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Title: Structural Study of the Complex of the Mutant Cholera Toxin A1 Subunit with Human ADP-Ribosylating Factor 6 Bound to Agmatine
Creator: Chang, Shih-chia
Date: 2012-07-12, 2012-07
Description: Cholera is a severe disease that causes devastating diarrhea when Vibrio cholerae infects the human intestine. Once the bacterium gets the...
Show moreCholera is a severe disease that causes devastating diarrhea when Vibrio cholerae infects the human intestine. Once the bacterium gets the access into the intestinal cell, adenosine diphosphate (ADP)- ribosylation of the human signaling protein Gs!!is catalyzed by the cholera toxin A1 subunit (CTA1). According to the previous researches, this reaction is activated allosterically by a GTP-bound human ADP-ribosylation factor six (ARF6). Arginine is one of the substrates for cholera toxin A1 subunit. In the thesis, the crystal structure of agmatine, a L-arginine analogue, bound to the protein complex of the mutant CTA1 with ARF6-GTP reveals the possibility of how arginine might interact with the complex.
M.S. in Molecular Biochemistry and Biophysics, July 2012
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Title: STUDY OF THE FUNCTION AND TARGET GENES OF THE MIR-17-92 CLUSTER IN MLL-ASSOCIATED LEUKEMIA
Creator: Wiley, Anissa
Date: 2011-04-19, 2011-05
Description: MicroRNAs (miRs) have recently been found to be important regulatory agents in cancers. More specifically, the miR-17-92 cluster, which is...
Show moreMicroRNAs (miRs) have recently been found to be important regulatory agents in cancers. More specifically, the miR-17-92 cluster, which is overexpressed in acute leukemias with fusions of the mixed lineage leukemia (MLL) gene, such as MLL-AF9 and MLL-ELL, has been shown to affect regulation of normal genes in human acute leukemias. Studies show that the miR-17-92 cluster targets genes in acute leukemias that play roles in cell differentiation and proliferation. To study this, a luciferase reporter assay was performed showing that miR-17-92 targets transforming growth factor beta induced (TGF!I) and suppressor of zeste 12 (SUZ12). Studies have shown TGF!I to be downregulated in MLL fusion acute leukemias and SUZ12 to have an inverse correlation of expression with miR-17-92. Both genes are important in the regulation of cell differentiation and proliferation. Furthermore, previous studies have shown that the miR- 17-92 cluster partially blocks cell differentiation and induces cell proliferation, and when combined with MLL fusions, these effects are amplified. To further validate the results of these studies, a colony forming assay was performed confirming that a knock-in version of the miR-17-92 cluster promotes formation of colonies of non-differentiating cells, especially in the presence of MLL-AF9 and MLL-ELL. These studies implicate the miR- 17-92 cluster as an important regulator in acute leukemias with MLL fusions given its effect on cell differentiation and proliferation.
M.S. in Biology, May 2011
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Title: EFFECTS OF ENGINEERING USING BACTERIAL HEMOGLOBIN ON THE ABILITY OF ETHANOLOGENIC E. COLI TO FERMENT LIGNOCELLULOSIC HYRDOLYSATE TO ETHANOL
Creator: Kunkel, Stephanie
Date: 2011-05-03, 2011-05
Description: E. coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol has been further engineered in our laboratory using...
Show moreE. coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol has been further engineered in our laboratory using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The resulting strain (TS3) expresses VHb at a moderate level under microaerobic conditions in shake flasks and produces more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the carbon source. In the work reported here, this research was extended, focusing particularly on fermentation of lignocellulosic hydrolysate. The increased ability of the VHb-bearing strain to produce ethanol from pure glucose or xylose was confirmed. A VHb-correlated increase in ethanol production was also noted for lignocellulosic hydrolysate produced in our laboratory, but not for ligncellulosic hydrolysate prepared by the National Laboratory for Renewable Energy (NREL). Experiments were performed at several hydrolysate levels for both prepartories, in an attempt to determine the cause of the difference. One possibility is differences in levels of toxic hydrolysis byproducts in the two preparations as well as increased sensitivity to these toxins due to VHb expression.
M.S. in Biology, May 2011
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Title: SUBCLONING AND ACTIVITY EXAMINATION OF VARIANT VITREOSCILLA HEMOGLOBINS PRODUCED BY RANDOM MUTAGENESIS
Creator: Raba, Daniel A.
Date: 2015, 2015-05
Description: A comparison of mutant versus wild type versions of the Vitreoscilla hemoglobin gene (vgb) were the focus of this study. E. coli DH5a cells...
