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Title: BIOLOGICAL STRATEGIES FOR ENHANCEMENT OF LIQUID FUELS: SULFUR REMOVAL FROM PETROLEUM AND BIOETHANOL PRODUCTION
Creator: Wang, Jia
Date: 2013, 2013-12
Description: Rhodococcus baikonurensis CW25 was transformed with the Rhodococcus erythropolis strain IGTS8 desulfurization operon (dszABC, which encodes...
Show moreRhodococcus baikonurensis CW25 was transformed with the Rhodococcus erythropolis strain IGTS8 desulfurization operon (dszABC, which encodes the enzymes of the “4S” desulfurization pathway) or this operon modified to contain a synthetic cysteine-methionine rich “sulpeptide” gene (S1) (dszAS1BC). The two CW25 derivatives were subjected to directed evolution to select faster growing cells using the key 4S pathway substrate dibenzothiophene (DBT) as the sole source of sulfur. Data of cell doubling times verified the success of selection of cultures with increasingly rapid growth. The desulfurization activities of resting cells of early passages demonstrated improvements, and the highest activity of the dszAS1BC-bearing CW25 derivative was 115% higher than that of the CW25 derivative without S1. In addition, a trend of initial high activity was followed by a decrease in subsequent passages. Rates of DBT metabolism of growing cells demonstrated a different trend, probably because the activity of growing cells concurrently reflects the activity of DszABC enzymes and the growth rates of the recombinants. Dry cell weights fluctuated during the evolution process, probably because of variations in the efficiency of the conversion of the sulfur in DBT into sulfite, then into sulfate or biomass, or, for the S1-bearing cells, because the secretion of the S1 peptide from cells might have variable efficiency. A mixed culture of two Paenibacillus species (“W” and “Y”) was isolated that can metabolize DBT at temperatures up to 54 ºC. Strain Y is the only one of the two with desulfurization activity, while strain W enhances the desulfurization ability of Y. The W-Y culture may be a useful starting point for selection of desulfurization cultures with even greater thermal stability. xiii Ethanologenic Escherichia coli strain FBR5 was compared with Vitreoscilla hemoglobin (VHb)-expressing FBR5 (TS3) regarding the concentrations of ATP, NAD+, NADH, NAD+/NADH ratio; and growth and ethanol production at various points during growth. The significant finding was that the NAD+/NADH ratio for TS3 was lower in early growth, but higher in later growth compared to that for FBR5. This is probably because more NADH was required by TS3 for its enhanced ethanol production and VHb-related increased respiration under microaeration conditions.
PH.D in Biology, December 2013
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Title: PROBING THE PAN-GENOME OF LISTERIA MONOCYTOGENES
Creator: Deng, Xiangyu
Date: 2011-04-26, 2011-05
Description: Bacterial pathogens often show significant intra-species variations in ecological fitness, host preference and pathogenic potential to cause...
Show moreBacterial pathogens often show significant intra-species variations in ecological fitness, host preference and pathogenic potential to cause infectious disease. The species of Listeria monocytogenes, a facultative intracellular pathogen and the causative agent of human listeriosis, consists of at least three distinct genetic lineages. Two of these lineages predominantly cause human sporadic and epidemic infections, whereas the third lineage has never been implicated in human disease outbreaks despite its overall conservation of many known virulence factors. The genomes of 26 L. monocytogenes strains representing the three lineages are compared based on both in silico comparative genomic analysis and high-density, pan-genomic DNA microarray hybridizations. We uncover 86 genes and 8 small regulatory RNAs that likely make L. monocytogenes lineages differ in carbohydrate utilization and stress resistance during their residence in natural habitats and passage through the host gastrointestinal tract. We also identify 2,330 to 2,456 core genes that define this species along with an open pan-genome pool that contains more than 4,052 genes. Phylogenomic reconstructions based on 3,560 homologous groups allowed robust estimation of phylogenetic relatedness among L. monocytogenes strains. The pan-genome approach enables accurate co-analysis of DNA sequence and hybridization array data for both core gene estimation and phylogenomic reconstruction. Application of our method to the pan-genome of L. monocytogenes sheds new insights into the intra-species genomic diversification, niche expansion and evolution of this important foodborne pathogen.
