• Secure Routing in Mobile Ad-hoc Networks

      Jadhav, Sumedh; Hash, Larry; Adviser (2016-05-01)
      The detailed study investigates the various problems faced by Mobile Ad hoc Networks because of their inherent characteristics. The characteristics make these networks vulnerable to various attacks. The attack known to be performed on MANETs were researched in order to gain better understanding and insight on how they can be defended against. The focus of this project was to secure the routing protocols, since routing plays a vital role in the operation of network and is the target of most attacks. Various research papers were referenced for collecting the information needed. The existing routing protocols were systematically categorized and compared. Additionally, advanced routing protocols that integrate cryptographic methods into them were studied to come to a conclusion that they are the most robust protocols that effectively protect the Mobile Ad hoc Networks against some dangerous attacks. Securing the routing protocols is indeed the most important aspect for securing MANETs. However, there are other techniques that can be used as extensions for strengthening these networks, in addition to using a robust routing protocol. Recommendations have been made for the same.
    • SECURITY CHALLENGES IN SDN IMPLEMENTATION

      Patil, Pradnya; Hash, Larry; Advisor; White, Joshua; Reviewer; Tekeoglu, Ali, Reviewer (2018-05)
      This study analyzes how security challenges caused by data and control layer separation in the SDN, such as Denial of Service attacks and unauthorized access attacks, limit SDN deployment. This study also offers network engineers’ views on preventing those security issues and whether implementing SDN is a good idea in the first place. This study was conducted in order to answer three questions: 1. How does data and control layer separation in SDN cause DoS and unauthorized access attacks? 2. What are the best practices and measures to minimize such security threats from the engineer’s point of view? 3. Do security threats at the lower layer affect the decision to implement SDN? These questions were answered by reviewing research papers and interviewing engineers from the telecommunication field. DoS and unauthorized access attacks are due to vulnerabilities in OpenFlow, SDN switches and SDN controllers. Table 6 presents solutions for preventing DoS and unauthorized access attacks. Most of the network engineers said SDN should be implemented based on cost, limited risk, customers’ positive views, and company projects, despite the current security challenges.
    • SELF-ASSOCIATION IS A COMMON PROPERTY IN BOVINE, MOUSE AND XENOPUSARRESTIN

      Calvert, Peter; Qiu, Shuang (2014)
      The light-driven translocation of arrestin from rod inner segment to outer segment was indicated to involve free diffusion with binding affinityto light-activated phosphorhodopsin, however, it is still debatable how arrestin is excluded from the dark-adapted outer segment. Previousstudies demonstrated that bovine visual arrestin had the property of self-association. The self-association propertyof both wild-type and mutated purified visual arrestin of several species (bovine, mouse and Xenopus laevis) was studiedby performing analytical ultracentrifugation experimentswhich providedthe oligomer formation information and association constants.Theself-association parameters of purified bovine and mousevisual arrestinwere investigated and compared with other studies. Results showed that arrestin of both species could self-associate, forming dimersin a concentration-dependent manner, but tetramerswere not detected at the highest concentrations examined.Xenopus arrestin was shown to self-associateas well, existing in a monomer-dimer equilibrium with the dimer dissociation constantKD,dim=80.8μM, which suggestedthat self-association was also a feature of Xenopus visual arrestin. Interestingly, homologous mutations (F78A/Y84A/F193A)of Xenopusarrestin, which were supposed to beconstitutive monomers, failed to completely disrupt the oligomerizationof Xenopusarrestinwith the dimer dissociation constantKD,dim=200.7μM , indicating that these regions were not conserved in amphibian visual arrestin, includingXenopuslaevis. The percentage of the dimer was higher than that of monomer at physiological concentrations in all species of arrestins tested.
    • Sense Making Methodology: A Study of the Impact of a New Technology on Banking Culture

