• FH2-dependent Localization of FHOD Formins in the Sarcomere

      Blystone, Scott; Hamilton, Elisabeth (2016)
      Formins are a class of actin nucleating factors containing a highly-conserved FH2 domain, which binds actin. There are 15 mammalian formin proteins in seven sub-families that have been found to nucleate, cap, sever, bundle, and polymerize linear actin filaments. An expression analysis of all 15 human formins across 22 different cell and tissue types showed high levels of expression for one formin sub-family in striated muscle: FHOD. While FHOD1 is highly expressed across many cell and tissue types, FHOD3 is only found at comparatively high levels in striated muscle cells. The mature structure of skeletal muscle is a very recognizable periodic repetition of filaments that allows muscles to contract. The sarcomere is the smallest contractile unit of skeletal muscle, with the two main filaments, the thick filament containing myosin, and the thin filament containing actin moving back and forth via the interaction of myosin heads with the thin filament, which allows muscles to contract. While the molecular functioning and the mature structure of skeletal muscle is well understood, the exact mechanism by which the sarcomere is assembled, and specifically, how the actin-core of the thin filament is formed and incorporated into the thin filament remains unknown. The high levels of expression of FHOD formins in striated muscle, combined with their ability to interact with filamentous actin warranted a closer look at FHOD1 and FHOD3 in the sarcomere. In this study we found that FHOD1 and FHOD3 have distinct sarcomeric localizations in C2C12 cells. FHOD1 localizes to the barbed end of the actin filament at the Z-disk and FHOD3 localizes to the pointed end of the actin filament near the center of the sarcomere. Full-length cDNA constructs for FHOD1 and FHOD3 were introduced into skeletal muscle cells, and we were able to recreate the endogenous localization of FHOD3 with the exogenous cDNA. The FHOD1 cDNA localized not to the barbed end of the actin filament, as endogenous FHOD1 does, but instead to the pointed end of the actin filament, where endogenous and exogenous FHOD3 were found. We hypothesized that FHOD binding to actin is dependent upon two highly conserved actin-binding sites in the FH2 domain. Mutations of the actin-binding residues in the FH2 domain impaired the actin-binding ability of both exogenous FHOD1 and FHOD3 and showed that the localization of FHOD1 and FHOD3 in the sarcomere is actin-binding dependent.
    • The Formin FMNL1 Contributes to the Macrophage Inflammatory Response by Regulating Podosome-dependent Adhesion and Migration.

      Blystone, Scott; Miller, Matthew (2015)
      Macrophages are indispensible white blood cells (leukocytes) that contribute to both the innate and adaptive immune response. They are crucial for resolving inflammatory events by clearing pathogens and cellular debris, in addition to promoting wound repair. Macrophages are derived from peripherally circulating monocytes, which after stimulation undergo diapedesis from the vasculature into the underlying complex extracellular matrix, where they can become fully differentiated macrophages and migrate to inflammatory loci. Tissues also contain residential populations of macrophages that aid in immediate immune responses and maintain tissue homeostasis. Conversely, unwarranted macrophage activation largely contributes to the onset and progression of inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, in addition to aiding cancer metastasis and facilitating organ transplant rejection. In order for macrophages to effectively resolve inflammatory events or contribute to disease pathology, they must be able to undergo directional migration, which is mediated by integrin-dependent adhesion complexes termed podosomes. Macrophage podosomes are the most prominent structure of the macrophage actin cytoskeleton, containing a pillar-like core of dense filamentous actin that is tethered to the cortical actincytoskeleton via radial actin filaments. Podosomes also contain a variety of proteins that are circumferentially arranged orassociated with the core, and thatare involved in signaling, linking, and scaffolding,as well as modulating the actin cytoskeleton.Historically, our lab has been interested in leukocyte integrin biology and understanding how these receptors mediate adhesion and migration through complex extracellular matrices. Previous studies in our lab demonstrated the novel podosomal association of an actin modulating protein with the ability to processively elongate unbranched linear actin filaments. Subsequent studies determined this protein to be the formin FMNL1, which is predominantly expressed in hematopoietic cells. Consequently, we further revealed that FMNL1 localizes to the apex of the dense actin core, and is required for podosome stability and macrophage adhesion.The work described in this dissertation has greatly expanded on these findings, demonstrating for the first time that primary macrophage migration is dependent on the formin FMNL1. Utilizing in vitro and in vivotechniques with aid of a novel conditional murine FMNL1 KO, we have observed that macrophage podosome formation, migration, and tissue distribution are dependent on FMNL1. Additionally, we have indicated that FMNL1 is required for embryonic development. Remarkably, our findings also suggest that FMNL1-dependent macrophage migration and podosome localization rely on the specific isoform FMNL1γ. Foremost, we have demonstrated that barbed end binding by the FMNL1γ FH2 domain is dispensable for its cellularfunction in macrophages, which has not been previously shown for any other cellular formin function. Thus, these findings, in addition to current formin knowledge, have allowed us develop a working model for FMNL1 function at macrophage podosomes. This work has distinguished FMNL1 as a unique therapeutic target to restrict macrophage migration that contributes to macrophage-mediated diseases. Furthermore, this could translate to treatment of certain cancers, since FMNL1 has been suggested to promote leukemic cell migration.
    • Functional alterations and rhythmic disturbances by pan-histone deacetylase inhibition in the heart.

