Life Sciences Commons^™

A Role Of Yeast Adhesin Amyloids In Force-Dependent Adhesion And Biofilm Formation, Cho Xiao Juan Chan

Dissertations, Theses, and Capstone Projects

Candida albicans adhesins have amyloid-forming sequences (Ramsook et al. 2010, Otoo et al. 2008). Similarly, Tango and Waltz predicted that amyloid-forming sequences are also present in Saccharomyces cerevisiae flocculins, Flo1p and Flo11p. The cell surface of Flo1p- and Flo11p-expressing cells have ordered domains that are thioflavin T fluorescent and Congo red birefringent, two hallmarks of amyloids. Flo1p and Flo11p amyloids were important for activities of the flocculins including cell-to-cell adhesion, cell-to-substrate adhesion, and agar invasion. In addition, amyloid-perturbing dyes thioflavin S and Congo red inhibited aggregation, biofilm formation and agar invasion.

Force-induced formation and propagation of adhesion nanodomains in Als5p-expressing …

Design And Optimization Of A De Novo Protein Charge Separation Dyad, Andrew C. Mutter

Dissertations, Theses, and Capstone Projects

The ever-increasing demand for cheap, plentiful energy to fuel the needs of a growing population requires research into alternative clean energy. Solar irradiation has the potential to power the planet many times over; the challenge is efficient capture and conversion of this energy source. Nature has already solved this problem with photosynthesis, which harvests solar irradiation converting it to stored chemical energy and is the source of the energy for life. The goal of my dissertation is to use de novo designed protein to mimic the charge separation system in photosynthesis. A stable protein scaffold will be designed and used …

Development Of Cell-Active Inhibitors And Activity-Based Probe Of Cysteine Cathepsins, Dibyendu Dana

Dissertations, Theses, and Capstone Projects

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. Still however, many of their cellular function remain poorly understood. Chemical biology approaches employing small molecules can be utilized for this purpose. Unfortunately small molecule probes that are cell-permeable and non-peptidyl in nature are scarcely available.

In this work, first a library of sulfonyloxiranes is synthesized. From this library, 2-(2-ethylphenylsulfonyl)oxirane is identified as a selective inhibitor of cysteine cathepsins. Cell-based study reveals that 2-(2-ethylphenylsulfonyl)oxirane is a cell-permeable, covalent, and irreversible inhibitor of cathepsin B with …

Identification And Characterization Of Protein Kinase C Substrates In Human Breast Cells, Xin Zhao

Dissertations, Theses, and Capstone Projects

Aberrations in PKC signaling can lead to the development of multiple human diseases and the most prominent association of PKC with disease has been in tumor growth and metastasis. PKC and its related pathways have been recognized as promising targets for blocking the malignancy of breast cancer cells. To better understand PKC-mediated pathway in breast cancer cells, it is important to identify the cellular substrates of PKC. The main focus of this work is to identify physiologically relevant cellular substrates of PKC in human breast cells and to characterize their roles in cancer-related phenotypes. The work to be described consists …

The Role Of The Striatal Neuropeptide Neurotensin In The Methamphetamine-Induced Neural Injury In Mice, Qingkun Liu

Dissertations, Theses, and Capstone Projects

Methamphetamine (METH) is a widely abused psychostimulant that induces neurotoxicity to several brain regions, including the striatum. Similar to dopamine (DA) in chemical structure, METH can be transported into DA pre-synaptic terminals, evoking the neurodegeneration in DA terminals and post-synaptic striatal neurons. Despite the critical role of DA in METH-induced neurodegeneration, no pharmaceutical therapeutics has been approved for these conditions. It is therefore essential to investigate the endogenous factors regulating the dopaminergic system. The neuropeptide neurotensin has emerged as a potential modulator of METH-induced striatal neurodegeneration mainly due to its intimate interactions with dopamine in the striatum.

In this study, …

Venomic Characterization Of The Terebridae And Novel Terebrid Neuropeptides, Mary Elizabeth Wright

Dissertations, Theses, and Capstone Projects

Unravelling the complex mixture of neuropeptides produced by the terebrid venom duct holds the promise of discovering tomorrow's therapeutics. Cone snails have already demonstrated the potential found in the venom of these unusual marine organisms, through the commercial approval of drugs for pain and other indications. Terebrids, as the sister family to the cone snails, have been much less investigated, but have a species richness that makes them very attractive in the search for novel neuropeptides. The venomics research described in this work encompasses the first comprehensive characterization of the terebrid venom duct transcriptomes of two species, Cinguloterebra anilis and …

Fabrication And Characterization Of Sol-Gel Based Nanoparticles For Drug Delivery, Reeta Yadav

