• T Cell Factor-1 (TCF-1) Regulates Mature Alloactivated T Cells to Separate GVHD From GVL

      Mobin Karimi; Harris, Rebecca (2021)
      Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment used for patients with cancer or other hematological malignancies. However, widespread use of this treatment is hindered by development of graft-versus-host disease (GVHD), a life-threatening complication of allo-HSCT. Mature donor T cells in the graft mediate GVHD, but also help kill residual malignant cells in the patient by the graft-versus-leukemia (GVL) effect. Depletion of mature T cells from the graft eliminates this beneficial anti-tumor response. Mature T cells are also needed for proper stem cell engraftment. Therefore, current work has focused on how to modulate T cell signaling and function to separate GVHD from GVL. T Cell Factor-1 (TCF-1) is a T cell developmental transcription factor that is also important in some contexts for T cell activation. The role of TCF-1 in alloactivated mature T cells is completely unknown. To examine the role of TCF-1 in this context, a mouse model of allo-HSCT leading to GVHD/GVL was used to study T cells from mice with a T cell-specific deletion of TCF-1. This work showed that loss of TCF-1 separates GVHD from GVL, with reduced disease severity and persistence yet maintained GVL effects. TCF-1 affects alloactivated T cell phenotypes and suppressive profiles, as well as the major T cell functions (proliferation, migration, and cytokine/cytotoxic mediator production). TCF-1 also controls alloactivated T cell survival, apoptosis, and gene expression programs. The regulation of these functions and programs by TCF-1 is distinct in CD4 versus CD8 T cells. TCF-1 also controls two unique T cell subsets - stem-like CXCR5+ CD8+ T cells, and CD25- noncanonical Tregs. Therefore, TCF-1, or these two unique T cell types, may be a therapeutic target to improve allo-HSCT outcomes by separating GVHD from GVL effects. Expansion of CD25- Tregs during TCF-1 deficiency may also be useful for treatment of other T cell-mediated disorders as well.
    • Targeting SHIP paralogs to promote microglial effector function in the CNS

      Thomas, Stephen J.; Pedicone, Chiara (2022-06)
      The two SH2-containing inositol 5'-phosphatases , SHIP1 (INPP5D) and SHIP2 (INPPL1), play an essential role in modulation of cellular signaling by transforming the PI3K product PI(3,4,5)P3 into PI(3,4)P2. PI3K signaling triggers activation of downstream signaling cascades that drive survival, effector functions, differentiation, and proliferation. SHIP1 can also mask the cytoplasmic tails of key receptors or their adaptor proteins such as DAP12, thus preventing PI3K recruitment to Trem2, a critical receptor for microglial function. Several GWAS studies correlated single nucleotide polymorphisms (SNPs) in INPP5D with Alzheimer's Disease (AD). However, it remains unclear whether these SNPs are deleterious or protective in AD and how they alter SHIP1 protein expression. SHIP2 overexpression has also been correlated with AD, suggesting that both SHIP1 and SHIP2 might be therapeutic targets. To study how SHIP1 and SHIP2 modulate microglial functions we used small molecule inhibitors and agonists of these enzymes. In our initial study we found that both SHIP paralogs are expressed in murine microglia and that Pan- SHIP1/2 inhibition increases lysosomal size and enhances microglial phagocytosis of Ab1-42 fibrils and dead neurons using both flow cytometry and confocal microscopy. Our lead Pan-SHIP1/2 inhibitor, K161, showed Blood Brain Barrier penetration as detected in the cerebral cortex of treated mice with mass spectrometry. K161 treatment of WT mice showed no difference in microglial frequency or lysosomal content in vivo; however, we observed a significant 2 increase in Ab1-42 and dead neurons phagocytosis ex vivo in microglia in K161- treated mice versus controls. Subsequently, we discovered a novel and highly potent SHIP1 selective agonist (K306) via artificial intelligence guided computational screening. We found that K306 can reduce the release of inflammatory cytokines in macrophages and microglial cells stimulated with LPS or Ab1-42. Interestingly, K306 didn't alter microglial phagocytic uptake of cargo, but did promote degradation of phagocytosed lipid-laden cargo - defining a novel role of SHIP1 in degradation of lipid cargo in microglia. These results highlight the importance of SHIP1 and SHIP2 in microglial biology and their modulation as therapeutics in different stages of neurodegenerative disease where microglia play a major role, such as AD.
    • Targeting wild-type and mutant p53 for cancer treatment

