Biochemistry, Biophysics, and Structural Biology Commons^™

Repsa-Directed Identification Of Dna-Binding Specificity For Orphan Transcription Factors, Kamir Hiam

Symposium of Student Scholars

The function of many genes and the biological roles of their encoded products are still not well characterized. Given the sequence-specific DNA-binding properties of transcription factor proteins, it is possible to purify them, identify the responsible polypeptide(s), determine their consensus binding sequences, and identify their genomic binding sites. Thus, one can go from cellular extract to proposed biological regulatory roles in relatively short order. Our goal is to identify and characterize orphan DNA-binding proteins in the model organism E. coli K12 using the novel combinatorial technique, REPSA (Restriction Endonuclease Protection Selection Amplification), as well as further develop the REPSA to …

Determining Lung Cancer Subtypes, Classifications, And Potential Gene Markers, Daniel L. Johnson

Student Theses and Dissertations

Lung Cancer is the leading cause of cancer-related deaths in the United States. The key to surviving this disease is early discovery and treatment. Precise diagnosis is critical when selecting the treatment for the patient. Diagnosis of the patient will depend upon the location of the tumor, the stage of the tumor, and the class of the tumor. Clinically, lung cancer is divided into two classes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Non small-cell lung cancer accounts for 85% of all lung cancer. NSCLC can be separated into three major subtypes: adenocarcinoma, squamous-cell, and large-cell …

Nmr Spectroscopy Reveals The Presence And Association Of Lipids And Keratin In Adhesive Gecko Setae, Dharamdeep Jain, Alyssa Y. Stark, Peter H. Niewiarowski, Toshikazu Miyoshi, Ali Dhinojwala

Polymer Science Faculty Research

Lipid and protein aggregates are one of the fundamental materials of biological systems. Examples include cell membranes, insect cuticle, vertebrate epidermis, feathers, hair and adhesive structures known as ‘setae’ on gecko toes. Until recently gecko setae were assumed to be composed entirely of keratin, but analysis of footprints left behind by geckos walking on surfaces revealed that setae include various kinds of lipids. However, the arrangement and molecular-level behavior of lipids and keratin in the setae is still not known. In the present study we demonstrate, for the first time, the use of Nuclear Magnetic Resonance (NMR) spectroscopy techniques to …

Urine Proteomics Identifies Preeclampsia Subphenotypes, Michelle R. Axe

Undergraduate Honors Thesis Projects

Preeclampsia is a perinatal complication of unknown etiology, resulting in 76,000 maternal deaths and 500,000 infant deaths annually₁. The Nationwide Children’s Center for Perinatal Research was the first cohort to propose that preeclampsia is rooted in protein misfolding. We aimed to characterize the misfolded protein components of maternal urine samples and to determine if distinct molecular profiles exist for clinical preeclampsia phenotypes. A total of 86 maternal urine samples from Yale-New Haven Hospital were analyzed for this study, from six clinical preeclampsia subphenotypes- pregnant control (n=10), idiopathic proteinuria (n=15), mild preeclampsia (n=14), superimposed preeclampsia (n=12), typical severe preeclampsia …

The Effect Of Red Maple Leaf Toxicosis On Reduced Glutathione Levels In Equine Erythrocytes In Vitro, Madeline A. Rohl

Undergraduate Honors Thesis Projects

Red maple leaf toxicosis is an equine blood disorder resulting from the consumption of wilted red maple (Acer rubrum L.) leaves by horses. Compounds within the leaves of red maple have oxidative effects on equine erythrocytes and can cause hemolysis of erythrocytes, the conversion of hemoglobin to methemoglobin, and the production of Heinz bodies. Reduced glutathione is important in the protection of equine erythrocytes from these oxidative events; however, in the presence of red maple toxin, glutathione is rapidly oxidized and is unavailable. The objective of this study is to determine whether the presence of vitamin C alters levels …

Biotransformations: Reaction Conditions, Antimicrobial Properties, And Enzyme Isolation, Jasmine Moreland, Marianne Burnett, Amey Gonzalez, Michele Harris

Undergraduate Research Conference

Biotransformation reactions using whole cell or organisms have been extensively studied. Most of the studies have focused on producing and characterizing the enantiospecific products. Benefits of biotransformation reactions include the use of water and other environmentally friendly conditions. We decided to focus on a single reaction to determine the best reaction conditions for the biotransformation, explore possible antimicrobial activity of the product, and isolate and characterize the protein involved in catalyzing the reaction.

