• A Qualitative Study on Web Product Development: Based on Experiences of Professionals

      Plano, Krista A. (2011-08-01)
      The purpose of this thesis is to discuss experiences of web professionals and how these professionals interact with one another and with clients to create web products. I used relevant literature and qualitative research to present the themes that exist in the production of web products. When I began my thesis I was ans aspiring Web Project Manager with minimal experience in the digital field and decided to research the experiences of web professionals to learn about the process of creating web-based products from beginning to end. Research and interviews provided information on the steps involved in web product development: I learned that the steps are dependent on the scope of a project and the requirements of stakeholders, clients, and users. I conducted semi-structured, in-depth interviews with seven professionals and extracted themes that include making a functional team, professional roles, the merging roles of web developer and web designer, problem solving, marketing strategies, the process, team interaction, client relations, and products versus projects. Professionals discussed experiences related to team and client interaction that help define the overall development process and answer the research question, how do web professionals create web products? I learned that these steps vary and overlap depending on experiences and professional roles.
    • Rab4acontrol over metabolism and mTOR drives disease progression in Systemic Lupus Erythematosus

      Perl, Andras; Huang, Nick (2020-05-15)
      Endosomal trafficking is key to intercellular communication and metabolic regulation of immunological development. Rab4a, an endosomal trafficker, is elevated in lupus T cells and polymorphisms of the Rab4a gene have been linked to disease susceptibility. Here, we report the constitutive activation of Rab4a increases susceptibility and severity to lupus nephritis in the genetic SLE1.2.3. model of lupus and is corrected by the deletion of Rab4a in T cells. Alternatively, in a pristane model of induced autoimmunity, the deletion of Rab4a in T cells magnifies the pulmonary manifestations of diffuse alveolar hemorrhage that is otherwise protected by the constitutive activation of Rab4a. Rab4a mediates these changes through control over mTOR, mitochondrial function and homeostasis, and immunological development. In particular, inactivation of Rab4a in T cells reduces expression of activation signals, mitochondrial mass and electrochemical potential. Alterations to Rab4a activity drives the aberrant development and function of anti-inflammatory regulatory T cells and pro-inflammatory double-negative T cells. These data provide new insights into the regulation of metabolism and immunological development through endosomal trafficking. As such, the targeting of Rab4a is a novel therapeutic approach in the treatment of autoimmune diseases such as lupus, which has lacked new targeted therapeutics for more than half a century.
    • Racial disparities in substance abuse treatment and the ecological fallacy.

      Melnick, Gerald; Duncan, Alexandra; Thompson, Azure; Wexler, Harry K; Chaple, Michael; Cleland, Charles M
      This study examined engagement in treatment in substance abuse treatment programs that treated primarily either African American or White clients. Findings showed higher levels of engagement in White programs; however, engagement of African Americans in White programs was similar to that of Whites and was greater than Whites in African American programs. No significant differences emerged when a mixed model analysis considered additional variables of staff consensus (regarding treatment elements), treatment climate, acceptance of Medicaid clients, the proportion mandated to treatment, and the quality of the physical space. Although African American programs may show poorer levels of engagement than White programs, attribution of engagement in treatment to client level race/ethnicity should be made with caution.
    • Random Addition Concatenation Analysis: A Novel Approach to the Exploration of Phylogenomic Signal Reveals Strong Agreement between Core and Shell Genomic Partitions in the Cyanobacteria

      Narechania, Apurva; Baker, Richard H.; Sit, Ryan; Kolokotronis, Sergios-Orestis; DeSalle, Rob; Planet, Paul J. (Oxford University Press (OUP), 2011-11-16)
      Recent whole-genome approaches to microbial phylogeny have emphasized partitioning genes into functional classes, often focusing on differences between a stable core of genes and a variable shell. To rigorously address the effects of partitioning and combining genes in genome-level analyses, we developed a novel technique called Random Addition Concatenation Analysis (RADICAL). RADICAL operates by sequentially concatenating randomly chosen gene partitions starting with a single-gene partition and ending with the entire genomic data set. A phylogenetic tree is built for every successive addition, and the entire process is repeated creating multiple random concatenation paths. The result is a library of trees representing a large variety of differently sized random gene partitions. This library can then be mined to identify unique topologies, assess overall agreement, and measure support for different trees. To evaluate RADICAL, we used 682 orthologous genes across 13 cyanobacterial genomes. Despite previous assertions of substantial differences between a core and a shell set of genes for this data set, RADICAL reveals the two partitions contain congruent phylogenetic signal. Substantial disagreement within the data set is limited to a few nodes and genes involved in metabolism, a functional group that is distributed evenly between the core and the shell partitions. We highlight numerous examples where RADICAL reveals aspects of phylogenetic behavior not evident by examining individual gene trees or a "'total evidence" tree. Our method also demonstrates that most emergent phylogenetic signal appears early in the concatenation process. The software is freely available at http://desalle.amnh.org.
    • A randomized controlled efficacy trial of an mHealth HIV prevention intervention for sexual minority young men: MyPEEPS mobile study protocol.