Ph.D. in Biology, May 2011
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Title: BIOPHYSICAL AND BIOCHEMICAL STUDY OF NATIVE AND EDITED DYSTROPHIN ROD REGION
Creator: Mangat, Khushdeep
Date: 2014, 2014-12
Description: Duchenne Muscular Dystrophy (DMD) is a severe X-linked recessive disease affecting 1 in 3500 boys that is characterized by the degeneration of...
Show moreDuchenne Muscular Dystrophy (DMD) is a severe X-linked recessive disease affecting 1 in 3500 boys that is characterized by the degeneration of muscle function and strength. The cause of this disease lies in gene defects that eliminate expression of the protein dystrophin. Becker Muscular Dystrophy, BMD is a milder form of disease that has a later onset and much longer survival (up to the 7th decade of life, compared to median survival of 25 years for DMD patients) because of the presence of low levels of modified dystrophin protein. BMD is very heterogeneous, however, and many cases are nearly as severe as DMD. A major therapy for DMD involves exon skipping, which produces modified forms of dystrophin that are very similar to BMD. However, how these edits impact the function of dystrophin, and how they are linked to the severity of BMD or the BMD-like state produced in DMD exon skip therapy is unknown. We investigated this in two specific cases involving a specific panel of BMD defects linked to a major cause of death, dilated cardiomyopathy (DCM). We also investigated the contribution of various exons to interaction with a signaling partner of dystrophin, neuronal nitric oxide synthetase (nNOS).
Ph.D. in Biological and Chemical Sciences, December 2014
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Title: FUNCTIONAL ANALYSIS OF UBIQUITIN-LIKE PROTEIN 4A
Creator: Zhao, Yu
Date: 2015, 2015-12
Description: Ubiquitin-like protein 4A (Ubl4A) was identified as a housekeeping gene at X chromosome. It involves in the guided entry of tail-anchored (GET...
Show moreUbiquitin-like protein 4A (Ubl4A) was identified as a housekeeping gene at X chromosome. It involves in the guided entry of tail-anchored (GET) protein pathway in which tail-anchored (TA) proteins are transported to endoplasmic reticulum (ER). However, Ubl4A also involves other functions not related to GET pathway, such as tumor suppression and DNA damage-mediated apoptosis. Up to date, the function of Ubl4A in mammals is still largely unknown. We found that either overexpression or knockdown of Ubl4A promoted cell death in cell culture system. Using an in vivo genetic knockout system, we found that Ubl4A knockout mice displayed a high neonatal mortality and had a defect in glycogen synthesis, which is mainly controlled by a key protein kinase Akt. Loss of Ubl4A resulted in the impairment of insulin-induced Akt translocation to the plasma membrane, an essential step for Akt activation. We demonstrated that Ubl4A directly interacted with actin-related protein 2/3 (Arp2/3) complex, further accelerated Arp2/3 complex-dependent actin branching, thereby bringing Akt to proximity into the plasma membrane for activation. Furthermore, we showed that Ubl4A-mediated actin branching also played important roles in other cellular activities, such as formations oflamellipodia and filopodia, macrophage phagocytosis, wound healing, and neutrophil chemotaxis. These findings provide us a new insight into understanding the roles of Ubl4A in cellular function and a molecular basis for treatment of related human diseases.
Ph.D. in Biology, December 2015
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Title: STRUCTURE AND FUNCTION OF NATIVE AND EDITED DYSTROPHIN RODS
Creator: Sahni, Neha
Date: 2011-05-10, 2011-05
Description: The purpose of this study is to examine the biophysical properties of the rod region of the dystrophin protein. This is important due to the...
