYMPHOMA
Creator: Sakthivel, Dharaniya
Date: 2015, 2015-05
Description: Diffuse large B cell lymphoma (DLBCL) is a heterogeneous malignancy distinguished by aggressive clinical symptoms and abnormal B cell receptor...
Show moreDiffuse large B cell lymphoma (DLBCL) is a heterogeneous malignancy distinguished by aggressive clinical symptoms and abnormal B cell receptor signaling. Current treatments are shown to have refractory or relapsed outcome post initial treatment signifying the need for novel chemotherapeutic targets. ERM family proteins have been shown to modify B cell immune response to antigen. Previous studies in our lab reveal that ERM proteins are phosphorylated in both DLBCL tumors and cell lines. Alteration of ERM function with Ezrin dominant negative mutant (EZ-DN) decreased the growth of DLBCL cells. This reduction of cell growth is thought to be mediated by interfering with the NF-kB and PI3K signaling. To understand the molecular mechanism of EZ-DN in a time dependent manner without impairing the cell growth immediately, Tet-on 3G based inducible expression system was utilized. Molecular cloning of the EZ-DN into the inducible system was performed, sequencing confirmed the insert of EZ-DN into the response vector (pTRE3G-BI-ZsGreen1) at desired locations. We have further evaluated the effect of doxycycline on the expression of fluorescent protein (Zsgreen1) and EZ-DN, also optimized its concentration for maximal expression of the proteins in DLBCL cell lines. Higher dose of doxycycline (above 5 μg/ml) induced reduction in cell viability. The expression of EZ-DN and Zsgreen1 is considered to be much stronger in the double stable cell lines containing the co-transfected regulator vector (pEF1α- TET3G) and response vector (pTRE3G-BI-ZsGreen1) with a selection marker. In conclusion, our data demonstrated the successful creation of inducible system that expresses EZ-DN, which can be employed to validate Ezrin as a therapeutic target in DLBCL.
M.S. in Biology, May 2015
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Title: DISTRIBUTION OF CRP/FNR FAMILY TRANSCRIPTION FACTORS IN SELECT BACTERIAL GENOMES
Creator: Yang, Shengning
Date: 2012-05-07, 2012-05
Description: Cyclic adenosine monophosphate (cAMP, cyclic AMP or 3'-5'-cyclic adenosine monophosphate) receptor protein (abbreviated as CRP), also known as...
Show moreCyclic adenosine monophosphate (cAMP, cyclic AMP or 3'-5'-cyclic adenosine monophosphate) receptor protein (abbreviated as CRP), also known as catabolite gene activator protein, is one type of regulatory protein in bacteria. It acts as a secondary messenger in many intracellular pathways, especially the signal transduction by binding cAMP, which allows the protein to bind DNA sequence in the promoters. Fumarate/nitrate reduction transcriptional regulator, in short Fnr, is another signal transduction molecule which transfers from oxygen status to nifL (Nitrogen fixing bacteria L) protein. Both Crp and Fnr can be grouped into a Crp-Fnr family of transcription factors in many bacteria strains. In this study, we identified Crp-Fnr family genes from different bacteria genomes. Using the Crp-Fnr gene sequences we reconstructed phylogenetic trees for 250 select bacterial genomes which contains at least one Crp-Fnr family gene. We also selected representative Escherichia coli and Salmonella enteriaca genomes for phylogenetic analysis. The circular maps for the four select bacterial genus, Listeria, Salmonella, Escherichia and Brucella were created to demonstrate the genomic locations of Crp-Fnr family genes. Our results showed that the location of Crp-Fnr family transcription factor genes are syntenic amongdifferent bacteria bacterial genomes, indicating their early ancestry acquisition prior to the speciation events.
M.S. in Biology, May 2012
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Title: Cannabidiol Inhibits Breast Cancer Growth by Generation of Reactive Oxygen Species
Creator: Sarma, Pranamee
Date: 2012-10-31, 2012-12
Description: Cancer cells possess heightened rates of cell proliferation due to aberrant cellcycle checkpoints. Cannabidiol (CBD) has been shown to down...
Show moreCancer cells possess heightened rates of cell proliferation due to aberrant cellcycle checkpoints. Cannabidiol (CBD) has been shown to down-regulate expression of the inhibitor of DNA-1 (Id-1), a basic helix-loop-helix transcription regulator that controls breast cancer metastasis. CBD is a cannabinoid that does not act efficiently with the known cannabinoid (CB1 and CB2 ) receptors. We studied the link between the ability of CBD to stimulate reactive oxygen species (ROS) production and its ability to inhibit cell viability. The CB1/CB2 receptor partial agonist Δ⁹- Tetrahydrocannabinol could not produce significant amounts of ROS except at a very high concentration demonstrating the unique ability of CBD to generate ROS is not due to CB1 and CB2 activation. Using multiple breast cancer cell lines, we determined the effects of CBD on ROS production were a concentration-dependent and occurred across a majority of breast cancer cell lines evaluated (human and mouse). We also analyzed whether the CB2-receptor selective antagonist SR144528 (SR2) and an ROS scavenger, α-tocopherol (TOC or vitamin E) could reverse the effects of CBD. The CB2 receptor antagonist could only partially reverse the inhibition of cell viability and stimulation of ROS production produced by CBD. TOC however could completely reverse the effects of CBD. To determine whether CBD stimulation of ROS was an effect specifically produced in breast cancer cell lines, we studied this effect in non-transformed human fibroblast cells. CBD produced negligible amount of ROS in non-transformed cells and xii was significantly less effective at inhibiting the viability of the fibroblast, suggesting that CBD must be acting through mechanisms unique to transformed cells. Here, we report that CBD inhibits breast cancer cell viability through efficient stimulation of ROS which is possibly mediated through unique interaction with the cannabinoid system, other than the mechanism of CB1 and CB2 receptor activation.