      Kuhns, Lisa M; Garofalo, Robert; Hidalgo, Marco; Hirshfield, Sabina; Pearson, Cynthia; Bruce, Josh; Batey, D Scott; Radix, Asa; Belkind, Uri; Jia, Haomiao; et al. (2020-01-15)
      Background: Young sexual minority men in the United States have a high incidence rate of HIV infection. Early intervention among this group, that is timed to precede or coincide with sexual initiation, is of critical importance to prevent HIV infection. Despite this, there are very few published randomized controlled efficacy trials testing interventions to reduce sexual vulnerability for HIV acquisition among racially/ethnically diverse, very young, sexual minority men (aged ≤18 years). This paper describes the design of a mobile app-based intervention trial to reduce sexual risk for HIV acquisition and promote health protection in this group. Methods: This study is a randomized controlled trial of an mHealth-based HIV prevention intervention, MyPEEPS Mobile, among diverse sexual minority cisgender young men, aged 13-18 years. The mobile intervention was adapted from a prior group-based intervention curriculum with evidence of efficacy, designed to be specific to the risk contexts and realities of young sexual minority men, and to include psychoeducational and skill-building components with interactive games and activities. Participants are recruited locally within four regional hubs (Birmingham, AL, Chicago, IL, New York City, NY, Seattle, WA) and nationwide via the Internet, enrolled in-person or remotely (via videoconference), and randomized (1:1) to either the MyPEEPS Mobile intervention or delayed intervention condition. Post-hoc stratification by age, race/ethnicity, and urban/suburban vs. rural statuses is used to ensure diversity in the sample. The primary outcomes are number of male anal sex partners and frequency of sexual acts with male partners (with and without condoms), sex under the influence of substances, and uptake of pre-and post-exposure prophylaxis, as well as testing for HIV and other sexually transmitted infections at 3-, 6- and 9-month follow-up. Discussion: Behavioral interventions for very young sexual minority men are needed to prevent sexual risk early in their sexual development and maturation. This study will provide evidence to determine feasibility and efficacy of a mobile app-based HIV prevention intervention to reduce sexual risk among this very young group. Trial registration: ClinicalTrials.gov number, NCT03167606, registered May 30, 2017.
    • RASD2, MYH9, and CACNG2 Genes at Chromosome 22q12 Associated with the Subgroup of Schizophrenia with Non-Deficit in Sustained Attention and Executive Function

      Liu, Yu-Li; Fann, Cathy Shen-Jang; Liu, Chih-Min; Chen, Wei J.; Wu, Jer-Yuarn; Hung, Shuen-Iu; Chen, Chun-Houh; Jou, Yuh-Shan; Liu, Shi-Kai; Hwang, Tzung-Jeng; et al. (Elsevier BV, 2008-11)
      Background: In a previous linkage study of schizophrenia that included Taiwanese samples, the marker D22S278 (22q12.3) was significantly linked to schizophrenia (p .001). Methods: We conducted fine mapping of the implicated genomic region, with 47 validated single nucleotide polymorphism (SNP) markers around 1 Mb of D22S278, in a Taiwanese sample of 218 pedigrees with at least 2 siblings affected with schizophrenia. We examined the association of these SNPs and their haplotypes with schizophrenia and with subgroups defined by the presence and absence of deficits in sustained attention as assessed by undegraded and degraded continuous performance tests (CPTs). We also examined subgroups defined by deficits in categories achieved in the Wisconsin Card Sort Test (WCST). Results: Three of five candidate vulnerability genes (RASD2, APOL5, MYH9, EIF3S7, and CACNG2), which had marginally significant associations with schizophrenia, had significant associations with schizophrenic patients who did not have deficits in sustained attention on the undegraded CPT (RASD2 gene SNP rs736212; p .0008 with single locus analysis) and the degraded CPT (MYH9 gene haplotype 1-1-1-1 of SNP rs3752463 - rs1557540 - rs713839 - rs739097; p .0059 with haplotype analysis). We also found a significant association for patients who showed no deficits in executive function as measured by categories achieved in the WCST (CACNG2 gene haplotype 2-1-1-1 of SNP rs2267360 - rs140526 - rs1883987 - rs916269; p .0163 with haplotype analysis). Conclusions: The genes RASD2, MYH9, and CACNG2 might be vulnerability genes for neuropsychologically defined subgroups of schizophrenic patients.
    • Rational design of a genetically encoded fluorescent protein color switch using a modular, entropy-driven mechanism