Consumption Of Oral Nicotine: Its Effects On The Reproductive System And Nicotine Metabolism Of Female Sprague-Dawley Rats, Swapnali Halder

Student Theses and Dissertations

Smoking is a major preventable cause of death throughout the world and the need for cessation is unanimously supported by clinicians and scientists. Nicotine in cigarettes is believed to cause tobacco dependence and addiction and low doses of nicotine are found in nicotine replacement therapies (NRT) to mitigate withdrawal symptoms and improve cessation efforts. In addition to smoking associated health risks that are common for men and women, women smokers are more likely to suffer from pregnancy or other reproductive health related complications than non-smokers. Studies show that female smokers are more vulnerable to smoking, and find it harder to …

Investigating The Initial Detection Stage Of Meiotic Silencing By Unpaired Dna In The Model Organism Neurospora Crassa, Pegan A. Sauls

Theses and Dissertations

In an attempt to neutralize transposable elements or retrovirus invasions Neurospora crassa will rely on one of its many genome defense mechanism, Meiotic Silencing by Unpaired DNA (MSUD). MSUD works in a two-step process that first detects unpaired sequences between homologous chromosomes followed by downstream silenced expression of the sequence. The ultimate silencing stage of MSUD is widely accepted to operate through an RNAi-like system. However, the mechanics of the detection step of MSUD remains elusive. The research presented attempts to elaborate on how the initial stage of MSUD occurs and its specifics. First, a genetic approach is utilized to …

Methods In Extracting Dna: Using Phenol-Chloroform On Formalin Fixed Human Brain Tissue, Stefanie Rigg, Nathaniel Kennedy

Undergraduate Research Conference

Genetic markers of diseases can be studied post-mortem with the isolation of DNA from tissues. While protocols exist for DNA extraction from fresh and formalin-fixed-paraffin embedded tissue and formalin fixed hard tissues such as bone, there are no published methods for formalin fixed soft tissue such as brain. Extractions from formalin fixed soft tissues are more difficult due to the higher amount of cellular degradation that results from fixation. The development of efficient DNA extraction protocols for formalin fixed soft tissues will allow post-mortem study of genetic markers and aid in identification of genetic diseases. Existing techniques of phenol-chloroform extraction …

Transcriptional Activity Of The Islet Β Cell Factor Pdx1 Is Augmented By Lysine Methylation Catalyzed By The Methyltransferase Set7/9, Aarthi V. Maganti, Bernhard Maier, Sarah A. Tersey, Megan L. Sampley, Amber L. Mosley, Sabire Özcan, Boobalan Pachaiyappan, Patrick M. Woster, Chad S. Hunter, Roland Stein, Raghavendra G. Mirmira

Molecular and Cellular Biochemistry Faculty Publications

The transcription factor Pdx1 is crucial to islet β cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass …

Reagent Pencils: A New Technique For Solvent-Free Deposition Of Reagents Onto Paper-Based Microfluidic Devices, Haydn T. Mitchell, Isabelle C. Noxon, Cory A. Chaplan, Samantha J. Carlton, Cheyenne H. Liu, Kirsten A. Ganaja, Nathaniel W. Martinez, Chad Immoos, Philip Costanzo, Andres W. Martinez

Chemistry and Biochemistry

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the …

Generating A Focused View Of Disease Ontology Cancer Terms For Pan-Cancer Data Integration And Analysis., Tsung-Jung Wu, Lynn M. Schriml, Qing-Rong Chen, Maureen Colbert, Daniel J. Crichton, Raja Mazumder, Ying Hu, + 10 More

Biochemistry and Molecular Medicine Faculty Publications

Bio-ontologies provide terminologies for the scientific community to describe biomedical entities in a standardized manner. There are multiple initiatives that are developing biomedical terminologies for the purpose of providing better annotation, data integration and mining capabilities. Terminology resources devised for multiple purposes inherently diverge in content and structure. A major issue of biomedical data integration is the development of overlapping terms, ambiguous classifications and inconsistencies represented across databases and publications. The disease ontology (DO) was developed over the past decade to address data integration, standardization and annotation issues for human disease data. We have established a DO cancer project to …