Show moreA comparison of mutant versus wild type versions of the Vitreoscilla hemoglobin gene (vgb) were the focus of this study. E. coli DH5a cells were transformed with recombinant pUC plasmids containing either the wild type or one of a series of mutated versions of the vgb gene. The study was conducted in two pmis. In the first part, eight strains in total were tested (one plasmid-free negative control, one vector-only (pUC18) negative control, one wild type positive control (pUC8: 16), and five mutants (pUC vgb M1-4 and pUC18 vgb M3)) and comparisons were made to reveal growth advantage or possible increased Vitreoscilla hemoglobin protein (VHb) expression due to vgb gene mutation. Three different growth assays were carried out to compare growth rates among the eight strains under different oxygen concentration conditions: (1) Luria-Bertani (LB) medium, aerobic growth assay, (2) Terrific Broth (TB) medium, low-oxygen growth assay, and (3) TB medium, microaerobic growth assay. Additionally, a series of carbon monoxide (CO) difference spectra were run to quantify functioning VHb protein concentrations in the different strains grown under each of the aforementioned oxygen concentration conditions. In the second part of the study, the two vgb gene mutants that exhibited the highest growth rates and the wild type version were subcloned into the vector pUC8, transformed into E. coli DH5a cells, and then compared using the CO difference spectrum assay. Five E. coli DH5a strains were tested (one plasmid-free negative control, one vector-only negative control (pUC8), one vgb wild type positive control (pUC8 vgb WT), and two vgb mutants (pUC8 vgb M1 and M3)). Transfmmation of plasmids containing the mutated or unmutated version of the vgb gene was verified through E.Z.N.A. plasmid miniprep with gel electrophoresis and additionally through growth on selective medium containing ampicillin (Amp) [50 micrograms (ug)/ milliliter (ml)]. Contrary to the results obtained by our Australian collaborators, our growth assays and CO difference spectra revealed no growth advantage or increased expression of functioning VHb protein due to any of the vgb mutations.
M.S. in Biology, May 2015
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Title: MOLECULAR MECHANISMS OF ESTROGEN RECEPTOR BETA ACTION IN COLORECTAL CARCINOGENESIS
Creator: Vaz Saleiro, Diana Nora
Date: 2013, 2013-07
Description: Colorectal cancer (CRC) is a major public health problem in the world population, which emphasizes the need to study novel molecular targets...
Show moreColorectal cancer (CRC) is a major public health problem in the world population, which emphasizes the need to study novel molecular targets in order to develop alternative chemopreventive and chemotherapeutic strategies against it. Estrogens have been shown to exert a protective role against CRC and its actions seem to be mediated by estrogen receptor β (ERβ). However, further studies are required to elucidate the role of ERβ in the colon. To achieve this goal, we evaluated the effects of ERβ deficiency in sporadic CRC and colitis-associated CRC (CAC) by using two in vivo models. In addition, we stably transfected RKO colon cancer cells to overexpress ERβ and identify novel molecules regulated by ERβ signaling in vitro. In the sporadic CRC in vivo model, azoxymethane (AOM)-treated ERβ knockout (βERKO) mice showed a significantly higher incidence and multiplicity of colonic preneoplastic lesions compared to AOM-treated ERα knockout and wild-type (WT) mice. These results were associated with a loss of normal colonic crypt organization and a decrease in apoptosis rates suggesting that ERβ is the ER subtype with a protective role against sporadic CRC progression. Confirming this hypothesis, we observed that AOM-treated βERKO mice presented a significantly higher incidence of adenomas than WT mice. By real time-polymerase chain reaction and immunohistochemistry analyses, ERβ-deficient adenomas were shown to be more proliferative and less differentiated than adenomas in WT mice. Furthermore, in the CAC in vivo model, βERKO mice developed more severe clinical colitis compared to WT mice, as evidenced by significantly higher disease activity index, weight to length ratio of the colons, inflammation score, and grade of dysplasia. ERβ-deficient tumors were characterized by a significant increase in pro-inflammatory xv molecules, suggesting that ERβ might exert anti-inflammatory effects in CAC. Moreover, in vitro results revealed a novel molecular mechanism by which ERβ might drive differentiation of goblet cells in the colonic epithelium. Altogether these findings highlight the importance of ERβ in protection against colorectal carcinogenesis and provide a clinical and translational potential to use ERβ selective agonists in order to prevent and/or treat CRC.
PH.D in Biology, July 2013
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Title: The Development of T7 Phage Based Imaging Probe and the Nature of Floating Cells in Human Embryonic Stem Cell Culture
Creator: Dasa, Siva Sai Krishna
Date: 2011-05-16, 2011-05
Description: Target specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes...
Show moreTarget specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes were synthesized by conjugating both affinity reagent and chelator molecules on the particle surface chemically. Subsequently, the isotope ions were attached to the particles via metalchelator interactions. Due to the nature of bio-conjugation process, the number of the chelator molecules and the geometry of the affinity reagent are hard to control. To overcome these limitations, a new approach of constructing an imaging probe was developed. In specific, T7 phage was used as a vehicle to synthesize a particle based imaging probe by displaying peptide that had both metal chelating (6 histidine) and targeting (RGD domains on the phage surface. It was demonstrated that the attachment of copper ions to the metal chelating domain does not interfere with target binding. Furthermore, by reducing the metal ions to metal, we were able to generate a very stable peptide templated metal hybrid T7 phage particle as potential target specific imaging probe. The behavior of the probes against both normal and tumor cells was investigated. The presence of floating cells is a common phenomenon in human embryonic stem (hES) cell cultures. Current assumption for the source of floating cells is that they are from apoptosis / senescence, cellular differentiation and other unavoidable imperfections in culture conditions. Inspired by recent studies on mitotic activities in stem cell colony, we believe the existence of floating cells is resulting from essential events required for hES cells proliferation. It was discovered that not all floating cells are dead and the percentage of floating cells over the attached cells was significantly increased with culture time possibly due to the space limitation for cells in the central part of the colonies. By providing more horizontal or vertical space through colony cutting and overlaid membrane insert in the Z direction, the percentages of floating cells were significantly reduced. All these results indicate that continuous cell division across the colonies is responsible for the emergence of floating cells during hES cell culture.