      Veenstra, Richard; Patel, Dakshesh (2016)
      Histone acetyl transferases (HATs) and histone deacetylases (HDACs) maintain a dynamic balance of acetylation and deacetylation of histone and non-histone proteins. HDAC inhibitors are small molecules anti-cancer therapeutics that exhibited dose limiting cardiac toxicities during clinical and preclinical trials. Multiple instances of abnormal T-waves, ST segment depression, QT prolongation, grade 3 sinus bradycardia and non- circumstantial deaths have been observed in patients. The underlying electrophysiological and molecular mechanism of these cardiac side-effects are poorly understood. In our in vivo ECG monitoring using Data Science International® telemetry transmitters, mice injected with panobinostat showed ventricular tachychardic and atrial fibrillation episodes with significant prolongation of ST, QT and QTc intervals. In whole cell patch clamp studies, we observed no significant change in transient and steady state K currents in myocyte ventricular cultures suggesting no role of hERG currents in ventricular arrhythmias. The majority (>90%) of congenital and drug induced QT prolongation is caused by alterations of hERG (IKr, Kv11.1) current. Interestingly, we observed significant reductions of INa and gap junctional conductance along with reductions in protein expression of Nav1.5 & Cx43 in vivo and in vitro. We conclude that pan-HDAC inhibition reduced cardiac INa density and gap junctional coupling with unaltered late INa and K+ currents explaining the cardiac abnormalities exhibited by panHDAC inhibitors. Decreased gap junctional coupling can enhance triggered activity by limiting electrotonic inhibition, combined with reduced INa density which can lead to slow myocardial conduction. Both of them taken together, increases the vulnerability to reentrant arrhythmias.
    • Functional Studies of Tumor Suppressor ECRG2 in the Regulation of Cancer Cell Death and Drug Resistance

      Huang, Ying; Lucchesi, Chris (2015)
      Esophageal Cancer Related Gene 2 (ECRG2) is a newer tumor suppressor whose mRNAhas previously been shown to be decreased in multiple human malignanceswhencompared to normal/adjacent tissues.Of importance, ECRG2 has previously been revealedto possess tumor suppressive attributes,including the ability to induce cell death in cancer cells. However, how ECRG2 is able to activate the apoptotic machinery has yet to be elucidated. In the present study,we highlight multiple angles that ECRG2 leverages in order to sensitize cancer cells to apoptosis. Moreover, we report for the first time,that ECRG2 protein expression in lung cancer patientsamples is lost/decreased in upwards of 90% ofthecancer tissues evaluatedcompared to normal tissue. Additionally, a single somatic point mutant found in patient tumor samples, V30E, was shown to lose tumor suppressive abilities and acquired resistance against multiple anticancer drugs. Our results demonstrate that ECRG2is upregulated in response to DNA damage, andis capable of inducing the activation of both caspase cascades (intrinsic and extrinsic) leadingto cancer cell death. We further illustratedthat ECRG2-mediated cell deathwasattributed to a reduction in the levels of apoptosis inhibitor, X chromosome-linked inhibitor of apoptosis protein (XIAP). ECRG2 was revealedto regulate XIAP protein levels via RNA-binding protein,human antigen R(HuR).We further highlight that ECRG2 causes increased HuR ubiquitination,subsequently leading to its degradation. Thus, we demonstrate that ECRG2 sensitizescancer cells to apoptosis through the downregulation of HuR, and consequent downregulation of XIAP. Next, we have identified ECRG2 as a potent positive regulator of death receptor 5 (DR5) gene expression.ECRG2-mediated upregulation of DR5 was shown to be facilitated through the upregulation of tumor suppressor p53 and transcription factors ATF3 and NFⱪB. Together, in a cooperative fashion, increased levelsof p53, ATF3 and NFⱪB stimulateDR5 gene expression. Contrastingly, silencing of ECRG2 not only decreased the levels of NFⱪB and DR5, but also resulted in decreased cancer cell sensitivity to genotoxic stressandTRAIL treatment. Collectively,our work establishes that ECRG2 is capable of inducing apoptosis in cancer cells by increasing the expression of pro-apoptotic proteins, while also negatively influencing anti-apoptotic proteins.Further, the loss of ECRG2’stumor suppressive abilities, as wehaveshownby the loss of ECRG2in lung patient tumor samples,and through the somatic point mutantV30E, illuminates possible mechanismsin which cancer cells can acquiremultiple drug resistance.
    • Further evidence of an association between maternal smoking during pregnancy and attention deficit hyperactivity disorder: Findings from a high-risk sample of siblings

