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Bacteria with antibiotic resistance are becoming a growing concern as the number of infections they are causing continue to increase. Many potential solutions are being researched in order to combat these pathogens. One such microbe is Pseudomonas aeruginosa, which causes acute and chronic human infections. It frequently colonizes the lungs of cystic fibrosis patients and is deadly. For these reasons, P. aeruginosa has been heavily studied in order to determine a solution to antibiotic resistance. One possible solution is the development of synbodies, which have been developed at the Biodesign Institute at Arizona State University. Synbodies are constructed from peptides that have antibacterial activity and were determined to have specificity for a target bacterium. These synbodies were tested in this study to determine whether or not some of them are able to inhibit P. aeruginosa growth. P. aeruginosa can also form multicellular communities called biofilms and these are known to cause approximately 65% of all human infections. After conducting minimum inhibitory assays, the efficacy of certain peptides and synbodies against biofilm inhibition was assessed. A recent study has shown that low concentrations of a specific peptide can cause biofilm disruption, where the biofilm structure breaks apart and the cells within it disperse into the supernatant. Taking into account this study and peptide data regarding biofilm inhibition from Dr. Aurélie Crabbé’s lab, screened peptides were tested against biofilm to see if dispersion would occur.
ContributorsPatel, Justin Hemant (Author) / Diehnelt, Chris (Thesis director) / Stafford, Phillip (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor)
Created2015-05
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
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
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
Approximately 248 million people in the world are currently living with chronic Hepatitis B virus (HBV) infection. HBV and HCV infections are the primary cause of liver diseases such as cirrhosis and hepatocellular carcinomas in the world with an estimated 1.4 million deaths annually. HBV in the Republic of Peru was used as a case study of an emerging and rapidly spreading disease in a developing nation. Wherein, clinical diagnosis of HBV infections in at-risk communities such the Amazon Region and the Andes Mountains are challenging due to a myriad of reasons. High prices of clinical diagnosis and limited access to treatment are alone the most significant deterrent for individuals living in at-risk communities to get the much need help. Additionally, limited testing facilities, lack of adequate testing policies or national guidelines, poor laboratory capacity, resource-limited settings, geographical isolation, and public mistrust are among the chief reasons for low HBV testing. Although, preventative vaccination programs deployed by the Peruvian health officials have reduced the number of infected individuals by year and region. To significantly reduce or eradicate HBV in hyperendemic areas and countries such as Peru, preventative clinical diagnosis and vaccination programs are an absolute necessity. Consequently, the need for a portable low-priced diagnostic platform for the detection of HBV and other diseases is substantial and urgent not only in Peru but worldwide. Some of these concerns were addressed by designing a low-cost, rapid detection platform. In that, an immunosignature technology (IMST) slide used to test for reactivity against the presence of antibodies in the serum-sample was used to test for picture resolution and clarity. IMST slides were scanned using a smartphone camera placed on top of the designed device housing a circuit of 32 LED lights at 647 nm, an optical magnifier at 15X, and a linear polarizing film sheet. Tow 9V batteries powered the scanning device LED circuit ensuring enough lighting. The resulting pictures from the first prototype showed that by lighting the device at 647 nm and using a smartphone camera, the camera could capture high-resolution images. These results conclusively indicate that with any modern smartphone camera, a small box lighted to 647 nm, and optical magnifier; a powerful and expensive laboratory scanning machine can be replaced by another that is inexpensive, portable and ready to use anywhere.
ContributorsMakimaa, Heyde (Author) / Holechek, Susan (Thesis director) / Stafford, Phillip (Committee member) / Jayasuriya, Suren (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Monoclonal antibody therapy focuses on engineering immune cells to target specific peptide sequences indicative of disease. An impediment in the continued advancement of this market is the lack of an efficient, inexpensive means of characterization that can be broadly applied to any antibody while still providing high-density data. Many characterization methods address an antibody's affinity for its cognate sequence but overlook other important aspects of binding behavior such as off-target binding interactions. The purpose of this study is to demonstrate how the binding intensity between an antibody and a library of random-sequence peptides, otherwise known as an immunosignature, can be evaluated to determine antibody specificity and polyreactivity. A total of 24 commercially available monoclonal antibodies were assayed on 125K and 330K peptide microarrays and analyzed using a motif clustering program to predict candidate epitopes within each antigen sequence. The results support the further development of immunosignaturing as an antibody characterization tool that is relevant to both therapeutic and non-therapeutic antibodies.
ContributorsDai, Jennifer T. (Author) / Stafford, Phillip (Thesis director) / Diehnelt, Chris (Committee member) / School of Life Sciences (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
In this project, biochemical characteristics of peptide binding agents, synthetic antibodies or synbodies, were examined with respect to the capture efficiency and specific binding ability to norovirus. Norovirus, although generally not a deadly pathogen, is the most common cause of acute gastroenteritis and outbreaks present a large social and financial burden to the healthcare and food service industries. With Dr. Diehnelt's laboratory group, a platform has been developed that enables us to rapidly construct peptide-based affinity ligands that can be characterized for binding to norovirus. The design needed to display clear results, be simple to operate, and be inexpensive to produce and use. Four synbodies, originally engineered with a specificity to the GII.4 Minerva genotype were tested with different virus strains varying in similarity to the GII.4 Minerva between 43% and 95.4%. Initial assays utilized norovirus-like particles to qualitatively compare the capture efficiency of the different synbodies without utilizing limited resources. To quantify the amount of actual virus captured by the synbodies, western blots with RT-PCR and RT-qPCR were utilized. The results indicated the synbodies were able to enrich the dilute solutions of the different noroviruses utilizing a magnetic bead pull-down assay. The capture efficiencies of the synbodies were comparable to currently utilized binding agents such as aptamers and porcine gastric mucine magnetic beads. This thesis presents data collected over nearly two years of research at the Center for Innovations in Medicine at the Biodesign Institute located at Arizona State University.
ContributorsSlosky, Rachael Marie (Author) / Diehnelt, Chris (Thesis director) / Stafford, Phillip (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Nucleic acid polymers have numerous applications in both therapeutics and research to control gene expression and bind biologically relevant targets. However, due to poor biological stability their clinical applications are limited. Chemical modifications can improve both intracellular and extracellular stability and enhance resistance to nuclease degradation. To identify a potential candidate for a highly stable synthetic nucleic acid, the biostability of α-L-threofuranosyl nucleic acid (TNA) was evaluated under simulated biological conditions. TNA contains a four-carbon sugar and is linked by 2’, 3’ phosphodiester bonds. We hypothesized that this distinct chemical structure would yield greater nuclease resistance in human serum and human liver microsomes, which were selected as biologically relevant nuclease conditions. We found that TNA oligonucleotides remained undigested for 7 days in these conditions. In addition, TNA/DNA heteropolymers and TNA/RNA oligonucleotide duplexes displayed nuclease resistance, suggesting that TNA has a protective effect over DNA and RNA. In conclusion TNA demonstrates potential as a viable synthetic nucleic acid for use in numerous clinical and therapeutic applications.
ContributorsCulbertson, Michelle Catherine (Author) / Maley, Carlo (Thesis director) / Mangone, Marco (Committee member) / Larsen, Andrew (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12