Show moreThe purpose of this study is to examine the biophysical properties of the rod region of the dystrophin protein. This is important due to the severity of the disease Duchenne Muscular Dystrophy, (DMD), which is associated with the malfunction of this protein. DMD is one of the most serious single gene genetic defects of man. This rod region consists of a number of repeat motifs called spectrin type repeats or STRs. The thermodynamical and biochemical stability analysis shows, which single motifs are unstable on their own and which ones become more stable when linked to their appropriate tandem neighbors. This knowledge will impact strategies to produce modified mini dystrophins for gene therapy. Exon skipping therapy is an emerging approach to treat such genetic diseases. This is done by the administration of modified antisense oligonucleotides, AONs, which can interfere with exon splicing process and eliminate certain exons from the mature transcript. Furthermore, the rod region has a number of ancillary functions, such as providing secondary binding sites for actin, neuronal NO synthetase and phospholipids, which may be adversely perturbed by the edits.
Ph.D. in Biological, Chemical, and Physical Sciences, May 2011
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Title: Eleven Words That Sound Like "Orange"
Creator: Monica, Samelson
Date: 2012, 2012-04

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: 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: ANTIBODY AND PEPTIDE CONJUGATES OF BIFUNCTIONAL CHELATORS FOR TARGETED CANCER THERAPY AND IMAGING
Creator: Kang, Chi Soo
Date: 2014, 2014-07
Description: A bifunctional ligand that can rapidly form a stable complex with a metal while conjugated to a tumor-targeting moiety is essential for...
Show moreA bifunctional ligand that can rapidly form a stable complex with a metal while conjugated to a tumor-targeting moiety is essential for targeted therapy and imaging of cancer. New bifunctional chelators in the NETA and DEPA series were developed for targeted radiation cancer therapy. The chelators showed rapid complexation kinetics with 90Y and 177Lu, and the corresponding radiolabeled complexes were stable in vitro and in vivo. Trastuzumab, Panitumumab, and c(RGDyK) conjugates (targeting HER-2, HER-1, and integrin αvβ3, respectively) of the novel bifunctional ligands were prepared. The conjugates displayed fast complexation kinetics with 90Y, 177Lu, and 205/6Bi, and the antibody and peptide conjugates labeled with 90Y, 177Lu, or 205/6Bi exhibited excellent stability and tumor targeting in mice bearing human colorectal cancer (LS-174T). Transferrin (Tf) and bile acid (BA) conjugates of novel chelators in the NE3TA series were prepared for targeted iron chelation therapy (ICT) and positron emission tomography (PET) imaging applications of cancer. Tf and BA conjugates of NE3TA had high cytotoxicity on HeLa, HT29, and PC3 cancer cells. The Tf-N-NE3TA and BA-N-NE3TA also displayed rapid complexation kinetics with 64Cu, and the Tf and BA conjugates of N-NE3TA labeled with 64Cu were stable and had high cellular uptake in HeLa and PC3 cancer cells. Tf-N-NE3TA was further conjugated with a fluorescence dye (Cy5.5) to develop targeted theranostic agent of cancer. Tf-N-NE3TA-Cy5.5 showed great radiolabeling efficiency with 64Cu, and the Tf-N-NE3TA-Cy5.5 labeled with 64Cu exhibited high cellular uptake in HeLa, HT29, and PC3 cancer cells. In summary, we have developed new bifunctional ligands having excellent chelation chemistry of 90Y, 177Lu, 205/6Bi, and 64Cu, and the tumor-specific antibody and peptide conjugates of the novel bifunctional chelators have a great potential for targeted cancer therapy and imaging as well as theranostic applications of cancer.
Ph.D. in Biology, July 2014
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Title: MULTIPLE SIGNAL TRANSDUCTION EVENTS AND MODULATION OF OLFACTORY RESPONSES IN MOUSE OLFACTORY SENSORY NEURONS
Creator: Yu, Yiqun
Date: 2012-04-30, 2012-07
Description: In the mammalian olfactory system, one olfactory sensory neuron (OSN) expresses a single olfactory receptor (OR) gene. I studied two subsets...
