Life Sciences Commons^™

First Course Portfolio For Bioc205: Scientific Analysis And Technical Writing, Lindsey B. Crawford

UNL Faculty Course Portfolios

BIOC205: Scientific Analysis and Technical Writing is a core course for the Department of Biochemistry at the University of Nebraska-Lincoln. This course is geared for second year (sophomore students) with minimal prerequisites and as a foundation for future classes. Challenges include 1) teaching the needed scientific background and elements of scientific analysis and writing to a broad cohort of students with diverse backgrounds and interests, 2) building student skills towards analysis of primary peer-reviewed scientific literature, 3) developing students as writers of scientific information for different audiences. This course used Genetic Engineering as a broad topic choice to capture students …

Body Fluid Identification Using Dna Extraction Waste Product, Rachel M. Tolces

FIU Electronic Theses and Dissertations

In many situations, the amount of DNA evidence recovered at a crime scene is at trace levels, limiting the amount of testing that can be done on the evidence. Because genetic profiling is generally considered the most imperative assay to be completed on a DNA sample, in situations where yield is low, DNA extracts may not be utilized for any other purpose. Fortunately, an alternate source of DNA may exist by utilizing the waste products resulting from DNA extraction.

The goal of this project was to a protocol for recovery of DNA from robotic extraction waste and utilizing this DNA …

Expression And Purification Of E. Coli Yoaa, A Putative Helicase, Mark Gregory, Vincent Sutera Mr., Susan Lovett Dr.

Medical Student Research Symposium

All cells must maintain their genomic integrity to survive, which they achieve through several repair mechanisms that necessitate unwinding the damaged DNA by helicases. In Escherichia coli (E. coli), YoaA has been genetically shown to be involved in DNA repair and shares conserved sequences with helicase DinG. The goal of our study was to purify YoaA for further biochemical characterization. For expression, YoaA was fused to a His tag and overexpressed in MG1655 E.coli under the lacZ or T7 promoters for 2 hours, 4 hours, or overnight at 24oC, 30oC or 37oC. For purification, crude lysate was applied to a …

Investigation Of The Role Of Overexpression Of Psbs Under Stress Inducible And Constitutive Promoters To Improve Water Use Efficiency., Annie Nelson

Honors Theses

As climate change continues to impact environmental growth conditions, it has become increasingly more important to identify potential mechanisms of crop development to resist these changes. Previous studies have identified the role of PsbS in the non-photochemical quenching (NPQ) mechanism in the plant by identifying its direct effect on the rate in which excitation energy absorbed by photosystem II is dissipated as heat. It was identified also that PsbS via NPQ oxidizes chloroplastic quinone A (QA) which is a signal for stomatal opening in response to light. By identifying this relationship between PsbS and the signal for stomatal opening in …

Analysis Of Single-Site Cysteine Mutation, I412c, In Human A Glycine Receptor States To Further Refine Structure And Allostery, Leah Engquist

Honors Theses

The glycine receptor (GlyR) is the major inhibitory receptor in the brain and spinal cord. A member of the pentameric ligand gated ion channel superfamily, crystal structures are available but there are still unresolved areas, specifically the C-terminal tail and TM3-TM4 intracellular loop. Further refinement can provide deeper understanding of the molecular mechanism and allow the creation of novel therapeutics to modulate its function. We propose to insert a single cysteine mutation, I412C, into a Cys null background (C41S/C290A/C345S) to study non- conducting states (resting, desensitized) or with F207G/A288G mutations to study the open state. Purified, reconstituted GlyR is crosslinked …

Development Of High Value Oil Traits Using The Model Oilseed Crop Camelina Sativa, Evan Updike

Department of Biochemistry: Dissertations, Theses, and Student Research

Plant oils are an important source of food, fuel, and feed in our society today. The oil found in the seeds of plants is composed almost entirely of triacylglycerol (TAG) molecules, which consist of three fatty acids esterified to a glycerol backbone. As crude oil supplies decline, vegetable oils are gaining traction as a renewable substitute to petroleum-based materials in fuels, lubricants, and specialty oleochemicals. However, as it currently stands vegetable oils do not possess the properties necessary to fill the void of a petroleum free world.