      Stewart N Loh; Blayney, Alan John (2021)
    • Targetingof PIM1 KinaseinMyeloproliferative NeoplasmsInduced by JAK2V617F

      Mohi, Golam; Stuver, Matthew (2017)
      Myeloproliferative neoplasms (MPNs) arestem cell-derivedblood disorders. The most common mutation found in MPN patients is the JAK2V617Fmutation. JAK2 is anon-receptor tyrosine kinase involved in STAT signaling. The JAK2V617F mutation is asingle amino acid substitution of a phenylalanine for valine, whichcauses JAK2 to be constitutively activated. This mutation can cause ahematopoietic transformation. Eventuallythis transformationcan lead to the development of one of thethree different Philadelphia-negative MPN diseases: Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF). The JAK2V617F mutationhas been identified in 95% PVpatients,and 50-60% ofETand PMF patients.A JAK1/2 inhibitor (ruxolitinib) has been approved for MF and PV patients and,though it provides initial benefits, it is not effective enough to causelong-termremission in patients. This creates a critical need to identify new therapeutic targets for MPN patients. We found that PIM1 levels were significantly increased inMPN patients, as well asour JAK2V617F mouse modelof MPN.We observedthatknockdown of PIM1 caused a significant decrease in proliferationof JAK2V617F expressing cells. We also found that PIM1 knockdownhad no effect on the proliferation of hematopoietic cells not expressing JAK2V617F, leading us to believe PIM1 is only required in JAK2V617F mediated proliferation. Pharmacological inhibition of PIM kinases,using TP-3654,(kindly provided by Tolero pharmaceuticals)also led to a significant decrease in proliferation of JAK2V617F-expressing cells, but had no effect on cellslacking the mutation. We also found thatthePIM inhibitor,TP-3654,workssynergistically with ruxolitinibto achieve an even greater decrease in proliferation. We found that using the combination of ruxolitiniband TP-3654,we could use both drugs at lower concentrations andachieve an even greater decrease in proliferation and an increase apoptosis. Furthermore,we found that inhibition of PIM kinasesusing TP-3654can resensitize ruxolitinib-resistant cells to ruxolitinibtreatment.These important findingsshow that PIM1 plays animportantrolein the proliferation of hematopoietic cells expressing the JAK2V617F mutation, but is dispensable for the maintenance of cells lacking the mutation. We also found that targeting PIM kinases with TP-3654,significantly decreasedthe proliferation, and increaseapoptosisactivationof JAK2V617Fexpressing cells. We also showedthat TP-3654 and ruxolitinibcan work synergistically. Lastly, we showed that inhibition of PIM kinases,using TP-3654,caused ruxolitinib-resistant cells tobecome resensitized toruxolitinib. These findings helpedus come to the conclusionthatPIM1 kinase, is an importanttherapeutictargetin JAK2V617F-induced MPNs.
    • Teaching of SQL Through a Game

      Ward, Patrick T. (2015-05-01)
      The project seeks to provide an effective alternate method for teaching SQL through the use of a Game. There is value in learning SQL, as SQL skills are still in the top ten list of sought after IT skills for 2015 (Greenspan, 2014). However, lecture based teaching may not fully engage the learner. Therefore, the game was constructed with the MDA framework and Problem-Based Learning in mind. These methods help the learner bridge the gaps between lesson content, problems, and solutions. The game itself was constructed with MSSQL, Adobe Cold Fusion, HTML, CSS, and JavaScript. The game presents lessons on SQL concepts and quiz-based challenges for students to solve. Students faced increasingly difficult challenges as their level SQL knowledge expanded. The project may be found here: http://www.patrickward.net/SQLGame/
    • Technology Case Study in Storage Area Networks

      Marsh, John ; Thesis Advisor; Hash, Larry J.; Climek, David; Bull, Ronny; Pethe, Ameya (2014-05)
      In today's world we need immediate access to data. The demand for networked data access has increased exponentially in the last 20 years. With that demand the importance and volume of networked data has also grown exponentially. The speed at which the data can be accessed has increased and with that the data has moved from individual workstations to a networked location. Over the last decade there has been a trend to move mission critical data away from individual workstations to a centralized data center. A centralized data center removes the location constraint for accessing the data. If critical data is stored on individual servers, a failure will cause the data to be inaccessible. Today, mission critical applications are spanned over multiple servers for redundancy. With this topology, having the data in a central location allows the individual servers to better work with data. With the addition of virtualization, servers can be moved online from one physical server to another. If the data is centralized, it can be presented to all hosts in the cluster. This allows servers to move efficiently between hosts without losing access to the critical data. Many businesses in various industries like finance, airline, hospital, research, etc. depend on the speed and secure availability of their centralized data to function efficiently.
    • A Technology Case Study on Integrating Open Stack with SDN for Internet Connectivity using BGP

