Matching Items (110)
Description
Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.
ContributorsHansen, Debra (Author) / Robida, Mark (Author) / Craciunescu, Felicia (Author) / Loskutov, Andrey (Author) / Dorner, Katerina (Author) / Rodenberry, John-Charles (Author) / Wang, Xiao (Author) / Olson, Tien (Author) / Patel, Hetal (Author) / Fromme, Petra (Author) / Sykes, Kathryn (Author) / Biodesign Institute (Contributor) / Innovations in Medicine (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor)
Created2016-02-24
Description
In eukaryotes, most messenger RNA precursors (pre-mRNA) undergo extensive processing, leading to the cleavage of the transcript followed by the addition of a poly(A) tail. This process is executed by a large complex known as the Cleavage and Polyadenylation Complex (CPC). Its central subcomplex, the Cleavage and Polyadenylation Specificity Factor (CPSF) complex is responsible for recognizing a short hexameric element AAUAAA located at the 3’end in the nascent mRNA molecule and catalyzing the pre-mRNA cleavage. In the round nematode C. elegans, the cleavage reaction is executed by a subunit of this complex named CPSF3, a highly conserved RNA endonuclease. While the crystal structure of its human ortholog CPSF73 has been recently identified, we still do not understand the molecular mechanisms and sequence specificity used by this protein to induce cleavage, which in turn would help to understand how this process is executed in detail. Additionally, we do not understand in additional factors are needed for this process. In order to address these issues, we performed a comparative analysis of the CPSF3 protein in higher eukaryotes to identify conserved functional domains. The overall percent identities for members of the CPSF complex range from 33.68% to 56.49%, suggesting that the human and C. elegans orthologs retain a high level of conservation. CPSF73 is the protein with the overall highest percent identity of the CPSF complex, with its active site-containing domain possessing 74.60% identity with CPSF3. Additionally, we gathered and expressed using a bacterial expression system CPSF3 and a mutant, which is unable to perform the cleavage reaction, and developed an in vitro cleavage assay to test whether CPSF3 activity is necessary and sufficient to induce nascent mRNA cleavage. This project establishes tools to better understand how CPSF3 functions within the CPC and sheds light on the biology surrounding the transcription process as a whole.
ContributorsGallante, Christina (Author) / Mangone, Marco (Thesis director) / Sharma, Shalini (Committee member) / Hrach, Heather (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Dielectrophoresis has been shown in the recent past to successfully separate bioparticles of very subtle differences at high resolutions using biophysical forces. In this study, we test the biophysical differences of methicillin resistant and susceptible Staph. aureus that are known to have very similar genomes by using a modified gradient insulator-based dielectrophoresis device (g-iDEP). MRSA is commonly seen in hospitals and is the leading killer of infectious bacteria, claiming the lives of around 10,000 people annually. G-iDEP improves many capabilities within the DEP field including sample size, cost, ease of use and analysis time. This is a promising foundation to creating a more clinically optimized diagnostic tool for both separation and detection of bacteria in the healthcare field. The capture on-set potential for fluorescently tagged MRSA (801 ± 34V) is higher than fluorescently tagged MSSA (610 ± 32V), resulting in a higher electrokinetic to dielectrophoretic mobility ratio for MRSA. Since the strains have proven to be genomically similar through sequencing, it is reasonable to attribute this significant biophysical difference to the added PBP2a enzyme in MRSA. These results are consistent with other bacterial studied within in this device and have proven to be reproducible.
ContributorsSmithers, Jared (Author) / Hayes, Mark (Thesis director) / Woodbury, Neal (Committee member) / School of Criminology and Criminal Justice (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The successful reduction of CO2 and protons by a light-induced cobalt porphyrin/cytb562 hybrid metalloenzyme in water is reported. Incorporation of the porphyrin into a protein scaffold results in increases in CO and H2 production over naked porphyrin. Rational point mutations to the CoPPIX binding site of cytb562 modulate production, indicating possible further improvements in catalytic activity.
ContributorsGwerder, Noah D (Author) / Ghirlanda, Giovanna (Thesis director) / Williams, Peter (Committee member) / Mangone, Marco (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Diabesity is a global epidemic affecting millions worldwide. Diabesity is the term given to the link between obesity and Type II diabetes. It is estimated that ~90% of patients diagnosed with Type II diabetes are overweight or have struggled with excess body fat in the past. Type II diabetes is characterized by insulin resistance which is an impaired response of the body to insulin that leads to high blood glucose levels. Adipose tissue, previously thought of as an inert tissue, is now recognized as a major endocrine organ with an important role in the body's immune response and the development of chronic inflammation. It is speculated that adipose tissue inflammation is a major contributor to insulin resistance particular to Type II diabetes. This literature review explores the popular therapeutic targets and marketed drugs for the treatment of Type II diabetes and their role in decreasing adipose tissue inflammation. rAGE is currently in pre-clinical studies as a possible target to combat adipose tissue inflammation due to its relation to insulin resistance. Metformin and Pioglitazone are two drugs already being marketed that use unique chemical pathways to increase the production of insulin and/or decrease blood glucose levels. Sulfonylureas is one of the first FDA approved drugs used in the treatment of Type II diabetes, however, it has been discredited due to its life-threatening side effects. Bariatric surgery is a form of invasive surgery to rid the body of excess fat and has shown to normalize blood glucose levels. These treatments are all secondary to lifestyle changes, such as diet and exercise which can help halt the progression of Type II diabetes patients.