M.S. in Biology, December 2012
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Title: ENHANCEMENT OF DESULFURIZATION BY DIRECTED EVOLUTION UTILIZING THE SULPEPTIDE AND CHLORAMPHENICOL RESISTANCE GENES
Creator: Yu, Qingfei
Date: 2013, 2013-12
Description: With the increasing demand of upgrading the quality of petroleum, optimizing the efficiency of biodesulfurization (BDS) is necessary. R....
Show moreWith the increasing demand of upgrading the quality of petroleum, optimizing the efficiency of biodesulfurization (BDS) is necessary. R. erythropolis 5F [pRESX dszABC] encodes the enzymes of the 4S pathway (on the dszABC operon) which catalyze the conversion of dibenzothiophene (DBT) to hydroxybiphenyl (2-HBP) plus sulfite. Our research attempts to increase the nutritional requirement of the cell for sulfur and to use selective pressure to obtain improved desulfurization. The genetic engineering strategy involves inserting the sulpeptide 1 (S1) gene, which encodes a sulfur-rich polypeptide, between dszA and dszBC in the dszABC operon to increase sulfur demand and force the strain into a spiral of ever increasing dszABC expression and thus desulfurization ability. Insertion of the chloramphenicol resistance (Cm) gene between dszAS1 and dszBC provides an additional method to select strains with higher desulfurization ability by their increasing resistance to chloramphenicol. We completed directed evolution for 22 passages for all three strains (those containing pRESX dszABC, pRESX dszAS1BC, or pRESX dszAS1CmBC transformed into strain 5F) and measured the desulfurization activity at selected passages. The results show that directed evolution did improve the desulfurization abilities in the first ten passages in the strains expressing dszABC and dszAS1CmBC, but reduced the desulfurization ability in the strain expressing dszAS1BC. For all three strains and all passages tested, the highest desulfurization activity was 83.6 μmol DBT/g DCW/hour in the dszABC bearing strain. Thus, the presence of S1 had a negative effect on the rate of desulfurization. A possible explanation for these results is that mutations occurring during the selection process enhance the efficiency of sulfur utilization and S1 alone can thus not force the strains to improve desulfurization.
M.S. in Biology, December 2013
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Title: EFFECTIVENESS OF CLEANlNG REGIMENS FOR REMOVlNG MILK RESIDUE FROM A PILOT -SCALE HTST PROCESSING LINE
Creator: Du, Qian
Date: 2011-11-30, 2011-12
Description: Undeclared allergens can be introduced into foods due to cross-contact during manufacture. Effective cleaning is essential for preventing...
Show moreUndeclared allergens can be introduced into foods due to cross-contact during manufacture. Effective cleaning is essential for preventing allergen cross-contact on shared processing lines. The objectives of this project were to evaluate the effectiveness of cleaning treatments on removing milk residue from a pilot-scale HTST processing line, and measure the levels of milk transferred into juice processed on an inadequately cleaned processing line. Nonfat milk was processed (81ºC,17 sec) on an HTST processing line followed by several cleaning regimens including 1) a 15 min water flush, 2) a 15 min water flush + a 60 min wash using full-strength chlorinated alkaline detergent (CAD) at 81ºC and a flow rate of 55-60 gal/h , 3) a 15 min water flush + a 60 min wash with ¼-strength CAD at 81ºC and flow rate of 55-60 gal/h, 4) a 15 min water flush + a 15 min full-strength CAD at 81ºC and flow rate of 55-60 gal/h, 5) a 15 min water flush + a 60 min full-strength CAD at 70ºC and flow rate of 55-60 gal/h, 6) a 15 min water flush + a 60 min full-strength CAD at 81ºC and flow rate of 27.7 gal/h, 7) a 15 min water flush + a 60 min full-strength CAD containing 1% milk at 81ºC and flow rate of 55-60 gal/h, 8) a a full cleaning cycle (15 min water flush + 60 min full-strength CAD at 81 ºC + 15 min water flush + 30 min acid detergent at 70 ºC + 15 min water flush + 15 min flush with 200 ppm sodium hypochlorite sanitizer at room temperature, flow rate of 55-60 gal/h). After each cleaning treatment, simulated apple juice was processed on the same processing line, collected and tested for presence of milk using a quantitative ELISA. The adequacy of the cleaning treatments was also assessed by determining the absence/presence of milk residue in sampling ports located in the processing line with ATP swabs, protein swabs and a milk-specific lateral flow kit. All clean treatments and analyses were done in triplicate. Milk was detected at levels of 59-150 μg/mL (ppm) in simulated apple juice processed on the HTST line after a 15 min water flush. No milk was detected in juice processed on the line cleaned with full-strength CAD or a full cleaning cycle. Lower milk levels in apple juice were detected with some of the intermediate cleaning regimens (¼-strength CAD, shorter cleaning time, reduced temperature of CAD, lower flow rate 27.7 gal/h of CAD cleaning and CAD with 1% milk). Swabs of sampling ports revealed the presence of milk/protein residue and high ATP levels after the water flush. Cleaning treatments using full-strength CAD reduced ATP levels and resulted in the no detectable of protein/milk residue in most sampling ports and in simulated apple juice. Reuse of CAD containing high levels of milk may result in milk cross-contact into juice subsequently processed on the milk processing line. This work illustrates the importance of validated cleaning procedures to prevent allergen cross-contact on shared processing lines.