Dissertations, Theses, and Capstone Projects

Nanogels are cross linked polymeric sol-gel based nanoparticles that offer an interior network for incorporation and protection of biomolecules, exhibiting unique advantages for polymer based delivery systems. We have successfully synthesized stable sol-gel nanoparticles by means of [a] silicification reactions using cationic peptides like polylysine as gelating agents, and [b] lyophilization of sol-gels. Macromolecules such as Hemoglobin and Glucose Oxidase and small molecules such as Sodium Nitroprusside (SNP) and antibiotics were encapsulated within the nanogels. We have used transmission electron microscopy, dynamic light scattering, zeta potential analysis, and spectroscopy to perform a physicochemical characterization of the nanogels resulting from the …

Deriving High-Resolution Protein Backbone Structure Propensities From All Crystal Data Using The Information Maximization Device, Armando D. Solis

Publications and Research

The most informative probability distribution functions (PDFs) describing the Ramachandran phi-psi dihedral angle pair, a fundamental descriptor of backbone conformation of protein molecules, are derived from high-resolution X-ray crystal structures using an information-theoretic approach. The Information Maximization Device (IMD) is established, based on fundamental information-theoretic concepts, and then applied specifically to derive highly resolved phi-psi maps for all 20 single amino acid and all 8000 triplet sequences at an optimal resolution determined by the volume of current data. The paper shows that utilizing the latent information contained in all viable high-resolution crystal structures found in the Protein Data Bank (PDB), …

Recruitment Of The Ribosomal 40s Subunit To The 3'Untranslated Region Of A Viral Mrna, Via The Eif4 Complex, Facilitates Cap-Independent Translation, Sohani Das Sharma

Dissertations, Theses, and Capstone Projects

Translation of uncapped plant viral RNAs can be facilitated by either an internal ribosomal entry site (IRES) in the 5' untranslated region (UTR) or a cap-independent translation element (CITE) in the 3' UTR. Barley yellow dwarf virus (BYDV) mRNA, which lacks both cap and poly(A) tail, has a translation element (3'BTE) in its 3' UTR that is essential for efficient translation initiation at the 5'-proximal AUG. This mechanism requires binding of the eukaryotic initiation factor 4G (eIF4G) subunit of the heterodimer eIF4F to the 3'BTE and base pairing between the 3'BTE and the 5' UTR. Here we investigate how this …

Interactions Of Eukaryotic Translation Initiation Factors And 3' Untranslated Region Of Barley Yellow Dwarf Virus Mrna During Protein Synthesis: A Study Of Equilibrium Binding, Kinetics And Thermodynamics, Bidisha Banerjee

Dissertations, Theses, and Capstone Projects

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley Yellow Dwarf Virus (BYDV) employs a cap-independent mechanism of translation initiation which is mediated by a structural element BTE (BYDV translation element) located in the 3’ UTR of its mRNA. eIF4F bound the BTE and a translational inactive mutant with high affinity; thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5-164% compared to WT were studied. Using fluorescence anisotropy …

Mechanisms Of Regulation Of Mrna 3' Processing By P53 Pathway, Emral Cakmak Devany

Dissertations, Theses, and Capstone Projects

Although the p53 network has been intensively studied, genetic analyses long hinted at the existence of components that remained elusive. This dissertation focuses on the study of the regulation of mRNA 3' processing during DNA damage response (DDR) by the p53 pathway and the regulation of p53 expression by the mRNA 3' processing machinery. The results in this dissertation revealed new roles of tumor suppressor p53 in mRNA 3' processing. In Chapter II, I showed that p53 inhibits the cleavage step of polyadenylation reaction and that cells with different levels of p53 expression have different mRNA processing profiles. As part …

Structure And Function In Bacteriophage Phi6, James Carpino

Dissertations, Theses, and Capstone Projects

The present study of bacteriophage Phi6 has been preceded by a great number of exploratory studies of its structure and function, and these studies have formed a basis for Phi6's development into a model organism. In this study, two aspects of the model organism have been examined. 1. There are several uncharacterized and presumed untranslated regions (UTRs) in Phi6's 13.3 kilobase-pair dsRNA genome. I examined the impact of specific modification to the 3' UTR of the small segment of bacteriophage Phi6. I determined that modification to the purported UTR of the small segment resulted in severe fitness costs, supporting a …

Role Of The Polyadenylation Factor Cstf-50 In Regulating The Brca1/Bard1 E3 Ubiquitin (Ub) Ligase Activity, Danae Fonseca

Dissertations, Theses, and Capstone Projects

The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. The studies presented in this dissertation focus on the determination and characterization of the role of mRNA processing factor CstF-50 and escort protein p97 in the regulation of the BRCA1/BARD1 E3 ubiquitin (Ub) ligase activity during the DNA damage response (DDR).

