Browsing by Subject "Biochemistry"
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Item Application Of Superparamagnetic Suspension As A Hemostatic Agent: A Physical Study(2009) Corcoran, Henry Robert; Williams, Conrad M; Physics; Master of ScienceHemorrhaging remains the leading cause of preventable death in modern wars. The military invests heavily on solutions that increase the survivability of troops injured with puncturing wounds. The hemostatic utility of capturing superparamagnetic nanoparticles dispersed in the circulatory system with magnets was studied. The research investigated the utility of the technique by examining nanoparticle suspensions in a precision flow instrument modeled after the human circulatory system and comparing the results against a Brownian diffusion model.Item Biochemical Assays Of The Neisseria Meningitidis Serogroup W Capsule Polymerase(2018) Sharyan, Abeer; McCarthy, Pumtiwitt; Chemistry; Master of ScienceThe leading cause of bacterial meningitis is one of three bacteria: Streptococcus pneumonia, Haemophilus influenza or Neisseria meningitidis. Our goal in this work is to gain insight into the molecular mechanism of Neisseria meningitidis serogroup W capsule polymerase enzyme. This enzyme is crucial for building the carbohydrate-rich capsule that surrounds the outer surface of the bacteria. Capsular polysaccharides are an important structure that help the bacteria protect itself against the human immune system. The serogroup W capsule polymerase creates sialic acid galactose heteropolymers and uses CMP-sialic acid and UDP-galactose as nucleotide sugar donors. Better understanding of how this enzyme works can pave the way for drug and vaccine development against N. meningitidis. One goal of this research was to determine suitable fluorescent acceptors of this enzyme to follow product formation using HPLC with fluorescence detection. Many acceptors were investigated but the best acceptor was found to be fluorescently labelled serogroup W polysaccharide fragments. An-other goal of this research was to determine the kinetic parameters (Km and Vmax) for serogroup W capsule polymerase enzyme with its donor sugars. These kinetic parameters were determined for UDP-Galactose however further work is necessary for determining those for CMP-Sialic Acid. Finally, initial studies were performed to use molecular biology techniques to introduce mutations into the serogroup W capsule polymerase enzyme. Future work will build upon these studies to use more sensitive assays to determine kinetic parameters and create site-directed mutants of the enzyme.Item Characterization of the DNA binding activity of NZF-1 zinc binding domains upon replacement of zinc with iron.(2011-05-18) Lue Sue, Niall D.; Cymet, Holly J.; Master of ScienceItem Metal-Assisted And Microwave-Accelerated Evaporative Crystallization: Application To Lysozyme Protein(2015) Mauge-Lewis, Kevin A.; Aslan, Kadir; Seifu, Dereje; Biology; Master of ScienceIn response to the growing need for new crystallization techniques that afford for rapid processing times along with control over crystal size and distribution, the Aslan Research Group has recently demonstrated the use of Metal-Assisted and Microwave-Accelerated Evaporative Crystallization MA-MAEC technique in conjunction with metal nanoparticles and nanostructures for the crystallization of amino acids and organic small molecules. In this study, we have employed the newly developed MA-MAEC technique to the accelerated crystallization of chicken egg-white lysozyme on circular crystallization platforms in order to demonstrate the proof-of-principle application of the method for protein crystallization. The circular crystallization platforms are constructed in-house from poly (methyl methacrylate) (PMMA) and silver nanoparticle films (SNFs), indium tin oxide (ITO) and iron nano-columns. In this study, we prove the MA-MAEC method to be a more effective technique in the rapid crystallization of macromolecules in comparison to other conventional methods. Furthermore, we demonstrate the use of the novel iCrystal system, which incorporates the use of continuous, low wattage heating to facilitate the rapid crystallization of the lysozyme while still retaining excellent crystal