Biochemistry Commons^™

Microscopy Techniques For Investigating Interactions In Microbial Systems, Amanda Nicole Edwards

Doctoral Dissertations

Biological interactions occur on multiple length scales, ranging from molecular to population wide interactions. This work describes the study of two specific areas of biological interactions in microbial systems: intracellular protein-protein interactions and cell-to-cell interactions. The implementation of optical and atomic force microscopy and the methodologies developed during this study proved to be invaluable tools for investigating these systems.

Identifying and characterizing protein interactions are fundamental steps toward understanding complex cellular networks. We have developed a unique methodology which combines an imaging-based protein interaction assay with a fluorescence recovery after photobleaching technique (FRAP). Protein interactions are readily detected by co-localization …

Chemical Tools To Characterize Membrane-Protein Binding Interactions Using Synthetic Lipid Probes, Meng Meng Rowland

Doctoral Dissertations

Signaling lipids such as diacylglycerol (DAG) and the phosphatidylinositol polyphosphates (PIP_ns) play crucial roles in numerous cellular pathways. However, characterization of their activities is hindered by the complexity of associated signaling pathways and of the membrane environment. To address this issue, we have developed lipid probes that are effective for characterizing biological events using different applications, including activity-based probing (PIP_ns and DAG) and microarray analysis (PIP_ns). The activity-based probes have been applied to label receptor targets in multiple cancer cell proteomes through photocrosslinking followed by click reactions. The probes were found to label several …

Novel Constitutively Active Point Mutations In The Nh2 Domain Of Cxcr2 Capture The Receptor In Different Activation States, Giljun Park

Doctoral Dissertations

Chemokines are structurally and functionally related 8-10 kDa proteins defined by four conserved cysteine residues. They consist of a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and other cell types through binding to their respective chemokine receptor, a member of the GPCR family. Abnormal control of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the “on” position. This can lead to cellular transformation/tumorigenesis. A viral CAM receptor, ORF74, that can cause tumors in humans, also has homology to …

Dynamics Of The Toc Gtpases: Modulation By Nucleotides And Transit Peptides Reveal A Mechanism For Chloroplast Protein Import, Lovett Evan Reddick

Doctoral Dissertations

The chloroplast is the green organelle in the plant cell responsible for harvesting energy from sunlight and converting it into sugars and ATP. Origins of this organelle can be traced back to an endosymbiotic event in which a primitive eukaryotic cell capable of oxidative phosphorylation engulfed a free-living cyanobacterium capable of photosynthetic respiration (1). Immediately following this event the details are not clear, however what is known is that over the course of evolution, the engulfed cyanobacteria relinquished approximately 97% of its protein coding sequences to the host cell nucleus, thus making the newly formed chloroplast reliant on its host …

Nodulin 26-Like Intrinsic Protein Nip2;1 And Nip7;1: Characterization Of Transport Functions And Roles In Developmental And Stress Responses In Arabidopsis, Won-Gyu Choi

Doctoral Dissertations

Nodulin-intrinsic proteins (NIPs) are plant-specific, water and solute transporters with homology to soybean nodulin 26. In this study, it is shown that Arabidopsis NIP2;1 (AtNIP2;1) expression is acutely stimulated upon waterlogging (70-fold in whole seedlings within 1 hr) and hypoxia (> 1000-fold in roots within 2 hr). Subcellular localization of a AtNIP2;1::YFP fusion protein shows localization to the plasma membrane. Analysis of AtNIP2;1 protein in Xenopus oocytes shows that it is a transporter of lactic acid, a fermentation end product.

Experiments with T-DNA insertional mutants in the AtNIP2;1 promoter showed that reduced AtNIP2;1 expression induced higher lactic acid …

Micro, Nano Encapsulation Methods For Sustained Release Drug Formulations And Biomimetic Applications, Shantanu Balkundi

Doctoral Dissertations

The Layer-by-Layer (LbL) assembly technique was used to obtain a new type of protein/polyphenol microcapsule based on naturally occurring polyphenol (-)-epigallocatechin gallate (EGCG) and gelatin, type A. The dependence of permeability on the molecular weight of permeating substances was studied and compared with commonly used polyallylamine/polystyrene sulfonate capsules. A quartz crystal microbalance was used to monitor the regularities of EGCG adsorption in alternation with type A and B Gelatins and electrophoretic mobility measurements were used that indicated that the nature of assembly was dependent on Gelatin properties. It was shown that EGCG retains its antioxidant activity in the LbL assemblies. …

Gene Synthesis, Cloning, Expression, Purification And Biophysical Characterization Of The C2 Domain Of Human Tensin, Kiran Sukumar Gajula

