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Articles 1 - 30 of 37
Full-Text Articles in Life Sciences
Caulobacter Clpxp Adaptor Popa’S Domain Interactions In The Adaptor Hierarchy Of Ctra Degradation, Thomas P. Scudder
Caulobacter Clpxp Adaptor Popa’S Domain Interactions In The Adaptor Hierarchy Of Ctra Degradation, Thomas P. Scudder
Masters Theses
The degradation and recycling of protein is a process essential for the maintenance and regulation of cellular function. More specifically, in Caulobacter crescentus, the ClpXP protease is responsible for driving progression through the cell cycle and protein quality control. This protease utilizes three known adaptors to selectively degrade proteins that initiate different stages of development. This thesis will elaborate on the specific binding interface on one of these adaptors, PopA, with another, RcdA, and focus in on specific residues on PopA and investigate their roles in adaptor binding and delivery of CtrA, the master regulator of Caulobacter. Finally, I …
Combining Simulation And The Mspa Nanopore To Study P53 Dynamics And Interactions, Samantha A. Schultz
Combining Simulation And The Mspa Nanopore To Study P53 Dynamics And Interactions, Samantha A. Schultz
Masters Theses
p53 is a transcription factor and an important tumor suppressor protein that becomes activated due to DNA damage. Because of its role as a tumor suppressor, mutations in the gene that encodes it are found in over 50% of human cancers. The N-terminal transactivation domain (NTAD) of p53 is intrinsically disordered and modulates the function and interactions of p53 in the cell. Its disordered structure allows it to be controlled closely by post-translation modifications that regulate p53’s ability to bind DNA and interact with regulatory binding partners. p53 is an attractive target for developing cancer therapeutics, but its intrinsically disordered …
Atomistic Simulations Of Intrinsically Disordered Protein Folding And Dynamics, Xiping Gong
Atomistic Simulations Of Intrinsically Disordered Protein Folding And Dynamics, Xiping Gong
Doctoral Dissertations
Intrinsically disordered proteins (IDPs) are crucial in biology and human diseases, necessitating a comprehensive understanding of their structure, dynamics, and interactions. Atomistic simulations have emerged as a key tool for unraveling the molecular intricacies and establishing mechanistic insights into how these proteins facilitate diverse biological functions. However, achieving accurate simulations requires both an appropriate protein force field capable of describing the energy landscape of functionally relevant IDP conformations and sufficient conformational sampling to capture the free energy landscape of IDP dynamics. These factors are fundamental in comprehending potential IDP structures, dynamics, and interactions. I first conducted explicit solvent simulations to …
Investigation Of Kinase Conformational Dynamics And Analytes Detection With Protein Nanopore, Fanjun Li
Investigation Of Kinase Conformational Dynamics And Analytes Detection With Protein Nanopore, Fanjun Li
Doctoral Dissertations
Protein nanopores are pore-forming proteins which have been developed as single-molecule biosensors. Due to the high sensitivity, selectivity, label-free and real-time detection methodology, protein nanopores have been used for a wide variety of applications. In this dissertation, we use ClyA nanopore to investigate kinase conformational dynamics and develop a kinase/nanopore system for the specific detection of kinase allosteric inhibitors. Besides, we engineer OmpG nanopore to be a sensor for nucleic acid detection. Protein kinases play essential roles in cellular regulation by catalyzing the phosphorylation of target proteins and are promising drug targets. The conformational dynamics are critical for kinase functions. …
Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward
Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward
Doctoral Dissertations
Molecular motors, such as myosin, have evolved to transduce chemical energy from ATP into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction in these motors is a root cause of many pathologies necessitating the application of intrinsic control over molecular motor function. We hypothesized that altering the myosin’s energy substrate via minor positional changes to the triphosphate portion of the molecule will allow us to control the protein and affect its in vitro function. We utilized positional isomers of a synthetic non-nucleoside triphosphate, azobenzene triphosphate, and assessed whether myosin’s force- and motion-generating capacity could …
Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni
Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni
Doctoral Dissertations
Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions. Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis …
Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri
Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri
Doctoral Dissertations