As part of the studies presented in Chapter II, I determined that the polyadenylation factor CstF plays a direct role in DDR, specifically in transcription-coupled repair (TCR), and that it localizes with RNA polymerase II (RNAP II) and BARD1 to sites of …

An Optogenetic Gene Expression System With Rapid Activation And Deactivation Kinetics, Laura B. Motta-Mena, Anna Reade, Michael J. Mallory, Spencer Glantz, Orion D. Weiner, Kristen W. Lynch, Kevin H. Gardner

Advanced Science Research Center

Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range, or slow activation/deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach utilizes an engineered version of EL222, a bacterial Light-Oxygen-Voltage (LOV) protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (< 10 s) and deactivation kinetics (< 50 s), and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

Mechanism Of Phospho-Alpha-Tubulin-Driven Motility In Human Breast Epithelial Cells, Shatarupa De

Dissertations, Theses, and Capstone Projects

Protein kinase C (PKC), an enzyme important in signaling pathways that give rise to various cell phenotypes, has been closely associated with metastatic phenotypes of breast cancer. Tissues from patients of varying degrees of tumorgenicity have shown an elevated expression of the PKCα isoform as well as other diacylglycerol (DAG)-sensitive isoforms. Moreover, independent studies with highly invasive breast cell lines, such as MDA-MB-231 cells, have also shown an elevated level of PKCα. Therefore, PKCα has long been a therapeutic target for breast cancer. Among its known substrates (recently identified by our laboratory), α6-tubulin will, upon phosphorylation by PKC, enhance migration …

Iron Regulates Mrna Translation Initiation Through Rna Iron Responsive Element (Ire), Jia Ma

Dissertations, Theses, and Capstone Projects

The non-coding IRE-RNA structure, a 30 nt stem loop structure, regulates synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA binding with iron regulatory protein (IRP) proteins inhibits ribosome accessing mRNA. Increasing iron concentration decreases IRP binding with IRE-RNA. Previous models of IRE-mRNA translation regulation concentrate on Fe-S binding to IRP and IRP degradation after release from IRE-RNA. These models lack information on the details of decreasing IRE-RNA/IRP protein binding with iron concentration elevation. This research shows 1. Eukaryotic initiation factor 4F (eIF4F) binds to IRE-RNA with high affinity and works as a positive control element …

Polymerase Alpha Components Associate With Telomeres To Mediate Overhang Processing, Raffaella Diotti

Dissertations, Theses, and Capstone Projects

Telomeres consist of TTAGGG repeats, which end with a 3' G-overhang and are bound by a six-protein complex, known as Shelterin. In humans, telomeres shorten at each cell division, unless telomerase is expressed and able to add telomeric repeats to the 3' G-overhang. However, for effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. In this study, I focused on the Polα/primase complex, in particular the subunits p68 (POLA2, the regulatory subunit) and p180 (Polα, the catalytic subunit), and their potential roles at telomeres. I was able to detect p180, p68 and OBFC1, a …

Lipid Dependence In Ras-Driven Tumors, Darin Salloum

Dissertations, Theses, and Capstone Projects

Over past decade, metabolic alterations in cancer cells have received a substantial amount of interest. It had been established that cancer cells undergo a significant amount of metabolic alterations, and some of these alterations are similar to those in normal highly proliferative cells. However, it is becoming more apparent that many of the metabolic alterations are specific to particular oncogenic signaling pathways. Although altered metabolic machinery makes cancer cells more efficient at promoting growth when nutrients are supplied at the sufficient amounts, the dependency of cancer cells on particular metabolic reprogramming deems cancer cells susceptible to disruptions within metabolic network. …

Conformational Features Of The Human U2-U6 Snrna Complex, Ravichandra Bachu

Dissertations, Theses, and Capstone Projects

The splicing of precursor messenger (pre-m) RNA, during which noncoding intervening sequences are excised and flanking coding regions ligated, is an integral reaction of gene expression. In eukaryotes, it is carried out by a dynamic RNA-protein complex called the spliceosome, in which five small nuclear (sn) RNA components are actively involved in recognition and chemical aspects of the process. A complex formed between U2 and U6 snRNAs is implicated in the chemistry of pre-mRNA splicing. The catalytic activity of the U2-U6 snRNA complex is dependent on the presence of Mg2+ ions, and the complex has been shown to have several …

Mechanisms Of Deadenylation Regulation Under Different Cellular Conditions, Xiaokan Zhang

Dissertations, Theses, and Capstone Projects

Control of gene expression by regulating mRNA stability after DNA damage has the potential to contribute to the cells rapid response to stress. The main focus of this dissertation is to elucidate the role(s) of nuclear PARN deadenylase in controlling mRNA stability, hence gene expression, of factors in the p53 signaling pathway during the DNA damage response (DDR). Understanding the mechanisms of these regulatory pathways will provide new insights on how the control of gene expression upon DNA damage decides cellular fate, offering new opportunities for therapeutics. In Chapter II, I presented evidence that PARN along with the cleavage factor …