Doctoral Dissertations

Tensin is a large "docking" protein found in the adhesive junctions of animal cells and recruited early in the development of cell-substrate contacts. There it binds to the cytoplasmic domain of integrin β1 and caps the barbed ends of filamentous actin. This forms a rational basis for its implication in a direct role in the mechanics of membrane-cytoskeleton interactions. Tensin provides a physical link between the actin cytoskeleton, integrins, and other proteins at the cell-substrate contacts. Its overall biochemical properties are a function of its domain composition and architecture, i.e., the domains that are present and their relative positions in …

High Resolution X-Ray And Neutron Crystallographic Studies Of Escherichia Coli Dihydrofolate Reductase, Brad C. Bennett

Doctoral Dissertations

Dihydrofolate Reductases (DHFRs) have been identified in nearly every proteome and are essential for most biosynthetic pathways involving one-carbon transfer reactions due to their recycling of tetrahydrofolate (THF). They catalyze the NADPH-dependent reduction of dihydrofolate (DHF), producing THF. Inhibition of DHFR ultimately depletes cellular pools of THF; causing a reduced supply of thymine nucleotides for DNA synthesis, resulting in genomic instability and cell death. Therefore, DHFRs remain important drug targets in antimicrobial and chemotherapeutic treatments. Despite exhaustive investigation of E. coli chromosomal DHFR, controversy persists over the dynamics of regulatory loops (the Met20, the βF-βG, and the βG-βH) and the …

Microcapsule Biosensors Based On Competitive Binding And Fluorescence Resonance Energy Transfer Assays, Swetha Chinnayelka

Doctoral Dissertations

Fluorescent sensing systems offer the potential for minimally invasive monitoring with implantable devices, but they require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility while maintaining adequate response characteristics. Towards the development of this goal, a general design of a biosensor with the capability of detecting different metabolites was investigated. The approach is based on the encapsulation of a competitive binding assay in microcapsules and monitoring the changes in fluorescence resonance energy transfer (FRET) in the presence of analyte. To experimentally demonstrate this type of sensing system, glucose was chosen as the model target analyte. The design, fabrication, and …

Structure–Function Studies Of The Yeast Saccharomyces Cerevisiae Α–Mating Factor Pheromone Receptor Ste2p, Ayça Akal–Strader

Doctoral Dissertations

G protein–coupled receptors (GPCRs) are seven transmembrane domain cell surface proteins that respond to a variety of environmental cues. Response of these receptors to their cognate stimuli on the extracellular region of the cell results in a concurrent activation of a complex series of intracellular signaling pathways that prepare the cell for the required adjustments through regulation of gene expression levels. Participation of GPCRs in such intricate signal transduction pathways renders them important players in human diseases. The GPCR family of proteins therefore represents one of the largest classes of proteins to be targeted in the development of drug design …

Layer-By-Layer Self -Assembly For Enzyme And Dna Encapsulation And Delivery, Amish Patel

Doctoral Dissertations

Thin wall microcapsules were formed via Layer-by-Layer Self-Assembly of alternate adsorption of oppositely charged polyelectrolyte on microcores. After the core dissolution, empty polymeric shells with 20–25 nm thick walls were obtained. These microcapsules were loaded with Myoglobin, Hemoglobin and Glucose Oxidase by opening capsule pores at low pH and closing them at higher pH. The native structure of the enzyme was not affected due to different treatments. Biocompatible nanoshells were also prepared for encasing DNA. Using the same Layer-by-Layer Self-Assembly approach nanoparticle were constructed containing DNA as one of the layers. The nanoparticles of different architecture were used to deliver …

Investigation Of Biochemical Mechanisms Associated With Insulin Resistance In The Non-Insulin-Dependent Diabetes Mellitus, To-Yu Huang

Doctoral Dissertations

Non-insulin dependent diabetes mellitus (NIDDM) is a heterogeneous disease arising from metabolic defects in insulin secretion and insulin action. The inability of insulin to trigger full metabolic responses results in impaired glucose transport, which elicits compensatory hyperinsulinemia with increased lipogenesis. In order to identify the molecular defects responsible for insulin resistance, we have investigated three different model systems: the ability of sulfonylurea receptor (SUR) ligands to modify insulin regulated events in cultured rodent adipocytes; the ability of agouti and/or agouti related polypeptide (AGRP) to modify insulin regulated events; and the regulation of the activity and amount of the HNF-1a transcription …

Analysis Of Events Governing The Meiotic Division In Mouse Spermatocytes, Shannon Stewart Eaker

Doctoral Dissertations

The meiotic division is essential for successful gametogenesis. However, many events occurring during male and female meiotic development remain poorly understood. While it is known that chromosomes must pair, recombine, and segregate to form gametes, critical questions remain. How and when do these events occur with respect to each other? What mechanisms monitor their developmental success? Insight into these questions is provided in this dissertation, using the mouse spermatocyte as a model. The purpose of this work is to aid in the overall understanding of mammalian meiosis.

After an introduction into mammalian meiosis in Part I, a temporal order of …