      Milberger, Sharon; Biederman, Joseph; Faraone, Stephen V.; Jones, Janice (Informa UK Limited, 1998-09)
      The authors investigated the relationship between attentiondeficit/byperactivity disorder (ADHD) and cigarette smoking in siblings of ADHD and non-ADHD probands. They conducted a 4-year follow-up of siblings from ADHD and control-group families. In the siblings of ADHD probands, ADHD was associated with higher rates and earlier onset of cigarette smoking. There was also a significant positive association between cigarette smoking and conduct disorder, major depression, and drug abuse in the siblings, even after adjusting for confounding variables. Moreover, smoking was found to be familial among ADHD families but not control-group families. Our findings indicate that ADHD is a risk factor for early initiation of cigarette smoking in the high-risk siblings of ADHD probands
    • Further Evidence of Association Between Behavioral Inhibition and Social Anxiety in Children

      Biederman, Joseph; Hirshfeld-Becker, Dina R.; Rosenbaum, Jerrold F.; Hérot, Christine; Friedman, Deborah; Snidman, Nancy; Kagan, Jerome; Faraone, Stephen V. (American Psychiatric Association Publishing, 2001-10)
      Objective: The authors sought to examine psychopathological correlates of behavioral inhibition in young offspring of parents with panic disorder and/or major depression. Method: Behavioral inhibition, determined by using standard laboratory observations, was assessed in four groups of children (age 2–6 years): 129 children of parents with both panic disorder and major depression, 22 children of parents with panic disorder alone, 49 children of parents with major depression alone, and 84 comparison children of parents with neither panic disorder nor major depression. Psychopathology in children ≥5 years was compared between children with behavioral inhibition (N=64) and without (N=152). Results: Social anxiety disorder (social phobia or avoidant disorder) was significantly more likely to be found in the children with behavioral inhibition (17%) than in those without (5%). Noninhibited children were significantly more likely than inhibited children to have disruptive behavior disorders (20% versus 6%, respectively) and had higher scores on the attention problems scale of the Child Behavior Checklist (mean=52.1 versus 50.8). Conclusions: This study adds to the growing literature suggesting an association between behavioral inhibition and social anxiety disorder and an inverse relationship between inhibition and disruptive behavior disorders.
    • Further investigation of a chromosome 15 locus in schizophrenia: Analysis of affected sibpairs from the NIMH genetics initiative

