This work comprises analyses of cell fate decision-making in response to DNA damage. DNA damage is a ubiquitous threat to genomic stability, and depending on the type and extent of the damage, can lead to widespread changes in cell function as well as cell death. How apoptosis, or programmed cell death, is triggered in damaged cells was studied in different cell types for different types of damage.

<p> Lithium ion battery technology has flourished since its introduction into the consumer market. Not only has it helped revolutionize consumer electronics, it also compliments R&amp;D into clean forms of energy harvest e.g. solar, wind, and hydro-electric. As demand for the technology grows, innovative approaches have been taken to improve capacity, output, and lifetime in Li-ion batteries. The approach studied in this research involves the inclusion of nanostructures, which have the potential to significantly increase capacity. While several techniques to fabricate nanostructures are understood, underlying phenomena governing ion transport in and around these nanostructures is only partially understood, which could directly impact design principles for such devices. </p><p> This thesis examines a variety of model systems which could serve to simulate environments found in proposed devices and answer questions regarding ion transport phenomena. The main components we studied from such battery systems were electrolyte and cathode materials. The electrolyte experiences different ion transport phenomena arising from the nanoconfinement of the cathode structures both around and inside the electrode material. Thus, having model systems to examine electrolyte and cathode material separately and in tandem is useful for elucidating phenomena without the challenge of deconvolution resulting from other current-carrying mechanisms. </p><p> Our main tools for carrying out our research were synthetic nanopores. The nanopore structures afforded means to access nanoscale, control environment, and even fabricate components for study. By studying the current-voltage curves in these systems, we were able to draw meaningful conclusions about mechanisms of ion transport in these model systems. The main findings of this research include the inducement of positive surface charge on nanopore structures by organic solvent-based electrolytes by means of dipole and/or ion adsorption, positive evidence of gel electrolyte fitting current models of ion current rectification, and the impact of oxidation state and cycling in cathode material on ion transport through its porous media. Each of these findings is directly related to the thrust of the research and potentially provide insights for future battery design.</p><p>

The administration of glucocorticoids has been shown to induce atrophy and weakness in whole skeletal muscle. These effects frequently appear to be more pronounced in pale muscle than in red muscle. The present study was undertaken to examine the effects of steroid treatment on the contractile, electrical, and fatigue properties of single meter units in pale and red cat hindlimb muscles. Single motor units in the medial gastrocnemius (MG) and soleus muscles were isolated using techniques developed by McPhedran, et al., (1965). The properties of a large number of motor units were examined in normal animals and were compared to those of single units from the muscles of steroid-treated (3-4 mg. of triamcinolone acetonide per kg. per day for 10 -16 days) animals. Motor units examined in both control and experimental animals were classified by type according to criteria developed by Burke, et al., (1973,1974). The results show that steroids produce alterations in the strength- and speed-related properties of motor units which are more pronounced in fast-twitch than in slow-twitch units. The mean maximum tetanic tension (Pmax) for types FF (fast-twitch, readily fatiguable) and FR (fast-twitch, fatigue-resistant) units in MG were reduced by 63% and 71% respectively; The Pmax for type S units in MG was reduced by 25% as a result of steroid treatment but was unchanged for type S units in soleus. The mean maximum rate of rise of tetanic tension (dP/dt) for the three classes of motor units in MG was reduced in a pattern similar to that for tetanic tension. The dP/dt for types FF, FR, and S units in MG were reduced by 79%, 54%, and 25% respectively. The dP/dt for soleus units was increased by 73% as compared to control soleus units. Steroid treatment lengthened the mean twitch contraction time (CF) of FF units and shortened the CT for type S units in both MG and soleus. The integrated EMG signals elicited from FF and FR units were unchanged following steroid treatment but were significantly increased for type S units in both pale and red muscles. Steroid administration did not markedly alter the susceptibility to fatigue of the three classes of units in MG or in soleus units. There were however fewer MG units with intermediate fatigue indeces in steroid-treated animals. These finding suggest that those units most frequently activated in muscular contractions are the least susceptible to steroid-induced changes in their contractile properties and vice versa. Since the overall frequency of activation of motor ufiits is determined by cell size, these results imply that the Size Principle (Hennemann, et al., 1965) can be extended to not only explain the recruitment order of motor units in muscular contractions but also to account for the patterns of motor unit involvement in steroid-induced myopathy.