      Loh, Stewart; John, Anna (2022-06)
      Engineered protein conformational switches have applications in cellular and in vitro biosensing, molecular diagnostics and artificial signaling systems in synthetic biology. They broadly consist of an input module and an output module that communicate via a conformational change. The overarching goal of this thesis is to tackle two major challenges in protein switch design - signal transduction, by coupling a target recognition domain to an output domain to produce a robust change in signal in addition to modularity, which allows the facile creation of sensors binding novel targets. Here, we attempted to test a rational design strategy that exploits two key protein engineering principles (1) loop entropy, by which long insertions into a loop of a host protein destabilizes the host due to an entropic cost associated with loop closure unless the inserted sequence adopts a folded structure; and (2) alternate frame folding (AFF), which allows a protein - green fluorescent protein variants(GFP), in this case - to switch between two mutually exclusive folds. Toward this goal, we first studied the effect of loop entropy at two different insertion sites in a GFP variant (chapter 2) using a well-characterized ribose binding protein as the input domain. We provide stability measurements using circular dichroism and fluorescence data to support our hypothesis of the application of the loop entropy principle in a GFP beta barrel scaffold. To provide a proof-of-concept of the combination of loop entropy and the AFF mechanism in a genetically encodable GFP scaffold, we chose an unstable, circularly permuted FK506-binding protein (cpFKBP) as the input recognition domain and inserted it in one of the two mutually exclusive folds of the GFP-AFF fusion protein (chapter 3). Upon addition of ligand, binding induced folding of the cpFKBP domain effects a conformational change in which the tenth beta strand of GFP exchanges, replacing Thr203 (green state) with Tyr203 (yellow state). We confirmed this mechanism in vitro by a ratiometric change in fluorescence output and observed that the process is slow and irreversible. We elucidate the biophysical principles underlying this mechanism by using denaturant and temperature to modulate the relative populations of the two folds in vitro. We also observed a faster and higher intensiometric response in mammalian cells which may be attributed to an alternate mechanism. We then harnessed this intensiometric response in a single fold of the fluorescent protein combined with a previously engineered monobody scaffold capable of binding a variety of targets (chapter 4). Altogether this work may have the potential to create a novel class of fluorescent protein biosensors comparable to existing single fluorescent protein-based biosensors currently available.
    • Rational Design of Protein-Based Biosensors Using Engineered Binding-Induced Conformational Switches

      Loh, Stewart; ZHENG, HUIMEI (2014)
      Biosensor development continues to be driven by the growing need to accurately detect and monitor analytes with many biotechnology, clinical, agriculture, and military applications. With their well-established capacity for molecular recognition, proteins are the go-to choice of binding elements in many conventional sensor designs. Switchable proteins offer the potential of integrating analyte binding and signal transduction within a single molecule, thus reducing the need for complex and expensive detection equipment and opening the door to miniaturization and in vivo applications. The principal challenge is that the majority of natural binding proteins do not undergo a large-scale change in conformation upon target binding. This work describes two complementary protein design strategies for the rational conversion of ordinary binding proteins into ligand induced conformational switches for biosensing purposes. In the first approach, we applied the Alternate Frame Folding (AFF) mechanism to the human sulfiredoxin (hSrx) and the fibronectin (FN3) monobody scaffold towards the creation of an ATP biosensor and a customizable biosensor platform, respectively. In a second novel approach, the Protein Fragment Exchange (FREX) mechanism was demonstrated in a proof-of principle study that converts the FN3 scaffold into a biosensor, capable of genetic encoding and application in mammalian cells. While these designs were based on well established principles of protein folding and thermodynamics, the results obtained from these studies also offer important insights regarding protein sequence-structure-function relationships.
    • Reading from an Electronic Reading Device versus Hardcopy Text