      Leonard, Sherry; Gault, Judith; Moore, Theodore; Hopkins, Jan; Robinson, Misi; Olincy, Ann; Adler, Lawrence E.; Cloninger, C. Robert; Kaufmann, Charles A.; Tsuang, Ming T.; et al. (Wiley, 1998-07-10)
      Linkage of a neurophysiological deficit associated with schizophrenia, i.e., the failure to inhibit the auditory P50 response, was previously reported at chromosome 15q14. The marker with the highest pairwise lod score, D15S1360, was isolated from a yeast artificial chromosome containing a candidate gene, the α7-nicotinic acetylcholine receptor gene. In the present study, this linkage was further investigated in a subset of the NIMH Genetics Initiative schizophrenia families. These families have not been studied neurophysiologically, as were the families in the original report. Therefore, the DSMIII-R diagnosis of schizophrenia was used as the affected phenotype. Twenty families fulfilled the criteria of at least one sibpair concordant for schizophrenia, along with their two parents or another affected relative outside the nuclear family, available for genotyping. Sibpair analysis showed a significant proportion of D15S1360 alleles shared identical-by-descent (0.58; P < 0.0024). The results further support the involvement of this chromosomal locus in the genetic transmission of schizophrenia. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:308–312, 1998. © 1998 Wiley-Liss, Inc.
    • GENESIS AND MAINTENANCE OF LONG-TERM IgM+ T-BET+ B CELLS

      Papillion, Amber (2017)
      IgM memory cells are recognized as an important component of B cell memory, based on several studies both in mice and humans. Our studies of B cells elicited in response to ehrlichial infection identified a population of CD11c/T-bet-positive IgM memory cells and an IgM T-bet-positive bone marrow antibody-secreting cell population (ASCs). The origin of these populations was unknown, although an early T-independent spleen CD11c-and T-bet-positive IgM plasmablast population precedes both, suggesting a linear relationship. The majority of IgM memory cells detected after day 30 post-infection had undergone somatic hypermutation, indicating that they expressed activation-induced cytidine deaminase (AID). Therefore, to identify early AID-expressing precursor cells, we infected an AID-regulated tamoxifen-inducible Cre-recombinase-EYFP reporter strain. Tamoxifen administration led to labeling of both the IgM memory cells and bone marrow ASCs on day 30 and later post-infection. High frequencies of labeled cells were identified on day 30 post-infection,following tamoxifen administration on day 10 post-infection. Both IgM memory cells and IgM bone marrow ASCs were labeled when tamoxifen was administered as early as day 4 post-infection. We also identified mechanisms involved in maintenance of the IgM bone marrow ASCs and IgM+ memory cells, namely proliferation and FcγRIIb respectively. BrdU studies revealed that the bone marrow IgM ASCs were maintained by proliferation, unlike the IgM memory cells. RNAseq analysis revealed a 2-fold higher expression of inhibitory Fc receptor, FcγRIIb. Because FcγRIIb inhibits B cell activation, we hypothesized that FcγRIIb negatively regulates IgM+ memory B cells by binding immune complexes present during low-level chronic infection. E. murisinfection of FcγRIIb-deficientmice revealed a 3-fold expansion of the IgM+ memory 30 days post-infection. We further demonstrated that the expansion of the IgM+ memory cells was not due to increased proliferation, but a decrease of apoptosis, due to a lack of Fas expression in FcγRIIb-deficient mice. This result was mimicked in AID-deficient mice, which lack the ability to class switch to IgG and make immune complexes, revealing a role for immune complexes in regulating IgM+ memory. Altogether, these studies demonstrate a novel germinal center-independent pathway for the generation of two distinct long-term IgM-positive B cell populations.
    • Genome-wide association study of response to methylphenidate in 187 children with attention-deficit/hyperactivity disorder

      Mick, Eric; Neale, Benjamin; Middleton, Frank A.; McGough, James J.; Faraone, Stephen V. (Wiley, 2008-12-05)
      We conducted a genome-wide association study of symptom response in an open-label study of a methylphenidate transdermal system (MTS). All DNA extraction and genotyping was conducted at SUNY Upstate Medical University using the Affymetrix Genome-Wide Human SNP Array 6.0. All quality control and association analyses were conducted using the software package PLINK. After data cleaning and quality control, there were 187 subjects (72% (N¼135) male) with mean age 9.2 2.0 years and 319,722 SNPs available for analysis. The most statistically significant association (rs9627183 andrs11134178;P¼3 10 6) fell short of the threshold for a genome-wide significant association. The most intriguing association amongsuggestivefindings(rs3792452;P¼2.6 10 5) was with the metabotropic glutamate receptor 7 gene (GRM7) as it is expressed in brain structures also previously associated with ADHD. Among the 102 available SNPs covering previously studied candidate genes, two SNPs within the norepinephrine transporter gene (NET, SLC6A2) were significant at P 1 10 2. These results should be considered preliminary until replicated in larger adequately powered, controlled samples but do suggest that noradrenergic and possibly glutaminergic genes may be involved with response to methylphenidate.
    • Genome-wide search for schizophrenia susceptibility loci: The NIMH genetics initiative and millennium consortium

