Life Sciences Commons^™

Telomere Length Dynamics In Aging Mice, Paul J. Comartin

Electronic Thesis and Dissertation Repository

Leukocyte telomere length (TL) shortens with age and is associated with age-related pathologies. However, inherited and acquired variation in telomere length in individuals complicates clinical interpretations of TL as a biomarker of aging and age-related pathologies. Therefore, it is critical to identify a post-mitotic tissue as a surrogate marker of TL at birth. In my thesis project, I used quantitative PCR to trace TL dynamics of a variety of tissue types of inbred mice during 1st year of life. I found that TL of smooth muscle of aortic media did not shorten with age and represents birth TL. Notably, birth …

Structural And Bioinformatic Analysis Of The B Subunit Of F-Atp Synthase, Ardeshir Goliaei

Electronic Thesis and Dissertation Repository

F₁F_o ATP synthases are rotary enzymes that produce most ATP in living organisms. The enzymes’ b₂δ subunits form a stator stalk that holds the F₁ sector against the torque of the rotor. In Escherichia coli, b₂ is an asymmetric homodimer. However, ATPases from some species have a heterodimer of subunits b and b’. Here, a modified E. coli ATP synthase containing a heterodimeric stator stalk was engineered by replacing residues 34-110 of E. coli b with sequences of Rhodobacter capsulatus b and b’, and expressing both chimeras. This produced a …

Structural Motifs Of Novel Metallothionein Proteins, Duncan E K Sutherland

Electronic Thesis and Dissertation Repository

Metallothioneins (MT) are a family of small cysteine rich proteins, which have been implicated in toxic metal detoxification, protection against oxidative stress, and as a metallochaperone. The most well studied member of the family is the mammalian MT, which consists of two domains: a β-domain with 9 cysteine residues, which sequesters 3 Cd²⁺/Zn²⁺, and an α-domain with 11 cysteine residues, which sequesters 4 Cd²⁺/Zn²⁺. The exact functions of MT are unknown but must relate to its metalation status. Several areas that could lead to the assignment of function include 1) the determination …

Mass Spectrometry-Based Proteomics Analysis Of The Matrix Microenvironment In Pluripotent Stem Cell Culture, Christopher Hughes

Electronic Thesis and Dissertation Repository

The stem cell microenvironment contains soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to effect cellular behavior. Mass spectrometry based proteomics offers the opportunity to directly assay components of extracellular microenvironments, thereby providing a sensitive means for obtaining insight into the stem cell niche. In this study we present the generation and analysis of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) matrix microenvironments using an MS-based proteomics approach.

One of the primary limitations in the proteomics analysis of hESCs and hiPSCs is the reproducible generation of sufficient cell numbers amenable …

Molecular Dynamics Simulations Of Peptide-Mineral Interactions, Susanna Hug

Electronic Thesis and Dissertation Repository

We present molecular dynamics (MD) simulations providing information about the mechanisms of biomineralization. We focus on osteopontin-related peptides, which inhibit the growth of calcium oxalate monohydrate (COM) the primary constituent of kidney stones.

First, we performed two ab initio MD simulations: aspartic acid (Asp) and the dimer of aspartic acid and phosphoserine (Asp-pSer) interacting with a fully hydrated COM crystal slab exposing the {100} face. For Asp we found that one of the carboxyl and the amine group both interact with the crystal surface but neither forms a stable contact during the simulation. Asp-pSer interacts preferably with its carboxyl groups …

Atpase Regulation In The Maltose Transporter, Alister D. Gould

Electronic Thesis and Dissertation Repository

This thesis investigates the mechanism of activity-coupling in the maltose transporter of Escherichia coli (MalFGK₂); the way ATP hydrolysis is prevented in the absence of maltose, and then enabled to drive maltose transport. Like other ATP binding cassette importers, MalFGK₂ requires substrate to be presented by a peripheral substrate-binding protein, in this case the maltose binding protein (MBP). MBP predominantly adopts an ‘open’ resting state, but undergoes a rotation of its two domains to a ‘closed’ state after maltose binding. In the closed state MBP is able to activate MalFGK₂ to stimulate ATP hydrolysis and maltose …

Structural Characterization Of Hip2 Enzyme Interactions In Ubiquitination, Benjamin W. Cook

Electronic Thesis and Dissertation Repository

The ubiquitin proteolysis pathway utilizes three enzymes, an E1 activating enzyme, an E2 conjugating enzyme and an E3 ligating enzyme, to respectively activate, transfer and ligate ubiquitin (Ub) onto a substrate protein. The creation of a K48-linked poly-Ub chain on a substrate will target this protein to be degraded by the 26S proteosome. E2 conjugating enzymes are central proteins in this pathway and interact with the E1 and E3 enzymes to perform Ub transfer. The mechanism by which Ub molecules are interconnected remains poorly understood. The E2 enzymes HIP2 and Ubc1 have been shown to create poly-Ub chains in the …

Structure-Function Analysis Of Enzymes Of The Polyisoprenyl-Phosphate Hexose-1-Phosphate Transferase Family, Kinnari B. Patel