ContributorsRobles, Alondra Maria (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Allen, James (Committee member) / Hendrickson, Kirstin (Committee member) / Sanford School of Social and Family Dynamics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease which occurs in approximately 1 in 3,500 male births. This disease is characterized by progressive muscle wasting and causes premature death. One of the earliest symptoms of this disease is mitochondrial dysfunction. Dystrophin is a protein found under the sarcolemma. The N terminus binds to actin and the C terminus binds to dystrophin glycoprotein complex (DGC). DMD is caused by mutations in the dystrophin gene. C. elegans possess an ortholog of dystrophin, DYS-1. Though there is evidence that C. elegans can be used as a model organism to model DMD, nematode DGC has not been well characterized. Additionally, while we know that mitochondrial dysfunction has been found in humans and other model organisms, this has not been well defined in C. elegans. In order to address these issues, we crossed the SJ4103 worm strain (myo-3p::GFP(mit)) with dys-1(cx18) in order to visualize and quantify changes in mitochondria in a dys-1 background. SJ4103;cx18 nematodes were found to have less mitochondrial than SJ4103 which suggests mitochondrial dysfunction does occur in dys-1 worms. Furthermore, mitochondrial dysfunction was studied by knocking down members of the DGC, dys-1, dyb-1, sgn-1, sgca-1, and sgcb-1 in SJ4103 strain. Knock down of each gene resulted in decrease in abundance of mitochondria which suggests that each member of the DGC contributes to the overall health of nematode muscle. The ORF of dyb-1 was successfully cloned and tagged with GFP in order to visualize this DGC member C. elegans. Imaging of the transgenic dyb-1::GFP worm shows green fluoresce expressed in which suggests that dyb-1 is a functional component of the muscle fibers. This project will enable us to better understand the effects of dystrophin deficiency on mitochondrial function as well as visualize the expression of certain members of the DGC in order to establish C. elegans as a good model organism to study this disease.
ContributorsObrien, Shannon Nishino (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Hrach, Heather (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cleavage and polyadenylation is a step in mRNA processing in which the 3’UTR is cleaved and a polyA tail is added to create a final mature transcript. This process relies on RNA sequence elements that guide a large multimeric protein complex named the Cleavage and Polyadenylation Complex to dock on the 3’UTR and execute the cleavage reaction. Interactions of the complex with the RNA and specific dynamics of complex recruitment and formation still remain largely uncharacterized. In our lab we have identified an Adenosine residue as the nucleotide most often present at the cleavage site, although it is unclear whether this specific element is a required instructor of cleavage and polyadenylation. To address whether the Adenosine residue is necessary and sufficient for the cleavage and polyadenylation reaction, we mutated this nucleotide at the cleavage site in three C. elegans protein coding genes, forcing the expression of these wt and mutant 3’UTRs, and studied how the cleavage and polyadenylation machinery process these genes in vivo. We found that interrupting the wt sequence elements found at the cleavage site interferes with the cleavage and polyadenylation reaction, suggesting that the sequence close to the end of the transcript plays a role in modulating the site of the RNA cleavage. This activity is also gene-specific. Genes such as ges-1 showed little disruption in the cleavage of the transcript, with similar location occurring in both the wt and mutant 3’UTRs. On the other hand, mutation of the cleavage site in genes such as Y106G6H.9 caused the activation of new cryptic cleavage sites within the transcript. Taken together, my experiments suggest that the sequence elements at the cleavage site somehow participate in the reaction to guide the cleavage reaction to occur at an exact site. This work will help to better understand the mechanisms of transcription termination in vivo and will push forward research aimed to study post-transcriptional gene regulation in eukaryotes.
ContributorsSteber, Hannah Suzanne (Author) / Mangone, Marco (Thesis director) / Harris, Robin (Committee member) / LaBaer, Joshua (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The Heliobacterial Reaction Center (HbRC) is the simplest Type I Reaction Center (RC) known today. However, upon illumination it has been found to produce menaquinol, and this has led to experiments investigating the function of this reduction scheme. The goal of the experiment was to investigate the mechanisms of menaquinol production through the use of Photosystem II (PSII) herbicides that are known to inhibit the QB quinone site in Type II RCs. Seven herbicides were chosen, and out of all of them terbuthylazine showed the greatest effect on the RC in isolated membranes when Transient Absorption Spectroscopy was used. In addition, terbuthylazine decreased menaquinone reduction to menaquinol by ~72%, slightly more than the reported effect of teburtryn (68%)1. In addition, terbuthylazine significantly impacted growth of whole cells under high light more than terbutryn.
ContributorsOdeh, Ahmad Osameh (Author) / Redding, Kevin (Thesis director) / Woodbury, Neal (Committee member) / Allen, James (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.
ContributorsBrooks, Meilia Catherine (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Ghirlanda, Giovanna (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The free-base tetra-tolyl-porphyrin and the corresponding cobalt and iron porphyrin complexes were synthesized and characterized to show that this class of compound can be promising, tunable catalysts for carbon dioxide reduction. During cyclic voltammetry experiments, the iron porphyrin showed an on-set of ‘catalytic current’ at an earlier potential than the cobalt porphyrin’s in organic solutions gassed with carbon dioxide. The cobalt porphyrin yielded larger catalytic currents, but at the same potential as the electrode. This difference, along with the significant changes in the porphyrin’s electronic, optical and redox properties, showed that its capabilities for carbon dioxide reduction can be controlled by metal ions, allotting it unique opportunities for applications in solar fuels catalysis and photochemical reactions.
ContributorsSkibo, Edward Kim (Author) / Moore, Gary (Thesis director) / Woodbury, Neal (Committee member) / School of Molecular Sciences (Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05