Protein secretion is a selective and regulated process that is essential in all organisms. In bacteria the preprotein translocase SecA, either free in the cytosol or associated with the SecYEG translocon, recognizes and binds most post-translational secretory proteins containing an N-terminal signal sequence. In Gram-negative bacteria, the molecular chaperone SecB binds many of the preproteins to keep them in a translocation-competent state. Subsequently, SecB delivers the preproteins to the translocon-associated SecA, which binds the signal sequence and also interacts with mature regions of the preprotein. After the preprotein/SecA/SecYEG complex has formed, the energy derived from ATP hydrolysis by SecA coupled with the proton motive force drives the insertion of the preprotein through the translocon pore. During the translocation reaction, the conformation of SecA dramatically changes from an inactive closed form (c-SecA) to one more active and open states. The various crystal structures of SecA have provided many structural details about c-SecA. The recent low resolution crystal structure of a fragment of SecA bound to SecYEG (Zimmer et al., 2008) has provided a starting point for structural analysis of the active and open conformation of SecA. Previous work in our laboratory demonstrated that an N-terminal proteolytic fragment of SecA, SecA64, is an activated form of SecA that with higher affinity signal peptides better than c-SecA (Triplett et al., 2001). To correlate the SecA64 results with full-length SecA, we determined that SecA in the presence of low concentrations of urea has an enhanced ATPase activity similar to translocation level, which is comparable to what was observed with SecA64. Analysis by CD and Trp fluorescence indicates the presence of an intermediate at 2.2 M urea at 22°C (termed u-SecA). Using limited proteolysis, we determined that u-SecA is in an protease-sensitive conformation that mimics the translocation-active form of SecA. These structural rearrangements occur primarily in the C-terminal one-third of the protein. Next, we sought to understand the signal sequence interactions with c-SecA and translocation-active u-SecA. Using a photoactivatable cross-linking approach along with limited proteolysis, two-dimensional gels, and domain mapping with region-specific antibodies, the signal sequence-binding site was mapped to the interface of NBF II, PPXD, and HSD. The site is the same in both forms of SecA but in our data suggests u-SecA that the binding groove as expanded.

RNA polymerases carry out a process, which at times carries contradictory mechanistic requirements. During initiation, an RNA polymerase should be highly sequence specific, binding only to and initiating only from specific promoter sequences. However, during processive elongation, an RNA polymerase should show no sequence specificity. Thus after sequence specific initiation, it must break initial tight binding contacts in a process known as promoter escape. An RNA polymerase must also carry out de novo initiation, starting from a 2mer, to a 3mer, etc. RNA. We have shown that elongating RNA polymerases achieve their high stability in part from a topological locking of the RNA around the DNA template strand. But a 4 base RNA transcript has insufficient length for stability from either topological or base pairing considerations; the complex must maintain substantial protein-DNA interactions. Not surprisingly then, RNA polymerases retain strong promoter contacts during synthesis of the first 8-10 bases. Also not surprisingly, short RNA (abortive) products are released along the path to a stable elongation complex. What governs the probability of abortive synthesis? How is subsequent promoter release achieved? What energetics drive that release? How is the translocational timing of release achieved? The current study builds on a model in which growth of the RNA-DNA hybrid drives a small rotation of the promoter binding domain in the protein, leading to 1) a physical separation of two components (the specificity loop and the N-terminal platform) and/or 2) disruption of the open complex stabilizing intercalating loop selectively weakening promoter binding at translocation past position +8. Probing complexes halted at specific positions, we explore a kinetic pathway in which translocating polymerases, release promoter contacts at different positions, with different rates. Current models for abortive cycling propose that the enzyme has difficulty achieving the structural transition to elongation. A mutant P266L, which shows dramatically reduced abortive cycling, has been proposed to possess a lower barrier for the transition to elongation and thus to transition sooner. We show instead that the mutant transitions to the promoter-released elongation configuration later than wild type does. We propose that the mutant shows a delay in the barrier to rotation, resulting in a delay in the transition to elongation. Conversely, lower initial barriers to rotation lead to lower destabilizing "push back" against the hybrid, leading to reduced abortive dissociation. As a direct test of this model, mutations (insertions and deletions) in the connecting C-terminal "leg" show the same phenotype (delayed transition and lower abortive dissociation) as the initial P266L mutant, as predicted.