quality. With the incorporation of the iCrystal system, we observe crystallization times that are even shorter than those produced by the MA-MAEC technique using a conventional microwave oven in addition to significantly improved crystal quality.Item Neisseria Meningitidis Serogroup W Capsule Polymerase: Determining Enzyme Kinetics Of Donor Sugars And Development Of A Chemoenzymatic Method To Produce A Photo-Crosslinking Derivative(2017) Gonzalez, Cendy I.; McCarthy, Pumtiwitt; Biology; Master of ScienceMost cases of bacterial meningitis are caused by one of three bacteria, Streptococcus pneumonia, Haemophilus influenza or Neisseria meningitidis. The overall aim of our work is to better understand the molecular mechanism of Neisseria meningitidis serogroup W capsule polymerase enzyme. The capsule that surrounds the outer surface of the bacteria is carbohydrate-rich. Capsular polysaccharides help protect the bacteria against the human immune system.The serogroup W capsule contains sialic acid-galactose heteropolymers and uses CMP-sialic acid and UDP-galactose as sugar donors during synthesis. Our long-term goal is to understand how this enzyme performs catalysis. This knowledge can provide new insight for drug and vaccine development against N. meningitidis. One goal of this thesis work was to determine the kinetic parameters (Km, Vmax, kcat) for each nucleotide donor sugar with the recombinant protein. An absorbance-based multi-enzyme method was used in which activity of the N. meningitidis serogroup W enzyme was linked to NADH oxidation. Kinetic parameters were established for both donors, however initial work suggests further testing is required for CMP-sialic acid. The second goal of this work was to develop a chemoenzymatic method to produce a photo-crosslinking derivative of CMP-sialic acid (diazirine modified, CMP-SiaDAz). A strategy, using two recombinant enzymes from the sialic acid biosynthetic pathway, was used first to synthesize CMP-Sialic acid. Once success was confirmed this same system was used to synthesize CMP-SiaDAz. Light-activated crosslinking of the product with N. meningitidis serogroup W protein was performed. Afterwards the serogroup W capsule polymerase was incubated with endoproteinase and peptide fragments were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry. Endoproteinase digestion was successful; however crosslinking analysis was inconclusive. This thesis work is an important step towards understanding catalysis by the N. meningitidis serogroup W capsule and future work will focus on optimizing current methods and repeating experiments.Item Synthesis And Fluorescence And In Vitro Cytotoxicity Studies Of Some Re(I) Diimine Complexes(2010) Orsa, David Hagar; Mandal, Santosh K.; Chemistry; Doctor of PhilosophyCancer is the leading cause of death in the world. There are conventional methods for cancer treatment, but each has its limitations or drawbacks. These circumstances have led researchers to look for new cytotoxic agents which could be used for cancer treatment. Here, convenient one-pot synthesis of several photoluminescent rhenium (I) diimine complexes and their screening for anticancer properties are presented. The rhenium(I) complexes include fac- CO3 (Bipy)ReCl (1), fac,- CO3 (Phen)ReCl (2), fac,-CO3 (2,9-Me2Phen)ReCl (3), fac,- CO3 (2,9-Me2-4,7-Ph2Phen)ReCl (4),CO3(Quinox)Re(&mu- O C 5 H11 ) 2 Re CO3 - (Quinox) (5), [CO3 Re((&mu- O C 5 H11 ) 2 Re CO3] 2 (&mu-Pyrazine) 2 (6), [CO35 H11 ) 2 Re(CO3 ] 2 (&mu-4,4'-Bipy) 2 (7), and [(CO32-Phen)Re(&µ-H)Re(2,9- Me2Phen)( CO3 ]+[( CO3) Re (&µ- O C 5 H11 ) Re CO3 ]- (8). The complexes have been characterized using spectroscopic techniques and the molecular structures of 3-8 have been confirmed through X-ray crystal structure determinations. As expected, these complexes exhibit fluorescence. The anticancer properties of 1-8 are evaluated using the human prostate cancer cell line (PC-3) and breast cancer cell line (MCF-7A). The results of this study demonstrate that these complexes have significant anticancer properties. For example, the complex 6 is highly effective against MCF-7A and the complex 1 is highly effective against PC-3. Therefore, these complexes can potentially find applications in the treatment of some cancers.