An Active Site Loop Mutant Of A Zinc Metallo-Deubiquitinase Suggests The Importance Of Loop Dynamics To Catalysis, Amy N. Bueno

Open Access Theses

AMSH is a conserved zinc metalloprotease that functions with endosomal sorting complexes required for transport (ESCRT) machinery in order to down regulate and degrade cell-surface receptors. It has been shown to have high specificity toward Lys63-linked polyubiquitin chains, a signal used for receptor down regulation by ESCRT machinery. It has previously been found that Sst2, an AMSH orthologue from fission yeast, contains a flexible "loop" near the proximal ubiquitin binding site, composed of residues L402 and F403. The different mobility of the side chains of these residues contribute to the opening and closing conformation of the catalytic cleft which allow …

Support Needs Of Young Ovarian Cancer Survivors: A Pilot Study, Aurelie Chuong

Open Access Theses

Purpose. Young ovarian cancer survivor (18 years old or older but diagnosed before the age of 34 years old) support needs (i.e., physical and daily living, psychological, sexual, patient care and support, health system and information) were examined to guide the design and implementation of future young adult and adolescent cancer support groups and interventions. Method. This cross-sectional pilot study included 11 young ovarian cancer survivors. The Supportive Care Needs Survey Short Form 34 (SCNS-SF34) was used and open-ended questions were added. Results. The calculated standardized scores for the SCNS-SF34 survey results showed that sexuality needs were ranked highest (41.7 …

Developmentally Regulated Sumoylation In The Ciliate Tetrahymena Thermophila, Amjad M. Nasir

Open Access Dissertations

The Small Ubiquitin-like MOdifier (SUMO) protein regulates numerous nuclear events such as transcription, mitosis and meiosis and DNA repair. These processes are critical to the programmed nuclear events of conjugation in ciliates and provide the potential to investigate developmentally regulated SUMOylation. We predicted a developmental increase in SUMOylation during late conjugation based on the extensive genome remodeling in the developing macronucleus (anlagen) ofTetrahymena thermophila. Immunoblotting of cell lysates from vegetative and mating cells using anti-SUMO antibodies revealed distinct developmental differences and an increased signal correlated with formation of the anlagen. Immunofluorescence of mating Tetrahymena cells with the same antibody …

Magnetic Manipulation And Multimodal Imaging For Single Cell Direct Mechanosensing, Robert L. Wilson

Open Access Theses

The study of internal mechanics of single cells is paramount to understand mechanisms of mechanoregulation. External loading and cell-mediated force generation result in changes in cell shape, rheology, and the deformation of subcellular structures such as the nucleus. Moreover, alterations in the processes that regulate these responses have been further correlated to specific pathologies. Cellular deformation is often studied through application of forces in the environment of the cell, relying on strain and stress transfer through focal adhesions and the cytoskeletal system. However, the transfer of these external forces to internal mechanics can introduce uncertainties in the interpretation of subcellular …

“Analyze, Acquire, Apply, And Write” As A New Learning Model In Science, Jeong Choe

Faculty Publications & Research

I have developed a new teaching and learning model called AAAW, which stand for Analyze, Acquire, Apply and Write. This model grows from action research and unique experience in teaching a biochemistry course to high school students who are talented in math and science. In this model, students first "Analyze" lab data to generate questions that lead them to "Acquire" background knowledge. Students then go back to the data and "Apply" their new knowledge to better understand the data. Finally, students "Write" about the connections they make from their reading, data analysis, and application of the data. The rationale behind …

Structural Analysis Of The 14-3-3Ζ/Chibby Interaction Involved In Wnt/Β-Catenin Signaling, Ryan C Killoran, Jingsong Fan, Daiwen Yang, Brian H Shilton, Wing-Yiu Choy