Mass spectrometry, featured at high sensitivity, is an indispensable tool in the field of biophysics for studying intact protein and protein complex. Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer offers unparalleled resolving power and measured mass accuracy among various types of mass spectrometer. Further increase in mass resolving power can be achieved by increasing magnetic field strength and/or improving the performance of the mass analyzer (i.e., FTICR ion trap). Increasing the magnetic field strength is nontrivial in terms of fabrication techniques and economic aspect. This work is to increase the resolving power by the latter approach: developing a new FTICR ion trap of superior performance to increase the resolving power without increasing the magnetic field strength. The new FTICR ion trap development has been conducted in three aspects corresponding to the mass analyzer's three basic functions: trapping, excitation and detection. Chapter Two describes comprehensive comparisons of two latest FTICR ion traps for which the trapping electric fields are generated based on two different principles: statically harmonized electric potential and dynamically harmonized electric potential. The evaluation of the two FTICR ion traps is carried out by simulation and experiments. The simulation is based on finite difference method (FDM). The experiments are conducted on a custom-built 9.4 Tesla FTICR instrument. All of the advantages of the two ion traps have been incorporated into the new FTICR ion trap design. Chapter Three describes a novel electrical circuit which enables conducting excitation and detection functions on the same electrodes of FTICR ion trap. This circuit eliminates the restriction on excitation and detection electrode angular extents, which permits more efficient choice of electrode geometries for excitation and detection simultaneously. The circuit laid the foundation for the new FTICR ion trap design. Chapter Four demonstrates the new FTICR ion trap which possesses optimal excitation electric fields and ability of conducting the second frequency-multiple detection, founded on the optimized geometry and the associated circuit. The optimized geometry improves the radial and axial uniformity of excitation electric field which promotes large post-excitation radii desired by frequency-multiples detection. The circuit for conducting excitation and detection on same electrodes establishes the feasibility for such intended geometry modifications. The design of the new FTICR ion trap is initiated by theoretical analysis, guided by simulation and evaluated by experiments. The new FTICR ion trap not only meet the design target (i.e., doubling the resolving power under the same magnetic field strength), but also provide an approach of conducting even higher frequency-multiples detection for FTICR MS in future. Chapter Five describes an application of ultra-high resolving power mass spectrometry in the field of biophysics: the ultra-high resolution Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) experiments. Compared to the conventional HDX-MS experiments, ultra-high resolution MS can eliminate the potential errors in deuterium uptake calculation and largely accelerate the data processing. Chapter Six introduces one of the future research projects: the third frequency-multiple detection for FTICR MS. It is a follow-up of this doctoral work and is being investigated in our group. The corresponding circuit diagram is presented. / A Dissertation submitted to the Institute of Molecular Biophysics in partial fulfillment of the Doctor of Philosophy. / Spring Semester, 2015. / January 28, 2015. / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; Linda S. DeBrunner, University Representative; Timothy M. Logan, Committee Member; William S. Oates, Committee Member; Scott Stagg, Committee Member.

The second leading cause of death in the United States is cancer, and approximately 90% of cancer related deaths are due to metastasis. When cancer metastasizes, cell from the tumor enter the circulation where they are exposed to hemodynamic forces. One of the main mechanical forces of the circulation is fluid shear stress (FSS), which was thought to be the main reason for metastatic inefficacy. However, recent studies have shown that in vitro cancer cells are more resistant to FSS than non-transformed epithelial cells. Additionally, that loss of viability cancer cells experience is biphasic in nature. Investigations into this adaptive response have shown that the Young’s Modulus of cancer cells is increased. Further investigating the adaptive phenomena, RhoA activity is shown to be increased in cancer cells and not non-transformed cells after exposure to two brief pulses of FSS. Also, extracellular calcium is also essential to maintain resistance upon exposure to FSS, although, through unknown mechanisms. Additionally, inhibiting myosin II sensitizes cell to FSS both in vivo and in vitro.

The multifunctional calcium and calmodulin dependent protein kinase II (CaMKII) is implicated in both animal models and human forms of cardiovascular disease. CaMKII is activated by elevated neurohormonal signals including enhanced â-adrenergic stimulation and angiotensin II signaling, whereas CaMKII inhibition is cardioprotective from these pathologic triggers. In addition to â-blockers and angiotensin II inhibitors, aldosterone antagonist drugs are the third and most recent class of pharmacologic agents comprising the frontline therapy for heart disease patients. Here, I show that CaMKII activation is important for cardiac aldosterone signaling in the post-myocardial infarction (MI) mouse model. Aldosterone infusion to MI mice increases cardiac rupture, a lethal and nearly untreatable clinical problem. CaMKII inhibition protects from aldosterone enhanced post-MI rupture. We previously reported microarray analysis of genes upregulated after MI but downregulated in the presence of cardiac CaMKII inhibition. Surprisingly, a number of these genes are involved in extracellular matrix remodeling. Here, I validated the microarray findings for matrix metalloproteinase 9 (MMP9), an extracellular matrix remodeling enzyme known to contribute to the rupture phenotype. My results support a sequence where aldosterone infusion after MI recruits NADPH oxidase-derived reactive oxygen species to enhance CaMKII oxidation and subsequent myocyte enhancer factor 2 driven increases in MMP9 expression in myocytes. I found that oxidative activation of CaMKII is critical for this rupture phenotype through a new transgenic mouse model that overexpresses methionine sulfoxide reductase A, which modulates CaMKII oxidation and therefore activation. These results implicate CaMKII activation in cardiac aldosterone signaling and reinforce the importance of CaMKII hyperactivity in acute cardiac remodeling after MI. Overall, this work supports myocardial CaMKII as a novel mediator of cardiac aldosterone stimulation of post-MI matrix remodeling and suggests potential efficacy for molecularly targeted anti-oxidant therapy in the treatment of patients after acute MI.

