Biochemistry, Biophysics, and Structural Biology Commons^™

Sirt1 Regulation Of The Heat Shock Response In An Hsf1-Dependent Manner And The Impact Of Caloric Restriction, Rachel Rene Raynes

USF Tampa Graduate Theses and Dissertations

The heat shock response (HSR) is the cell's molecular reaction to protein damaging stress and is critical in the management of denatured proteins. Activation of HSF1, the master transcriptional regulator of the HSR, results in the induction of molecular chaperones called heat shock proteins (HSPs). Transcription of hsp genes is promoted by the hyperphosphorylation of HSF1, while the attenuation of the HSR is regulated by a dual mechanism involving negative feedback inhibition from HSPs and acetylation at a critical lysine residue within the DNA binding domain of HSF1, which results in a loss of affinity for DNA. SIRT1 is a …

Regulation Of The Tumor Suppresser P53 And Survivin By Ras And Ral Gtpases:Implications For Malignant Transformation, Awet G. Tecleab

USF Tampa Graduate Theses and Dissertations

Abstract

Although the critical role of the small GTPases Ras and Ral in oncogenesis has been well documented, much remains to be investigated about the molecular mechanism by which these GTPases regulate malignant transformation. The work under this thesis made two major contributions to this field. The first is the discovery that K-Ras, RalA and/or RalB are required for the maintenance of the high levels of the anti-apoptotic protein survivin in some human cancer cells, and the second is the demonstration that down regulation of K-Ras, RalA and/or RalB, but not Raf-1 or Akt1/2, stabilizes the tumor suppressor p53 and …

Epigenetic Modifiers To Augment The Immunogenicity Of Chronic Lymphocytic Leukemia, Jason A. Dubovsky

USF Tampa Graduate Theses and Dissertations

Cancer cells employ a litany of immunosuppressive and immunevasive strategies to avoid detection and elimination by the various arms of the innate and adaptive immune system. Many hematologic and solid tumors progressively develop a specialized microenvironment which promotes tissue restructuring inflammation while masking the immune signature of the tumor cells themselves. Chronic lymphocytic leukemia, a malignancy of mature B lymphocytes must constantly balance on the precipice of immune recognition. A mature antigen presenting cell themselves, CLL clonal growth is dependent on the very interactions which, if effective, could potentially lead to their demise. To circumvent this, CLL clones set up …

Structure, Dynamics, And Evolution Of The Intrinsically Disordered P53 Transactivation Domain, Wade Michael Borcherds

USF Tampa Graduate Theses and Dissertations

in numerous disease states, including cancers and neurodegenerative diseases. All proteins are dynamic in nature, occupying a range of conformational flexibilities. This inherent flexibility is required for their function, with ordered proteins and IDPs representing the least flexible, and most flexible, respectively. As such IDPs possess little to no stable tertiary or secondary structure, they instead form broad ensembles of heterogeneous structures, which fluctuate over multiple time scales. Although IDPs often lack stable secondary structure they can assume a more stable structure in the presence of their binding partners in a coupled folding binding reaction.

The phenomenon of the dynamic …

Optical Investigations Of Neurohypophysial Excitability And Amyloid Fibril Formation, Joseph Leo Foley

USF Tampa Graduate Theses and Dissertations

This dissertation describes the work done on two distinct projects. In the first part I sought to unravel the mechanisms that underlie the activity-dependent modulation of response in the excitation-secretion coupling of the neurohypophysis. In the second part, I optically monitored and analyzed the secondary structure changes accompanying amyloid fibril formation along multiple pathways, under both denaturing and near-physiological conditions.

Neuronal plasticity plays an important role in regulating various biological systems by modulating release of hormones or neurotransmitters. The changing response to the same stimulus, depending on the context and previous stimulation events, is also the basis of learning and …

Immobilization And Characterization Of Physisorbed Antibody Films Using Pneumatic Spray As Deposition Technique, Jhon J. Figueroa

USF Tampa Graduate Theses and Dissertations

The immobilization of antibodies on silica surfaces has been a wide and common practice via cross-linking with the formation of covalent bonds between surface and antibody. The formation of antibody thin films on solid surfaces using pneumatic spray (PS) as the deposition technique and the analysis of the surface morphology of these films were investigated during this study. The pneumatic spray method was compared with the covalent bonding method Avidin-Biotin Bridge (ABB). The intensities and capture efficiency tests showed similar results for both techniques with a lower signal-to-noise ratio (SNR) for the PS deposited films. Specificity tests suggested that the …

Determination Of Structural Stabilities Of Metal-Free Porphyrin Cytochrome C, Zinc Cytochrome C, And Cobalt Cytochrome C, Usha K. Tottempudi

Masters Theses

No abstract provided.