      Hue, Jennifer E. (2013-06-24)
      The use of electronic reading devices has become more prevalent. Many individuals of all ages are using personal electronic readers (e.g., Kindle, Nook, E-Reader) in place of hardcopy printed materials. Previous work in our laboratory has demonstrated that symptoms when reading from a computer screen are significantly greater than those experienced when reading printed text. Accordingly, the aim of the present study was to examine both symptoms and task performance when reading from a Kindle e-reading device, and to compare the findings with those from hardcopy, printed materials.
    • The Real maccoyii: Identifying Tuna Sushi with DNA Barcodes – Contrasting Characteristic Attributes and Genetic Distances

      Lowenstein, Jacob H.; Amato, George; Kolokotronis, Sergios-Orestis (Public Library of Science (PLoS), 2009-11-18)
      Background: The use of DNA barcodes for the identification of described species is one of the least controversial and most promising applications of barcoding. There is no consensus, however, as to what constitutes an appropriate identification standard and most barcoding efforts simply attempt to pair a query sequence with reference sequences and deem identification successful if it falls within the bounds of some pre-established cutoffs using genetic distance. Since the Renaissance, however, most biological classification schemes have relied on the use of diagnostic characters to identify and place species. Methodology/principal findings: Here we developed a cytochrome c oxidase subunit I character-based key for the identification of all tuna species of the genus Thunnus, and compared its performance with distance-based measures for identification of 68 samples of tuna sushi purchased from 31 restaurants in Manhattan (New York City) and Denver, Colorado. Both the character-based key and GenBank BLAST successfully identified 100% of the tuna samples, while the Barcode of Life Database (BOLD) as well as genetic distance thresholds, and neighbor-joining phylogenetic tree building performed poorly in terms of species identification. A piece of tuna sushi has the potential to be an endangered species, a fraud, or a health hazard. All three of these cases were uncovered in this study. Nineteen restaurant establishments were unable to clarify or misrepresented what species they sold. Five out of nine samples sold as a variant of "white tuna" were not albacore (T. alalunga), but escolar (Lepidocybium flavorunneum), a gempylid species banned for sale in Italy and Japan due to health concerns. Nineteen samples were northern bluefin tuna (T. thynnus) or the critically endangered southern bluefin tuna (T. maccoyii), though nine restaurants that sold these species did not state these species on their menus. Conclusions/significance: The Convention on International Trade Endangered Species (CITES) requires that listed species must be identifiable in trade. This research fulfills this requirement for tuna, and supports the nomination of northern bluefin tuna for CITES listing in 2010.
    • Reconciling Apparent Conflicts between Mitochondrial and Nuclear Phylogenies in African Elephants

      Ishida, Yasuko; Oleksyk, Taras K.; Georgiadis, Nicholas J.; David, Victor A.; Zhao, Kai; Stephens, Robert M.; Kolokotronis, Sergios-Orestis; Roca, Alfred L. (Public Library of Science (PLoS), 2011-06-08)
      Conservation strategies for African elephants would be advanced by resolution of conflicting claims that they comprise one, two, three or four taxonomic groups, and by development of genetic markers that establish more incisively the provenance of confiscated ivory. We addressed these related issues by genotyping 555 elephants from across Africa with microsatellite markers, developing a method to identify those loci most effective at geographic assignment of elephants (or their ivory), and conducting novel analyses of continent-wide datasets of mitochondrial DNA. Results showed that nuclear genetic diversity was partitioned into two clusters, corresponding to African forest elephants (99.5% Cluster-1) and African savanna elephants (99.4% Cluster-2). Hybrid individuals were rare. In a comparison of basal forest "F" and savanna "S" mtDNA clade distributions to nuclear DNA partitions, forest elephant nuclear genotypes occurred only in populations in which S clade mtDNA was absent, suggesting that nuclear partitioning corresponds to the presence or absence of S clade mtDNA. We reanalyzed African elephant mtDNA sequences from 81 locales spanning the continent and discovered that S clade mtDNA was completely absent among elephants at all 30 sampled tropical forest locales. The distribution of savanna nuclear DNA and S clade mtDNA corresponded closely to range boundaries traditionally ascribed to the savanna elephant species based on habitat and morphology. Further, a reanalysis of nuclear genetic assignment results suggested that West African elephants do not comprise a distinct third species. Finally, we show that some DNA markers will be more useful than others for determining the geographic origins of illegal ivory. These findings resolve the apparent incongruence between mtDNA and nuclear genetic patterns that has confounded the taxonomy of African elephants, affirm the limitations of using mtDNA patterns to infer elephant systematics or population structure, and strongly support the existence of two elephant species in Africa.
    • Reconstitution and Characterization of RNA Polymerase I Upstream Activating Factor.

