Biochemistry, Biophysics, and Structural Biology Commons^™

A Dna-Peptide Crosslink (Dpc) Increases Mutagenicity In Sos-Induced Escherichia Coli, Alessandra Bassani

Honors Scholar Theses

Bacteria, such as Escherichia coli, have an inducible system in response to DNA damage termed the SOS response. This system is activated when the replicative DNA polymerase (Pol) III encounters a lesion, uncouples from DNA helicase, and single-stranded DNA (ssDNA) accumulates at the replication fork. In this study, we investigated DNA-peptide crosslink (DpC), a common lesion that results from cross-linking of proteins or peptides, UV irradiation, and alkylating agents. To increase survival following formation of a lesion, the SOS response can utilize homologous recombination, translesion synthesis (TLS), or excision repair. With TLS, the levels of DNA Pol II, IV, …

Identifying The Cell Composition And Clonal Diversity Of Supratentorial Ependymoma Using Single Cell Rna-Sequencing, James He

Honors Scholar Theses

Ependymoma is a primary solid tumor of the central nervous system. Supratentorial ependymoma (ST-EPN), a subtype of ependymomas, is driven by an oncogenic fusion between the ZFTA and RELA genes in 70% of cases. We introduced this fusion into neural progenitor cells of mice embryos via in utero electroporation of a non-viral binary piggyBac transposon system containing ZFTA-RELA. From preliminary data in the LoTurco lab, inducing the expression of ZFTA-RELA into different neural progenitor cells produces tumors of varying lethality and cellular composition. To define the cellular composition and subclonal diversity of ST-EPN tumors, we used single cell RNA-sequencing …

Single-Fluorophore Sensors For Mechanical Force In Living Cells, Sarah Kricheff

Honors Scholar Theses

Mechanotransduction is the process by which a mechanical stimulus is converted to a cellular signal. This process is heavily influential of cell morphology, differentiation, and behavior. However, altered levels of mechanical stimuli are also found in many pathological contexts. For example, cancerous cells have stiffer surrounding tissue than healthy cells, and research suggests that this alters cell behavior and promotes metastasis. Despite these findings, the cellular processes behind these signaling alterations remain widely unknown. Understanding these cascades is critical, as involved proteins can give us a deeper understanding of the role of mechanotransduction, and certain proteins can potentially be targeted …

Toxicity Analysis Of 2’-Deoxyguanosine-N2-6-Aminopyrene And 2’-Deoxyguanosine-N2-8-Aminopyrene In Escherichia Coli, Emily Janeiro

Honors Scholar Theses

Cancer is a disease that stems from genomic errors that are not corrected properly by cellular repair mechanisms. Errors are more likely to form when organisms are subjected to DNA damage by mutagenic compounds. 1-Nitropyrene, a nitrated polycyclic aromatic hydrocarbon (nitro-PAH), has been shown to be a potent mutagen that causes cancer. Nitro-PAHs can arise from diesel exhaust products in the environment. Out of all nitro-PAHs, 1-nitropyrene is found in largest quantities in the environment. This poses a great need to study its effects biochemically in order to address its toxicity in DNA. Other nitropyrene derivatives, including 1,6-dinitropyrene and 1,8-dinitropyrene, …

An Analysis Of Crispr-Cas Gene Editing In Agriculture, Ashley Laliberte

Honors Scholar Theses

The CRISPR-Cas system is a promising form of gene editing, especially for the agriculture industry. The ability to make single-nucleotide edits within a gene of interest, without the need to introduce foreign DNA, is a powerful tool for designing healthier and more efficient crops and food animals. This system provides opportunity for increased nutritional value, decreased food waste, and more economically and environmentally sustainable food production. Though this biotechnology is facing mechanistic limitations due to off-target effects and inefficient homology-directed repair, vast improvements have already been made to improve its efficacy. The CRISPR-Cas system is already the most advanced form …

Development Of A Sonically Powered Biodegradable Nanogenerator For Bone Regeneration, Avi Patel

Honors Scholar Theses

Background: Reconstruction of bone fractures and defects remains a big challenge in orthopedic surgery. While regenerative engineering has advanced the field greatly using a combination of biomaterial scaffolds and stem cells, one matter of difficulty is inducing osteogenesis in these cells. Recent works have shown electricity’s ability to promote osteogenesis in stem cell lines when seeded in bone scaffolds; however, typical electrical stimulators are either (a) externally housed and require overcomplex percutaneous wires be connected to the implanted scaffold or (b) implanted non-degradable devices which contain toxic batteries and require invasive removal surgeries.