Biochemistry Publications

The partially disordered Chibby (Cby) is a conserved nuclear protein that antagonizes the Wnt/β-catenin signaling pathway. By competing with the Tcf/Lef family proteins for binding to β-catenin, Cby abrogates the β-catenin-mediated transcription of Wnt signaling genes. Additionally, upon phosphorylation on S20 by the kinase Akt, Cby forms a complex with 14-3-3 to facilitate the nuclear export of β-catenin, which represents another crucial mechanism for the regulation of Wnt signaling. To obtain a mechanistic understanding of the 14-3-3/Cby interaction, we have extensively characterized the complex using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and isothermal titration calorimetry (ITC). The crystal structure …

Molecular Effects Of Cancer-Associated Somatic Mutations On The Structural And Target Recognition Properties Of Keap1., Halema Khan, Ryan C Killoran, Anne Brickenden, Jingsong Fan, Daiwen Yang, Wing-Yiu Choy

Biochemistry Publications

Kelch-like ECH-associated protein 1 (Keap1) plays an important regulatory role in the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress response pathway. It functions as a repressor of Nrf2, a key transcription factor that initiates the expression of cytoprotective enzymes during oxidative stress to protect cells from damage caused by reactive oxygen species. Recent studies show that mutations of Keap1 can lead to aberrant activation of the antioxidant pathway, which is associated with different types of cancers. To gain a mechanistic understanding of the links between Keap1 mutations and cancer pathogenesis, we have investigated the molecular effects of a …

Functional Screening Of The Cronobacter Sakazakii Baa-894 Genome Reveals A Role For Prop (Esa_02131) In Carnitine Uptake, Audrey Feeney, Roy D. Sleator

Department of Biological Sciences Publications

Cronobacter sakazakii is a neonatal pathogen responsible for up to 80% of fatalities in infected infants. Low birth weight infants and neonates infected with C. sakazakii suffer necrotizing enterocolitis, bacteraemia and meningitis. The mode of transmission most often associated with infection is powdered infant formula (PIF) which, with an aw of ∼0.2, is too low to allow most microorganisms to persist. Survival of C. sakazakii in environments subject to extreme hyperosmotic stress has previously been attributed to the uptake of compatible solutes including proline and betaine. Herein, we report the construction and screening of a C. sakazakii genome bank and …

Evaluating Breast Cancer Stem Cell Response To Antiangiogenic Therapy, Connor J. Holloway

Open Access Theses

Angiogenic inhibitors function by blocking tumor cell signals used to recruit host tissue vasculature to the tumor site, thereby depriving the cancer of the nutriment needed for further expansion. The development and implementation of angiogenic inhibitors in conjunction with standard chemotherapy agents has increased progression-free survival but not overall patient survival. It is hypothesized that chronic exposure to large doses of AAT drugs worsens hypoxic conditions within the tumor mass, selectively stimulating aggressive cancer stem cell populations to grow and proliferate. ^ In this study, the expression of the CSC biomarkers ALDH1, DLL1, and EpCAM were evaluated in breast cancer …

Exploring The Effect Of Climate Change On Biological Systems, Nardos Sori

Chemistry & Biochemistry Theses & Dissertations

The present and potential future effect of global warming on the ecosystem has brought climate change to the forefront of scientific inquiry and discussion. For our investigation, we selected two organisms, one from cyanobacteria and one from a cereal plant to determine how climate change may impact these biological systems. The study involved understanding the physiological and adaptive responses at both the genetic and protein function levels to counteract environmental stresses. An increase in atmospheric carbon dioxide is a key factor in global climate change and can lead to alterations in ocean chemistry. Cyanobacteria are important, ancient and ubiquitous organisms …

Identification Of Perilipin 5 Interaction Partners, Emily E. Burton

Undergraduate Honors Thesis Projects

Perilipin 5 is one of the more recently discovered in the PAT protein family. Several studies on this protein have shown that it resides in multiple intracellular locations such as the endoplasmic reticulum, the cytosol, the mitochondria, and most interestingly on lipid droplets. These lipid droplets have proven not to be just clumps of fat, but to be very complex intracellular organelles, with a core of lipids surrounded by a phospholipid monolayer coated by specific proteins, one of those being perilipin 5. Some proteomic studies have revealed these lipid droplets to be involved in intracellular trafficking, signaling, cytoskeletal organization and …

Functional Genomic Analyses Of Switchgrass Developmental Processes, Nathan Palmer

Department of Biochemistry: Dissertations, Theses, and Student Research

Switchgrass (Panicum virgatum L.), a C_4-perennial grass species, is being developed as a bioenergy crop. Although much is known from a breeding perspective, there is limited information on the functional genomics of this crop, specifically regarding molecular mechanisms controlling aerial senescence, winter dormancy, and traits that confer winter hardiness. Using functional genomics to generate a transcriptional roadmap underpinning senescence and winter dormancy will provide researchers with a molecular understanding that can be applied to improve switchgrass germplasm.