      Knutson, Bruce; Smith, Marissa (2018)
      RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and one of the most critical steps in ribosome biosynthesis. Pol I transcription initiation is coordinated by four Pol I factors that include the Upstream Activating Factor (UAF), TATA-binding protein (TBP), Core Factor (CF), and Rrn3. These factors work together to recruit Pol I to the rDNApromoter and to initiate transcription.UAF is a six-subunit complex composed of Rrn9, Rrn5, Uaf30, Rrn10, and histones H3 and H4.To investigate the importance of each UAF subunit in UAF complex formation and complex integrity, we developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex. Here, we found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit. Last, wedetermined the stoichiometry of the subunits of the UAF complex, revealing there are two copies of histoneH3 and one copy of the remaining UAF subunits, including histone H4. Together, our results provide a new model suggesting that UAF contains a hybrid H3–H4 tetramer-like subcomplex.The results from this thesiswill help to reveal key mechanisms in Pol Itranscription activation.
    • Redesigning Adirondack Adventures: Analyzing Crowd Culture to Develop Social Media Marketing Strategies

      McFadden, Hannah (2016-12-01)
      The purpose of this thesis is to analyze present day crowd culture to better understand consumer behaviors and engagement to help redesign the brand and marketing strategy for Adirondack Adventures, a small white water rafting business in upstate New York. With small businesses struggling to take full advantage of social media platforms and the potential they offer, consumers and potential customers are left disengaged and consequently uninterested. By researching the correlation between Web 2.0 technology and the emergence of crowd culture it is my goal to develop a social media marketing strategy for Adirondack Adventures that will help establish their brand, increase their exposure, and grow their customer base to become a top competitor in their industry.
    • REELIN SIGNALING COORDINATES DENDRITICINITIATION AND CELLULAR POSITIONING BY NEURITESTABILIZATION

      Olson, Eric; O’Dell, Ryan (2015)
      The laminar organization characteristic of the adult mammalian neocortex is a product of the precise coordination of neuronal proliferation, migration, and differentiation. Among these processes, the biological signals controlling apical dendrite initiation and targeting are not completely understood.The secreted ligand Reelin is a largeextracellular matrix glycoprotein localized to the axonal plexus of themarginal zone, and mutations areassociated with severe disruptions in cellular organization in laminated brain regions. Although the Reelin signaling pathway has been traditionally describedas a modulator of neuronal migration, recent evidence suggests Reelin controlsneuronal orientation and subsequent dendritogenesis into the overlying marginal zoneduring a period of early cortical development known as preplate splitting.To explicitly characterize how Reelin coordinates the transition between migration and dendritogenesis and controls polarized apical dendrite initiation and growth, an ex uteroexplant model of early cortical developmentwas used for fixed tissue and multiphoton live imaging analysis. Our investigations revealed the apical dendrite of cortical neurons emerges via direct transformation of the leading process during terminal translocation.Both throughoutand after this migratory phase, the dendriticarbor demonstrated significant increases in growth and branching, typically initiatedafter leading process entryinto the Reelin-rich marginal zone.In the absence of Reelin signaling, mutant cortices demonstrated a significant proportion of neurons that successfully translocated, but showed unstable arbors and marginal zone avoidance after migration arrest. Application of exogenous Reelin protein rescued dendritekinetics and polarity within4 hours, resultinginthe retraction of tangentially orienteddendritessimultaneous with the extension of a highly branched,apicallyoriented primary process. These findings suggesta precise role of Reelin signaling in early cortical development in proper neuronal polarization and stabledendrite outgrowth into the marginal zone, an area otherwiseexclusionary for dendrites. Furthermore, it is suggested that appropriate dendritic arbor elaborationinto the marginal zone may not only promote terminal translocation, but also definesthe final position of migration arrest.Thisbody of work thus offers an important advancement in understanding Reelin’s role in polarized dendritic outgrowth and the subsequent knock-on effectsassociated withperturbationsof this signaling pathway.
    • Reelin Signaling in Oligodendrocyte Progenitor Cell Migration