Electronic Thesis and Dissertation Repository

Enzymes of the polyisoprenyl-phosphate hexose-1-phosphate transferase (PHPT) family are integral membrane proteins that initiate the synthesis of glycans by catalyzing the transfer of a hexose-1-phosphate sugar from UDP-hexose to the lipid carrier undecaprenyl phosphate (Und-P). These glycans such as O antigen and exopolysaccharide (EPS) provide bacteria with protection and adaptation to the environment and host immune factors. The role of PHPT proteins in initiation and the absence of any eukaryotic homologues make them an attractive target for novel antimicrobials; however study of these proteins is difficult due to the presence of multiple transmembrane helices. A requirement of the C-terminal domain …

Mitochondrial Metabolic Suppression And Reactive Oxygen Species Production During Hypometabolism In Mammals, Jason Cl Brown

Electronic Thesis and Dissertation Repository

During hibernation, daily torpor, and fasting, mammals reduce metabolic rate (MR) up to 99%, 95%, and 30%, respectively, compared to resting levels. Mitochondrial metabolic suppression likely contributes to this MR reduction, and the first objective of this study was to determine the relative contributions of active, regulated inhibition and passive thermal effects as body temperature (T_b) falls, to mitochondrial metabolic suppression, and to examine the mechanisms involved using top-down elasticity analysis and novel statistical approach. The second objective of this study was to determine how mitochondrial metabolic suppression affects mitochondrial reactive oxygen species (ROS) production, a topic which …

Regulation Of G Protein Signaling By Goloco Motif Containing Proteins, Peishen Zhao

Electronic Thesis and Dissertation Repository

Signal transduction via heterotrimeric G proteins in response to transmembrane G protein-coupled receptors plays a central aspect in how cells integrate extracellular stimuli and produce biological responses. In addition to receptor-mediated activation of heterotrimeric G proteins, during the last few decades, accessory proteins have been found to regulate G protein activity via different mechanisms. Several proteins have been identified that contain multiple G protein regulatory domains. Using various molecular and biochemical approaches, we have characterized the effects of two such proteins, G18 and RGS14, on G protein activity. Both proteins contain a second G protein binding domain in addition to …

Dissecting The Molecular Role Of Distinct Binding Interfaces On The Retinoblastoma Tumor Suppressor In Growth Control And Tumorigenesis., Matthew J. Cecchini

Electronic Thesis and Dissertation Repository

The retinoblastoma tumor suppressor protein (pRB) functions to maintain proliferative control and act as a barrier to tumorigenesis. pRB is capable of regulating E2F transcription factors to mediate control of proliferation through transcriptional regulation of S-phase target gene expression. In addition, pRB can stabilize the CDK inhibitor p27 through an interaction with two ubiquitin ligase complexes. Further, pRB is capable of forming a unique interaction with E2F1 termed the ‘specific’ interaction that is capable of blocking E2F1 induced apoptosis. These functions of pRB are mediated by distinct binding interfaces and their contributions to the overall functionality of pRB are not …

Transactivation By Human Adenovirus Early Region 1a-Conserved Region Three, Jailal Ng Ablack

Electronic Thesis and Dissertation Repository

One of the critical functions of human adenovirus (hAd) early region 1A (E1A) protein is to activate transcription of the early viral genes. The largest isoform of E1A contains a unique region termed conserved region 3 (CR3), which includes a Cysteine-4 (C4) zinc finger domain. This region activates viral gene expression by interacting with and recruiting cellular transcription machinery to the regulatory regions of early viral genes. Although this process has been studied at length with hAd type 5 E1A, far less is known about how the E1A proteins from other hAd types activate transcription. There are dramatic differences in …

The Role Of The Mcm2 Subunit In Regulating The Activities Of The Mcm2-7 Helicase, Brent E. Stead

Electronic Thesis and Dissertation Repository

The transmission of genetic information from parental to daughter cells requires the faithful duplication of an organism’s genome. Uncontrolled DNA replication can result in proliferative diseases, such as cancer. DNA replication requires a single-stranded DNA template to be produced from duplex DNA. In eukaryotes, DNA unwinding for replication is performed by the heterohexameric replicative helicase complex comprised of the minichromosome maintenance proteins 2 through 7.

Each of the Mcm2-7 subunits likely has a unique role in DNA binding and unwinding by the Mcm2-7 complex. The present study examines the role of the Saccharomyces cerevisiae Mcm2 subunit in regulating the activities …

Identification Of Regions Responsible For The Open Conformation Of S100a10 Using Chimaeric S100a11/S100a10 Proteins, Liliana Santamaria-Kisiel

Electronic Thesis and Dissertation Repository

S100A11 is a dimeric, EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2), and facilitate membrane vesiculation events. In contrast to other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an “open” conformation that is very similar to S100A11 in its calcium-bound state (Ca2+-S100A11). To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimeric proteins were constructed where regions …

Mitotic Regulation Of Protein Kinase Ck2, Nicole A. St. Denis

Electronic Thesis and Dissertation Repository

Protein kinase CK2 is a serine/threonine kinase with a multitude of substrates and roles in many cellular processes, including mitosis. CK2 is constitutively active, yet we hypothesize that CK2 is indeed regulated in mitosis through subtle means, enabling CK2 to perform its functions unique to cell division. Our aims were to examine the roles of mitotic phosphorylation, subcellular localization, and interplay with mitotic kinases in the regulation of CK2 activity.

We first examined the role of four highly conserved mitotic phosphorylation sites located in the unique C-terminus of CK2α. Phosphospecific antibodies generated against the sites show that CK2α phosphorylation is …