Mechanical forces are an integral part in biology, they regulate several cellular properties, such as morphology, proliferation, migration. These forces are also involved in receptor signaling and the differentiation of different cell types. Different proteins and biomolecules such as cadherin, integrin, notch proteins are essential elements of these processes. Measuring these intercellular forces are challenging considering the minimal intensity (piconewton-level) of these molecular forces. In our lab, we have developed a membrane DNA tension probe (MDTP) that uses a DNA hairpin module to sense tensile forces and has a lipid anchor to modify onto live-cell membranes. The programmability of DNA …
Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo
Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo
Doctoral Dissertations
Long-term memory and learning are still poorly understood from a molecular and cellular standpoint. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric kinase that is involved in this remarkable process. However, the molecular details of its specific roles in these processes remains elusive. CaMKII activation-triggered subunit exchange presents a novel possible mechanism involved in long-term memory and learning by exchanging active subunits with other CaMKIIs. CaMKII subunit exchange also shows that exchanged CaMKIIs spread their phosphorylation state to newly synthesized CaMKIIs. This provides a long-lasting signal that might possibly be involved in long-term memory by escaping a cell’s …
Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac
Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac
Doctoral Dissertations
High yields of RNA (e.g., mRNA, gRNA, lncRNA) are routinely prepared following a two-step approach: high yield in vitro transcription using T7 RNA polymerase, followed by extensive purification using gel or chromatic methods. In high yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer than desired, (partially) double stranded impurities. Current purification methods often fail to fully eliminate these impurities which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. This study establishes …
Investigating Structural Proteins By Light Scattering, Uma Nudurupati
Investigating Structural Proteins By Light Scattering, Uma Nudurupati
Masters Theses
This thesis evaluates the organization of the structural proteins, Human Gamma D crystallin and Collagen type II, into higher-order structures using light scattering. Specifically, it evaluates the natures of incipient aggregation in Human Gamma D crystallin and the nature of its interactions with CAPEGn, an electrostatic blocker. Additionally, this thesis evaluates whether Collagen type II growth kinetics follows Classical nucleation theory.
Controlled Membrane Remodeling By Nanospheres And Nanorods: Experiments Targeting The Design Principles For Membrane-Based Materials, Sarah Zuraw-Weston
Controlled Membrane Remodeling By Nanospheres And Nanorods: Experiments Targeting The Design Principles For Membrane-Based Materials, Sarah Zuraw-Weston
Doctoral Dissertations
In this thesis we explore two experimental systems probing the interactions of nanoparticles with lipid bilayer membranes. Inspired by the ability of cell membranes to alter their shape in response to bound particles, we report two experimental studies: one of nanospheres the other of long, slender nano-rods binding to lipid bilayer vesicles and altering the membrane shape. Our work illuminates the role of particle geometry, particle concentration, adhesion strength and membrane tension in how membrane morphology is determined. We combine giant unilamellar vesicles with oppositely charged nanoparticles, carefully tuning adhesion strength, membrane tension and particle concentration. In the case of …
Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma
Micro-Physiological Models To Mimic Mucosal Barrier Complexity Of The Human Intestine In Vitro, Abhinav Sharma
Doctoral Dissertations
The mucosal barrier in the intestine is vital to maintain selective absorption of nutrients while protecting internal tissues and maintaining symbiotic relationship with luminal microbiota. This bio-barrier consists of a cellular epithelial barrier and an acellular mucus barrier. Secreted mucus regulates barrier function via in situ biochemical and biophysical interaction with luminal content that continually evolves during digestion and absorption. Increasing evidence suggests that a mucus barrier is indispensable to maintain homeostasis in the gastrointestinal tract. However, the importance of mucus barrier is largely underrated for in vitro mucosal tissue modeling. The major gap is the lack of experimental material …
Characterization Of Β-2-Microglobulin Pre-Amyloid Oligomers And Their Role In Amyloid Inhibition, Tyler M. Marcinko
Characterization Of Β-2-Microglobulin Pre-Amyloid Oligomers And Their Role In Amyloid Inhibition, Tyler M. Marcinko
Doctoral Dissertations