In an initial study, a de novo assembly of the crown and rhizome transcriptome from an upland cultivar Summer was …

In Vivo Near Ir Fluorescent Imaging Of Hollow Polymeric Nanocapsules, Venkata Suresh Patthipati

Biological Sciences Theses & Dissertations

Medical Imaging has been an integral component in medical diagnosis and therapeutics. It is frequently used in conjunction with other treatment modalities and has been used for developing applications that range from cancer management to clinical practice. Nanotechnology and its applications are foreseen to have a profound impact on life sciences. Nanomaterial based imaging has a significant potential in addressing medical challenges such as tumor localization, targeted delivery of drugs and real time diagnosis and management of circulation pathology. In particular, polymer based nanoparticles are increasingly being developed as they can be customized according to the diagnostic and/or therapeutic requirements. …

Development Of Experimental And Instrumental Systems To Study Biological Systems, Amanda J Hemphill

Open Access Dissertations

Chapters 1-4 of this thesis describes the development of an experimental system to measure diffusion-limited reaction kinetics in a biological environment. About 100 years ago, the relationship between reaction rate and diffusion in homogenous solution, ie water or buffer, was described as a linear relationship by Smoluchowski. Applying this theory naively would suggest that since the diffusion coefficients drop by factors of 4-100 then the rates of reaction would drop by the same amount. However, recent theory and simulations suggest that this does not hold. Even though biological diffusion coefficients drop to 0.1-20% of that in buffer, these recent studies …

Characterization Of Hessian Fly From Israel, Alisha J Johnson

Open Access Dissertations

Mayetiola destructor Say, the Hessian fly, is a gall midge and a member of the Dipteran family Cecidomyiidae. It is a common pest of wheat found throughout all of the major wheat growing areas of the world and poses a serious economic threat to the United States (US), particularly in the Southeast winter wheat region. Damage to wheat is done solely by feeding first and second in-star larvae. Hessian fly (Hf) infestations result in a loss in grain yield by the stunting and/or killing of seedling wheat plants in the winter and by causing breakage at the nodes of the …

Role Of Syk In The Regulation Of Cytoskeleton And Stress Granules In Breast Cancer, Mariya Krisenko

Open Access Dissertations

The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than …

A Novel In Vivo Tumor Oxygen Profiling Assay: Combining Functional And Molecular Imaging With Multivariate Mathematical Modeling, Chung-Wein Lee

Open Access Dissertations

Purpose: The objective of this study is to develop and test a novel high spatio-temporal in vivo assay to quantify tumor oxygenation and hypoxia. The assay implements a biophysical model of oxygen transport to fuse parameters acquired from in vivo functional and molecular imaging modalities. ^ Introduction: Tumor hypoxia plays an important role in carcinogenesis. It triggers pathological angiogenesis to supply more oxygen to the tumor cells and promotes cancer cell metastasis. Preclinical and clinical evidence show that anti-angiogenic treatment is capable of normalizing the tumor vasculature both structurally and functionally. The resulting normalized vasculature provides a more efficient and …

Quantitative Mrna Detection With Advanced Nonlinear Microscopy, Jing Liu

Open Access Dissertations

Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. While current techniques are not capable to visualize single mRNA transcript beyond the diffraction limit. In this thesis, two nonlinear technologies, second harmonic super-resolution microscopy (SHaSM) and transient absorption microscopy (TAM), are developed to detect and quantify single Human edimer receptor 2 (Her2) mRNA transcripts. The SHaSM is used to detect single mRNA transcript beyond the diffraction limit, while the TAM is employed to detect mRNA without the interference of fluorescence background. The …