Ph.D. in Biology, May 2011
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Title: CLONING AND CHARACTERIZATION OF THE SNTA: NNOS: DYSTROPHIN INTERACTION SITES
Creator: Shi, Chenyue
Date: 2014, 2014-07
Description: Duchenne Muscular Dystrophy (DMD) is a lethal genetic disease caused by malfunction of the protein dystrophin. The main function of dystrophin...
Show moreDuchenne Muscular Dystrophy (DMD) is a lethal genetic disease caused by malfunction of the protein dystrophin. The main function of dystrophin is to connect the sarcolemma to the machinery of muscle function via specific binding domains near the C-terminus and N-terminus. These binding domains are connected by a large central rod region that had previously thought to have been biologically inert, but has recently been shown to contain binding sites for various biological partners, including a specific rod region that binds to nNOS, which is an important molecule in modulating vasodilation and increasing blood flow by relax vessel. Removal of this nNOS binding eliminates NO signaling during exercise, causing exercise-related ischemia leading to muscle damage. A major therapeutic strategy being studied to treat DMD is exon skipping, which results in modifications to the dystrophin protein. However, it remains unclear exactly where the binding sites on dystrophin protein and nNOS are, or how they interact with each other. This project is embarked upon more fully characterize the D1617 binding site on the nNOS PDZ domain. Previous studies show that, in neural tissue, PDZnNOS binds to two other proteins (PSD95 and N-methyl-D-aspartate receptor, NMDAR) forming a ternary complex in which the nNOS: PSD95 is a typical PDZ type interaction, but in which the nNOS :NMDAR binding occurred in an unusual fashion, through a unique “finger” region present in PDZnNOS but not in most other PDZ domains. Interestingly, in muscle cells, nNOS also interacts with two proteins, dystrophin and syntrophin (SNTA). Using this as an analogy, we hypothesize that in this case, the binding may also involve both the canonical and finger regions of PDZnNOS, one binding to each protein. We are testing this by constructing PDZnNOS variants with specific amino acid changes designed to xi disrupt each of these interactions (canonical and finger region) independently, and will then examine the impact on both the PDZSNTA and dystrophin D1617 interactions. It is hoped fully understanding the dystrophin nNOS interaction will allow therapies that require modification to dystrophin to conduct these modifications in a way that retains nNOS signaling required for proper muscle function.
M.S. in Cell and Molecular Biology, July 2014
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Title: THE LENGTH-TENSION CURVE OF AND LATTICE SPACING CHANGES IN SKINNED FLIGHT MUSCLE IN MANDUCA SEXTA
Creator: Shaoshuai, Chen
Date: 2016, 2016-05
Description: The synchronous indirect flight muscle (DLM1) of the Hawk moth, Manduca Sexta, has similarities to cardiac striated muscle in its...
Show moreThe synchronous indirect flight muscle (DLM1) of the Hawk moth, Manduca Sexta, has similarities to cardiac striated muscle in its physiological properties. In particular, both operate in vivo on the so-called ascending limb of the length-tension curve. The length-tension curve is a classical experiment to study the physiological properties of muscle. The length tension curve of DLM1 of Manduca sexta has previously been reported with a descending limb that is steeper than what would be expected given likely dimensions for the thick and thin filaments. Excessive rundown of the muscle preparations is a likely cause of these observations. Factors caused muscle rundown are many, such as the time allowed for the muscle to relax, the time for muscle spends contracting or the time spent on sarcomere length adjusting. Insights into the factors mentioned above were obtained by conducting a series of experiments designed to systematically explore the causes of rundown. These included Constant Sarcomere Length Experiments, Reciprocating Stretch Experiments and Back to SL 3.25 μm Experiment. Then a modified protocol for carrying out the length-tension experiment was developed based on these findings. A new length-tension curve was plotted and shows a shape closer to what might be expected theoretically. The force went to zero at about SL 5.7μm. This result is constant with other measurements of the length of the myofilaments. Finally, the lattice spacing experiment was carried out to figure out how the interfilament lattice spacing changes across the SL growing. Result shows that the lattice spacing did not change much over the plateau region of the length-tension curve (SL 2.9- 3.1) but increased substantially as SL decreases further in the ascending limb of the length tension curve.
M.S. in Biology, May 2016
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Title: STRUCTURAL ANALYSIS OF MUTANT TYPES OF VITREOSCILLA HEMOGLOBIN
Creator: Bai, Dongyang
Date: 2016, 2016-07
Description: The aerobic, Gram-negative bacterium Vitreoscilla spp. produces an oxygen-storage protein, which is a hemoglobin. Vitreoscilla hemoglobin (VHb...
Show moreThe aerobic, Gram-negative bacterium Vitreoscilla spp. produces an oxygen-storage protein, which is a hemoglobin. Vitreoscilla hemoglobin (VHb) was the first microbial hemoglobin to be characterized. Like mammalian hemoglobins, it can be described in terms of the distal (ligand-binding) and proximal sides of the heme group. VHb is regarded as a model to study the structure and function of hemoglobin. After the crystal structure of wild-type VHb was obtained, along with several mutation studies on its distal site, it was proposed that residue Tyr29 might play a vital role in ligand binding. In a recent study, a Tyr29Ala mutant displayed functional properties similar to those of the wild-type protein, evidently because the space in the ligand-binding domain occupied by the aromatic ring of Tyr29 in wild type is occupied by the aliphatic ring of Pro54 residue in the mutant which results in a similar ring structure at the ligand-binding domain. This project is aimed to characterize structural changes when Pro54 is mutated to Ala, either with or without the accompanying mutation of Tyr29 to Ala. In an earlier effort, the Y29A/P54A and P54A mutant vgb gene was constructed, but mutant proteins could not be crystallized. In the current study, a hexahistidine tag was added to the C terminus of VHb to aid in protein purification. The Y29A/P54A and P54A mutant protein was purified by affinity chromatography, ion-exchange chromatography and hydrophobic interaction chromatography. The purity of the protein was determined on SDS-PAGE. Circular dichroism data indicated that both Y29A and Y29A/P54A mutants’ α-helix content decreased significantly. Thermal denaturation studies suggest that there are no major changes in the Tm for the Y29A/P54A mutant, and a slight increase for the P54A mutant.