      Osterhout, Donna; BHATTI, HARNEET (2016)
      Oligodendroglial progenitor cells (OPCs) are the precursors to the myelinating oligodendrocytes in the central nervous system (CNS). These cells are produced in the ventral neuroepithelium at later stages of cortical development, migrating into the cortex where they contact axons and differentiate, ultimately forming a myelin membrane. During the process of differentiation, OPCs undergo significant morphological changes, extending many processes which will make contact with axons. Once in contact with an axon, the oligodendrocyte process expands and begins to form the myelin membrane which will ensheathe the axon. Reelin is a highly conserved secretory glycoprotein, which has acritical role in directing neuronal migration. Reelin orchestrates the proper cortical layer formation and neuronal organization during brain development. In the absence of Reelin, the cerebral crotex is disorganized, with inverted cortical layers, generating devastating biological effects. Reelin acts through several cellular receptors, activating numerous downstream effectors and complex signaling cascades. If elements of the Reelin signalling pathway are disrupted,similar defects in migration can occur.Oligodendroglial cells, from the early progenitor cells to the mature myelinating cells secrete Reelin, but also express a receptor for Reelin and criticalelements of the intracellular Reelin signaling pathways. It is not known if these cells canrespond to Reelin. In this thesis, we examined the effects of Reelin on oligodendroglial cells, using both in vitroand in vivomethods. We demostrate a potentialrole for Reelin in modulating oligodendrocyte migration, but also identify a novel aspect ofReelin signalling in the biology of oligodendroglia.
    • REGULATION AND FUNCTION OF TUMOR SUPPRESSOR ECRG2 IN RELATION TO DNA DAMAGE AND MICROTUBULE DYNAMICS IN HUMAN MALIGNANCIES

      Huang, Ying; Patel, Harsh (2020)
      phageal Cancer-Related Gene 2 (ECRG2) is a novel tumor suppressor which is frequently mutated or downregulated in multiple human cancers. Previous studies have demonstrated that ECRG2 inhibits growth of cancer cells by inducing apoptotic death. However, the molecular basis of its regulation and involvement in DNA damage response remain to be elucidated. The function of tumor suppressor p53 in cellular response to stress conditions, such as DNA damage, has been well-established. In the present study, we report for the first time, that ECRG2 is a novel pro-apoptotic transcriptional target of p53 and ECRG2 expression is induced by DNA damage in a p53-dependent manner. Moreover, we demonstrate that disruption of ECRG2 leads to reduced apoptosis and improved survival following the treatment with DNA damage-inducing anticancer agent despite p53 activation in cancer cells. Significantly, we characterized a natural variant in ECRG2promoter (rs3214447) that is found in the genomes of ~38.5% of world population and showed that ECRG2 promoter with rs3214447 variant is defective in responding to p53 and DNA damage. Thus, ECRG2 is an important executor of p53-mediated apoptosis in response to DNA damage. We also report a novel biological function of ECRG2 and demonstrate that ECRG2 interacts with and stabilizes microtubules. ECRG2 was shown to protect the microtubules against the destabilization induced by cold and nocodazole treatment. In addition, we show that ECRG2 increases acetylation of microtubules, which is associated with more stable microtubules. Importantly, we demonstrate that ECRG2 disruption give rise to increased cell proliferation by elevated activation of Akt. Taken together, our findings ascribe a novel function to ECRG2 in the regulation of microtubule dynamics and cancer cell proliferation. ECRG2-mediated tumor suppressor activities elucidated in this dissertation are clinically significant. Our database analyses reveal that cancer patients with lower ECRG2expression in their tumors had poor prognosis and reduced disease-free survival as compared to their counterparts. These observations suggest that loss of ECRG2 expression and function confers survival advantage to cancer cells. Collectively, this dissertation highlights novel aspects of ECRG2 regulation and function in cancer cell sensitivity to DNA damage-inducing anticancer therapy, microtubule dynamics and cell proliferation.
    • REGULATION OF CELLULAR BIOENERGETICS IN CNS DEMYELINATING DISEASE