      Gonuguntla, Raja Bhushan Rao; Hash, Larry; Advisor (2016-12)
      There were many developments in Internet usage, which resulted in significant increase in Internet routing. With existing networking infrastructure, it is difficult to meet these requirements and causing more cost and less performance. Since network devices are hardware modules, processing them requires more power and more memory. However, if network protocols are developed using software modules, flexibility can be achieved in various programming applications and reduces dependency on hardware. The concept of using networking protocols as a software module can be explained using “Software Defined Networking (SDN).” With SDN, existing infrastructure can be integrated with various applications and centralized control protocols can be developed. One of the key components of SDN is integrating with Cloud Computing, where many applications can be built, which can be used for on-demand services. Integrating cloud computing with SDN will create dynamic networks and reduces infrastructure costs. In this paper, a case was considered for providing better internet connectivity by building public & private networks using Open source cloud technology (OpenStack) and existing distribution environments. For connectivity, BGP was used as routing protocol as it is known to be well- suited for such environments. Both public and private networks were integrated with SDN for centralized control. OpenStack was used to build various network topologies using different plugins through SDN controller. This method allowed to develop SDN controller with global view of OpenStack networks. The same controller was connected to distributed layers using Open Flow protocol. Since, both OpenStack and distributed networks were attached to SDN controller, centralized control of network protocols could be achieved. This model of centralized networks could be very useful in reducing costs and improving network efficiency, especially in large scale deployments.
    • Technology Case Study on Web Real-Time Communications (WebRTC)

      Karnati, Nagarjuna; Hash, Larry; Adviser (2016-05-01)
      Web real-time communication (WebRTC) is the latest technology standard which enables web browsers to communicate directly without having to install any internal or external plug-ins. WebRTC fills a critical gap in the web platform where a native proprietary app like Skype could do something which is media communication that World Wide Web just couldn’t. Now, this can be done form web using WebRTC technology. This paper starts with a brief introduction of WebRTC and how it got started. Moving on, it provides information about the WebRTC technical goals, architecture and protocols involved. This paper highlights the network address translation (NAT) traversal where STUN, TURN and ICE protocols are involved. Also, this paper highlights about the peer to peer to media flows with reference to WebRTC protocol stack and application program interface (API). In the end, this paper discusses about implemented security features, tools available for WebRTC development and provides enterprise use cases.
    • Testing the Effectiveness of an International Conservation Agreement: Marketplace Forensics and CITES Caviar Trade Regulation

      Doukakis, Phaedra; Pikitch, Ellen K.; Rothschild, Anna; DeSalle, Rob; Amato, George; Kolokotronis, Sergios-Orestis (Public Library of Science (PLoS), 2012-07-25)
      Background: The international wildlife trade is a key threat to biodiversity. Temporal genetic marketplace monitoring can determine if wildlife trade regulation efforts such as the Convention on International Trade in Endangered Species (CITES) are succeeding. Protected under CITES effective 1997, sturgeons and paddlefishes, the producers of black caviar, are flagship CITES species. Methodology/principal findings: We test whether CITES has limited the amount of fraudulent black caviar reaching the marketplace. Using mitochondrial DNA-based methods, we compare mislabeling in caviar and meat purchased in the New York City area pre and post CITES listing. Our recent sampling of this market reveals a decrease in mislabeled caviar (2006-2008; 10%; n = 90) compared to pre-CITES implementation (1995-1996; 19%; n = 95). Mislabeled caviar was found only in online purchase (n = 49 online/41 retail). Conclusions/significance: Stricter controls on importing and exporting as per CITES policies may be having a positive conservation effect by limiting the amount of fraudulent caviar reaching the marketplace. Sturgeons and paddlefishes remain a conservation priority, however, due to continued overfishing and habitat degradation. Other marine and aquatic species stand to benefit from the international trade regulation that can result from CITES listing.
    • Text Detection from an Image