      Smith, Daniel J. (2012-12-01)
      The purpose of this paper is to explore the possibility that Remote Deposit Capture technology has enhanced productivity and culture at ABC Credit Union. This study used relevant literature as well as qualitative research methodology to investigate how this new technology has changed relationships between financial institution employees and members of the financial institution. More specifically, this qualitative study used a grounded theory concept developed by Glaser and Strauss (Merriam, 2009, pp. 199) to “constantly compare” employees and their productivity and workflow at ABC Credit Union in Upstate, NY. Using interviews, field notes and documents from employees has determined if ABC Credit Union has benefited from the implementation of Remote Deposit Capture. I also performed a coding analysis defined by Corbin & Strauss (Merriam, 2009, pp. 180) as “coding that comes from interpreting and reflection on meaning” to evaluate the responses provided to me by my participants regarding the implementation of Remote Deposit Capture at ABC Credit Union. Remote Deposit Capture technology is the ability to deposit checks directly into your ABC Credit Union account via mobile devices such as an iPhone or Android device. Based on the interviews I have been able to answer the research question: Does the implementation of Remote Deposit Capture enhance the culture at Financial Institutions? If so, how?
    • Separating Attention Deficit Hyperactivity Disorder and Learning Disabilities in Girls: A Familial Risk Analysis

      Doyle, Alysa E.; Faraone, Stephen V.; DuPre, Emily P.; Biederman, Joseph (American Psychiatric Association Publishing, 2001-10)
      Objective: Familial risk analysis was used to clarify the relationship in girls between attention deficit hyperactivity disorder (ADHD) and learning disabilities in either mathematics or reading. Method: The authors assessed the presence of ADHD and learning disabilities in 679 first-degree relatives of three groups of index children: girls with ADHD and a comorbid learning disability, girls with ADHD but no learning disabilities, and a comparison group of girls without ADHD. Results: The risk for ADHD was similarly higher in families of ADHD probands with and without learning disabilities; both groups had significantly higher rates of ADHD than did families of the comparison girls. In contrast, only among relatives of ADHD probands with a learning disability was there a higher risk for learning disabilities. A strong (although statistically nonsignificant) difference emerged that suggested at least some degree of cosegregation of ADHD and learning disabilities in family members. There was no evidence of nonrandom mating between spouses with ADHD and learning disabilities. Conclusions: These results extend previously reported findings regarding the relationship of ADHD and learning disabilities to female subjects and raise the possibility that, in girls, the relationship between ADHD and learning disabilities is due to shared familial risk factors.
    • A seq2seq model to forecast the COVID-19 cases, deaths and reproductive R numbers in US counties

      Zhang-James, Yanli; Hess, Jonathan; Salekin, Asif; Wang, Dongliang; Chen, Samuel; Winkelstein, Peter; Morley, Christopher P; Faraone, Stephen V. (Cold Spring Harbor Laboratory, 2021-04-20)
      The global pandemic of coronavirus disease 2019 (COVID-19) has killed almost two million people worldwide and over 400 thousand in the United States (US). As the pandemic evolves, informed policy-making and strategic resource allocation relies on accurate forecasts. To predict the spread of the virus within US counties, we curated an array of county-level demographic and COVID-19-relevant health risk factors. In combination with the county-level case and death numbers curated by John Hopkins university, we developed a forecasting model using deep learning (DL). We implemented an autoencoder-based Seq2Seq model with gated recurrent units (GRUs) in the deep recurrent layers. We trained the model to predict future incident cases, deaths and the reproductive number, R. For most counties, it makes accurate predictions of new incident cases, deaths and R values, up to 30 days in the future. Our framework can also be used to predict other targets that are useful indices for policymaking, for example hospitalization or the occupancy of intensive care units. Our DL framework is publicly available on GitHub and can be adapted for other indices of the COVID-19 spread. We hope that our forecasts and model can help local governments in the continued fight against COVID-19.
    • Shared polygenic risk for ADHD, executive dysfunction and other psychiatric disorders