Show moreIn the mammalian olfactory system, one olfactory sensory neuron (OSN) expresses a single olfactory receptor (OR) gene. I studied two subsets of mouse OSNs, hereafter refered to as Ho-OSNs and AB-OSNs, in intact mouse olfactory turbinates using calcium imaging. Both of Ho-OSNs and AB-OSNs were located in the most ventral olfactory receptor zone. Ho-OSNs were specifically responsive to 2-heptanone (Ho), heptaldehyde (H) and cis-4-heptenal (cH). Dose-dependent analysis indicated that their responses to individual odorant was saturated within one logarithm unit of concentrations, a typical characteristic of isolated OSNs. Binary mixture and crossadpatation studies showed that these three odorants bound to the same binding sites in Ho-OSNs. However, these three structurally similar odorants activated distinct signaling pathways in Ho-OSNs. In detail, 2-heptanone-evoked intracellular calcium elevation was mediated by AC-cAMP signaling, while heptaldehyde triggered the PLC-DAG pathway. The cis-4-heptenal-evoked [Ca2+]i increases resulted from a combination of cAMP mediated activation and suppression involving PLC signaling. Furthermore, the PLCmediated intracellular calcium alteration was independent of IP3 signaling. A further complexity is that these olfactory receptors were able to interact with other types of Gprotein coupled receptors (GPCRs), such as purinergic receptors. I determined that both the P2X3 and P2Y2 receptor subtypes were expressed in Ho-OSNs. Application of purinergic agonists elevated [Ca2+]i increases in Ho-OSNs. It was discovered that the ATP-induced calcium response required either intracellular or extracellular calcium, while depletion of intracellular calcium stores blocked the UTP-evoked [Ca2+]i increases. Purinergic agonists were able to modulate the odor response in Ho-OSNs, and purinergic antagonist experiments showed that modulation of heptaldehyde-induced calcium responses was due to activation of P2X3 receptor subtypes while heptanone-induced calcium responses was not. Although AB-OSNs were found adjacent to Ho-OSNs, they had complete separate response profiles. AB-OSNs were sensitive to acetophenone (Ace) and benzaldehyde (Ben). In AB-OSNs, both acetophenone and benzaldehyde activated the PLC signaling pathway. Pharmacological characterization indicated that in AB-OSNs, P2X1 and P2Y2 receptors were present, which is different from that in Ho-OSNs. P2X and P2Y agonists modulated odorant responses in AB-OSNs. Purinergic signaling differentially regulated the various odorant responses in AB-OSNs. The acetophenoneevoked [Ca2+]i increases were negativly modulated through activation of the P2Y2 receptor, while the calcium response induced by benzaldehyde was modulated by P2X1 receptor activation. Collectively, these studies suggest that a complex signaling mechanism exists in OSNs, which has important implications for understanding the mechanism of information process in the olfactory system.
Ph.D. in Biology, July 2012
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Title: THE MOLECULAR MECHANISM OF BAXΔ2-MEDIATED CELL DEATH AND ITS TISSUE DISTRIBUTION IN COLON CANCER
Creator: Zhang, Honghong
Date: 2014, 2014-07
Description: Bax is a pro-death tumor suppressor in the Bcl-2 family, and is frequently mutated in microsatellite instable tumors, especially Hereditary...