M.S. in Biology, July 2016
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Title: REGENERATIVE POTENTIAL OF HUMAN CD34+ STEM CELLS MOBILIZED AND ENRICHED FROM BONE MARROW
Creator: Cohen, Amy
Date: 2014, 2014-12
Description: This thesis provides an in-depth investigation of the heterogeneous population of CD34+ cells obtained through granulocyte-colony stimulating...
Show moreThis thesis provides an in-depth investigation of the heterogeneous population of CD34+ cells obtained through granulocyte-colony stimulating factor (G-CSF) mobilization, apheresis, and an immuno-magnetic selection process. The results provide a better understanding of the phenotypic characteristics, functional migratory capabilities and paracrine secretory performance when exposed to normal and low-oxygen environments in vitro. The overall composition of the population acquired through the cells selection process and the CD34+ cells were characterized for maturity and commitment to various cell types as well as their potential for migration, adhesion and proliferation. The detailed phenotypic characteristics included positivity for more primitive, undifferentiated hematopoietic stem cells (HSCs) as well as markers indicating enrichment of long term HSCs. A functional quantitative bioassay was used to measure the migratory ability of the CD34+ cells. On average, 18.7% of the CD34+ cells migrated after 3 hours of incubation in the presence of 200ng/mL of Stromal Cell-Derived Factor One alpha (SDF-1α) as compared to 0.6% of the CD34+ cells incubated without chemoattractant. The migration was successfully neutralized by using monoclonal antibodies to CXCR4 and a CXCR4 antagonist, AMD3100. An evaluation of the migratory cell population showed that the cells exhibited an enhanced commitment to a monocyte or lymphocyte lineage and a larger percentage were CXCR4+. CD34+ cells were incubated in normoxic and hypoxic conditions and the proliferation, viability, immunophenotypic profile, migratory potential and secreted cytokines were evaluated and compared to incubation in normoxic conditions. The CD34+ cells proliferated in both the normoxic and hypoxic conditions over three days of incubation. The proliferation was higher in the hypoxic condition after one day of incubation, but after three days of incubation the normoxic conditions resulted in more proliferation. An increase in CD164 and a decrease in CD99 in the Day 3 hypoxic condition suggest that hypoxia was stimulating an adhesion pathway and suppressing the functional migration of the CD34+ cells. The three hour hypoxia incubation resulted in a significant (63%) reduction in the migration of the CD34+ cells as compared to the 3 hour normoxia condition, but many of the cells continued to migrate in hypoxia over the 24 hour period while the normoxic cells continued to migrate, but at a much slower rate. Culture media from CD34+ stem cells that had been incubated in normoxic or hypoxic conditions for one day and three days was assayed for sixty cytokine proteins known for possessing angiogenic qualities. Twenty five angiogenesis cytokines were increased in the culture media incubated with CD34+ cells, including tissue inhibitor of metalloproteinases-1 and 2 (TIMP-1 and TIMP-2), angiopoietin-1 (ANG-1) and insulin-like growth factor-1 (IGF-1). Thirteen angiogenesis cytokines were elevated in hypoxia as compared to normoxia, including TIMP-1, IGF and growth-regulated oncogene-β (Gro-β).
Ph.D. in Biological and Chemical Sciences, December 2014
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Title: EFFECT OF METABOLIC INHIBITION ON THE GROWTH AND BIOFILM PRODUCTION OF VIBRIO CHOLERAE AND PSEUDOMONAS AERUGINOSA
Creator: Bunn, Dakota C.
Date: 2017, 2017-05
Description: V. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is...
Show moreV. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is especially prevalent in developing countries that lack proper water treatment and in areas struck by natural disasters such as hurricanes. P. aeruginosa is an opportunistic pathogen that is ubiquitous in nature, and increasingly found in hospitals burn wards, sinks, catheters and other surgical equipment. Both bacteria are developing increased antibiotic resistance through several mechanisms, with one of the most common ones being the formation of a complex exopolysaccharide matrix known as a biofilm. In this study, using metabolic inhibition, we determined that Na+-NQR is essential for the growth of V. cholerae and P. aeruginosa in both nutrient rich and physiological conditions. We were also able to confirm that inhibition of this enzyme, in both growth conditions, resulted in decreased biofilm production, subsequently eliminating one of the main mechanisms for antibiotic resistance of these bacteria.
M.S. in Biology, May 2017
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Title: GENETIC MODIFICATION OF PAENIBACILLUS UTILIZING VITREOSCILLA HEMOGLOBIN TO IMPROVE BIODESULFURIZATION
Creator: Bai, Yu
Date: 2015, 2015-05
Description: Biodesulfurization (BDS) is a type of microbial biodegradation involving the removal of organic sulfur compounds from fossil fuels that is...