      Cloninger, C. Robert; Kaufmann, Charles A.; Faraone, Stephen V.; Malaspina, Dolores; Svrakic, Dragan M.; Harkavy-Friedman, Jill; Suarez, Brian K.; Matise, Tara C.; Shore, David; Lee, Hang; et al. (Wiley, 1998-07-10)
      chizophrenia has a complex pattern of inheritance, indicative of interactions among multiple genes and environmental factors. The detection and replication of specific susceptibility loci for such complex disorders are facilitated by the availability of large samples of affected sib pairs and their nuclear families, along with standardized assessment and systematic ascertainment procedures. The NIMH Genetics Initiative on Schizophrenia, a multisite collaborative study, was established as a national resource with a centralized clinical data base and cell repository. The Millennium Schizophrenia Consortium has completed a genome-wide scan to detect susceptibility loci for schizophrenia in 244 individuals from the nuclear families of 92 independent pairs of schizophrenic sibs ascertained by the NIMH Genetics Initiative. The 459 marker loci used in the scan were spaced at 10-cM intervals on average. Individuals of African descent were higher than those of European descent in their average heterozygosity (79% vs. 76%, P < .0001) and number of alleles per marker (9.2 vs. 8.4, P < .0001). Also, the allele frequencies of 73% of the marker loci differed significantly (P < .01) between individuals of European and African ancestry. However, regardless of ethnic background, this sample was largely comprised of schizophrenics with more than a decade of psychosis associated with pervasive social and occupational impairment. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:275–281, 1998. © 1998 Wiley-Liss, Inc.
    • GENOME-WIDESTUDIES OF AUTISM IDENTIFY ALTERED IMMUNOLOGIC AND NEURODEVELOPMENTAL PATHWAYS AND HIGHLIGHT PUTATIVE DIAGNOSTIC BIOMARKER SIGNALS

      Glatt, Stephen; Tylee, Daniel (2017)
      Autism is a complex neurodevelopmental syndrome that can be challenging to identify in young children. Family-based and genetic studies indicate that autism has a strong genetic component, though immunologic processes may also contribute to altered neurodevelopment. Over the past two decades, genome-wide investigations have provided critical insights into the genetic causes and molecular correlates of autism at the levels of both the individual and the population. Studies of DNA have identified highly penetrant genetic factors that appear to explain a sizable minority (20-40%) of autism-affected individuals. However, patho-developmental mechanisms are less-clearly understood for the remaining majority of individuals for whom no highly penetrant factors are identifiable(i.e., idiopathic autism). The present body of work contains three studies that used genome-wide assessment methods to predict the diagnosis of autism and to characterize its molecular correlates using samples that predominantly reflect idiopathic etiology. In Chapter 1, we demonstrate that large numbers of low-penetrance genetic factors(i.e., commonly varying single nucleotide polymorphisms; SNPs) can be harnessed with machine-learning methods to predict autism risk. Throughout this document, we provide a review and critical evaluation of DNA-and RNA-based autism biomarkers. In Chapters 2 and 3, we use microarray and RNA sequencing to identify consistent patterns of autism-related gene expression in peripheral blood samples. These patterns shed light on altered immunologic signaling and also implicate signaling pathways that are known to be involved in neurodevelopment and to influence autism-related clinical and neurobiological phenotypes. Importantly, our findings indicate that the molecular correlates of idiopathic autism may converge with mechanisms understood from high-penetrance genetic causes. Furthermore, our findings support the idea that immunologic mechanisms could contribute to perturbations of neurodevelopmental pathways. We integrate our findings in the context of existing literature, highlight current gaps in knowledge, and propose future experiments to address these questions.
    • Heterogeneity and the genetics of bipolar disorder

      Faraone, Stephen V.; Tsuang, Ming T. (Wiley, 2003-10-30)
    • IDENTIFICATION OF p53-MEDIATED NEUROGENOMIC RESPONSES TO ETHANOL USING IN VIVO AND IN VITRO MODELS OF FETAL ALCOHOL SPECTRUM DISORDER