Objective: Here, we establish a biodegradable, piezoelectric …

Modeling And Analyzing An Optogenetic System For Photoactivatable Protein Dissociation, Anvin Thomas, James Schaff

Honors Scholar Theses

Computational modeling of cell-cell interactions can grant clues and can answer questions about an experiment, especially for observations about binding interactions and kinetics. This approach was used to investigate an interaction between a light-oxygen-voltage (LOV) domain and an engineered protein called Zdark (Zdk). The LOV domain is membrane-bound while Zdk is cytosolic. The LOV domain and Zdk bind strongly in dark (K_d 26.2 nM), and weakly upon exposure to blue light (K_d > 4 μM). Total internal reflection fluorescence (TIRF) images are acquired of Zdk, the fluorescent species bound to a mCherry tag, and the loss of fluorescence is …

Reconstitution Of Gabaergic Postsynapses In Host Cells, Karthik Kanamalla

Honors Scholar Theses

Type A GABA receptors (GABAARs) can be found embedded in postsynaptic membranes or in a variety of extrasynaptic locations. Receptors with synaptic function are recruited to the postsynapse by submembranous scaffolds composed of gephyrin and collybistin (CB). This study was aimed at assessing whether the ability to interact with the scaffold differentiates synaptic from non-synaptic receptors. Using HEK293 cells as an expression system, and indirect immunofluorescence (IF), co-localization of extrasynaptic receptors α1β3δ and α4β3δ with the CB-gephyrin scaffold was assessed and compared with that of the synaptic receptor α1β3γ2. Results indicated that both extrasynaptic receptors were able to colocalize with …

Alternate Formulations For Optimization Of Plga Microsphere-Comprised Glucose Biosensor Coating Against Foreign Body Inflammation., Klair Lubonja

Honors Scholar Theses

In this study, dexamethasone loaded PLGA microsphere/PVA hydrogel composites were explored as an outer drug-eluting coating for implantable biosensors to provide defense against acute inflammation of the foreign body response. The microspheres, with a dexamethasone release target period of approximately two weeks, were manufactured under various conditions: different co-solvents and homogenization speeds. Particle size measurement, scanning electron microscopy and differential scanning calorimetry were performed for all the microsphere formulations prepared. The addition of acetone as a co-solvent for dexamethasone, at normal homogenization speeds, shows promise for the improvement of drug loading and the attainment of superior release profiles. PLGA microsphere/PVA …

The Development Of Skeletal Muscle In Young Horses: An Ultrasonography And Satellite Cell Approach, Emma K. Lavigne

Honors Scholar Theses

Muscle growth in young horses is characterized by an increase in muscle cross-sectional area, which can be accomplished through the activation and differentiation of satellite cells. Satellite cells can be stimulated or inhibited in response to different cytokines and growth factors and are key mediators of muscle hypertrophy and regeneration. The aim of this study was to investigate the growth of the longissimus dorsi (LD) muscle in horses under 5 years of age and to obtain preliminary data on satellite cell behavior in foals. The area, width, height, and subcutaneous fat were measured using ultrasonography at 6-month increments over the …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Post-Injury Calcium Chelation Rescues Skeletal Muscle Regeneration In Mice, Matthew D. Magda

Honors Scholar Theses

During aging the ability of skeletal muscle to regenerate after injury wanes until muscle integrity cannot be maintained. In this study it is shown that calcium chelation can restore young-like regenerative ability in an aged mouse model of skeletal muscle regeneration.

