Biochemistry, Biophysics, and Structural Biology Commons^™

Hiv-1 Transcription Elongation By Tat-Mediated Recruitment Of P-Tefb, Elizabeth Griggs

Honors Theses

Over 38.0 million people live with the human immunodeficiency virus (HIV) as of 462019. HIV hijacks the host's cellular machinery to replicate its viral DNA and transcribe the corresponding RNA. HIV-1 transcription relies on both cellular and viral transcription factors for proper regulation. The viral transcriptional activator Tat is a primary regulator. Transcription activation and elongation is controlled through the interaction of Tat with Positive Transcription Elongation Factor b (P-TEFb), a cellular transcriptional activator. The focus of this paper is 1) an in-depth understanding of the interaction between P-TEFb and Tat in HIV transcription, and 2) a review of recent …

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 …

Analysis Of Caveolin-1 Expression And Interaction With Fshr In Lipid Raft Domains, Joseph Bott

Honors Theses

For thousands of women in the United States ovarian cysts, tumors, and hormonal imbalances contribute to reduced fertility and fecundity. In addition to these factors, many other unexplained physiological mechanisms may play a role in women’s reproductive health. Specifically, human follicle stimulating hormone (hFSH) binds to a G protein coupled receptor (GPCR) called human follicle stimulating hormone receptor (hFSHR). This interaction helps to regulate the reproductive system in both males and females indicating that it may be a key player to target in order to promote fertility. Previous work in our lab has demonstrated that hFSHR, like other GPCR, has …

Covalent Modification Of Genetically Engineered Polymers With Bodipy Fluorophores, Emilie Smith

Honors Theses

Osteoarthritis and other debilitating diseases cause chronic pain that is difficult to manage. Nonsteroidal Anti-inflammatory Drugs (NSAIDs) are the current standard of treatment for many long-term pain management, but there are potential systemic effects when using NSAIDs long-term. Therefore, it isnecessary to have an alternative option for NSAID delivery that doesn’t include these systemic effects. Hydrogels can be that alternative option. Hydrogels’ distinct characteristics allow them to be viable in the body, degrade under unique conditions, and be modified through many different chemistries. Additionally, hydrogels can be engineered and their properties optimized for the use as a drug depot inside …

Chemical And Co-Solute Effects Of Polyethylene Glycol On I-Motif Formation, Lindsey Rutherford

Honors Theses

DNA typically forms Watson and Crick double helix structures in which adenine, thymine, guanine, and cytosine pair with their complimentary DNA base. However, DNA i-motif structures can form in cytosine rich DNA, typically under slightly acidic conditions (~pH 6). DNA i-motifs are four stranded secondary structures in which cytosine pairs with cytosine to form a quadruplex. The i-motifs are typically formed in acidic conditions because of the protonation in the C•C base pair between one of the three hydrogen bases. Recent studies have suggested i-motifs can also form under neutral conditions, which is more realistic for a cell. It is …

Effects Of Crowding Agents On I-Motif Dna, Hayden Brines

Honors Theses

Deoxyribonucleic acid (DNA) is a well-known double stranded, helical, biological molecule. In addition to its more commonly known structure, DNA can also form more complicated structures like G-quadruplexes and i-motifs (iM). The iMs are formed by cytosine rich DNA and are a four stranded structure that is typically looped around itself. The iM formation is typically pH-dependent and is favored in more acidic conditions; the pKa value is approximately 6.5. This pKa value allows for potential in vivo formation, since the cells have a pH of approximately 7.3. Due to this, iMs are thought to be powerful, innovative molecules for …

Construction And Analysis Of Three Multi-Partite Synthetic Microbial Communities, Alexander J. Lazzara, Jacob K. Fanning

Honors Theses

Microbial Communities are of interest to molecular biologists hoping to understand the nature of metabolic interactions between co-existing, or possibly mutualistic, organisms. These interactions are ubiquitous in nature, but understanding the molecular mechanisms involved remains challenging and not well understood. Here, we design three tri-partite microbial circuits based on possible interactions among involved microbes, which are discussed and may suggest mutualistic interactions. Carbon and nitrogen molecular pathways and the intracellular metabolism of each microbe is discussed. We present minimal growth media that will ensure that organisms utilize available resources, which may originate from metabolic processes in neighboring microbes, simulating a …

Molecular Dynamic Simulation Of The Complex Folding Patterns Of Apolipoprotein A1 In Various Concentrations Of Potassium Chloride, Hannah Holmberg