      Camargo, Maria (2016)
      Fetal Alcohol Spectrum Disorder (FASD) is a serious public health concern affecting 3.6% of the US population. One avenue to achieve a decrease in the prevalence of FASD is for scientific research to identify cellular mechanisms of action of imbibed alcohol and propose solutions to treat or prevent the damage done. Here we present our investigation into the molecular consequences of ethanol exposure in mouse brain cells and mouse neural stem cell cultures. Specifically, we tested the hypothesis that p53 mediates the neurogenomic response to ethanol exposure in brain cells in the somatosensory cortex, hippocampus and neural stem cells. p53 is a versatile transcription factor well known for inducing cell death in cancer cells. We identified the apoptosis pathway as being changed in a p53-related manner only in the CA1 subregion of the hippocampus, based on expression changes in Casp2, Cdk1, and Stat1. Overall, the regions interrogated revealed that p53’s cellular response is heterogeneous. In the somatosensory cortex and hippocampus a subset of gene expression changes occurred depending on both ethanol exposure and the presence of p53: Ephb1in layer 2/3; Ctgf in layer 5; Camk1 in layer 6; Cdk1, Casp2, Cdk1, and Stat1 in the CA1; and Camk1 in the DG. In regards to the specific mRNAs that changed, they differed in the brain regions and cell cultures, but we did observe that neuronal and developmental genes were the most significantly changed upon ethanol exposure. In addition, we also identified that the category of genes whose methylation pattern was changed after ethanol exposure are related to basic neuronal functions. Neural cells also appeared to be engaged in a challenging response to ethanol because DNA repair proteins Ercc1, Hus1, and Rad51 alter their DNA binding after ethanol exposure. In addition, we identified that p53 transcription factor changes its DNA binding in response to ethanol exposure. In conclusion, we identified that neural p53 signaling is measurably perturbed by ethanol exposure.
    • Identification of TIMP2 as the first secretory co-chaperone of eHSP90

      Dimitra Bourboulia; Baker-Williams, Alexander J. (2021)
      Heat Shock Protein- 90 (HSP90) is an essential molecular chaperone. HSP90 relies on its intrinsic ATPase activity as well as interactions with co-chaperone proteins to chaperone its clients. HSP90 is also an extracellular protein, performing both a signaling and chaperoning role. Extracellular client, matrix metalloproteinase-2 (MMP2) relies on HSP90 for its stability. MMP2 mediates extracellular matrix remodeling through its gelatinolytic activity. MMP2 activity is also tightly regulated by its endogenous inhibitor, the Tissue Inhibitor of Metalloproteinase-2 (TIMP2). At present how HSP90 performs its chaperoning role in the extracellular matrix is uncertain. In this thesis, I describe that TIMP2 acts as the first bona fide extracellular co-chaperone of eHSP90, and show that TIMP2 is a stress inducible protein. I describe how TIMP2 directly interacts with HSP90, and how TIMP2 decelerates the HSP90 ATPase cycle. TIMP2 also sensitizes HSP90 to both ATP and N-terminal pharmaceuticals. Overall, TIMP2 acts as both a scaffold and a disruptor of the client/chaperone relationship between MMP2 and HSP90, performing both a HSP90 co-chaperone and MMP2 inhibitor role, non-mutually exclusively. The activatory co-chaperone AHA1 competes with TIMP2 for HSP90 binding. TIMP2 and AHA1 are able to form two independent ternary complexes with MMP2 and HSP90; as a result, the TIMP2 complex is MMP2 proteolytically inactive and the AHA1, active. This competition is further described in vivo where it can be inhibited by both _AHA1 antibodies and TIMP2 AHA1 antibodies and TIMP2 exogenous protein treatments, whilst induced following AHA1 protein and _AHA1 antibodies and TIMP2 TIMP2 antibody treatments. Finally, the role of phos-Y-TIMP2 was examined in relation to its interaction with HSP90. To address this, a novel methodology to purify hTIMP2 from E.Coli without previously necessary refolding strategies in a scale-able manner suitable for therapeutic TIMP2 treatments, was developed. Wild type recombinant human TIMP2 and phospho-mutants Y90E, Y90F, and TE (Y62E, Y90E, Y165E) were purified, were inhibitory towards MMP2, and modulated TIMP2 interaction with HSP90. Taken together, I demonstrate how extracellular HSP90 is regulated by co-chaperones to facilitate the chaperoning of pro-invasive client, MMP2. It further shows ways in which we can manipulate this system to promote an inactive MMP2 protease, a key strategy in cancer therapeutics.
    • The impact of mosquito salivary factors on Chikungunya virus transmission