Show moreBiodesulfurization (BDS) is a type of microbial biodegradation involving the removal of organic sulfur compounds from fossil fuels that is accomplished by utilizing the metabolism of certain microorganisms (McFarland et al., 1999). In past decades, many related genetic modifications or transformations have been made to improve biodesulfurization. In this study, Vitreoscilla hemoglobin (VHb) was used to enhance the ability of sulfur metabolism and growth in Paenibacillus naphthalenovorans. Paenibacillus naphthalenovorans wild type (32O-Y) is a thermophilic bacterium isolated in our lab that has also been transformed with recombinant plasmid pnW33N-vgb (producing strain 32O-Y[pNW33N-vgb]). This plasmid encodes both VHb and chloramphenicol (Chl) resistance. The Chl resistance gene provides a method to distinguish from wild type and select for transformed 32O-Y[pNW33N-vgb]. Presence of the plasmid in the transformed strain was verified utilizing PCR. Both 32O-Y and 32O-Y[pNW33N vgb] grew in minimal medium (CDM) that contained dibenzothiophene (DBT) as the sole sulfur source (while Chl was added to 32O-Y[pNW33N-vgb] cultures). DBT is a common organic sulfur-containing molecule in petroleum. It is metabolized by the dsz pathway, present in a number of soil bacteria, which produces 2-hydroxybiphenol (2-HBP) as the final product. Growth (OD600mm) of 32O-Y and 32O-Y[pNW33N-vgb] at 37°C, 45°C, 50°C were compared. The desulfurization activities of wild type and transformed Paenibacillus naphthalenovorans strains were monitored by the Gibbs assay, which measures the concentration of 2-HBP through absorbance at 610 nm. The results demonstrate that the expression of VHb in strain 32O-Y[pNW33N-vgb] correlated with increased growth, especially at 37°C. The desulfurization activities of 32O-Y[pNW33N-vgb] at various temperatures were also improved, compared to untransformed 32O-Y, as VHb was expressed. Efficiency of the VHb-related improvements at the three temperatures was calculated as the ratio between maximum 2-HBP produced and OD600nm at this point. The efficiency values indicated that when compared to 32O-Y, 32O-Y[pNW33N-vgb] had maximum increased productivity at 37°C and 50°C, and minimum at 45°C.
M.S. in Biology, May 2015
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Title: CHARACTERIZING THE ROLE OF PCL1 IN YEAST PHEROMONE RECEPTOR POLARIZATION
Creator: Pai, Chih-yu
Date: 2015, 2015-05
Description: Chemotropism, or directed cell growth in response to a chemical gradient, plays a vital role in many processes, including pollen tube...
Show moreChemotropism, or directed cell growth in response to a chemical gradient, plays a vital role in many processes, including pollen tube formation, axon guidance, angiogenesis, and fungal infections. One of the best-studied models of eukaryotic directional sensing is Saccharomyces cerevisiae, also known as budding yeast. Chemotropic growth relies on both pheromone receptor and its associated G-protein. In vegetative yeast cells, the pheromone receptor is uniformly distributed on the plasma membrane. Upon receptor activation, G~y is released from Ga and G~ is subsequently phosphorylated at multiple residues. Free G~y signals to the nucleus via a MAPK cascade that causes G1 cell cycle arrest and induction of mating specific genes. In addition, G~y recruits various polarity proteins that orient actin-directed secretion to a specific growth site, and newly synthesized receptors are delivered to it. This pheromone-induced receptor polarization appears as "crescents" where the mating projection later forms. However, how yeast cell interpret the external signals and establish the chemotropic growth site remains unknown. Here, we characterized the role of Pcl1, which is a cyclin for the CDK Ph085, and which has been implicated in polarization during budding. It is known that G~ phosphorylation plays a role in receptor polarization and chemotropism. Because G~ has a consensus phosphorylation motif for Ph085, and because G~ and Pcl1 showed a genetic interaction, we hypothesized that Ph085-Pcl1 regulates receptor polarity through G~ phosphorylation. Our results showed Pcl1 localized towards mating projection during mating response. Moreover, Pcl1 and G~ exhibit a direct interaction in response to pheromone. Lastly Ph085 inactivation also appears to affect receptor polarity. Hence, our data are consistent with the hypothesis that Pho85-Pcl1 facilitates receptor polarity establishment by phosphorylating G~.
M.S. in Biology, May 2015
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Title: Efforts to Obtain Desulfurization Competent Cultures from Environmental Samples with Increased Desulfurization Activity
Creator: Davaadelger, Batzaya
Date: 2011-04-18, 2011-05
Description: In this study, environmental samples were collected from different places to obtain various desulfurization competent cultures with increased...
Show moreIn this study, environmental samples were collected from different places to obtain various desulfurization competent cultures with increased desulfurization activity. In order to obtain the cultures the soil samples were inoculated in enriched medium and minimal medium containing DBT as the sole source of sulfur. The cultures grown in minimal medium with DBT were transferred multiple times with increasing temperatures, where as the cultures grown in enriched media were grown only one cycle at 37 degrees. After a certain growth period the cells were isolated and DNA was extracted. Efforts to amplify dsz genes from the obtained cultures with universal dszABC primers were mostly unsuccessful. DNA from bacterial strains isolated from the environmental samples were amplified with universal primers and amplicons were cloned. Samples #2, #6, #17, #21, #32 were amplified successfully but sequencing analyses showed little homology with dszABC genes. DNA from samples #32*, 32*Y and 32*W was isolated with Power Soil kit and PCR analysis showed a 3 kb amplicon. Sequencing analyses, however, showed less homology with dszABC and revealed homology instead with proteins such as those involved in Fe/S biogenesis and thiol:disulphide interchange. In addition, alternate universal primers were designed and PCR analyses were made with R .erythropolis IGTS8 and sample #32*. Alternate primer combination #3 (A1 and C1) and combination #8 (B1 and C2) amplified expected bands with IGTS8 DNA. Combination #8 amplified a 1 kb band from sample #32* DNA. Cultures #32*, #32*Y and #32*W were selected due to their ability to grow in minimal medium with DBT as high as 55 ˚C for multiple transfers. These isolated cultures from a soil sample from the Chicago area are able to catabolize dibenzothiophene (DBT) to 2-hydroxybiphenyl (2-HBP), which was detected by the Gibbs assay.