Show moreBax is a pro-death tumor suppressor in the Bcl-2 family, and is frequently mutated in microsatellite instable tumors, especially Hereditary Non-Polyposis Colorectal Cancer (HNPCC). The loss of apoptotic Bax contributes to tumor development and chemoresistance. We recently uncovered that the combination of a Bax microsatellite mutation with a specific alternative splicing generated a unique Bax isoform (BaxΔ2) in Bax-negative cells. Similar to the prototype Baxα, BaxΔ2 is a potent pro-apoptotic molecule. However, the pro-apoptotic mechanism, therapeutic implication, and tumor tissue distribution of BaxΔ2 protein remain elusive. In this thesis research, we isolated and analyzed isogenic sub-cell lines that represent different Bax microsatellite statuses from colorectal cancer cells. We found that the colon cancer cells harboring Bax microsatellite G7/G7 alleles produced low levels of endogenous BaxΔ2 transcripts and proteins. BaxΔ2-positive cells were selectively sensitive to a subgroup of chemotherapeutics in comparison with BaxΔ2-negative cells. Different from other Bax isoforms, which mostly act through targeting mitochondria, BaxΔ2 recruited caspase-8 into the aggregates for activation, and consequently induced cell death independent of the mitochondrial pathway. Furthermore, the distribution of BaxΔ2 protein was mostly found in well-differentiated epithelial cells in primary colon tumor tissues or in primary squamous buccal cells, which contain Bax G7 mutation. However, not all cells harboring the Bax G7 mutation had a detectable level of BaxΔ2 proteins. These data suggest that, similar to Baxα, BaxΔ2 protein is pro-apoptotic, but not toxic to normal cells; expression of BaxΔ2 protein restores apoptotic program in Bax negative cells via a non-classical signaling pathway. Importantly, BaxΔ2 may provide a selective chemotherapeutic advantage for certain Bax-negative colon tumors.
Ph.D. in Biology, July 2014
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Title: ANTI-TUMOR SPLICING: RESTORING THE TUMOR SUPPRESSOR BAX IN MICROSATELLITE UNSTABLE TUMORS
Creator: Haferkamp, Bonnie
Date: 2011-11, 2011-12
Description: Microsatellite instability (MSI) is a hallmark for many tumors, especially colon, endometrial, gastric and bladder. Bax, a tumor suppressor...
Show moreMicrosatellite instability (MSI) is a hallmark for many tumors, especially colon, endometrial, gastric and bladder. Bax, a tumor suppressor and pro-death Bcl-2 family member, is frequently mutated in MSI tumors. A microsatellite mutation produces a frameshift with premature termination, leading to “Bax-negative” tumors. Although low Bax expression in tumors is often associated with poor prognosis, several studies have correlated lack of Bax in MSI tumors with improved prognosis. However, the molecular explanation for this paradox is unknown. Here we show that “Bax-negative” tumors in fact generate a novel family of anti-tumor Bax-MSI isoforms through alternative splicing. The thesis includes two parts. In Chapter One, we fully characterize one Bax-MSI isoform, BaxΔ2. We show that BaxΔ2 is detrimental to cancer cells but through a non-conventional death pathway, with differential sensitivity to chemotherapeutics. In Chapter Two, we present an entire family of Bax-MSI isoforms, and illustrate a potential molecular mechanism behind its production. We show that the frequency of Bax alternative splicing is significantly higher in MSI than non-MSI tumors, and that BaxΔ2 trans splicing requirements are ubiquitous in human cell lines. The discovery of functional Bax isoforms in Bax-mutated tumors may help explain why the apparent loss of Baxα in tumors is sometimes associated with a better prognosis. In addition, the unique sequences of Bax-MSI isoforms can serve as biomarkers for diagnostic and treatment purposes. Importantly, identification of Bax-MSI isoforms will provide a great opportunity from a genetic approach or drug design for treatment of MSI cancer.
Ph.D. in Biology, December 2011
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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: 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: 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: PHYSIOLOGICAL AND BEHAVIORAL EVIDENCE THAT CHEMICALS IN THE ENVIRONMENT INFLUENCE DEVELOPMENT OF THE ZEBRAFISH OLFACTORY SYSTEM
Creator: Valesio, Eric
Date: 2013, 2013-12
Description: Olfactory imprinting is the process of producing life-long changes through neural modification that is independent of associative learning....