      Thangamani, Saravanan; Esterly, Allen (2022-08-12)
      Vector-borne diseases affect an estimated 3.9 billion people in over 128 countries annually (WHO, 2017b). Mosquito-borne viruses, such as Chikungunya virus (CHIKV), are transmitted to human hosts through the saliva of female blood-feeding mosquitoes. During blood-feeding, salivary molecules enhance blood-feeding efficiency, dampen specific inflammatory signals, and enhance virus infection and dissemination. Skin is the interface where mosquito salivary molecules and viruses interact with the human immune system. Our long-term goal is to understand the impact of mosquito saliva on the skin and the role of salivary molecules in potentiating virus infection. This thesis aims to model the human skin bite site, identify mosquito salivary molecules present during virus infection, and investigate their impact on virus replication. We developed a human skin arbovirus infection model that was viable for four days ex vivo (Esterly et al., 2022a). Arbovirus infection in human skin ex vivo allowed flavivirus and alphavirus replication and dissemination. Using this model, we will investigate mosquito salivary factors and their impact on virus transmission. Next, we identified mosquito salivary factors highly expressed in Ae. aegypti salivary glands during CHIKV infection. We hypothesize that salivary proteins that contain secretion signals and are found to be highly expressed during CHIKV infection are delivered to the bite site during mosquito feeding. Through a combination of RNA-seq and miRNA-seq analysis, we identified several salivary factors, protein and small non-coding RNAs, which warrant further investigation. Lastly, we assessed the impact of identified and recombinantly expressed proteins or specific small RNA inhibitors on CHIKV replication in vitro relative to Ae. aegypti salivary gland extract (SGE). We found that both protein and small RNAs can influence CHIKV replication in a significant manner; however, the effect observed by mosquito saliva is a combination of multiple factors and will require more investigation to characterize fully. In summary, mosquito salivary factors work in concert to enhance virus replication and dissemination. Although individual factors may not recapitulate the total effect of SGE, the discovery and isolation of these factors further our understanding of arbovirus transmission. These studies highlight the complexity of mosquito salivary secretion and further our knowledge of mosquito-borne virus transmission. The following thesis will describe our most recent findings and contribution to understanding mosquito saliva's role during arbovirus transmission.
    • Impact of Psychometrically Defined Deficits of Executive Functioning in Adults With Attention Deficit Hyperactivity Disorder

      Biederman, Joseph; Petty, Carter; Fried, Ronna; Fontanella, Jessie; Doyle, Alysa E.; Seidman, Larry J.; Faraone, Stephen V. (American Psychiatric Association Publishing, 2006-10)
      Objective: The association between deficits in executive functioning and functional outcomes was examined among adults with attention deficit hyperactivity disorder (ADHD). Method: Subjects were adults who did (N=213) and did not (N=145) meet DSMIV criteria for ADHD. The authors defined having deficits in executive functioning as having at least two measures of executive functioning with scores 1.5 standard deviations below those of matched comparison subjects. Results: Significantly more adults with ADHD had deficits of executive functioning than comparison subjects. Deficits of executive functioning were associated with lower academic achievement, irrespective of ADHD status. Subjects with ADHD with deficits of executive functioning had a significantly lower socioeconomic status and a significant functional morbidity beyond the diagnosis of ADHD alone. Conclusions: Psychometrically defined deficits of executive functioning may help identify a subgroup of adults with ADHD at high risk for occupational and academic underachievement. More efforts are needed to identify cost-effective approaches to screen individuals with ADHD for deficits of executive functioning.
    • Impact of Tic Disorders on ADHD Outcome Across the Life Cycle: Findings From a Large Group of Adults With and Without ADHD