Dikar-Induced Synthetic Lethality In A Drosophila Model Of Cag Repeat Diseases Does Not Result From An Expression Feedback Loop, Daniel Camacho

Honors Scholar Theses

Human CAG repeat diseases manifest themselves through the common pathology of neurodeneration. This pathological link is attributed to the property shared by all nine of these diseases: an expanded polyglutamine (polyQ) tract. The most evident result of polyQ expansion is protein aggregation, and it is believed that this phenomenon is partly responsible for conferring cytotoxic properties on the mutated protein. Apart from sequestering the mutated protein, cellular aggregates are able to incorporate native proteins via polyQ-mediated aggregation, thus disrupting important cellular pathways. Using Drosophila melanogaster as a disease model, researchers have been able to compile collections of these so-called disease …

Investigations In Enhancing The Reproducibility Of Implantable Amperometric Glucose Biosensors For Mass Production, Dipesh Manharbhai Patel

Honors Scholar Theses

Implantable glucose sensors for Diabetes management should possess several attributes such as linearity, sensitivity, selectivity, long life time and should elicit no negative tissue response. Based on this, the UConn implantable glucose sensor team has developed a 5-layer sensor architecture that simultaneously affords the aforementioned requirements. However, the large scale production of these 5-layer sensors is inhibited by the lack of high sensor-to-sensor reproducibility. Herein, we investigate the origin of sensor-to-sensor irreproducibility and develop methodologies for large-scale fabrication of these devices.

Characterizing The Role Of Cortactin In Actin Pedestal Assembly By Enterohemorrhagic Escherichia Coli (Ehec), Sarah E. Grout

Honors Scholar Theses

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne cause of bloody diarrhea and renal failure. During colonization of the intestine, EHEC injects the transmembrane receptor protein Tir and the cytoplasmic effector protein EspFU into host cells to reorganize the actin cytoskeleton into adhesion “pedestals.” EspFU has been shown to bind and activate the actin nucleation factor N-WASP to drive actin polymerization into pedestals. However, EspFU can still assemble pedestals in cells lacking N-WASP, suggesting that this effector protein is able to also trigger N-WASP-independent pathways of actin polymerization during infection. Cortactin is an atypical nucleation factor that localizes to pedestals, …

Elucidating The Mechanism Of Antimigratory Activity Of Cardiac Glycosides, Joshua H. Johnson

Honors Scholar Theses

The focus of this research is on cell migration and how it can be better understood through the use of small molecules that modulate cell migratory activity. The results have particular relevance in the realm of cancer pharmacology. Cardiac glycosides, which are known inhibitors of the eukaryotic Na⁺/K⁺-ATPase, have been determined to have antimigratory activities through the screening of several small molecule libraries. Here we investigate the antimigratory activities of the cardiac glycoside digitoxin as well as its analogs that we synthesized. Antimigratory activity was determined by conducting a wound closure assay with MDA-MB-231 human breast …

Identifying Progenitor Cells Of Heterotopic Ossification, Eileen E. Semancik

Honors Scholar Theses

Heterotopic Ossification (HO) is the abnormal formation of bone within extraskeletal soft tissues. The condition can occur through both genetic and acquired means. Acquired cases of HO result from invasive surgery or traumatic injuries, with increasing prevalence of ectopic skeletogenesis as a result of combat-related blast injuries. HO has been characterized to some extent, including the histological features and the mutation underlying the genetic form, but the cells resident in skeletal muscle that represent the progenitors of heterotopic bone have yet to be determined. Only a few publications have attempted to definitively determine the progenitor cells in this disorder. Findings …

Improvements To The Forensic Analysis Of Mitochondrial Dna Typing, Elizabeth Montano

Honors Scholar Theses

Sequence analysis of human mitochondrial DNA (mtDNA) is an effective and reliable tool for the genetic characterization of forensic samples. The nature of the mitochondrial genome (mtgenome), its high copy number and small size (~17kb) makes it more resistant to degradation and more stable than nuclear DNA. For this reason mitochondrial DNA is often the only feasible option for the forensic analysis of environmentally compromised samples. Currently the forensic analysis of the mtgenome is restricted to the hypervariable regions, also known as the Displacement loop (d-loop). Previous studies, confirmed in the Strausbaugh lab, have demonstrated an increased variability in the …