      Chang, Suhua; Yang, Li; Wang, Yufeng; Faraone, Stephen V. (Springer Science and Business Media LLC, 2020-06-09)
      Many psychiatric disorders are associated with impaired executive functioning (EF). The associated EF component varies by psychiatric disorders, and this variation might be due to genetic liability. We explored the genetic association between five psychiatric disorders and EF in clinically-recruited attention deficit hyperactivity disorder (ADHD) children using polygenic risk score (PRS) methodology. Genome-wide association study (GWAS) summary data for ADHD, major depressive disorder (MDD), schizophrenia (SZ), bipolar disorder (BIP) and autism were used to calculate the PRSs. EF was evaluated by the Stroop test for inhibitory control, the trail-making test for cognitive flexibility, and the digital span test for working memory in a Chinese ADHD cohort (n = 1147). Exploratory factor analysis of the three measures identified one principal component for EF (EF-PC). Linear regression models were used to analyze the association between each PRS and the EF measures. The role of EF measures in mediating the effects of the PRSs on ADHD symptoms was also analyzed. The result showed the PRSs for MDD, ADHD and BIP were all significantly associated with the EF-PC. For each EF component, the association results were different for the PRSs of the five psychiatric disorders: the PRSs for ADHD and MDD were associated with inhibitory control (adjusted P = 0.0183 and 0.0313, respectively), the PRS for BIP was associated with working memory (adjusted P = 0.0416), and the PRS for SZ was associated with cognitive flexibility (adjusted P = 0.0335). All three EF measures were significantly correlated with ADHD symptoms. In mediation analyses, the ADHD and MDD PRSs, which were associated with inhibitory control, had significant indirect effects on ADHD symptoms through the mediation of inhibitory control. These findings indicate that the polygenic risks for several psychiatric disorders influence specific executive dysfunction in children with ADHD. The results helped to clarify the relationship between risk genes of each mental disorder and the intermediate cognitive domain, which may further help elucidate the risk genes and motivate efforts to develop EF measures as a diagnostic marker and future treatment target.
    • SHP-1-dependent macrophage responses mediate virus-induced myositis and demyelinating disease.

      Massa, Paul; Watson, Neva (2015)
      Regulation of inflammatory immune responses to pathogenic microbial infections is critical for protecting against extensive tissue damage and chronic inflammation. Correspondingly, genes associated with inflammatory immune responses have been identified as potential genetic risk factors for chronic inflammatory diseases including multiple sclerosis (MS) and idiopathic inflammatory myopathies (IIMs). This dissertation will focus on characterizing how the key immune regulator, Src-homology 2 containing protein tyrosine phosphatase-1 (SHP-1), controls virus-induced inflammatory diseases in the central nervous system (CNS) and skeletal muscle. We previously reported that SHP-1 inhibits proinflammatory macrophage-mediated CNS demyelinating disease during Theiler’s murine encephalomyelitis (TMEV) infection in mice. Presently, we describe that SHP-1 controls TMEV-induced dystrophic calcification of skeletal muscle. Muscle-infiltrating macrophages displayed a proinflammatory M1-like phenotype and promoted muscle calcification in WT mice, whereas an increased infiltration of macrophages with a reduced M1 signature corresponded with absence of muscle disease in SHP-1-/-mice. These studies reveal SHP-1 as a key regulatory gene mediating CNS and skeletal muscle disease in response to a virus trigger.Proinflammatory macrophages promoted tissue damage in either skeletal muscle of WT mice or CNS of SHP-1-deficient mice following TMEV infection. We thus attempted to determine if SHP-1 activity within macrophages was sufficient to control the outcome of TMEV infection using multiple genetic approaches. However, since these cells have a high turnover rate, and SHP-1 was strongly induced in macrophages by TMEV infection, these approaches were not sufficient to address whether SHP-1 activity specifically within macrophages mediates tissue-specific disease outcomes following TMEV infection. The studies described here suggest that SHP-1 affects macrophage maturation in peripheral (muscle) and immune-privileged (CNS) tissues in opposite ways. However, SHP-1 inhibited inflammatory monocyte CCR2 expression and subsequent infiltration into both of these major sites of infection, indicating that additional environmental cues mediated by SHP-1 are needed to drive tissue-specific maturation of pathogenic M1-like macrophages in either the CNS or muscle, to explain tissue-specific disease outcomes in SHP-1-deficient mice. Thus, this dissertation characterized unique mechanisms by which SHP-1 mediates inflammatory responses to virus infections, and has revealed SHP-1 and proinflammatory M1-like macrophages as essential mediators of myositis and demyelinating CNS disease.
    • SipNrate: Mobile Application for New York State Wines