The Sex Of The Cell Dictates Its Response, Carlos Ganesh Penaloza

Dissertations, Theses, and Capstone Projects

Male and female differences in frequency of occurrence in disease have perplexed scientists for some time. This in part derives from limitations in the systems in which one can evaluate sex differences. At the organismal level, differences can be hidden by a myriad of extensive and complex factors. Additional limitations exist since most biomedical studies are performed almost exclusively on male subjects, as the female hormonal milieu is intrinsically more variable and too troublesome for routine inclusion in research protocols. Research documenting sex differences continues to grow, and while most researchers suggests that sex hormones are at the core of …

An Experimental Investigation Into The Mechanisms Of Bacterial Evolution, Zhenmao Wan

Dissertations, Theses, and Capstone Projects

This thesis studies the two fundamental mechanisms of bacterial evolution — horizontal gene transfer and spontaneous mutation, in the bacterium Escherichia coli through novel experimental assays and mathematical simulations. First, I will develop a growth assay utilizing the quantitative polymerase chain reaction (qPCR) to provide real-time enumeration of genetic marker abundance within bacterial populations. Second, I will focus on horizontal gene transfer in E. coli occurring through a process called conjugation. By fitting the qPCR data to a resource limited, logistic growth model, I will obtain estimated values of several key parameters governing the dynamics of DNA transfer through conjugation …

Identification And Characterization Of Protein Phopshatases Regulating The Sma/Mab Pathway In C. Elegans, Sheng Xiong

Dissertations, Theses, and Capstone Projects

TGF-beta signaling is a conserved signaling pathway among eukaryotes, which controls various normal cellular responses from cell proliferation to cell death. The mutations in its components are found in developmental disorders and cancer. Therefore, this signaling pathway is extensively investigated so that new therapeutic targets could be discovered and novel drugs could be developed. Previous studies suggested the involvement of phosphatases in regulation of TGF-beta signaling, but these studies were performed in cell culture rather than intact organisms. C. elegans is a tractable organism in which to study signaling in vivo. In C. elegans, growth is controled by a conserved …

Computational Insights Into The Oxygen Evolving Complex Of Photosystem Ιι, Muhamed Amin

Dissertations, Theses, and Capstone Projects

The Oxygen Evolving Complex (OEC) of Photosystem II (PSII) is a unique Mn4O5Ca2+ cluster that catalyzes the photoactivated water splitting reaction. The OEC is a model system for bio-inspired artificial systems to use solar energy to pull electrons from water to produce fuel. The OEC goes through a cycle of 5 S states storing 4 holes, via electron transfer to P680+, the primary electron donor in PSII to generate a high valence S4 state that oxidizes water. The key questions are what controls the order of oxidation and deprotonation of the OEC complex and how does the PSII protein modulate …

Metabolic Checkpoints In Cancer Cell Cycle, Mahesh Saqcena

Dissertations, Theses, and Capstone Projects

Growth factors (GFs) as well as nutrient sufficiency regulate cell division in metazoans. The vast majority of mutations that contribute to cancer are in genes that regulate progression through the G1 phase of the cell cycle. A key regulatory site in G1 is the growth factor-dependent Restriction Point (R), where cells get permissive signals to divide. In the absence of GF instructions, cells enter the quiescent G0 state. Despite fundamental differences between GF signaling and nutrient sensing, they both have been confusingly referred to as R and therefore by definition considered to be a singular event in G1. Autonomy from …

Roles Of Igh Intronic Enhancer Eμ In Clonal Selection At The Pre-B To Immature B Cell Transition And In The Elimination Of Autoreactive B Cells, Cheng Peng

Dissertations, Theses, and Capstone Projects

The immunoglobulin heavy chain locus (Igh ) intronic enhancer, Eμ, enhances transcription of recombined Igh genes. We have previously shown that in mice with an Eμ-deficient Igh allele (V _H δ^a ), Igμ is expressed at half of the wild-type levels in pre-B cells. We also described an Eμ-dependent "check-point", operating at the pre-B to immature B cell transition, for heavy chain allelic exclusion. We now show that deletion of Eμ results in a smaller immature B cell compartment, and the pre-BCR/BCR signaling is diminished in pre-B cells as a result of the reduced Igμ levels, making it …

Spatial Gaussian Markov Random Fields: Modelling, Applications And Efficient Computations, Yu Ryan Yue, Xiao-Feng Wang

Publications and Research

A powerful modelling tool for spatial data is the framework of Gaussian Markov random fields (GMRFs), which are discrete domain Gaussian random fields equipped with a Markov property. GMRFs allow us to combine the analytical results for the Gaussian distribution as well as Markov properties, thus allow for the development of computationally efficient algorithms. Here we briefly review popular spatial GMRFs, show how to construct them, and outline their recent developments and possible future work.