      Andriamanalimanana, Bruno R.; Thesis Advisor; Novillo, Jorge; Thesis Committee; Spetka, Scott; Thesis Committee; Goda, Piyush Jain (2020-12)
      Recently, a variety of real-world applications have triggered a huge demand for techniques that can extract textual information from images and videos. Therefore, image text detection and recognition have become active research topics in computer vision. The current trend in object detection and localization is to learn predictions with high capacity deep neural networks trained on a very large amount of annotated data and using a high amount of processing power. In this project, I have built an approach for text detection using the object detection technique. Our approach is to deal with the text as objects. We use an object detection method, YOLO (You Only Look Once), to detect the text in the images. We frame object detection as a regression problem to spatially separated bounding boxes and associated class probabilities. YOLO, a single neural network, that predicts bounding boxes and class probabilities directly from full images in one evaluation. Since the whole detection pipeline is a single network, it can be optimized end-to-end directly on detection performance. The MobileNet pre-trained deep learning model architecture was used and modified in different ways to find the best performing model. The goal is to achieve high accuracy in text spotting. Experiments on standard datasets ICDAR 2015 demonstrate that the proposed algorithm significantly outperforms methods in terms of both accuracy and efficiency.
    • The Effect of Multifocal Contact Lenses on Accomodation and Phoria in a Pediatric Population

      Gong, Celia (2017)
      The increasing prevalence of the use of distance-centered multifocal (MF) contact lenses as a method of myopia control in the pediatric population calls for a better understanding of binocularity and accommodation in children wearing these lenses. This was a prospective, randomized, crossover, single visit study that enrolled myopic children with normal accommodation and binocular vision and no history of myopia control treatment. All subjects were fitted with Coopervision Biofinity single vision (SV) and MF (+2.50D center distance add) contact lenses. Accommodative responses (photorefraction) and phorias (Modified Thorington) were measured at 4 distances (>3m, 100cm, 40cm, 25cm). Secondary measures included high and low contrast logMAR acuity, accommodative amplitude, and accommodative facility. Differences between MF and SV contact lenses were analyzed using repeated measures regression and paired t-tests. A total of 16 subjects, aged 10-15 years, completed the study. There was a small decrease in high (SV: -0.08, MF: +0.01) and low illumination (SV:-0.03, MF: +0.08) (both p<0.01) visual acuity, and contrast sensitivity (SV: 2.0 log units, MF: 1.9, p=0.015) with MFs. Subjects were more exophoric at 40cm (SV: -0.41 Δ, MF: -2.06 Δ) and 25cm (SV: -0.83 Δ, MF: -4.30 Δ) (both p<0.01). With MFs, subjects had decreased accommodative responses at distance (SV: -0.04 D; MF: -0.37 D, p=0.02), 100 cm (SV: +0.37 D; MF: -0.35 D, p<0.01), 40 cm (SV: +1.82 D; MF: +0.62 D, p<0.01), and 25 cm (SV: +3.38 D; MF: +1.75 D, p<0.01). There were no significant differences in accommodative amplitude (p=0.66) or accommodative facility (p=0.54). Children wearing MF contact lenses exhibited reduced accommodative responses and more exophoria at increasingly higher accommodative demands than with SV contact lenses. This suggests that children may be relaxing their accommodation and using the positive addition or increased depth of focus from added spherical aberration of the MF lenses. Further studies are needed to evaluate other lens designs, different amounts of positive addition and aberrations, and long-term adaptation to lenses.
    • THE ROLE OF DENGUE VIRUS NON-STRUCTURAL PROTEIN 1 IN DISEASE PATHOGENESIS