      Choquette, Rebecca E (2015-12)
      It is estimated that 893 million gallons of wine were consumed in 2014, according www.wineinstitute.org, which is an increase of 228 million gallons from ten years ago. In addition mobile phone usage has soared with 90% of American adults owning a cell phone and 50% of those cell phone users, use their phones to download mobile applications, according to the Pew Research Center. Due to increases in both wine consumption and cell phone usage/mobile downloads it showed a clear need for a mobile application to assist consumers in making an informed decision prior to purchasing wine. The prototype was created using mokeupphone.com, a free online tool. Cooley’s theory of Human Centered Design was used during the creation of this mock up to insure that it would be all around user friendly. The mock ups created aide in the understanding of why this mobile application is needed, furthermore this paper explores the need for this mobile application through research in wine consumption, wine marketing and cell phone usage. The protoypes created for this project are embedded as images throughout the paper with clearly labeled image numbers to coincide with the labeling in the paper.
    • Six-week, double-blind, placebo-controlled study of desipramine for adult attention deficit hyperactivity disorder

      Wilen, T E; Biederman, J; Prince, J; spencer, T J; Faraone, Stephen V.; Warburton, R; Schleifer, D; Harding, M; Linehan, C; Geller, D (American Psychiatric Association Publishing, 1996-09)
      O bjective: The factor structures of individual positive and negative symptoms as well as global ratings were examined in a diagnostically heterogeneous group of subjects. Method: Subjects were identified through a clinical and family study of patients with major psychoses at a VA medical center and evaluated with the Scale for the Assessment of N egative Symptoms and the Scale for the Assessment of Positive Symptoms. For the examination of global-level factor structures (N =630), both principal-component analysis and factor analysis with orthogonal rotation were used. Factor analysis was used for the examination of item-level factor structures as well (N =549). Results: The principal-component analysis of global ratings revealed three factors: negative symptoms, positive symptoms, and disorganization. The factor analysis of global ratings revealed a negative symptom factor and a positive symptom factor. The itemlevel factor analysis revealed two negative symptom factors (diminished expression and disordered relating), two positive symptom factors (bizarre delusions and auditory hallucinations), and a disorganization factor. Conclusions: The generation of additional meaningful factors at the item level suggests that important information about symptoms is lost when only global ratings are viewed. Future work should explore clinical and pathological correlates of the more differentiated item-level symptom dimensions
    • Smart Technologies in a Technology Classroom: Integration Investigation of Smart board and Smart Notebook into 7-12 Technology Education Classroom

      Owens, Travis H. (2012-04-01)
      The project explores how the different uses of Smart technologies (Smart Board and Smart Notebook) can aide in teaching technology education and address New York State and ITEEA (International Technology and Engineering Educators Association) Standards for Technical Literacy during instruction by the creation of three Smart Notebook documents to be used in a 9-12 grade technology education classroom of 8-14 students. The lengths of the three presentations vary from 10-40 minutes. One presentation is used as a teacher presentation(and student note-taking) tool and demonstrates some of the affordances that Smart Notebook has over using a traditional whiteboard and markers or Microsoft Office Power Point. The second presentation explores interactivity and demonstrates how the interactive whiteboard can be used to increase student participation and motivation in the through the use of games, activities, and interactive websites. Lastly, Smart Notebook is utilized as an assessment tool; allowing the teacher to create questions from multiple resources. The project also covers a review of literature that attempts to explain why Smart Technologies have become so popular and are often credited to improve student learning. The literature review covers a few basic theories of student learning and informational technology and design that explain how content should best be created and structured so it is learner-friendly and effective.
    • Social Media Emoji Analysis, Correlations and Trust Modeling