M.S. in Biology, May 2011
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Title: SCREENING AND COMPARING GENES OF INTEREST IN MICROBIAL SPECIES USING WHOLE GENOME SEQUENCING
Creator: Butler, Robert Raymond Iii
Date: 2016, 2016-12
Description: This study utilized the latest advances in whole genome sequencing, assembly and annotation to develop high quality curated genomes, which...
Show moreThis study utilized the latest advances in whole genome sequencing, assembly and annotation to develop high quality curated genomes, which were compared to related organisms with differential traits to identify or characterize the trait-associated genes. Additionally, we were able to infer potential origins of these traits, and present gene targets for further study. Here we examined two biological phenomena: the desulfurization capability of a Paenibacillus species, and the exceptionally high spore heat resistance of Clostridium sporogenes PA 3679. Microorganisms with the capability to desulfurize petroleum are in high demand with escalating restrictions currently placed on fuel purity. Thermophilic desulfurizers are particularly valuable in high temperature industrial applications. A culture containing Paenibacillus naphthalenovorans 32O-Y and Paenibacillus sp. 32O-W was isolated by repeated passage of a soil sample at up to 55°C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway, however 32O-W enhanced DBT metabolism by 32O-Y in a mixed culture. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties, with 32O-W lacking homologs for two necessary components of the 4S pathway. Both 32O-Y alone and the 32O-Y/32O-W mixed culture may be useful in development of an improved thermophilic petroleum biodesulfurization process. Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA sequence and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades. Clade I C. sporogenes isolates were genetically distinct from clade II isolates, and thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates; clade II demonstrating the typical phenotype of PA 3679. A pan-genomic analysis of clade I and clade II isolates identified genes associated with PA 3679’s exceptional heat resistance. The most significant difference was the acquisition of a second spoVA operon, spoVA2, whose products are responsible for dipicolinic acid transport into the spore core during sporulation. The small acid-soluble spore protein ssp4 potentially plays a role in spore heat resistance, though further exploration is needed. spoVA2 was also found in some C. botulinum species clustering phylogenetically with PA 3679. Most other C. sporogenes examined both lack the spoVA2 locus and are phylogenetically distant within the group I Clostridia, adding to the understanding that C. sporogenes are dispersed C. botulinum strains lacking toxin genes. C. sporogenes strains are thus a very eclectic group, and few strains possess the characteristic heat resistance of PA 3679. Analysis from both Paenibacillus and Clostridium models revealed some interesting insights into genomic analysis that extrapolate to other projects. Each of the four generations of sequencing technology has remained a necessary component of genomics. The delineating factors for which sequencing tool to use depends heavily on the application they are being used for. New software for assembly and annotation are developed and released daily, and the challenge has become deciding which tools are actually an improvement over existing methodology. In order to best facilitate the large amount of genomic data in need of analysis, pipelines that are consistent and comprehensive are of higher value. Our studies identified many useful tools for future comparative analysis, and explored some novel ways to represent data in a visually appealing manner. As these tools and new ones continue to be developed, the value of genomics will increase with the new insights it provides.
Ph.D. in Biology, December 2016
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Title: DEGUELIN AS A THERAPEUTIC DRUG FOR TRIPLE NEGATIVE AND METASTATIC BREAST CANCER
Creator: Kalra, Amit
Date: 2011-12-13, 2011-12
Description: Breast cancer is a second leading cause of cancer related deaths. 15-20 % of women with Breast cancer are diagnosed with Triple negative...