      King, Christine; Endy, Timothy; Barbachano-Guerrero, Arturo (2020)
      Dengue virus (DENV) causes an estimated 390 million infections worldwide annually, with severe forms of disease marked by vascular leakage and an over reactive inflammatory response. Endothelial cells (EC) are directly responsible for vascular homeostasis and are highly responsive to circulating mediators but are not commonly infected. Mast cells (MC) are potent cells of the innate immune system that play an important role in EC biology and inflammatory responses. DENV encodes 10 proteins; with only one, the non-structural protein 1 (NS1), secreted from infected cells and accumulating in the blood of patients.NS1 has been implicated in the pathogenesis of vascular permeability, but the mechanism is not completely understood. Using a complementary array of in vitroassays and disease relevant ECs and MCs, we described the possible roles for NS1 in dengue disease pathogenesis. Using microscopy and immunoblotting we observed that ECs internalize NS1 into endosomes, where it accumulates and is degraded overtime. Transcriptome and pathway analysis defined changes in global gene expression in ECs that are associated with cell dysfunction. We observed that NS1 induced an increase in multicellular rearrangements and a decrease in barrier function in ECs. We demonstrated that NS1-dependent activation of the p38 MAPK pathway controls the changes in EC permeability in vitro. Further, we discovered iiithat ECs and MCs respond to NS1 by secreting a specific array of proinflammatory cytokines and chemokines that may contribute to the cytokine storm in dengue disease. Finally, we found that NS1 internalization can mediate the uptake of bound antibodies into ECs. Together, these results suggest a vasoactive and proinflammatory role for DENV NS1 that may participate in the development of severe symptoms in dengue disease. The observed functions of NS1 could lead to the discovery of new therapeutic targets in dengue disease.
    • Therapeutic Targeting of Oncogenic Gain-of-Function Mutant p53 by Proteasome Inhibition

      Oduah, Eziafa I. (2022-07)
      Non-small cell lung cancer (NSCLC) is a molecularly complex and heterogenous disease. Recent advances in genomic profiling have changed the therapeutic landscape of NSCLC to incorporate targeted and immunotherapeutic approaches. Despite these advances, lung cancer remains the leading cause of cancer mortality in the United States and worldwide. This is partly because these novel treatments are not applicable to all patients and are often associated with primary or secondary resistance. This highlights the need for continued search for new therapeutic agents and strategies for NSCLC patients. However, the drug discovery and development pipeline is protracted and inherently expensive for new drugs. The projected timeline from identification of a new drug candidate from preclinical research to clinical trials and approval is estimated at about 12-15 years with an average cost of $1.3 billion [1]. Moreover, the failure rate for new drugs during the clinical development stage is high, reaching up to 96% by some estimates [2] and is partly due to adverse risk profiles of candidate molecules. Given the ongoing need for continued drug development in lung cancer, repurposing previously approved drugs for new indications when possible is advantageous. Such strategies decrease the cost and timeframe of drug development and pose a lower safety risk to patients since the toxicity profiles of the repurposed drugs are already well established. Drug repurposing has had success in cancer therapy. Some of these include the repositioning of thalidomide for use in multiple myeloma and the repurposing of rituximab from lymphoma to incorporate its use in rheumatoid arthritis [3]. Interestingly, the observations that led to many drug repurposing efforts were serendipitous by nature. However, recently more systematic approaches to repurposing drugs are being employed and include retrospective clinical analysis, genetic associations and pathway matching, binding assays to identify relevant target interactions, and large-scale in vitro drug screens with paired genomic data [3]. In this thesis compilation, I first and foremost lay the groundwork for repurposing proteasome inhibitors for therapeutic targeting of gain-of-function (GOF) oncogenic mutant p53 using lung cancer as a model disease. This has a potential for generalizability across cancers that bear GOF p53 mutations since alterations in TP53 are central to carcinogenesis and prevalent across tumor types. As the ‘guardian of the genome’, p53 maintains the genome integrity by inducing DNA damage repair or forcing aberrant cells into apoptosis or senescence. Failure of this function results in propagation of abnormal cells and the progression from normal to precancerous and malignant cells. Moreover, gain-of-function (GOF) activities of mutated TP53 related the acquisition of novel oncogenic properties are well described in the literature and are related to excess accumulation of the mutant protein. This work describes the mechanism of paradoxical destabilization of GOF p53 by proteasome inhibition in lung cancer and identifies ‘hyperactive’ proteasome genes in mutant p53 as targetable vulnerabilities in this subset of NSCLC. Since proteasome inhibitors are FDA approved drugs and prior drug candidates targeting p53 have not had success in clinical development, the final goal is to repurpose proteasome inhibitors to target GOF p53 mutant NSCLC.
    • The Ties That Bind: A blog project about the meaning of fandom