In dialysis patients, β-2 microglobulin (β2m) can aggregate and eventually form amyloid fibrils in a condition known as dialysis-related amyloidosis, which deleteriously affects joint, bone, and organ function, and eventually causes organ failure. To understand the early stages of the amyloid assembly process, we have employed a series of biophysical tools including chromatography, spectroscopy, and most especially, native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble pre-amyloid oligomeric species. We have also collaborated and integrated computational modeling to help better understand and rationalize the structural basis behind oligomerization. Recently, several small molecules have been identified …
Rna-Seq And Mechanistic Enzymology Confirm Rna Self-Templated Extension By T7 Rna Polymerase And Suggest Novel Approaches Towards Improved In Vitro Rna Synthesis, Yasaman Gholamalipour
Rna-Seq And Mechanistic Enzymology Confirm Rna Self-Templated Extension By T7 Rna Polymerase And Suggest Novel Approaches Towards Improved In Vitro Rna Synthesis, Yasaman Gholamalipour
Doctoral Dissertations
Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyzed in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirmed that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. …
The Effects Of Acidosis On Calcium Dependent Binding Of A Single Crossbridge, Matthew Unger
The Effects Of Acidosis On Calcium Dependent Binding Of A Single Crossbridge, Matthew Unger
Masters Theses
Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because acidosis depresses the calcium (Ca2+) sensitivity and force production of muscle (18, 50). However, the molecular mechanisms behind this depression in Ca2+ sensitivity and force production are unknown. This gap in knowledge poses a significant challenge in generating a complete understanding of the fatigue process. To close this gap, the ability of myosin to bind to a single actin filament was measured under acidic conditions, in a laser trap assay, with and without regulatory proteins. Decreasing pH from 7.4 to 6.5 reduced the frequency …
Multiscale Simulations Of Intrinsically Disordered Proteins, Xiaorong Liu
Multiscale Simulations Of Intrinsically Disordered Proteins, Xiaorong Liu
Doctoral Dissertations
Intrinsically disordered proteins (IDPs) lack stable secondary and/or tertiary structures under physiological conditions. The have now been recognized to play important roles in numerous biological processes, particularly cellular signaling and regulation. Mutation of IDPs are frequently associated with human diseases, such as cancers and neuron degenerative diseases. Therefore, it is important to understand the structure, dynamics, and interactions of IDPs, so as to establish the mechanistic basis of how intrinsic disorder mediates versatile functions and how such mechanisms may fail in human diseases. However, the heterogeneous structural ensembles of IDPs are not amenable to high resolution characterization solely through experimental …
Bio-Inspired Polymers That Bind And Deliver Protein Cargo, Nicholas D. Posey
Bio-Inspired Polymers That Bind And Deliver Protein Cargo, Nicholas D. Posey
Doctoral Dissertations
Delivering functional proteins and antibodies into cells can allow researchers to probe the intracellular environment, discover new cellular pathways, and pioneer new therapeutics. However, the entry of exogenous, charged molecules, like proteins, into the cell is usually restricted by the membrane, thereby hindering intracellular delivery. Membrane permeable molecules such as cell penetrating peptides (CPPs) and protein transduction domains (PTDs) can be used to bypass the cell membrane and deliver protein into the cell, but these peptides involve iterative and laborious syntheses and are limited in terms of their chemical diversity. This dissertation work overall focuses on the design and synthesis …
Probing Apoptotic Caspase Allostery And Exosite Interactions For Alternative Regulation, Derek J. Macpherson
Probing Apoptotic Caspase Allostery And Exosite Interactions For Alternative Regulation, Derek J. Macpherson
Doctoral Dissertations
Programmed cell death, or apoptosis is a critical homeostatic pathway that monitors the balance of cell life and death. Apoptosis is regulated by a class of enzymes known as the cysteine aspartic proteases, or the caspases. The 12 human caspases that play important roles in the progression and regulation of apoptosis and inflammation. Caspases are tightly regulated by numerous factors including enzymatic activation, post-translational modifications, metal ligand binding, and protein modulation. Aberrant caspase activation and regulation has been implicated in the progression of numerous diseases such as proliferative diseases and neurodegeneration. The deeply entwined nature of caspases and apoptosis makes …
Characterization Of The Motion Of Cellulose Synthase Protein Complexes In The Plant Cell Membrane, Nina Zehfroosh
Characterization Of The Motion Of Cellulose Synthase Protein Complexes In The Plant Cell Membrane, Nina Zehfroosh
Doctoral Dissertations