Show moreBreast cancer is a second leading cause of cancer related deaths. 15-20 % of women with Breast cancer are diagnosed with Triple negative breast cancer (ER, PR, and HER2 –ve) which is the most aggressive type showing higher incidence of recurrence at the local as well as distant sites. Conventional chemotherapy, which shows high levels of toxicity, is the only option available for triple negative breast cancer as hormonal therapy does not work. Thus new drugs which could successfully inhibit the growth of triple negative breast cancer along with the inhibition of metastasis are highly desired. Deguelin, originally isolated from an African plant Mundulea sericea is shown to be effective in skin, lung, breast and colon cancer, effect is thought to be mediated by downregulation of the PI3K/AKT pathway. However, its effect on triple negative breast cancer in vitro and in vivo has not been evaluated. There are few in vivo experimental models to study the effect of novel drugs for the therapy of TNBCs. Murine 4T1 mammary breast cancer cell model is so far the most appropriate experimental model to study efficacy of new drugs on not only tumor growth but also on metastasis to different visceral organs including lungs. This model is relevant and mimics stage IV breast cancer in women. We evaluated the effects of Deguelin on triple negative and highly metastatic breast cancer cell line MDA MB 231 and also In vivo in the 4T1 breast cancer model. We also evaluated effect of Deguelin in combination with low dose (IC30) Taxol. Deguelin treatment alone inhibited the growth of MDA MB 231 breast cancer cells in a time and dose dependent manner causing S-G2 arrest. Deguelin induced apoptosis and inhibited cell proliferation. Deguelin also induced changes in the cell shape and subcellular distribution of the cytoskeletal (F-actin and Tubulin) and cell-cell attachment protein beta-catenin. Growth inhibitory effect was enhanced when Deguelin was given along with low dose Taxol. In vivo, Deguelin (2 or 6mg/kg body weight) administered daily for 21 days significantly (P<0.05) inhibited growth of 4T1 cells transplanted s.c. in to 4-6 weeks old female BALB/c mice. Interestingly, as compared to vehicle only, Deguelin treatment also significantly (P<0.02) reduced the number of metastatic lesions from intravenously injected 4T1 cells. Western blot analysis of several key signaling proteins in MDA MB 231 cells suggested that Deguelin (250nM) reduces PI3K, pAKT, and NF-κB protein levels. Immunohistochemical studies in 4T1 xenografts and metastatic lung lesions obtained from vehicle and Deguelin treated animals suggested that Deguelin reduces pAKT, COX2 and HIF-1 alpha, major key players involved in angiogenesis, cell proliferation and metastasis. In conclusion, our results show that Deguelin has growth inhibitory effect on TNBC cell lines; it inhibits in vivo and in vitro growth of murine mammary cancer cell line. Deguelin has anti metastatic activity in 4T1 experimental model. Anti proliferative and anti metastatic effects of Deguelin are mediated through down regulation of pAKT, COX2 and HIF-1 alpha. Deguelin alone or in combination to Taxol showed promising results and could be further developed as potential drugs for the treatment of triple negative breast cancer.
M.S. in Biology, December 2011
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Title: ELASTIC ELEMENTS IN THE SARCOMERES OF STRIATED MUSCLE
Creator: Ma, Weikang
Date: 2016, 2016-07
Description: The flight muscle of the Hawkmoth, Manduca sexta, is an emerging model system for structure and function studies. M. sexta flight muscle shows...
Show moreThe flight muscle of the Hawkmoth, Manduca sexta, is an emerging model system for structure and function studies. M. sexta flight muscle shows several interesting properties such as its length tension curve is similar with cardiac muscle, but the detailed protein compositions of M. sexta flight muscle is not known. Here we identified proteins that might be responsible for the elastic properties of M. sexta flight muscle. 1% vertical SDS-agarose gel electrophoresis combined with western blot analysis was used to separate and identify high molecular weight proteins in M. sexta flight muscle. Two projectin isoforms as well as two kettin isoforms were found in M. sexta flight muscle. In addition, two high molecular weight proteins were seen in agarose gels which turned out to be Sallimus (Sls) proteins isoforms based on the sallimus (sls) gene map. The localization and orientation of projectin and Sallimus proteins were determined by immuno-localization using confocal microscopy. The thin and thick filament lengths were also determined, and shown to be consistent with the length tension curve data. Knowledge of myofilament compliance is critical in interpreting cross bridges kinetics interpretation and modeling. Here we used small angle X-ray diffraction to study thick filament compliance in intact mouse soleus muscle. The thick filament compliance was estimated by plotting the spacing changes of myosin based meridional reflections against tension generated by the muscle during contraction. A non-linear relationship of thick filament compliance was seen for the first time. Nebulin is a giant thin filament protein and has been proposed to play significant roles in muscle physiology, but the underlying mechanisms are largely unknown. A conditional nebulin gene knockout mouse model was used here to study any structural and functional changes caused by nebulin deficiency using small angle X-ray diffraction. The thin filament compliance was estimated, and the results showed that the thin filament compliance in nebulin deficient muscle was larger than muscle from control animals, whereas no difference was seen in thick filament compliance between knockout and control muscles. The inter-filament spacing was larger in knock out muscle than in control muscle, and less force was generated by each cross bridge. The larger interfilament spacing and less force per cross bridge might explain the muscle weakness seen in both the knock out mouse model, as well as in nemaline myopathy patients. Other structural changes caused by nebulin deficiency was also characterized by small angle X-ray diffraction.
Ph.D. in Molecular Biochemistry and Biophysics, July 2016
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Title: MEMBRANE-DRUG INTERACTION MECHANISMS OF PEPTOID-BASED ANTIMICROBIAL AGENTS
Creator: Andreev, Konstantin
Date: 2017, 2017-05
Description: Nature is a major source of inspiration for drug design. Bacteria are developing resistance towards conventional antibiotics. Utilizing...
Show moreNature is a major source of inspiration for drug design. Bacteria are developing resistance towards conventional antibiotics. Utilizing antimicrobial peptides (AMPs) – an essential component of innate immune system, as therapeutic agents, may be a viable alternative. Unfortunately, there are a number of serious hurdles on the way towards clinical application of AMPs, including their low bioavailability, costly manufacturing process and toxicity against host cells. To address this issues, current research is focused on the design of synthetic compounds mimicking natural peptides, among which oligo(Nsubstituted glycines), or peptoids, have shown great promise. Antimicrobial drug efficacy is defined by how it interacts with the membrane of invading pathogen. The physicochemical characteristics of peptoid molecule play a crucial role in these interactions, yet their detailed structure-activity relationships remain obscure. Herein, we have demonstrated that conformational flexibility, cationic charge or hydrophobicity, are critical for oligomeric peptoids to permeate bacterial cell membranes. The outer surface of membrane was modeled by Langmuir monolayers of desired lipid composition and subjected to the constant-pressure insertion assays, epifluorescence microscopy (EFM), synchrotron X-ray reflectivity (XR) and grazing incident-angle X-ray diffraction (GIXD). Our results shed light on the critical details in peptoid mode of action. We believe this will aid in the rational design and of novel anti-infective drugs. Additionally, we have applied our experimental system to model the processes occurring at the air-water interface in lungs. Alveoli are coated by a complex lipidprotein mixture referred to as pulmonary surfactant. This facilitates respiration and prevents alveolar collapse. Patients with respiratory distress receive surfactant replacement therapy that often has the serious drawbacks. X-ray scattering data shows that the structural organization of adsorbed films correlates with surfactant delivery methods onto the respiratory surface. We anticipate that our findings will contribute to the development of novel clinical approaches for treating respiratory diseases.