      Stam, Kathryn; Thesis Advisor; Lizardi, Ryan; Second Reader; Isgar, Eric (2021-05)
      Fandoms, or fan communities; groups of enthusiasts or ‘fans’ that have come together through their shared love of some kind of media, have been around for a long time in a number of different forms around the world, though in small numbers. When comic books became popular, those groups increased, and once again as movies became more commonplace. These communities and cultures are reliant on information technology, and the technology reliant on them, in a symbiotic relationship. It is my objective to research, analyze and observe fandoms and the culture related to the associated communities of fans within, and how they have formed, communicated and interacted, both prior to the development of modern information technology and after, as well as their continued growth on established and upcoming platforms. My actual project consists of a set of blog posts, or ‘mini-essays’, on Tumblr, around one hundred words per post, perhaps more, with ten posts in total.
    • Time Trends in Smoking Onset by Sex and Race/Ethnicity Among Adolescents and Young Adults: Findings From the 2006-2013 National Survey on Drug Use and Health.

      Thompson, Azure B; Mowery, Paul D; Tebes, Jacob Kraemer; McKee, Sherry A
      Introduction: During the 2000s the number of adolescents who became new smokers in the United States declined while the number of young adults who did so increased. However, we do not know among which demographic groups these changes occurred. Methods: We analyzed data from the 2006 to 2013 National Survey of Drug Use and Health (n = 180 079). Multivariate linear regression models were used to assess annual trends in smoking onset and log-binomial regression models to assess changes over time in the risk of smoking onset among young adults (18- to 25-years-old) relative adolescents (12- to 17-years-old). Results: From 2006 to 2013, the rate of onset among young adults (6.3%) was greater than among adolescents (1.9%). Time trends demonstrated that annual declines in smoking onset occurred among white young adult males and females. Rates of smoking onset increased among black and Hispanic young adult males with a lower rate of decline among black and Hispanic young adult females. There was a greater risk of smoking onset among young adults relative to adolescents that did not change over time. Conclusions: Smoking onset is becoming more concentrated in the young adult than adolescent years. Despite this trend, there were annual declines in young adult smoking onset but not uniformly across racial/ethnic groups. More effective strategies to prevent young adult smoking onset may contribute to a further decline in adult smoking and a reduction in tobacco-related health disparities. Implications: Smoking onset is becoming more concentrated in the young adult years across sex and racial/ethnic groups. The United States may be experiencing a period of increasing age of smoking onset and must develop tobacco control policies and practices informed by these changes.
    • TIMP-2 PHOSPHORYLATION BY EXTRACELLULAR c-SRC REGULATES proMMP-2 ACTIVATION

      Bourboulia, Dimitra; Sánchez Pozo, Javier (2018)
      Matrix metalloproteinases (MMPs) are secreted zinc-dependent endopeptidases that are involved in many extracellular biological processes due to their matrix-degrading function. The majority of theseenzymes are released intothe extracellular space in their inactive form and require activation. The tissue inhibitors of metalloproteinases (TIMPs) are also secreted proteins and mainly function to inhibit all members of the MMP family. Interestingly, TIMP-2 also participates in the activationprocessof proMMP-2. Although the interaction between TIMP-2 and proMMP-2 has been known for decades, the molecular signal that triggersthis association has only recently beendetermined. Studies in our lab haveshown that TIMP-2 is tyrosine phosphorylatedby the c-Srctyrosinekinase. Also, phosphorylation of TIMP-2 Tyr90is essential for its interaction with proMMP-2in vivo. Our hypothesis is that c-Src-mediated TIMP-2 phosphorylation happens outside the cell. Here, wedemonstratethat TIMP-2 and c-Src are secreted through different secretory pathways and that TIMP-2 phosphorylation takes place in the extracellular space. Our workalso showsthatextracellularc-Srcisactive, reinforcing the fact that phosphorylation can happen extracellularly. We also hypothesize that extracellular c-Src plays a critical role in facilitating TIMP-2:proMMP-2 interaction. We first confirmed thatTIMP-2 and proMMP-2 endogenously interact only in cells containing endogenous c-Src. This interaction,as well as TIMP-2 phosphorylation,was blocked by treating cells with acustom-made anti-c-Src polyclonal antibody (pAb)that targets amino acids 84-110. We also showthat ananti-c-Src antibody that targets the first 79 amino acids does not inhibit TIMP-2 phosphorylation and interaction with proMMP-2. Therefore, since TIMP-2:proMMP-2 complex formation promotes proMMP-2 activation, we hypothesize that c-Src is an essential player in this process. Our data showsthatthe non-phosphorylatable TIMP-2Tyr90mutant does not promote proMMP-2 activation. Furthermore, pretreatment with the anti-c-Src pAbblockedTIMP-2-mediated proMMP-2 activation, whereasthe anti-c-Src mAb6 did not affect proMMP-2 activation. Overall, these findings provide further evidence that secreted c-Src-mediated TIMP-2 phosphorylation occurs in the extracellular space, where thesecretedkinase is also active. Moreover, c-Src is essential for TIMP-2:proMMP-2 complex formation as well as proMMP-2 activation.
    • To beat or not to beat a tick: comparison of DNA extraction methods for ticks (<i>Ixodes scapularis</i>)