To address this problem, plant biotechnologists have done extensive work on genetic engineering …

High And Low Toxin Producing Strains Of Karenia Brevis Differ Significantly In The Redox Proteome, Lipid Profiles, And Xanthophyll Cycle Pigments, Ricardo Colon

FIU Electronic Theses and Dissertations

The dinoflagellate Karenia brevis, blooms annually in the Gulf of Mexico, producing a suite of neurotoxins known as the brevetoxins. The cellular toxin content of K. brevis, however, is highly variable between or even within strains. I investigated biochemical differences between high (KbHT) and low (KbLT) toxin producing cultures both derived from the Wilson strain, related to energy-dependent quenching (qE) by photosystem II, and the content of reduced thiols of the proteome. By characterizing the xanthophyll content of the two strains I was able to determine that KbLT performs qE inconsistently. To investigate the …

Structural Analysis Of Protein-Peptide Interactions, Melody Gao

WWU Honors College Senior Projects

Over the last three years in the Amacher lab, I have been fortunate to work on two amazing projects studying protein-peptide interactions: PDZ domains and Class A sortases. Both recognize a certain substrate motif, and we are interested in these proteins' selectivity and promiscuity of their substrate.

Engineering Src Homology 2 Domains With Improved Specificity For Sulfotyrosine, Anya Morozov

Honors Theses

Protein tyrosine O-sulfation (PTS) is a common post-translational modification that has been implicated in a variety of biological processes and human illnesses. Despite continued progress in the field of sulfoproteomics, the extent and function of sulfated tyrosine (sulfotyrosine) residues is a topic of ongoing research. Previous work in the Guo Lab has identified Src Homology-2 (SH2) mutants that have a high affinity for sulfotyrosine along with retained high affinity for their natural ligand, phosphorylated tyrosine (phosphotyrosine). In this thesis, I attempted to generate SH2 mutants that have high affinity and specificity for sulfotyrosine over phosphotyrosine. While I successfully generated …

Pedagogical Approaches To Enhance Q2s Conversion Teaching And Learning: Application Of The Acue Effective Practice Framework To Upper Division Biochemistry, Jason Burke

Q2S Enhancing Pedagogy

The Effective Practice Framework course by the Association of College and University Educators (ACUE), and offered through the CSUSB Teaching Resource Center (TRC), was applied to the upper division biochemistry course series (CHEM 436A and 437A) in preparation for the Q2S conversion. This essay includes five reflections on the application of enhanced pedagogical approaches curated from a total of twenty-five. The enhanced pedagogies covered in the course are: designing and effective course and class; establishing a productive learning environment; using active learning techniques; promoting higher-order thinking; and assessing to inform instruction and promote learning. The reflections that follow have been …

Investigating The Interactions Between Individual Calmodulin And Hiv-1 Protein Domains, Riley K. Kendall, Jerry Larue

Student Scholar Symposium Abstracts and Posters

The World Health Organization found that 37.9 million people were living with HIV by the end of 2018. HIV is a virus that weakens the immune system through viral replication and the destruction of CD4⁺ T-cells, which are white blood cells that detect infection and make antibodies. A cure for HIV has not yet been discovered. HIV-1 contains a Gag polyprotein which regulates the stages of viral replication. Previous studies suggest that the myristoyl group of a matrix protein peptide found on the Gag polyprotein, MA, forms a complex with a calcium-binding, multifunctional regulatory protein called Calmodulin (CaM). CaM …

Comparative Analysis Of The Human Serine Hydrolase Ovca2 To The Model Serine Hydrolase Homolog Fsh1 From S. Cerevisiae, Jessica S. Bun, Michael D. Slack, Daniel E. Schemenauer, R. Jeremy Johnson