Metallothionein Gene Dose And The Immune Response, Meaghan Roy-O-Reilly

Honors Scholar Theses

Metallothionein (MT) is a small, cysteine-rich protein with significant immunomodulatory activity. It has been shown to play a critical role in important cellular mechanisms including heavy metal detoxification, essential metal management and the inflammatory response. MT production can be induced by a number of cellular stressors and acts to lessen the harmful effects of oxidizing agents and heavy metal exposure. Previous studies have shown that the dose of the metallothionein gene present in an individual may have significant effects on the adaptive immune response, yet the mechanism behind this phenomenon remains unknown. We hypothesize that the gene dose of metallothionein …

Characterization Of Arginine-82 Mutants With Non-Native Chromophores, Vivek Alaigh

Honors Scholar Theses

Bacteriorhodopsin, found in most halobacteria, is an integral protein that contains seven transmembrane alpha helices and an organic chromophore, all-trans retinal. Light energy is captured by the protein and results in a series of spectrally discrete intermediates that conclude with a proton being pumped across the membrane from the cytoplasmic side to the extracellular milieu. The most blue-shifted photo-intermediate, the M state, has been of interest for protein-based holographic memory storage devices. Bacteriorhodopsin mutants were prepared with either a 4-hydroxy retinal or 3,4-dihydro retinal analog: R82A, R82C, R82H, R82K, R82N and R82Q. The objective of this research was to investigate …

Modeling Human Immune Response To The Lyme Disease-Causing Bacteria, Yevhen Rutovytskyy

Honors Scholar Theses

The purpose of this project is to develop and analyze a mathematical model for the pathogen-host interaction that occurs during early Lyme disease.

Based on the known biophysics of motility of Borrelia burgdorferi and a simple model for the immune response, a PDE model was created which tracks the time evolution of the concentrations of bacteria and activated immune cells in the dermis. We assume that a tick bite inoculates a highly localized population of bacteria into the dermis. These bacteria can multiply and migrate. The diffusive nature of the migration is assumed and modeled using the heat equation. Bacteria …

Expression Of The Porcine Reproductive And Respiratory Syndrome Virus Non-Structural Protein 3 (Nsp 3) In Escherichia Coli, Lidia Beka

Honors Scholar Theses

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is single-stranded, positive-sense RNA virus in the family Arteriviridae, order Nidovirales. PRRSV is the most economically significant viral infection of swine herds in the United States. The single-stranded RNA genome is 15 kb in length and encodes 9 open reading frames (ORF1a, ORF1b, ORF2a, ORF2b and ORFs 3 through 7). ORFs 1a and 1b encode for 13 non-structural proteins (nsp) that are suggested to be involved in transcription and viral genome replication. The exact role of non-structural proteins in PRRSV cycle is still unknown. Moreover, there is a limited availability of reagents such …

Design And Bio-Production Of A Nanoparticle Avian Influenza Vaccine, Kashif N. Ather

Honors Scholar Theses

Influenza is one of the most common diseases in the world and the cause for numerous deaths every year. The primary method of combating the disease is the influenza vaccine, which is produced by inoculating chicken eggs with inactivated virus. An emerging solution is to use Self-Assembling Polypeptide Nanoparticles (SAPN) to elicit an immune response in the body, rather than using inactivated viruses. This project focuses on the synthesis, purification, and refolding of two peptide constructs, BN5C and S43, which are specific protein sequences that under the right conditions will refold into the 3-dimensional structures necessary for producing an immune …

Quorum Sensing In Archaea, Charles Mackin

Honors Scholar Theses

Bacteria coordinate cell density dependent behaviors by communicating through chemical intermediaries in a process called quorum sensing. In a bacterial culture, individual cells will constitutively produce signal molecules, termed autoinducers, and export them into the environment. When the concentration of autoinducers reaches a threshold, the cells sense that they are in a specific situation, which requires the upregulation of certain genes. This upregulation causes the bacteria to produce proteins that allow them to take part in a coordinated population-wide behavior.