M.S. in Food Safety and Technology, December 2011
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Title: APPLICATION OF BIMOLECULAR FLUORESCENCE COMPLEMENTATION ASSAY FOR MONITORING BCL-2 AND BAX INTERACTION
Creator: Chayanam, Sudharani
Date: 2011-07, 2011-07
Description: Bimolecular Fluorescence Complementation is a modern method to monitor protein-protein interactions in live cells. The objective of this...
Show moreBimolecular Fluorescence Complementation is a modern method to monitor protein-protein interactions in live cells. The objective of this project is to employ this method for deciphering signaling complexes involved in androgen receptor mediated cell death. Bcl-2 and Bax were selected to serve as initial experimental targets since these proteins were known to interact with each other. In this method, split EGFP consisting of 1-157 amino acid N-terminal fragment was fused to Bcl-2 and 158-238 amino acid Cterminal fragment to that of Bax with a six amino acid linker (SGSGVD) to generate fusion expression cassettes. We tested two combinations of fusion expression cassette pairs NGFP/Bcl-2, Bax/CGFP and NGFP/Bcl-2, CGFP/Bax for a BiFC signal by cotransfecting them in Human Embryonic Kidney 293T cells. Our results show that fusion proteins NGFP/Bcl-2, Bax/CGFP and CGFP/Bax were well expressed as evident by the Western blots. But the efficiency of BiFC formation was better for NGFP/Bcl-2 and CGFP/Bax fusion proteins combination than NGFP/Bcl-2 and Bax/CGFP fusion proteins. Therefore indicate that the fluorescence complex formation imparting the BiFC signal is specific for Bcl-2 and Bax interaction. Though the BiFC signal obtained was brighter for image capturing but the efficiency was very low, only few fluorescing cells were detected under the fluorescence microscope. Despite this weakness, BiFC is still a promising technique for studying protein-protein interactions in living cells and at single cell level.
M.S. in Biology, July 2011
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Title: CHARACTERIZATION OF COAGULATION AND MUSCLE ATTACHMENT MUTATIONS IN DROSOPHILA MELANOGASTER LARVAE AND THE GENERATION OF A NOVEL TRANSGLUTAMINASE LOSS OF FUNCTION MUTANT LINE
Creator: Schubert, Nina H
Date: 2014, 2014-05
Description: It is known that the protein Fondue (Fon) is involved in clotting in Drosophila melanogaster. Consequently, Fon was being studied to...
Show moreIt is known that the protein Fondue (Fon) is involved in clotting in Drosophila melanogaster. Consequently, Fon was being studied to characterize its role in the clot. During this study, it was found that Fon and the protein Tiggerin have the same expression pattern. Additionally, mutants for both proteins develop into long, thin pupae (as compared to wild type). Due to this similarity in mutant phenotype and protein localization in combination with literature citing Tiggerin to be involved in both the clot and muscle attachment, Fon was looked at for a muscle attachment phenotype. Furthermore, whilst we do know that Tiggerin is involved in the clot as well as muscle attachment, its specific role in coagulation and its muscle attachment phenotype have not been previously characterized. Due to the pleotropic role of Fon and Tiggerin in muscle attachment and clotting, the long, thin pupal phenotype was used as an indicator of potential clotting mutants in a search at the Bloomington stock center. There, 722 lines were found to have long, thin pupae and from these the 9 lines with the strongest mutant phenotypes were selected to test for coagulation and/or muscle attachment abnormalities. Our collaborator took confocal micrographs of third instar larval fillets obtained from these lines as a means of visualizing actin structure within the muscles. From these images, it was found that the Cchl mutant line had the most severely detached muscles and, consequently, this line was selected as the best candidate for a clotting and muscle attachment mutation and was subjected to further study. The role of a third protein, transglutaminase (TG), which is also involved in clotting, has not been fully characterized due to the lack of a null line. To address this deficiency and fully characterize the role of TG in coagulation in Drosophila, a line with an inactivated TG gene was used as a starting line to generate a null line.
M.S. in Biology, May 2014
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