Honors Theses

Apopliprotein or ApoA-1 is a complex lipoprotein that functions in the removal of cholesterol from the blood, removing cholesterol from the area around white blood cells and promoting the excretion of lipids through the lymphatic system. Previous research has found that ApoA-1 shows both folded and unfolded conformations depending on the concentration of NaCl in solution in the water around it. The protein was studied using molecular dynamics simulations. Once this state of equilibrium was reached, various structural properties of the protein were measured including the radius of gyration and the radial distribution function. The goal of the project was …

Molecular Characterization Of Galectin From Amblyomma Americanum In Context Of Α-Gal Syndrome, Sumar Beauti

Honors Theses

The lone star tick Amblyomma americanum is a vector of various disease-causing pathogens and tick-borne alpha-gal syndrome (AGS) with rapidly expanding populations in the south- and northeast regions of the United States. This study aimed to molecularly characterize galectin and determine its involvement in galactose-α-1,3-galactose (α-gal) synthesis, transport, reproductive fitness, and microbial homeostasis in this tick. The lone star tick galectin possesses two conserved carbohydrate recognition domains and shares homology with other Ixodid tick galectins. Time and tissue-dependent expression data shows that galectin is constantly expressed in salivary glands, midgut, and ovary tissues. An RNA interference approach was used to …

Biochemical And Computational Characterization Of Small Regulatory Rnas In The Eastern Oyster, Crassostrea Virginica, Isabelle Townsend

Honors Theses

RNA interference (RNAi) is a process by which gene expression is regulated using small non-coding RNAs (ncRNAs). Three classes of ncRNAs, including micro-RNA (miRNA), short-interfering RNA (siRNA), and Piwi-interacting RNA (piRNA), are readily distinguishable in eukaryotic systems based on unique characteristics such as read sizes, overlap signatures, and mode of biogenesis. In this study, a method for purification of small RNAs was explored in the eastern oyster, Crassostrea virginica. This method involved the use of Sepharose beads for anion exchange chromatography to enhance purification of Argonaute associated small RNAs. Following RNA extraction and purification, small RNA libraries were created …

The Effect Of The Apolipoprotein A1 (Apoa1): The Stability And Folding In Potassium Chloride Environment, Alexandra Paladian

Honors Theses

Healthy levels of potassium chloride (KCl) can significantly affect the workings of the cholesterol level of the human body and how they pertain to an individual person. The search for a better salt additive for the human diet can provide a better option for people who experience high cholesterol levels and heart disease. The study focuses on the experimental design of the Molecular Dynamic (MD) simulation of the Apolipoprotein A1 (APOA1) in the potassium ion solution environment to determine the stability and folding of the protein. The study also compares its data to the previous experimental design of chloride ions …

Analyzing The Effects Of E-Hook Peptides On Kinesin-1, Ashton Ward Murrah, Baylee Hope Howard

Honors Theses

Cancer is the second leading cause of death in the United States. Cancerous growth is a result of oncogenes, or mutated genes that increase the rate of cell division in an uncontrolled manner. Cell division, which consists of mitosis and cytokinesis phases, is dependent upon the active movement of kinesin motor proteins along microtubules to rearrange the cytoskeleton for equitable distribution of genetic material to daughter cells. As kinesins are vital to this process, if we could prevent kinesin from binding to the microtubules, cell division would cease.

The goal of this study is to develop a method to prevent …

Finding Optimal Opa-Iblc Derivatized Amino Acid Analysis Parameters With Three-Dimensional Fluorometric Scans For Hplc Method Validation, Aaron Jacobs

Honors Theses

Three-dimensional fluorometric scans of OPA-IBLC derivatized glutamic acid and histidine solutions revealed that decreasing concentration maintained maximum λem but produced different maximum λex. In pH 0.4 M sodium borate buffer (pH 10.4), 50 μM derivatized amino acid solutions had maxima around 350- to 450-nm λex-λem where 12.5 μM solutions had maxima around 230- to 450-nm λex- λem. In the lower concentration, the λex peak at 230 nm was about 80% higher than 350 nm. Thus, collecting spectra with 230 nm λex would provide better LOD and LOQ. Results inform HPLC standard concentrations since differential quenching effects may produce non-linear calibration …