      Ammazzalorso, Alyssa D.; Zolnik, Christine P.; Daniels, Thomas J.; Kolokotronis, Sergios-Orestis (PeerJ, 2015-08-13)
      Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis-nymph and adult-that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.
    • To Upgrade or Not To Upgrade Application

      Stam, Kathryn; Thesis Advisor; Lizardi, Ryan; Second Reader; Francisco, Neil (2021-05)
      New Technology consists of new hardware devices, computational workflows, digital advances, and information systems. As technology continues to evolve over the years, this never-ending cycle of new devices and experiences will always be present amongst consumers. Traditionally, new hardware devices are intriguing because they are designed to improve our access to information, media, and a connection to the digital world, but does this mean our previous-gen devices are no longer valuable? This project involves creating a prototype application designed for both computer and mobile interfaces to help improve the accessibility to information and the overall user experience with an older device. The “To Upgrade or Not To Upgrade” app will inform end-users of their older technological device specifications and suggest hardware/software methods to unlock their full potential. The goal of this paper is to shed some light on consumers that upgrading to the following gen devices is not always necessary to receive the best human-to-computer interactions. It is likely the computer or mobile device that one owns now, with some slight modifications, is all that is needed to provide a pleasant experience.
    • A trancriptomics based approach reveals the functional consequences of RNase MRP RNA mutations in yeast.

      Schmitt, Mark; Shafiuddin, Md (2018)
      RNase MRP is a eukaryotic ribonucleoprotein complex involved in multiple cellular functions that includes ribosomal RNA processing, primer generation for mitochondrial DNA replication and degradation of cell cycle related mRNAs. In Saccharomyces cerevisiae, the RNA component of RNase MRP is encoded by NME1. We have performed random deletion mutagenesis of RNase MRP RNA gene and isolated a mutation, nme1-91, that causes temperature sensitive growth defect on glycerol media. RNA analysis of nme1-91 showed that this mutant is mildly deficient in the 5.8S rRNA processing function of RNase MRP. Growth analysis and northern blotting of RNase MRP RNA mutations generated based on nme1-91 allele suggested that 3’-end nucleotide sequences of the nme1-91 allele contribute to its phenotype. Highcopy suppression screen identified tRNA modification gene NCS6 as a suppressor of nme1-91. Additionally, primary mode of suppression by NCS6 was found to be non-mitochondrial since NCS6 partially suppressed the nme1-91 phenotype on fermentable carbon source. Strains carrying a deletion of NCS6 in combination with nme1-91 showed a synthetic sick phenotype. Polysome profile analysis of nme1-91 revealed that 80S monosomal fraction accumulates in this mutant. Differential gene expression analysis of nme1-91 by RNAseq indicated that rRNA processing and cell cycle related genes become mis-regulated due to this mutation. A similar high-throughput sequencing based approach was also employed to investigate the transcriptional basis of positive genetic interactions between components of RNase MRP and nonsense-mediated decay pathway. A yeast strain bearing the nme1-P6 mutation in the RNA component of RNase MRP exhibits temperature-sensitive growth defect. This phenotype can be suppressed by deletion of NMD components. Differential gene expression analysis identified several mis-regulated biological processes in nme1-P6 and Δupf1 strains. Comparative transcriptomic analysis suggested that suppression of nme1-P6 phenotype by Δupf1 is accompanied by large shift in gene expression pattern towards Δupf1 strain. Moreover, the majority of direct targets of NMD were not down-regulated in nme1-P6 indicating that the effect of NMD on nme1-P6 might be due to increased degradation on mRNAs that are not targeted by NMD in normal conditions. Taken together, these results show that mutations of RNase MRP RNA can modulate diverse biological processes.