      Massa, Paul T.; Minchenberg, Scott (2017)
      Multiple Sclerosis (MS) is a debilitating neurological disease characterized by sclerotic inflammatory demyelination of the white matter tracts in the central nervous system (CNS). There is no “cure” for MS but rather disease modifying treatments that decrease relapse rates and slow disease progression. Due to the lack of insight into the pathogenesis of MS, animal models have been developed to study demyelination in the CNS. Two widely used models of demyelination are experimental autoimmune encephalomyelitis (EAE), and Theiler’s murine encephalomyelitis virus (TMEV). Our studies focused on TMEV mediated demyelination, which was dependent on the expression of the protein tyrosine phosphate SHP-1. SHP-1 is a major negative regulator of cytokine/growth factor signaling and a global deficiency triggers an acute macrophage mediated demyelination in C3H mice. SHP-1 deficient mice are also highly susceptible to systemic inflammation and dysmyelination. Our overall goal was to identify how SHP-1 is mediating susceptibility to inflammatory demyelination. We first demonstrated that SHP-1 deficient oligodendrocytes had increased reactive oxygen species (ROS) production resulting in downregulation of myelin gene expression and oxidation of myelin, a common finding in MS patients. To determine a source of the ROS we investigated how SHP-1 controls metabolic pathways as ROS production is tightly linked to metabolism. To determine how SHP-1 impacts bioenergetics, oligodendrocyte glycolytic and mitochondrial metabolism were quantified using the Seahorse XFe96 analyzer. We determined that SHP-1 enhances oligodendrocyte metabolism, which correlates with its ability to suppress STAT1 activity in oligodendrocytes. We corroborated these results via activation of STAT1 in oligodendrocytes with the proinflammatory cytokine IFN-γ recapitulating the metabolic defects in SHP-1 deficient oligodendrocytes. Based the role of SHP-1 in oligodendrocyte bioenergetics and the importance of macrophage-derived cytokine production during demyelination; we investigated a role for SHP-1 in macrophage bioenergetics. In macrophages, enhanced glycolysis drives activation and proinflammatory cytokine production. TMEV infection specifically induced glycolysis in GM-CSF-derived macrophages lacking SHP-1. This finding may explain why SHP-1 confers susceptibility to macrophage-mediated demyelination after TMEV infection. Overall we demonstrate a novel role for SHP-1 in controlling oligodendrocyte and macrophage bioenergetics that is highly relevant in expanding our understanding of CNS demyelinating disease.
    • Regulation of fibrosis through the ubiquitin pathway

      Bernstein, Audrey; Phillips, Andrew (2022-08)
      Scarring in the cornea obstructs the refraction of incoming light onto the retina causing visual disability. Both acute scarring and chronic fibrosis are characterized by an accumulation of disorganized extracellular matrix (ECM). Disorganized ECM is deposited into the wound by specialized cells termed myofibroblasts. Pathological myofibroblasts are characterized by the expression of the highly contractile alpha smooth muscle actin (a-SMA) and the av-family of integrins (avb1,b3, b5, b6). The persistence of myofibroblasts in a healing wound promotes an autocrine loop of TGFb activity, over contraction of tissue, deposition of fibrotic ECM proteins, and ultimately the generation of scar tissue. My work is focused on the relative contribute of the deubiquitinase, USP10 to scarring in the cornea. I found that after wounding an increase in the expression of USP10 leads to deubiquitination of integrins and a subsequent increase in integrin recycling and matrix deposition. Knockdown of USP10 in vivo after corneal wounding significantly reduced the presence of myofibroblasts and immune cells in the healing wound, and corneal scarring. Through a yeast 2-hybrid screen I also identified a novel USP10 interacting protein, the formin Daam1. I found that Daam1 sequesters USP10 on actin stress fibers inhibiting its activity. Under pathological conditions, the expression of both USP10 and Daam1 are increased. My data suggest that Daam1 acts as a cellular reservoir, adding a layer of homeostatic control over USP10 activity and integrin function. Although defects in protein degradation have been identified as a major contributor to many diseases, together, my studies indicate that protein degradation (ubiquitin) pathways need to be considered in the context of integrin biology and in the pathogenesis of fibrotic healing.
    • The Relationship Between Meibomian Gland Morphology, Dry Eye Disease, and Electronic Device Use in Pediatric Patients