Thermodynamic Analysis Of Pamam Dendrimer Synthesis, Ryutaro Einar Jacobson

Honors Theses

The objective of this research was to determine kinetic and thermodynamic data about the reaction between ethylene diamine and methyl acrylate which is used in the production of PAMAM dendrimers. Establishing a rate law and finding values such as rate constant, activation energy, and Arrhenius coefficient will help in determining the best possible conditions for the reaction. A rate law was established through the method of initial rates, comparing the rates of reactions with different initial concentrations to one another. Additional reactions were run with active cooling to observe the effects on rate and reaction progress, and finally reactions were …

Construction Of E. Coli Strain With N-Terminal Domain Deletion In The Biotin Carboxyl Carrier Protein, Dara Khosravi

Honors Theses

No abstract provided.

Indolizine Donor-Based Dyes For Applications In Fluorescence Biological Imaging, William Meador

Honors Theses

NIR emissive fluorophores are intensely researched due to their potential to replace modern imaging procedures. Many molecular strategies have been employed in the literature to optimize fluorophores for deeper NIR absorption and emission, biocompatibility, and higher fluorescence quantum yields. Amongst the fluorophores studied to date, proaromatic indolizine donors are attractive alternatives to traditional alkyl amine and indoline based donors due to their 1) lower energy absorption and emission facilitated by proaromaticity, 2) large Stokes shifts due to increased dihedral angles about the π-system, 3) ease of functionalization and capacity for bioconjugation at the phenyl ring, and 4) potential for further …

Using Crispr-Cas9 To Construct Knockout Mutants In Dna-Repair Genes In Arabidopsis Thaliana, David Campbell

Honors Theses

The mitochondria are known as the powerhouse of the cell, and just like a real powerhouse, it can be a dangerous place to store sensitive information. Energy generation and redox reactions in the mitochondria can cause damage to the DNA stored there, resulting in a higher mutation rate. Compared to their animal counterparts, however, plant mitochondria exhibit a lower mutation rate and a higher recombination rate. It is hypothesized that the unique DNA repair methods present in plant mitochondria are responsible for the phenomena observed there. To study the mechanics of DNA-repair in this organelle, however, researchers must be able …

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 …

Structure Of Clostridium Perfringens Type Iv Pili, Alexander R. Meyer

Honors Theses

Type IV pili (T4P) are thin, hair-like bacterial appendages composed of protein subunits polymerized into a helical fiber. T4P perform diverse functions such as host cell adhesion, biofilm formation, natural competence, and twitching motility. While T4P are well characterized in Gram-negative bacteria, they have more recently been found in Gram-positive bacteria as well. In this work we aimed to solve the crystal structure of the type IV major pilin protein PilA2 from Clostridium perfringens, the predominant pilus subunit which makes up about 99% of the pilus fiber. We report expression, purification, and crystallization conditions which are sufficient for X-ray …

The Quantitative Assessment Of Pond Scum: An Examination Of The Biogeochemistry Of Phosphorus Cycling In The Belgrade Lakes, Abbey M. Sykes

Honors Theses

The internal recycling phosphorus in freshwater lake bottom sediments represents a significant source of hypolimnetic phosphorus (P) release for many of Maine’s lakes. In summer months, Maine lakes often thermally stratify and the lake hypolimnion develops anoxia, leading to a reduction in redox potential at the sediment-water interface. These reducing conditions facilitate the reductive dissolution of ferric iron, and, since phosphorus is often present in freshwater lake sediments as solid FeOOH-PO₄ complexes, results in release of soluble phosphorus into the water column. Our current study presents field and laboratory data from sediment fractionation extractions designed to quantify concentrations of …

Use Of Small Molecule Fanconi Anemia Pathway Inhibitors As Sensitizing Agents To Laromustine., Sam W. Marchant

Honors Theses

Laromustine is an experimental chemotherapeutic sulfonyl hydrazine prodrug shown in clinical trials to be effective against acute myeloid leukemia. The mechanism of action of laromustine involves interstrand crosslinking, via chloroethylation, and enzyme inhibition, caused by carbamoylation. The work described herein aims to investigate whether inhibition of the replication-dependent interstrand crosslink repair Fanconi Anemia pathway further sensitizes cells to laromustine. By measuring metabolic activity immediately after drug exposure, we find laromustine to be equally as cytotoxic towards Fanconi Anemia deficient and wild type cells. However, through clonogenic assays we show Fanconi Anemia mutations sensitize cells to laromustine’s anti-proliferative effect. Furthermore, we …