The polysaccharide cellulose is the main component of plant cell walls, so it is the most abundant polymer on Earth. While it is widely used in industry due to its remarkable properties, such as renewability and biodegradability, its biosynthesis is still not well understood. The large transmembrane protein Cellulose Synthase Complex (CSC) is responsible for synthesizing cellulose by polymerizing UDP glucose into the constituent glucan chains of cellulose. In this project, I used variable angle epi-fluorescence microscopy (VAEM) in combination with single-particle tracking to characterize the motion of GFP labeled CSCs in the hypocotyl of Arabidopsis thaliana (A. thaliana …
Feronia-Related Receptor Kinase 7 And Feronia And Their Role In Receiving And Transducing Signals, David Vyshedsky
Feronia-Related Receptor Kinase 7 And Feronia And Their Role In Receiving And Transducing Signals, David Vyshedsky
Masters Theses
Receptor kinases (RKs) are transmembrane proteins that have been shown to regulate an array of important processes in A. thaliana, including polar cell growth, plant reproduction, and many other plant growth processes. In this thesis, I examine RECEPTOR KINASE 7 (RK7) and FERONIA (FER), two closely related transmembrane RKs, and their effects on plant reproduction. The RK7 gene when knocked out (rk7) in conjunction with FER resulted in delayed plant growth, decreased seed yield, and a lower percentage of the seeds germinating as compared to the single FER knockout. Transgenic plants with GUS reporter driven by RK7 promoter …
Characterization Of She1 Spindle Role Using Ceullular, Biochemical, And Biophysical Methods, Yili Zhu
Characterization Of She1 Spindle Role Using Ceullular, Biochemical, And Biophysical Methods, Yili Zhu
Doctoral Dissertations
During development, metaphase spindles undergo large movement and/or rotation to determine the cell division axis. While it has been shown that spindle translocation is achieved by astral microtubules pulling and/or pushing the cortex, how metaphase spindle stability is maintained during translocation remains not fully understood. In budding yeast, our lab has previously proposed a model for spindle orientation wherein the mitotic spindle protein She1 promotes spindle translocation across the bud neck by polarizing cortical dynein pulling activity on the astral microtubules. Intriguingly, She1 exhibits dominant spindle localization throughout the cell cycle. However, whether She1 has any additional role on the …
Studies On The P. Aeruginosa T3s Translocon Assembly: Interaction Of Popd With Membranes, Yuzhou Tang
Studies On The P. Aeruginosa T3s Translocon Assembly: Interaction Of Popd With Membranes, Yuzhou Tang
Doctoral Dissertations
Type III secretion (T3S) system is deployed by a wide range of pathogens to manipulate host cell response and establish infection. The T3S system is a syringe-like apparatus that spans across the double membrane of bacteria, protruding 50nm-80nm into the extracellular space and connecting with target cell membrane. In Pseudomonas aeruginosa, the proteins PopB and PopD are secreted and found associated with the target eukaryotic cell membrane. These two proteins are believed to form a transmembrane complex or translocon to allow effector protein translocation. Despite its key role in pathogenesis, the assembly mechanism and structure of this critical transmembrane …
Observing The Molecular Basis Of Thin Filament Activation With A Three Bead Laser Trap Assay, Thomas Longyear
Observing The Molecular Basis Of Thin Filament Activation With A Three Bead Laser Trap Assay, Thomas Longyear
Doctoral Dissertations
Muscle contracts after calcium (Ca++) is released into the muscle cell, resulting from a cascade of events which result in myosin, the molecular motor of muscle, to produce force and motion. Myosin cyclically binds to a regulated thin filament, using the chemical energy of ATP to produce force and motion. Perturbations in muscle, such as a build-up of metabolic by-products or point mutations in key contractile proteins, can inhibit these functions in both skeletal and cardiac muscle either acutely or chronically. Despite the many years we have studied skeletal and cardiac muscle, we still do not have a clear picture …
The Role Of The Metallochaperone Hypa In The Acid Survival And Activities Of Nickel Enzymes In Helicobacter Pylori, Heidi Hu
Doctoral Dissertations
Helicobacter pylori is a bacterium that has colonized the human gastric mucosa of over 50% of the world population. Persistent infection can cause gastritis, peptic ulcers, and cancers. The ability of H. pylori to colonize the acidic environment of the human stomach is dependent on the activity of the nickel containing enzymes, urease and NiFe-hydrogenase. The nickel metallochaperone, HypA, was previously shown to be required for the full activity of both enzymes. In addition to a Ni-binding site, HypA also contains a structural Zn site, which has been characterized to alter its averaged structure depending on pH and the presence …
Dissecting Molecular Pathways That Ensure Proper Chromosome Segregation And Cell Division, Anna Ye
Dissecting Molecular Pathways That Ensure Proper Chromosome Segregation And Cell Division, Anna Ye