Scholarship and Professional Work - LAS

Over 100 metabolic serine hydrolases are present in humans with confirmed functions in metabolism, immune response, and neurotransmission. Among potentially clinically relevant but uncharacterized human serine hydrolases is OVCA2, a serine hydrolase that has been linked with a variety of cancer-related processes. Herein, we developed a heterologous expression system for OVCA2 and determined the comprehensive substrate specificity of OVCA2 against two ester substrate libraries. Based on this analysis, OVCA2 was confirmed as a serine hydrolase with a strong preference for long-chain alkyl ester substrates (>10-carbons) and high selectivity against a variety of short, branched, and substituted esters. Substitutional analysis …

The Role Of Inositol Polyphosphate-4-Phosphatase Type Ii B (Inpp4b) In Obese Models And Endocrine Cancers, Manqi Zhang

FIU Electronic Theses and Dissertations

INPP4B is a dual-specificity phosphatase and a tumor suppressor in prostate and breast cancers. Progression of the prostate and breast cancers depends on the androgen receptor (AR) or estrogen receptor alpha (ERα) signaling, respectively. In this work we demonstrated that INPP4B reprograms ERα transcriptional activity in breast cancer. INPP4B maintains expression and protein levels of progesterone receptor (PR), an ERα direct target gene required for mammary gland development. Consistently we demonstrated that Inpp4b knockout severely impairs lateral branching in the mammary gland of maturing virgin females. In advanced prostate cancer, activation and transcriptional reprogramming of AR frequently coincides with the …

Transition State Interactions In A Promiscuous Enzyme: Sulfate And Phosphate Monoester Hydrolysis By Pseudomonas Aeruginosa Arylsulfatase, Bert Van Loo, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Alvan C. Hengge, Florian Hollfelder

Chemistry and Biochemistry Faculty Publications

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PAS^_WT has a much less negative Brønsted coefficient (ß_{leaving group} ^obs-Enz=-0.33) than the uncatalyzed reaction (ß_{leaving group} ^obs=-1.81). This situation is diminished when cationic active site groups are exchanged for alanine. …

Structure And Mechanism Of Mycobacterial Topoisomerase I, Nan Cao

FIU Electronic Theses and Dissertations

The enzyme DNA topoisomerase I is an essential enzyme that plays an important role in eukaryotic and prokaryotic cellular processes such as DNA replication, transcription, recombination and repair. Mycobacterium tuberculosistopoisomerase I (MtTOP1) is a validated drug target for antituberculosis treatment. Mycobacterial topoisomerase I regulates the topological constraints in chromosomes and helps in maintaining the growth of mycobacteria. The N- terminal domain (NTD) of mycobacterial topoisomerase I contains conserved catalytic domains that along with the active site Tyrosine are involved in cleaving and rejoining a single strand of DNA. Magnesium is required in DNA cleavage activity of type IA topoisomerases. …

A Multisession, Undergraduate Molecular Biology Lab Experiment Using Green Fluorescent Protein Including Subcloning And Color Changing Mutagenesis, Nathan S. Winter

Chemistry Faculty Publications

This paper describes a series of experiments involving handling and manipulating the DNA coding for Green Fluorescent Protein (GFP) including the subcloning of this gene, and mutating the DNA so that Cyan Fluorescent Protein (CFP) or Blue Fluorescent protein (BFP) are expressed. The primers needed for the PCR based subcloning of GFP are presented, as are those needed to mutate the GFP to either CFP or BFP.