Ph.D. in Biology, May 2017
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Title: Elastic Proteins in the Flight Muscle of Manduca Sexta
Creator: Yuan, Chen-ching
Date: 2012-05-01, 2012-05
Description: Unlike the asynchronous flight muscles of Lethocerus or Drosophila, the flight muscles (DLM1) of the Hawkmoth Manduca sexta are synchronous,...
Show moreUnlike the asynchronous flight muscles of Lethocerus or Drosophila, the flight muscles (DLM1) of the Hawkmoth Manduca sexta are synchronous, requiring a neural spike for each contraction. While the asynchronous muscles can only extend a few percent, Manduca flight muscle can reversibly extend 50% in vitro and 9% in vivo. Furthermore, the dorsal and ventral regions of the DLM1 have different power output presumably due to the temperature gradient across from the cooler dorsal to the warmer ventral subunits. Together with the observation that length-tension curves of Manduca flight muscles resemble mammalian cardiac muscle, these observations suggest that Manduca muscle might be a useful model system to study some aspects of cardiac muscle contractility. The detailed protein composition of Manduca flight muscle is not known. Here we aimed to identify proteins which might be responsible for the unique properties of Manduca muscle. We used 1% vertical SDS-agarose gel electrophoresis (VAGE) to separate the high molecular weight proteins in Manduca, Lethocerus, and Drosophila flight muscle and bovine left ventricular myocardium. The Manduca samples showed two bands around 2MDa and 1.6MDa, smaller than the two titin isoforms in bovine cardiac muscle, but larger than the largest Lethocerus proteins. Projectin and Kettin are elastic proteins found in Lethocerus and Drosophila with sequence homologies to vertebrate titin. Using western blots, the Manduca sample showed two bands cross-reacting with projectin antibodies at ~800 kDa and ~1030 kDa. Ventral (warmer) DLM1 subunits expressed higher level of both projectin isoform. The 1030 kDa projectin is the predominant expressed isoform in both dorsal and ventral subunits. Kettin antibodies cross-reacted to bands at the same position in both Lethocerus and Manduca. We also used western blots from 4-15% PAGE gels to detect a flightin –cross reacting band at around 23kDa in Lethocerus and 30 kDa in Manduca. Flightin is a thick filament associated protein that presumably helps filament assembly and stability. However, toponin I and T antibodies did not cross-react with similar molecular weight proteins from Manduca flight muscle. Thus, Manduca flight muscle has not only proteins homologous to Lethocerus projectin, kettin, flightin, but also several unknown high molecular weight proteins which might play a role in stabilizing sarcomere structure and give the muscle its unique mechanical properties.
M.S. in Biology, May 2012
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Title: A TIME COURSE STUDY OF FIBRONECTIN MATRIX ASSEMBLY ON SURFACES ACTIVATED WITH CELL AND FIBRONECTIN BINDING DOMAINS
Creator: Chiang, Chun-yi
Date: 2011-07, 2011-07
Description: Fibronectin mediates cell adhesion, migration and initiates extracellular matrix assembly. Therefore, it plays critical roles on wound healing...
Show moreFibronectin mediates cell adhesion, migration and initiates extracellular matrix assembly. Therefore, it plays critical roles on wound healing processes. The purpose of the study is to elucidate the effects of the combination of two functional domains, III1-2 and III9-10 subunits, on the fibronectin matrix assembly. In the present study, NIH 3T3 cells were cultured on the functionalized polyurethane (0.5 μM III9-10 and the mixture with 0.5 μM III9-10 and 0.5 μM III1-2). Functionalization enabled the formation of the covalent linkages between the coverslip polyurethane and the recombinant proteins. Surface characterization was examined by ELISA. NIH3T3 cells were plated on the functionalized coverslips and harvested after 3, 6, 12, and 24 hours culture. Total fibronectin and extracellular matrix fibronectin which is insoluble in deoxycholic acid (DOC) were analyzed through western blotting. The quantification results showed that the mixture groups tended to have higher total fibronectin than the III9-10 alone groups at most time point (3, 6, and 12 hours). Especially obvious differences of DOC-insoluble fibronectin between the mixture group and III9-10 alone group were found in the early stage (3 hour) of the fibronectin matrix assembly. Immunostaining images also showed that fiber-like FN had been generated after three hours culture in the mixture group but FN still looked faint in III9-10 group. Longer and larger FN fibrils were identified in the mixture groups than in III9-10 groups at all time points. In conclusion, III1-2 enhances fibronectin matrix assembly when present with III9-10 through increase the rate of the formation of DOC-insoluble fibronectin in 24 hours culture.
M.S. in Biology, July 2011
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