Doctoral Dissertations
Equal segregation of the genome is a prerequisite for cell survival. During cell division the duplicated DNA is compacted into chromosomes and a multi-protein macrostructure, known as the kinetochore (Kt), is assembled on each copy of compacted DNA. Simultaneously, the mitotic spindle, which is made up of microtubules (MTs), is built to facilitate the equal distribution of the chromosomes between the resulting daughter cells. Kinetochores mediate the interaction between the MTs and the chromosomes, properly positioning them for segregation. To ensure that the DNA is equally divided in every cell division, cells have built a surveillance system to detect any …
Pore Forming Protein Assembly And The Use In Nanopore Sensing: A Study On E. Coli Proteins Clya And Ompg, Monifa Fahie
Pore Forming Protein Assembly And The Use In Nanopore Sensing: A Study On E. Coli Proteins Clya And Ompg, Monifa Fahie
Doctoral Dissertations
Pore forming proteins are typically the proteins that form channels in membranes. They have several roles ranging from molecule transport to triggering the death of a cell. This work focuses on two E. coli pore forming proteins that have vastly differing roles in nature. Outer membrane protein G (OmpG) is an innocuous β-barrel porin while Cytolysin A (ClyA) is an α-helical pore forming toxin. For OmpG we probed its potential to be a nanopore sensor for protein detection and quantification. A small high affinity ligand, biotin, was covalently attached to loop 6 of OmpG and used to capture biotin-binding proteins. …
Regulation Of Katanin Activity On Microtubules, Madison A. Tyler
Regulation Of Katanin Activity On Microtubules, Madison A. Tyler
Masters Theses
The cytoskeleton is a dynamic network of microtubules constantly being reorganized to meet the spatiotemporal demands of the cell. Microtubules are organized into subcellular highways to control cell processes such as cell division, cargo transport, and neuronal development and maintenance. Reorganization of this intricate network is tightly regulated by various stabilizing and destabilizing microtubule-associated proteins that decorate the network. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAA+) family. It can both sever and depolymerize microtubules. In order to sever microtubules, katanin recognizes the tubulin carboxy-terminal tails (CTTs) and hydrolyzes ATP. Using super-resolution microscopy …
Mechanistic Studies Of Peptide-Mediated Protein Transport Across Droplet-Interface Bilayers, Jing Huang
Mechanistic Studies Of Peptide-Mediated Protein Transport Across Droplet-Interface Bilayers, Jing Huang
Doctoral Dissertations
Pep-1 is a promising peptide tool that delivers proteins and peptides into cells with conserved bioactivity. Pep-1 has great potential because of the high efficiency and lack of toxicity. The mechanism of Pep-1-mediated transport is not fully understood. In my thesis, droplet-interface bilayer (DIB) has been used for the mechanistic studies of Pep-1. Here, DIB is developed for different functions such as quantitation of protein translocation, solution exchange to a formed bilayer and simultaneous observation of multiple membranes. Research work on Pep-1 with DIB reveals that the negative charge of the inner membrane leaflet plays a significant role in promoting …
Vitreous Gel Physics, Svetlana Morozova
Vitreous Gel Physics, Svetlana Morozova
Doctoral Dissertations
The transparent vitreous, which fills the posterior cavity of the eye, is incredibly engineered. The charged polyelectrolyte hyaluronic acid (HA) network swells to maintain the pressure in the eye, while stiff collagen type II bundles help absorb any external mechanical shock. Our investigations have contributed to a few key developments related to the physical properties of the vitreous: (1) The stiff collagen network that supports the soft gel network is self-assembled from single triple-helix collagen proteins. Electrostatic interactions drive this assembly, such that the size and concentration are optimized at physiological salt concentrations. The width of the assemblies remarkably changes …
X-Ray Characterization Of Mesophases And Phase Transitions Of Dna Analogues In Solutions, Mustafa Selcuk Yasar
X-Ray Characterization Of Mesophases And Phase Transitions Of Dna Analogues In Solutions, Mustafa Selcuk Yasar
Doctoral Dissertations
We think of DNA as double-stranded helices (duplex), but the polymer exists in many conformations. Several triplex and quadruplex DNA structures can be formed in laboratory settings and exist in nature. This thesis first provides a brief description of the nature of the order in arrays of duplex DNA under biologically relevant molecular crowding conditions. Then we compare the duplex DNA mesophases with the corresponding liquid crystalline phase behavior of the triplex and quadruplex DNA analogues. In particular, we focus on G-quadruplexes. Observed in the folds of guanine-rich oligonucleotides, G-quadruplex structures are based on G-quartets formed by hydrogen bonding and …