      Ribolla, Sofia (2022)
      "Purpose: The purpose of this systematic study was to establish preliminary comparisons of various morphological and clinical parameters between dry eye and normal subjects in a pediatric cohort. Methods: Children aged 5-17 were recruited for the study with no previous clinical diagnosis of dry eye disease (DED) or meibomian gland dysfunction (MGD). Diagnostic criteria for DED consisted of positive scoring on at least two of three components; subjective symptoms, abnormal tear function, and vital staining. All subjects completed SPEED questionnaires to assess dry eye symptoms; scores above 5 indicated positive symptomology. Tear film and ocular surface integrity were inspected using fluorescein and lissamine green dye with slit lamp miscroscopy. Corneal fluorescein, as well as temporal and nasal conjunctival lissamine green staining was graded from 1-4 (0=no staining; 4=coalesced). A staining score of more than 4 points across all 3 sections indicated positive vital staining. Abnormal tear function was defined by a TBUT ≤5s. Meibomian gland morphology, lipid layer thickness, and blink patterns were evaluated with the use of a Lipiview Interferometer. The 5-point meiboscale for gland atrophy was used for dropout grading, while tortuosity was defined by number of glands with ≥45° angles. Tear volume assessment was completed with phenol red test. Questionnaires administered to both the child and family member were used to assess electronic device usage in order to screen for possible associations with average daily screen time and aforementioned parameters. Results: A total of 24 subjects participated in the study. Dry eye was found in 41.7% of the subjects. Presence of meibomian gland dropout and tortuosity were 70.8% and 87.5% respectively. Dropout was significantly higher in the dry eye group (p=0.016), although tortuosity was similar between both groups (p=0.93). Tear breakup times were significantly lower in the dry eye group (5.30s vs 9.66s; p<0.001) along with total staining scores (8.00 vs. 3.21; p=0.043). Blink behavior and measurements of lipid layer thickness (LLT) did not vary between the two groups; partial blink ratios were 0.62 and 0.67 for DED and normal groups respectively (p=0.76), and lipid layer thicknesses were 55.9nm and 57.43nm (p=0.84). Electronic device use did not vary significantly between the two groups (p=0.99). Screen time was significantly correlated with higher rate of partial blinks (r=0.84). Higher lipid layer thickness significantly predicted higher partial blink fraction in the left eye (p=0.39) and approached significance in the right eye (p=0.08). Conclusion: The present study provides a current baseline data on ocular surface characteristics and meibomian gland anatomy in healthy children with clinically dry eye vs. those without dry eye. Our results indicate that MGD and DED are highly inter-related at a much earlier age than previously acknowledged, and that the significant rise in pediatric variations of DED represent a worthwhile cause for investigation into long-term risk factors for disease progression. Better understanding of baseline ocular surface and tear film characteristics will be crucial to identify the impact increasingly prevalent risk factors, such as visual device use, myopia interventions, and other changing environmental factors might have on the pediatric population."
    • RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD)

      Howell, Brian; Lammert, Dawn (2017)
      Autism spectrum disorder (ASD) affects approximately 1 in 45 people, and is characterized by deficits in social communication and repetitive behaviors. Sequencing advancements have enabled the identification of numerous candidate genes, but precisely how these genes contribute to ASD remains largely unknown. RELNis consistently implicated as a candidate gene for autism. The encoded secreted glycoprotein, Reelin is important for proper brain developmental and postnatal synapse function. Here we examine the molecular and cellular consequences of the de novo RELNmutation R2290C. This mutation falls in a conserved arginine-amino acid-arginine (RXR) motif that is found within the Reelin subrepeat structure. Several other ASD patient mutations fall with in this consensus and all examined reduce Reelin secretion. Based on this we tested two hypothesis: (1) that the mutations reduce Reelin signaling and (2) that they have a gain-of-function consequence, such as ER stress. Using an engineered cell line with a heterozygous RELNR2290C mutation and the RELN Orleans (Orl) mouse line that produces nearly full length Reelin that is defective for secretion, we found evidence for both increased Dab1 and increased PDIA1 expression. Since, like most genes implicated in ASD RELNlikely acts in a multifactorial manner, we investigated whether second site mutations might contribute to ASD-related behaviors. Towards this end we crossed the heterozygous Orl and Shank3b mice to model two hits that are present in at least one ASD proband. We found that the resulting double heterozygousmice had impaired socialization and altered ultrasonic vocalizations. Furthermore, forebrain and cerebellar lysates showed increased PSD-95, identifying a potentially common mechanism and therapeutic target for ASD. These studies are the first to investigate the biological relevance of RELNcoding mutations in ASD.