In cultured cells, the adaptor protein SH3PXD2B is capable of recruiting a variety of proteins involved in invadosome assembly and function. It is therefore considered as an essential organizer of invadosomes active in cellular responses that require extracellular matrix degradation. Despite increasing knowledge about its properties and functions at the molecular and cellular levels, its physiological role in whole animals has not previously been assessed. Here, we present that SH3PXD2B is essential for normal postnatal development and disrupting SH3PXD2B can lead to glaucoma. Our work on SH3PXD2B is based on nee, a spontaneous mutation in mice which arose on an inbred background. Mice homozygous for the nee mutation were initially noted to exhibit runted growth, craniofacial abnormalities and ocular defects. Our additional physiological characterization has uncovered skeletal abnormalities, hearing impairment, infertility and a form of lipodystrophy. Using genetic mapping and DNA sequencing, the cause of nee phenotypes was identified as a 1-bp deletion within the Sh3pxd2b gene on mouse Chromosome 11. The nee mutation is predicted to cause a frameshift and a protein truncation altering a portion of the third SH3 domain and deleting the entire fourth SH3 domain. Molecular analysis showed that the fourth SH3 domain of SH3PXD2B can interact with a transmembrane member of a disintegrin and metalloproteinase family of proteins, ADAM15, and that GFP tagged SH3PXD2B protein truncation mislocalizes to the nucleus. Therefore, the mutant allele likely disrupts the normal function of SH3PXD2B and leads to nee phenotypes. The initial ocular abnormalities of nee mutants suggested that they may develop glaucoma. To test this hypothesis, we performed detailed clinical and histological analyses. We find that nee mutants exhibit elevated intraocular pressure likely caused by a severe iridocorneal adhesion present early in development. During the same time that intraocular pressure becomes elevated, retinal ganglion cells are damaged and optic nerve degeneration ensues. These results demonstrate that nee mutants develop an early onset form of closed angle glaucoma. To test the potential involvement of SH3PXD2B in human diseases, DNA sequencing analysis has been initiated in patients with developmental, primary open angle and syndromic glaucoma, and several missense variations have been identified. In conclusion, my results demonstrate a critical role of SH3PXD2B in normal development of multiple tissues and suggest that mutations in SH3PXD2B may also contribute to glaucoma.

Protein aggregation has been implicated in several catastrophic diseases (neurodegeneration, diabetes, ALS) and its complexity has also become a major obstacle in large-scale production of protein-based therapeutics. Despite the generic behavior of proteins to aggregate, only a few globular proteins have known aggregation mechanisms. At present, there have been no clear connections between a protein folding, function and aggregation. We have tackled the challenge of understanding the links between a protein's natural tendency to fold and function with its propensity to misfold and aggregate. Using a predominantly β-sheet protein whose in vitro folding has been explored in detail: cellular retinoic acid-binding protein I (CRABP 1), as a model, we investigated sequence determinants for folding and aggregation. In addition, we characterized the aggregation-prone intermediate under native conditions. Our studies revealed similar contiguous aggregation cores in in vitro and in vivo aggregates of CRABP 1 validating the importance of sequence information under extremely different conditions. Hydrophobic stretches that comprise the interface in aggregates include residues surrounding the ligand binding portal and residues at the C-terminal strands of CRABP 1. Folding studies reveal that docking of the N and C terminals happen in the early stages of barrel closure of CRABP 1 emphasizing the role of folding in preventing exposure of risky aggregation-prone sequences. We further examined the intermediate that initiates aggregation under native conditions. We found that inherent structural fluctuations in the native protein, relevant to ligand binding of CRABP 1, expose aggregation-prone sequences. Binding of the ligand, retinoic acid decreases the aggregation of CRABP 1 illustrating the contribution functional interactions in avoiding aggregation. Our study implies that because of the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable risk.