      Preisendorfer, Matthew; Sengupta, Sam; Adviser; White, Joshua; Adviser; Tekeoglu, Ali; Adviser (2018-01-18)
      Twitter is an ever-growing social-media platform where users post tweets, or small messages, for all of their followers to see and react to. This is old news of course, as the platform first launched over ten years ago. Currently, Twitter handles approximately six thousand new tweets every second, so there is plenty of data to be analyzed. With a character limit of 140 per tweet, emojis are commonly used to express feelings in a tweet without using extra characters that more explaining might use. This is helpful in identifying the mood or state of mind that a person may have been in when writing their tweet. From a computing standpoint, this makes mood analysis much easier. Rather than analyzing a group of words and predicting moods from keywords, we can analyze single (or many) emoji(s), and then match those emojis to commonly expressed emotions and feelings. The objective of this research is to gather large amounts of Twitter data and analyze emojis used to find correlations in societal interactions, and how current events may drive social media interactions and behaviors. By creating topic models for each user and comparing it with the emoji distribution analysis, a trust ”fingerprint” can be created to measure authenticity or genuineness of a given user and/or group of users. The emoji distribution analysis also provides the possibility of demographic predictions. Analysis is not limited to Twitter of course but is used here because the API is free and generally easy to use. This paper aims to prove the validity of emoji analysis as a method of user identification and how their trust models can be used in conjunction with pre-existing models to improve success rates of these models.
    • Sodium hydrogen exchanger 9 NHE9 ( SLC9A9 ) and its emerging roles in neuropsychiatric comorbidity

      Patak, Jameson; Faraone, Stephen V.; Zhang‐James, Yanli (Wiley, 2020-05-13)
      Variations in SLC9A9 gene expression and protein function are associated with multiple human diseases, which range from Attention-deficit/hyperactivity disorder (ADHD) to glioblastoma multiforme. In an effort to determine the full spectrum of human disease associations with SLC9A9, we performed a systematic review of the literature. We also review SLC9A9's biochemistry, protein structure, and function, as well as its interacting partners with the goal of identifying mechanisms of disease and druggable targets. We report gaps in the literature regarding the genes function along with consistent trends in disease associations that can be used to further research into treating the respective diseases. We report that SLC9A9 has strong associations with neuropsychiatric diseases and various cancers. Interestingly, we find strong overlap in SLC9A9 disease associations and propose a novel role for SLC9A9 in neuropsychiatric comorbidity. In conclusion, SLC9A9 is a multifunctional protein that, through both its endosome regulatory function and its protein–protein interaction network, has the ability to modulate signaling axes, such as the PI3K pathway, among others.
    • Spanning Tree Protocol

      Kasu, Sivalasya; Hash, Larry; Adviser; Marsh, John; Reviewer; Bull, Ronny; Reviewer (2015-05-21)
      This technology case study focuses on Spanning Tree Protocol (STP). The Spanning Tree Protocol is a layer2 protocol that ensures a loop free topology for any LAN network. The basic function of STP is to prevent loops in the network. Spanning Tree Protocol is standardized as IEEE 802.1D. Spanning tree is created within a Layer 2 network of connected switches, leaves only one active path between two network devices. Spanning tree has evolutions and extensions such as; Per VLAN Spanning Tree Protocol (PVST), Rapid Spanning Tree Protocol (RSTP) and Multi Spanning Tree Protocol (MSTP).
    • Specific mutations in the α and ß subunits of the Kluyveromyces lactis F1-ATPase enhance ATP hydrolysis in the absence of the central γ-rotor

      Xin Jie Chen; Thuy La (2013)
      In eukaryotic cells, the mitochondria are vital organelles which are required for cell viability. Mitochondrial stresses such as oxidative stress, loss of membrane potential or loss of mitochondrial DNA are considered extreme and are associated with many neurodegenerative diseases and aging. The mitochondrial FoF1-synthase, where the majority of cellular ATP is synthesized, is composed of one inner membrane bound Fo domain and a water soluble F1 domain in the mitochondrial matrix. F1 contains the hexameric α3β3core and the centrally located γ subunit. The γ subunit is believed to play a key role in inducing conformational changes while rotating within the α3β3 core during ATP hydrolysis/synthesis. Previous studies have shown that the α3β3 core alone from the Thermophilic bacterium PS3 has a detectable hydrolyzing activity. In recent years, evidence of the rotary catalysis of Thermophilic Bacillus sp. PS3 F1-ATPase without its rotor - subunit γ - was shown using high-speed atomic force microscopy[1]. Moreover, previous study undertaken in our lab had utilized a unique genetic screen that allowed the identification of two specific mutations in the α and β subunits in the aerobic yeast Kluyveromyces lactis that stimulate ATP hydrolysis by the mitochondrial F1-ATPase in the absence of γ. This allows cells to survive upon the loss of mitochondrial DNA. In current work, we confirmed that the αF446I and βG419D mutations on the DELSEED loop are sufficient to allow ρ0 cells to survive in the absence of γ. Biochemical experiments showed that the γ -less F1-ATPase can be assembled to actively hydrolyze iv ATP in vivo, but this activity becomes extremely labile in vitro. These studies give insights into the catalytic mechanism of the α3β3 subcomplex and help to better understand the evolutionary origin of the mitochondrial F1-ATPase.
    • Sql Course Development Via Scaffolding, Social Constructivism, & Fantasy Football