Show moreOlfactory imprinting is the process of producing life-long changes through neural modification that is independent of associative learning. Here, I provide data to demonstrate that olfactory imprinting in zebrafish leads to neurobiological and behavioral changes. I treated zebrafish with an amino acid (AA) or a bile acid (BA) mixture from 4 days post-fertilization (dpf) to 40 dpf. Behavior studies showed that fish treated with odorants for 40 days exhibited preferential responses to treated odorants, which was different from the controls. These behavioral changes were retained 3 months after the odor treatment was ceased. Whole-mount immunohistochemistry was conducted using antibodies for parvalbumin (PV) and OTX. We discovered that treated fish had increased PV and OTX expression in the olfactory epithelium (OE) at 7 dpf and increased PV expression in the olfactory bulb (OB) at 12 dpf. Detailed analysis indicated that increased PV expression was observed in the OE apical region of treated groups while OTX was increased in both the apical and basal regions of the OE. In three regions of the OB analyzed, BA treated fish showed a doubling in PV expression in all regions while doubling was in two regions in AA treated fish. Increased OTX expression was in the three regions of AA treated fish but not in BA treated fish. These data demonstrate that exposure to AA or BA during zebrafish development leads to long-lasting physiological and behavioral changes. The report also includes a study of embryonic zebrafish treated with SP600125 (anthrapyrazolone), an inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway. Zebrafish embryos were treated with 1.25 μM, 5 μM, or 12.5 μM of SP600125 from 18 to 48 hours post-fertilization (hpf) followed by evaluation at 120 hpf. Zebrafish treated at 1.25 μM were not affected developmentally while embryos treated at xv 5 μM and higher displayed numerous morphological defects including edemas, eye malformations and reduction in olfactory organ size. Overall, it was observed that treatment at 5 μM, SP600125 caused severe developmental defects and that these defects worsened with increasing concentrations. Taken together, these data indicate that the environment has a profound influence on zebrafish development.
PH.D in Biology, December 2013
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Title: INVESTIGATING DIRECTED EVOLUTION AND GENETIC ENGINEERING WITH VITREOSCILLA HEMOGLOBIN TO PRODUCE CULTURES FOR LOW AERATION BIOLOGICAL WASTEWATER TREATMENT
Creator: Kunkel, Stephanie
Date: 2014, 2014-07
Description: The dominance of hemoglobin (Hb)-expressing bacteria in biological wastewater treatment systems could improve oxygen utilization under low...
Show moreThe dominance of hemoglobin (Hb)-expressing bacteria in biological wastewater treatment systems could improve oxygen utilization under low dissolved oxygen (DO) conditions. Hb-proteins are versatile molecules that have several biological functions. Here, Nitrosomonas europaea has been transformed with various plasmids; of particular interest is a recombinant plasmid bearing the constitutive Amo1 promoter and the gene (vgb) encoding the hemoglobin from the bacterium Vitreoscilla. Expression of VHb was assayed using various visible spectral methods, and VHb production seen in this recombinant strain. There were several positive effects on N. europaea metabolism related to VHb expression that were seen, specifically the ability of cultures to convert ammonia to nitrite at a slightly higher rate as well as higher specific oxygen uptake rates (SOUR) at both high (near saturation, 7 mg O2/ L) and low (< 2 mg O2/L) dissolved oxygen (DO) conditions. In parallel to this, two activated sludge cultures from the same source were cultivated using synthetic wastewater seeded with activated sludge from the same source and were operated at high DO (near saturation) and low DO (0.25 mg O2/L) concentrations for 370 days. There were significant changes in the bacterial species and phyla present in each of the cultures at various time points during the 370 day operational period. In the low DO culture, over time, there was a much greater expression of single domain and truncated Hbs which may enhance utilization and delivery of oxygen to various enzymes as well as to the respiratory chain. A larger increase in heme b was also observed which coincides with this observation. By the end of the acclimation period, the SOUR values were about 30% greater in the low DO culture compared to the high DO culture. This indicated the successful adaption of the low DO culture to respire more efficiently and eventually outperform the high DO culture.
Ph.D. in Biology, July 2014
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