Characterization Of Metabolic Networks In Differentiated Cd4+ T Cells, Bailee Lichter

Honors Theses

CD4+ T cells play a critical role in the immune system and protecting the body from infection. Cell differentiation of T-cells leads to the specialization of the immune system and has been determined to have plasticity. Differentiation of the CD4+ T cells depends on cytokines present in the environment, concentration of antigens, types of antigen – presenting cells (APCs), and costimulatory molecules (Luckheeram, 2012). Commonly known differentiated T-cells include the T-helper 1 (Th1) and the T-helper 2 (Th2) cells. Upon CD4+ T cell activation, the cells undergo metabolic changes that allow for cell growth and division. By characterizing the metabolic …

Purification Of Hepatocytes And Sinusoidal Endothelial Cells From Mouse Liver Perfusion, Fatima Cabral, Colton M. Miller, Katrina M. Kudrna, Blake E. Hass, Jocelyn G. Daubendiek, Brianna M. Kellar, Edward N. Harris

Department of Biochemistry: Faculty Publications

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated …

The Effects Of Mercury Exposure On The Cytochrome C Oxidase 1 Gene Of Larval Dragonflies, Megan C. Little

Honors College

Mercury is an environmental pollutant; its most toxic form is methylmercury. Once mercury is converted to methylmercury in a body of water it is able to bioaccumulate in organisms and biomagnify up the food chain. Mercury is able to cause DNA damage through the generation of free radicals and binding to sulfhydryl groups of cysteines in zinc finger DNA binding domains, inhibiting DNA repair machinery. In this study the potential mutagenic effects of mercury were investigated on larval dragonflies (Odonta: Anisoptera) collected from national parks across the United States. Since mercury is a known mutagen it was hypothesized that the …

Lipid Binding Studies Of Blood Coagulation Factor Viii C1 And C2 Domains, Rachel L. Blazevic

WWU Honors College Senior Projects

Blood coagulation factor VIII (fVIII) is an essential cofactor in the mammalian blood-clotting cascade. fVIII must bind the phospholipid membrane of activated platelets to function as a cofactor for fIXa. The blood coagulation cascade culminates in the formation of a stable blood clot. In humans, the C1 and C2 domains are implicated in binding phospholipid membranes, however the relative contribution of different residues in the lipid-binding mechanism is unclear. Using site-directed mutagenesis, expression of the isolated C1 and C2 domains in Escherichia coli cells, protein purification with metal affinity chromatography, electrospray ionization mass spectrometry, enzyme-linked immunosorbent assays, liposome sedimentation assays, …

A Polyaniline-Based Sensor Of Nucleic Acids, Partha Sengupta, Jared Gloria, Marcus K. Parker, Alex S. Flynt

Faculty Publications

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies rely on enzymatic modification that can introduce bias and artifacts. A critical element of next generation detection platforms will be direct molecular sensing, thereby avoiding a need for amplification or labels. Advanced nanomaterials may provide the suitable chemical modalities to realize label-free sensors. Conjugated polymers are ideal for biological sensing, possessing properties compatible with biomolecules and exhibit high sensitivity to localized environmental changes. In this article, a method is presented for detecting nucleic acids using the electroconductive …

Mutant Study Of Sinorhizobium Meliloti Proline Utilization A (Puta), Jacob E. Wilkinson, John J. Tanner, Donald F. Becker

UCARE Research Products

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or …

“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 …

Oxidative Dna Damage Modulates Trinucleotide Repeat Instability Via Dna Base Excision Repair, Meng Xu

FIU Electronic Theses and Dissertations

Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington’s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase β (pol b) then skipped over the hairpin creating a 5’-flap that was cleaved by flap …

An Ethnobotanical Approach To Finding Antimicrobial Compounds In Wooly Blue Curls (Trichostema Lanatum) Using A Kirby-Bauer Disc Diffusion Assay, Matthew C. Fleming, P. Matthew Joyner

Featured Research

Plants can be an important source of creativity and production of new drugs. In this study, extracts of wooly blue curls (Trichostema lanatum) were made using DMSO and tested for antimicrobial activity on a panel of bacteria commonly found in separate ecological niches. Wooly blue curls (WBC) was chosen due to its being recorded as a strong disinfectant by the Chumash people. It was found that WBC does exhibit antimicrobial activity against gram positive bacteria and not against gram negative bacteria. However, gram negative bacteria with reduced drug efflux function became susceptible to the WBC extract.