      Thornton, Shawna (2013-12-01)
      Applying scaffolding and social constructivist concepts in online learning environments requires consideration of multimedia design and the impact it has on learning achievement. Online learning is transforming students’ experiences from “learning from technology” to “learning with technology”. Social constructivism –theoretical model in which learners make meaning from experience – and scaffold –theoretical guidelines for learning through incremental assistance – approaches aid instructors in providing effective and responsible learning environments though balancing student support and engaging challenges to help students understand that, ultimately, they learn from and teach each other. This project seeks to execute scaffolding and social constructivist techniques in a prototype online undergraduate level course on Structured Query Language, a programming language, for computer science and related field students. The prototype focuses on utilizing datasets from professional football players and utilizing Gamification aspects to create a pseudo fantasy football league. Students manage their fantasy teams and compete via increasing difficult SQL statements.
    • Stability of executive function deficits into young adult years: a prospective longitudinal follow-up study of grown up males with ADHD

      Biederman, J.; Petty, C. R.; Fried, R.; Doyle, A. E.; Spencer, T.; Seidman, L. J.; Gross, L.; Poetzl, K.; Faraone, Stephen V. (Wiley, 2007-08)
      Objective: Although individuals with attention deficit‐hyperactivity disorder (ADHD) commonly exhibit deficits in executive functions that greatly increase the morbidity of the disorder, all available information on the subject is cross sectional. Method: Males (n = 85) 9–22 years with ADHD followed over 7 years into young adulthood were assessed on measures of sustained attention/vigilance, planning and organization, response inhibition, set shifting and categorization, selective attention and visual scanning, verbal and visual learning, and memory. A binary definition of executive function deficits (EFDs) was defined based on a subject manifesting at least two abnormal tests 1.5 standard deviations from controls. Results: The majority of subjects maintained EFDs over time (kappa: 0.41, P < 0.001; sensitivity: 55%, specificity: 85%, positive predictive value: 69%, and negative predictive value: 75%). Conclusion: Considering the morbidity of EFDs, these findings stress the importance of their early recognition for prevention and early intervention strategies. EFDs are stable over time.
    • A STRING OF LIGHTS: MATURIN EXPRESSION AND POTENTIAL ROLE DURING MOUSE RETINOGENESIS

      Ly, Christine (2020)
      During retinal development, a pool of progenitor cells divides to generate daughter cells that eventually differentiate into the seven retinal cell types, including horizontal cells (HCs) and retinal ganglion cells (RGCs). Much about how cells exit the cell cycle and maintain a differentiated state remain unknown. Dysregulation of this process can alter the cellular composition and function of the retina.Thus, by studying this developmental process, we can better understand the mechanisms by which progenitor cells become functional, differentiated cells. Our previous work determined that Maturin (Mturn)is highly conserved in its expression pattern and protein sequence across various vertebrate species. Furthermore, we concluded that it is required for differentiation of primary neurons in Xenopuslaevis. Preliminary work in mice revealed that in the absence of Mturn, extensive folds occur in the retina. I used this model to characterize the expression of Mturnin the mouse retina and ask if Mturn is required for normal mice retinogenesis. By immunostaining retinal sections with various cell type-specific antibodies, I found that Mturn is expressed in differentiating cells and not in proliferating cells. In addition to determining that its expression is maintained in mature HCs, I concluded that Mturn is not required for generating the proper number HCs. Our results from studies on Mturn in both frogs and mice have led us to hypothesize that Mturn may function to maintain HCs in differentiated state and prevent their reentry into the cell cycle. Although preliminary experiments testing this hypothesis were inconclusive, future work should continue to investigate the role of Mturn in retinogenesis.
    • Structural and functional characterization of the V-ATPase membrane sector