In bacteria that are naturally competent, or capable of importing DNA from the environment, the expression of competence genes is …

Characterizing The Role Of The Bacterial Metallothionein, Smta, In Mammalian Infection, Stephanie R. Davis

Honors Scholar Theses

Mammalian metallothioneins (MT) are induced by various immunomodulatory molecules and are involved in a spectrum of immune processes such as essential metal homeostasis, detoxification of certain heavy metals, inflammation, and immune cell trafficking [1-3]. A bacterial metallothionein, SmtA, shares some sequence homology with mammalian MT as well as its metal-binding capabilities [4]. In addition to its ability to sequester heavy metals, eukaryotic MT has also been shown to scavenge free radicals such as reactive oxygen and nitrogen species (ROS, RNS), interfering with their toxic effects on cells and potentially influencing their regulatory roles in cell proliferation and differentiation [5, 6]. …

Biomarker Signature Classification Of Various Stress Forms, Chana L. Rich

Honors Scholar Theses

Various types of stressful conditions can have unique and important effects on immunity and can lead to dramatic consequences to health. For my University Scholar project, the characteristic biomarker signatures produced from a set of diverse stressors (e.g. psychological, biological and chemical) are being investigated. A biomarker signature is a distinctive biological indicator of a specific condition. High-throughput tools for the measurement of different cellular products have the potential to further our understanding of human disease and facilitated the identification of new biomarkers in all areas of medicine. The hypothesis that each form of stress, psychological, chemical and physical, will …

Characterization Of The Putative Xyloglucan Glycosyltransferase Gt14 In Arabidopsis Thaliana, Najam R. Syed

Honors Scholar Theses

Plant cell walls largely consist of matrix polysaccharides that are linked to cellulose microfibrils. Xyloglucan, the primary hemicellulose of the cell wall matrix, consists of a repeating glucose tetramer structure with xylose residues attached to the first three units ('XXXG'). In Arabidopsis thaliana, the core XXXG structure is further modified by enzymatic addition of galactose and fucose residues to the xylose side chains to produce XLXG, XXLG, XLLG and XLFG structures. GT14 is a putative glycosyltransferase in the GT47 gene family. Initial predictions of GT14's hydrophobic regions, based on its translated amino acid sequence, are almost identical to its Arabidopsis …

Alkylphenol Contamination In Homarus Americanus, Jennifer Renee Urban

Honors Scholar Theses

Alkylphenols are pollutants that are present in marine sediments and fishes. In earlier work it has been discovered that alkylphenols are present in the Homarus americanus, or the American lobster. Research suggests that alkylphenols could behave as endocrine disruptors as they have been found to affect juvenile hormone activity. It has been hypothesized that lobsters may be able to rid themselves of alkylphenol contamination through secreting these compounds into the environment or sequestering them in their tissues. In this study, I address the question of how lobsters may rid themselves of alkylphenols by analyzing hemolymph, muscle, gill, and shell samples …

Determination Of The Myogenic Potential Of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells, Rory Coleman

Honors Scholar Theses

Human embryonic stem cells (hESCs) have the potential to

differentiate to all adult somatic cells. This property makes hESCs a very promising area of research for the treatment of disorders in which specific cell populations need to be restored. Despite this potential, research that focuses on producing mesodermally derived cell populations from hESCs is decidedly limited, notwithstanding the prevalence of disorders involving mesodermal tissues for which treatment options are limited. Skeletal muscle myoblasts are derivatives of mesodermal cells and are characterized by the expression of the MyoD gene. These cells are difficult to obtain from hESCs in a reproducible and …

Contribution Of The Novel C-Terminal Domain To The Ribosome Binding Activities Of Virulence Regulator Bipa, Heeren Makanji

Honors Scholar Theses

Bacterial GTPases regulate many cell functions, including the stress response, signal recognition, protein synthesis, and cell differentiation, through a molecular switch that is activated and deactivated depending on their nucleotide bound state (1). A member of the translational family of bacterial GTPases along with LepA and EF-G, BipA is a 67 kD protein that is essential for virulence and the stress response. Crystal structures from the Robinson lab have shown a unique C-terminal domain on BipA that has been implicated in ribosome binding. Using N-terminal deletion constructs, we have shown that the C-terminal domain is necessary, but not sufficient, to …