Integrating Art And Science In Undergraduate Education, Daniel Gurnon

Chemistry & Biochemistry Faculty publications

The prevailing vision for undergraduate science education includes increased collaboration among teachers of science, technology, engineering and math (STEM) and an overhaul of introductory courses [1]–[4]. But by staying within the borders of STEM, are we overlooking connections between the arts and innovative science? Likewise, are we missing an important opportunity to inspire and inform nonscientists? Here we explore how weaving the visual arts into a science curriculum can both help develop scientific imagination and engage nonscientists. As an example, we describe a recent collaboration between artists and scientists to create a series of science-inspired sculptures.

Structurally Diverse Hamigerans From The New Zealand Marine Sponge Hamigera Tarangaensis: Nmr-Directed Isolation, Structure Elucidation And Antifungal Activity, A. Jonathan Singh, Jonathan D. Dattelbaum, Jessica J. Field, Zlatka Smart, Ethan F. Woolly, Jacqueline M. Barber, Rosemary Heathcott, John H. Miller, Peter T. Northcote

Chemistry Faculty Publications

The NMR-directed investigation of the New Zealand marine sponge Hamigera tarangaensis has afforded ten new compounds of the hamigeran family, and a new 13-epi-verrucosane congener. Notably, hamigeran F (6) possesses an unusual carbon–carbon bond between C-12 and C-13, creating an unprecedented skeleton within this class. In particular, the structural features of 6, hamigeran H (10) and hamigeran J (12) imply a diterpenoid origin, which has allowed the putative biogenesis of three hamigeran carbon skeletons to be proposed based on geranyl geranyl pyrophosphate. All new hamigerans exhibited micromolar activity towards the HL-60 …

Her2 Targeted Molecular Mr Imaging Using A De Novo Designed Protein Contrast Agent, Jingjuan Qiao, Shunyi Li, Lixia Wei, Jie Jiang, Robert Long, Hui Mao, Ling Wei, Liya Wang, Hua Yang, Hans E. Grossniklaus, Zhi-Ren Liu, Jenny J. Yang

Chemistry Faculty Publications

The application of magnetic resonance imaging (MRI) to non-invasively assess disease biomarkers has been hampered by the lack of desired contrast agents with high relaxivity, targeting capability, and optimized pharmacokinetics. We have developed a novel MR imaging probe targeting to HER2, a biomarker for various cancer types and a drug target for anti-cancer therapies. This multimodal HER20targeted MR imaging probe integrates a de novo designed protein contrast agent with a high affinity HER2 affibody and a near IR fluorescent dye. Our probe can differentially monitor tumors with different expression levels of HER2 in both human cell lines and xenograft mice …

Novel Adaptor-Dependent Domains Promote Processive Degradation By Clpxp, Keith L. Rood

Masters Theses 1911 - February 2014

Protein degradation by ATP dependent proteases is a universally conserved process. Recognition of substrates by such proteases commonly occurs via direct interaction or with the aid of a regulatory adaptor protein. An example of this regulation is found in Caulobacter crescentus, where key regulatory proteins are proteolysed in a cell-cycle dependent fashion. Substrates include essential transcription factors, structural proteins, and second messenger metabolism components. In this study, we explore sequence and structural requirements for regulated adaptor mediated degradation of PdeA, an important regulator of cyclic-di-GMP levels.

Robust degradation of PdeA is dependent on the response regulator CpdR in vivo …

Resonance Assignments And Secondary Structure Predictions Of The As(Iii) Metallochaperone Arsd In Solution, Jun Ye, Yanan He, Jack Skalicky, Barry P. Rosen, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

ArsD is a metallochaperone that delivers As(III) to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Conserved ArsD cysteine residues (Cys12, Cys13 and Cys18) construct the As(III) binding site of the protein, however a global structural understanding of this arsenic binding remains unclear. We have obtained NMR assignments for ArsD as a starting point for probing structural changes on the protein that occur in response to metalloid binding and upon formation of a complex with ArsA. The predicted solution structure of ArsD is in agreement with recently published …