      Couoh-Carde, Sergio J. (2017)
      The vacuolar ATPase (V1VO-ATPase, V-ATPase) is a H+-pump involved in the acidification of organelles in eukaryotes. Under certain physiological conditions, the VATPase disassociates into an inactive soluble ATPase sector (V1) and a membrane sector (VO) that is impermeable to protons. Due to the lack of detailed structural and functional information, the auto-inhibition mechanism of VO is not well understood. Although the V-ATPase shares a similar structure and rotary catalysis mechanism with the F- and AATPases, V-ATPase’s increased structural complexity and unique mode of regulation suggest other functions beyond its canonical proton pumping. We purified Vo and Vo sub-complexes for structural and functional characterization. First, our ~18 Å cryo-EM model of Vo suggests that c-ring (c8c’c’’) is partially surrounded by the C-terminal membrane integral portion of subunit a (aCT). On the other hand, the soluble N-terminal portion of subunit a (aNT) interacts with subunit d that sits atop of the c-ring. Selective removal of subunit d (VoΔd) did not allow passive proton translocation. Second, the c-ring was isolated and its X-ray crystal structure was solved at ~4 Å resolution. Two c-rings interact to form a gap-junction like structure. The presence of c’’ disrupts the intrinsic and global symmetry of the c8c’ sub-complex, constituting a kinetic barrier during c-ring axial rotation. Third, we discovered that c-ring can act as a large-conductance ion-channel independently from its canonical function in proton pumping. Our biophysical, biochemical, and functional data suggest that exquisite kinetic barriers play a primary role in the auto-inhibition of Vo, and that Vo may have noncanonical functions in intercellular communication.
    • STRUCTURAL BASIS OF THE FUNCTIONAL ROLES OF HUMAN AROMATASE

      Ghosh, Debashis; Lo, Jessica (2015)
      Humanaromatase(AROM) catalyzes theconversion of androgens to estrogensand is a major breast cancer drug target. Structural investigation has provided insights intothe active siteandaromatization mechanism.Utilization of the structural data has permitted rational design of a series of novel steroidal inhibitors. Investigation ofthe roles of key amino acids is facilitated by a recombinant AROM identical in crystal structureto the placental AROM.We use mutagenesis, chromatography, ultracentrifugation, spectrophotometry, enzyme kinetics, and X-ray crystallography to probe the roles of critical residues and the molecular basis of oligomerization. Furthermore, weevaluate the potencies of novel inhibitors and determine the structural basis of inhibition andselectivity. A critical active site residue D309 withan elevated pKa remainsprotonated at neutral pHand facilitatessubstrate binding and catalysis.The “gatekeeper” R192, linked to D309 via a watermolecule, is postulated to have a role in proton relay and substrate selectivity. D309N and R192Q mutants are virtually inactive supporting thehypothesis that both play keyrolesin aromatization.AROM oligomerization is driven bytheD-E loop of one moleculeand heme-proximal region of another via hydrogen bonding, electrostatic interactions between E181 and K440, and shape complementarity.Del7, generated by deletionof 7 residues in the D-E loop, experiences 65% reductionin activitydue to the loss of oligomer formation. Mutants Del4, E181A, and E181K exhibit normal enzymatic activity,and maintain some oligomeric interactions. The heme-proximal interface is also the putative coupling site of the reductasethatsupplieselectronsfor aromatization. The siteis larger than the active site, and at least twice aslarge asother P450s.MutantsK440Qand Y361Fof this region are virtuallyinactive.Collectivelythe results suggestfunctional significanceof oligomerization. Several newly designedAIs are superiortoexemestane, the steroidal AI currently used as a drug, in inhibition and anti-proliferation assays. The C6β-(pent-2-yn-1-yloxy) side chains ofthe most potent compoundspenetrate the access channelunique to AROM and havethe sameconformation asin the enzyme-free state.Astructural-based approachcan improve drug efficacy by improving specificity and selectivity, and reducing sideeffects.