Proteolytic Processing Of The Amyloid Precursor Protein During Apoptosis And Cell Cycle: Implications For Alzheimer's Disease, Tina N. Fiorelli

USF Tampa Graduate Theses and Dissertations

Alzheimer's disease is characterized by the presence of amyloid plaques, made up primarily of Aϐ peptides, and neurofibrillary tangles, containing hyperphosphorylated tau. Aϐ is generated by sequential proteolysis of the amyloid precursor protein (APP) by beta and gamma secretases. The leading hypothesis of Alzheimer's disease pathogenesis is the amyloid cascade hypothesis, which suggests that amyloid is central to the disease process. However, tau pathology correlates more closely with cognitive dysfunction and follows a predictable anatomical course through the brain. We hypothesize that if Aϐ is upstream of tau pathology and tau pathology follows this predictable course through the brain, Aϐ …

Biochemical Aspects Of The Thermal Sensitivity And Energy Balance Of Polar, Tropical And Subtropical Teleosts, Eloy Martinez

USF Tampa Graduate Theses and Dissertations

The maintenance of a functional energy balance in ectothermic fauna could be challenging in a thermally disparate environment. Biochemical adaptations at the enzyme and membrane levels allows for a set compensatory mechanism that allow the individual to maintain an energetic surplus, thus allocating energy for growth and reproduction. The present work describes how the energetic machinery in the cell, particularly the mitochondrion, could be affected by temperature changes. More specifically, this work aimed to determine how environmental temperature affects the mitochondria energetic performance of fishes from disparate thermal regimes.

Mitochondrial ATP production efficiency was evaluated in fishes from polar, tropical …

Reaction Enthalpy And Volume Profiles For Excited State Reactions Involving Electron Transfer And Proton-Coupled Electron Transfer, William Antonio Maza

USF Tampa Graduate Theses and Dissertations

Electron transfer, ET, and proton-coupled electron transfer, PCET, reactions are central to biological reactions involving catalysis, energy conversion and energy storage. The movement of electrons and protons in either a sequential or concerted manner are coupled in a series of elementary reaction steps in respiration and photosynthesis to harvest and convert energy consumed in foodstuffs or by absorption of light into high energy chemi-cal bonds in the form of ATP. These electron transfer processes may be modulated by conformational dynamics within the protein matrix or at the protein-protein interface, the energetics of which are still not well understood. Photoacoustic calorimetry …

Mass Spectrometry-Based Methods For The Detection And Characterization Of Protein-Tyrosine Nitration, Kent W. Seeley

USF Tampa Graduate Theses and Dissertations

Protein tyrosine nitration (PTN) is a posttranslational modification resulting from oxidative/nitrosative stress that has been implicated in a wide variety of disease states. Characterization of PTN is challenging due to several factors including its low abundance in a given proteome, preferential site modification, multiple target site proximity within unique peptide sequences, and analytical method and instrument limitations. Current analytical techniques are insufficiently sensitive to identify endogenous nitration sites without incorporation of either nitrotyrosine or target protein enrichment. However, enrichment proficiency can also be inadequate. Chemical derivatization of the nitro- moiety can be incomplete or result in undesirable byproduct formation, while …

Phosphorylation Of Histone Deacetylase 6 Within Its C-Terminal Region By Extracellular Signal Regulated Kinase 1, Kendra Allana Williams

USF Tampa Graduate Theses and Dissertations

http://upload.etdadmin.com/etdadmin/pdfout/222759_supp_undefined_2A63E500-E9D7-11E2-925E-BE522E1BA5B1.PDF

Structural Basis For Ternary Complex Formation Between Tau, Hsp90, And Fkbp51, Alexander Steven Barrett

USF Tampa Graduate Theses and Dissertations

The accumulation of the microtubule associated protein tau has been implicated in several neurological disorders; however, its interaction with chaperones along its normal degradation pathway remains largely uncharacterized at single residue resolution. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to probe the interaction between tau, the molecular chaperone Hsp90, and the immunophilin FKBP51. Resonance intensity changes were observed for specific residues in the heteronuclear single quantum coherence (HSQC) spectra of 15N-labeled tau in the presence of Hsp90 and/or FKBP51. Analysis of the HSQC spectra identified the two hydrophobic hexapeptide motifs located at residues V275 - K280 and …

Molecular Mechanisms Involved In Anhydrobiosis Of Insect Cells, Diyagama Arachchi Ralalage Dilini Sewwandi Samarajeewa

Masters Theses

Animals possessing tolerance to extreme water stress are termed anhydrobiotes. Many desiccation tolerant organisms respond to water stress by intracellular accumulation of selected sugars such as trehalose and larger macromolecules such as Late Embryogenesis Abundant (LEA) proteins and thereby maintain the cell viability. Evidence indicates that the presence of trehalose and Late Embryogenesis Abundant (LEA) proteins may work synergistically to confer cellular protection during drying in eukaryotic cells. We evaluated any increase in cellular desiccation tolerance by expressing different LEA proteins in a non-desiccation tolerant insect cell line Drosophila melanogaster (fruit fly) (Kc167 cells) in the presence of trehalose.