      Spencer, Thomas J.; Biederman, Joseph; Faraone, Stephen V.; Mick, Eric; Coffey, Barbara; Geller, Daniel; Kagan, Jake; Bearman, Sarah Kate; Wilens, Timothy (American Psychiatric Association Publishing, 2001-04)
      Objective: The impact of tic disorders on the outcome of attention deficit hyperactivity disorder (ADHD) remains a subject of high scientific and clinical interest. To evaluate the impact of comorbid ADHD and tic disorders from a lifespan perspective, the authors systematically examined data from adults with and without ADHD. Method: They comprehensively evaluated 312 consecutively referred adults with ADHD and 252 comparison subjects without ADHD. Tic disorders were characterized along with a wide range of neuropsychiatric correlates, including other comorbid disorders as well as indexes of function in the domains of school, cognition, and interpersonal functioning. Results: A significantly greater proportion of adults with ADHD (12%) than those without ADHD (4%) had tic disorders. Tic disorders followed a mostly remitting course and had little impact on functional capacities. In addition, tic disorders were not associated with stimulant use. Conclusions: These findings in adults with ADHD confirm and extend previous findings in young subjects with ADHD, documenting that although individuals with ADHD are at greater risk for tic disorders, the presence of tic disorders has a limited impact on ADHD outcome.
    • Influence of Gender on Attention Deficit Hyperactivity Disorder in Children Referred to a Psychiatric Clinic

      Biederman, Joseph; Mick, Eric; Faraone, Stephen V.; Braaten, Ellen; Doyle, Alysa; Spencer, Thomas; Wilens, Timothy E.; Frazier, Elizabeth; Johnson, Mary Ann (American Psychiatric Association Publishing, 2002-01)
      Objective: The substantial discrepancy in the male-to-female ratio between clinic-referred (10 to 1) and community (3 to 1) samples of children with attention deficit hyperactivity disorder (ADHD) suggests that gender differences may be operant in the phenotypic expression of ADHD. In this study the authors systematically examined the impact of gender on the clinical features of ADHD in a group of children referred to a clinic. Method: The study included 140 boys and 140 girls with ADHD and 120 boys and 122 girls without ADHD as comparison subjects. All subjects were systematically assessed with structured diagnostic interviews and neuropsychological batteries for subtypes of ADHD as well as emotional, school, intellectual, interpersonal, and family functioning. Results: Girls with ADHD were more likely than boys to have the predominantly inattentive type of ADHD, less likely to have a learning disability, and less likely to manifest problems in school or in their spare time. In addition, girls with ADHD were at less risk for comorbid major depression, conduct disorder, and oppositional defiant disorder than boys with ADHD. A statistically significant gender-by-ADHD interaction was identified for comorbid substance use disorders as well. Conclusions: The lower likelihood for girls to manifest psychiatric, cognitive, and functional impairment than boys could result in gender-based referral bias unfavorable to girls with ADHD
    • The influence of genes on “positive valence systems” constructs: A systematic review

      Hess, Jonathan L.; Kawaguchi, Daniel M.; Wagner, Kayla E.; Faraone, Stephen V.; Glatt, Stephen J. (Wiley, 2015-09-14)
      The Research Domain Criteria (RDoC) address three types of aggression: frustrative non-reward, defensive aggression and offensive/proactive aggression. This review sought to present the evidence for genetic underpinnings of aggression and to determine to what degree prior studies have examined phenotypes that fit into the RDoC framework. Although the constructs of defensive and offensive aggression have been widely used in the animal genetics literature, the human literature is mostly agnostic with regard to all the RDoC constructs. We know from twin studies that about half the variance in behavior may be explained by genetic risk factors. This is true for both dimensional, trait-like, measures of aggression and categorical definitions of psychopathology. The non-shared environment seems to have a moderate influence with the effects of shared environment being unclear. Human molecular genetic studies of aggression are in an early stage. The most promising candidates are in the dopaminergic and serotonergic systems along with hormonal regulators. Genome-wide association studies have not yet achieved genome-wide significance, but current samples are too small to detect variants having the small effects one would expect for a complex disorder. The strongest molecular evidence for a genetic basis for aggression comes from animal models comparing aggressive and non-aggressive strains or documenting the effects of gene knockouts. Although we have learned much from these prior studies, future studies should improve the measurement of aggression by using a systematic method of measurement such as that proposed by the RDoC initiative. © 2015 Wiley Periodicals, Inc.