Transgenic …

New Tools For Real-Time Study Of Embryonic Development, Lauren M. Browning

Theses and Dissertations in Biomedical Sciences

Embryonic development represents one of the most complex and dynamic cellular processes in biology, and plays vital roles in understanding of functions of embryonic stem cells (ESCs) and design of ESC-based therapy. Conventional assays and fluorescence-based imaging methods have been widely used for the study of embryonic development. These conventional methods cannot effectively provide spatial and temporal resolutions with sufficient sensitivity and selectivity that are required to depict embryonic development in vivo in real-time at single-cell and single-molecule resolutions. In this dissertation, we have developed a wide range of innovative tools for real-time study of embryonic development. These new tools …

Attachment Ability And Melanoma Inhibitory Activity Mrna Expression Level Changes In Murine B16-F10 Melanoma Cells Post Nanosecond Electric Pulses, Hongxia Jia

Theses and Dissertations in Biomedical Sciences

The effects of high-voltage nanosecond electric pulses (nsEPs) on metastatic melanoma are still unclear. Hence, we applied one, two, three, and four 300 ns 40 kV/cm pulses to murine B16-F10 melanoma cells. Cell attachment ability was determined by comparing the number of floating cells and the percentage of attached cells. Melanoma inhibitory activity (MIA) is a secretory protein that is highly correlated with the malignancy and metastasis of malignant melanomas. We used MIA as our target to evaluate the effect of nsEPs on metastasis. Pulsed (experimental) and unpulsed (control) cells were incubated at 37°C under a 5% CO₂ atmosphere. …

Histone Deacetylation As A Mechanism Of Ypel3 Down-Regulation In Er-A Positive Breast Cancer Cell Lines, Remah Ali

Browse all Theses and Dissertations

YPEL3 is a growth inhibitory gene that was established by our laboratory to trigger senescence in both primary and tumor cells. We recently reported YPEL3 to be directly repressed by estrogen in estrogen receptor-a positive (ER-a +ve) breast cancer cell lines. Here, we set out to determine whether the repression of YPEL3 involves histone deacetylation and, if so, elucidate the molecular mechanism of this activity. MCF-7, T-47D, and ZR-75.1 (all ER-a +ve) breast cancer cell lines were treated with varying doses of the global histone deacetylase inhibitors (HDACIs), trichostatin A (TSA) or suberoyl anilide hydroxamic acid (SAHA), for 24, 48 …

Transmembrane Serine Mutations Reduce The Minimum Pore Diameter Of Channelrhodopsin-2, Robert Dempski, Ryan Richards

Robert E. Dempski

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin that, together with channelrhodopsin-1, mediates phototactic behavior in the green algae Chlamydomonas reinhardtii. Like all other microbial-type rhodopsins, ChR2 has seven transmembrane domains with the chromophore all-trans retinal bound to a single lysine residue. However, unlike other microbial-type rhodopsins, ChR2 functions as a non-selective cation channel and not an ion pump. A sequence alignment of ChR2 with the proton pump bacteriorhodopsin (bR) reveals that ChR2 lacks specific motifs within the transmembrane domains that facilitate non-covalent interactions and contribute to protein stability.

Structural Adaptations Of Proteins To Different Biological Membranes, Prof. Stephanie Tristram-Nagle, Irina Pogozheva, Harold Mosberg, Andrei Lomize

Prof. Stephanie Tristram-Nagle Ph.D.

To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/ polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30–31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic …

Periplasmic Response Upon Disruption Of Transmembrane Cu Transport In Pseudomonas Aeruginosa., José Argüello, Daniel Raimunda, Teresita Padilla-Benavides, Stefan Vogt, Sylvain Boutigny, Kaleigh Tomkinson, Lydia Finney

José M. Argüello

Pseudomonas aeruginosa, an opportunistic pathogen, has two transmembrane Cu(+) transport ATPases, CopA1 and CopA2. Both proteins export cytoplasmic Cu(+) into the periplasm and mutation of either gene leads to attenuation of virulence. CopA1 is required for maintaining cytoplasmic copper levels, while CopA2 provides copper for cytochrome c oxidase assembly. We hypothesized that transported Cu(+) ions would be directed to their destination via specific periplasmic partners and disruption of transport should affect the periplasmic copper homeostasis. Supporting this, mutation of either ATPase gene led to large increments in periplasmic cuproprotein levels. Toward identifying the proteins participating in this cellular response the …