• Preclinical Development of Anti-Cancer Drugs from Natural Products.

      Huang, Ying; Sun, Qing (2014)
      Cancer has been and will continue to be the common concern in the United States and worldwide. As a conventional treatment to fight cancer, new anti-cancer drugs with more efficiency and less toxicity are extremely required. In this study, we have identified two novel compounds with anti-cancer properties from two traditional Chinese medicinal plants. One is Lappaol F that was extracted from the seeds of the plant Actium Lapp L., which has been used in China for centuries as anti-viral and anti-bacterial medicine. Another is M-9 that was extracted from the stem of Marsdenia tenacissima,a plant that has been applied to treat inflammation and cancer in China. Our results showed that Lappaol F inhibited cancer cell growth by regulating a series of cell cycle related proteins and inducing cell cycle arrest at G1 and/or G2 phase. p21 played a critical role in Lappaol F-induced cyclin B1 and cyclin-dependent kinase 1 (CDK1) suppression as well as G2arrest. Lappaol F also induced cell death in a number of cancer cells through caspases activation. Lappaol F-mediated cell growth inhibition was p53-independent. Notably, results from animal studies showed that Lappaol F effectively inhibited tumor growth in vivo, while being well tolerated by the mice. Thus, Lappaol F has a strong potential to be developed as a novel anti-cancer chemotherapeutic. Our studies showed that M-9 successfully sensitized several tumor cells but not non-tumorigenic cells to paclitaxel (Taxol) treatment. Additionally, M-9 reversed chemotherapeutic resistance in a number of multidrug resistant cells. Further results suggested that M-9 functioned, at least to a certain extent, via inhibiting drug efflux by competitively binding to P-glycoprotein (P-gp), a protein that accounts for multidrug resistance. Importantly, results from the in vivostudies demonstrated that M-9 strongly enhanced Taxol-induced growth suppression against xenografts derived from HeLa cells. Moreover, mice tolerated the treatment of Taxol and M-9 well. Therefore, M-9 is a novel chemosensitizer candidate to overcome P-gp-mediated multidrug resistance. Taken together, our studies provide a solid basis for further development of these two compounds as anti-cancer remedies.
    • Psychoactive substance use disorders in adults with attention deficit hyperactivity disorder (ADHD): effects of ADHD and psychiatric comorbidity

      Biederman, J; Wilens, T; Mick, E; Milberger, S; Spencer, T J; Faraone, Stephen V. (American Psychiatric Association Publishing, 1995-11)
      Objective: The authors evaluated the association between attention deficit hyperactivity disorder (ADHD) and psychoactive substance use disorders in adults with ADHD, attending to comorbidity with mood, anxiety, and antisocial disorders. It was hypothesized that psychiatric comorbidity would be a risk factor for psychoactive substance use disorders. Method: Findings for 120 referred adults with a clinical diagnosis ofchildhood-onset ADHD were compared with those for non-ADHD adult comparison subjects (N=268). All childhood and adult diagnoses were obtained by structured psychiatric interviews for DSM-III-R. Rı ıiiIt.ıi There was a significantly higher lifetime risk for psychoactive substance use disorders in the ADHD adults than in the comparison subjects (52% versus 27%). Although the two groups did not differ in the rate ofalcohol use disorders, the ADHD adults had significantly higher rates ofdrug and drug plus alcohol use disorders than the comparison subjects. ADHD significantly increased the risk for substance use disorders independently ofpsychiatric comorbidity. Antisocial disorders significantly increased the risk for substance use disorders independently ofADHD status. Mood and anxiety disorders increased the risk for substance use disorders in both the ADHD and comparison subjects, but more demonstrably in the comparison subjects. Conclusions: Although psychiatric comorbidity increased the risk for psychoactive substance use disorders in adults with ADHD, by itself ADHD was a significant risk factor for substance use disorders. More information is needed to further delineate risk and protective factors mediating the development of substance use disorders in persons with ADHD.
    • 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.
    • 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.
    • 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.
    • 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.
    • 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.
    • Report from the second international meeting of the attention deficit hyperactivity disorder Molecular Genetics Network

      Faraone, Stephen V. (Wiley, 2001)
      Given evidence from twin, family, and adoption studies of genetic influence on attention deficit hyperactivity disorder (ADHD), a growing number of researchers have initiated molecular genetics studies to explore the influence of specific genes on this condition. In 1999, these investigators convened to discuss ways of sharing information and facilitating collaborations across research sites. Enthusiastic response to this first conference prompted an even larger group of investigators to come together this year. This recent meeting, held in London, began with a presentation of Hypescheme, an operational criteria checklist developed in an effort to promote the reliable communication of diagnostic and other relevant clinical information related to ADHD. The benefits and limitations of Hypescheme, as well as the continued challenges to collaboration, were discussed. A new ADHD-specific rating scale, developed to be of use in genetic analyses, was also presented. Focus then turned to collaborative projects proposed by investigators and practical suggestions regarding joint data analyses projects. Finally, new data from individual sites were presented. Because the mode of inheritance of ADHD is likely to be complex, efforts to collaborate and cross-validate findings remain an important priority for researchers studying the molecular genetics of this disorder. © 2001 Wiley-Liss, Inc.
    • Revisiting the factor structure for positive and negative symptoms: evidence from a large heterogeneous group of psychiatric patients

      Toomey, R; Kremen, W S; simpson, J C; Samson, J A; Seidman, L J; Lynons, M J; Faraone, Stephen V.; Tsuang, M T (American Psychiatric Association Publishing, 1997-03)
      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
    • Risk variants and polygenic architecture of disruptive behavior disorders in the context of attention-deficit/hyperactivity disorder

      Demontis, Ditte; Walters, Raymond K.; Rajagopal, Veera M.; Waldman, Irwin D.; Grove, Jakob; Als, Thomas D.; Dalsgaard, Søren; Ribasés, Marta; Bybjerg-Grauholm, Jonas; Bækvad-Hansen, Maria; et al. (Springer Science and Business Media LLC, 2021-01-25)
      Attention-Deficit/Hyperactivity Disorder (ADHD) is a childhood psychiatric disorder often comorbid with disruptive behavior disorders (DBDs). Here, we report a GWAS meta-analysis of ADHD comorbid with DBDs (ADHD + DBDs) including 3802 cases and 31,305 controls. We identify three genome-wide significant loci on chromosomes 1, 7, and 11. A meta-analysis including a Chinese cohort supports that the locus on chromosome 11 is a strong risk locus for ADHD + DBDs across European and Chinese ancestries (rs7118422, P = 3.15×10−10, OR= 1.17). We find a higher SNP heritability for ADHD + DBDs (h2 SNP = 0.34) when compared to ADHD without DBDs (h2 SNP = 0.20), high genetic correlations between ADHD + DBDs and aggressive (rg = 0.81) and anti-social behaviors (rg = 0.82), and an increased burden (polygenic score) of variants associated with ADHD and aggression in ADHD + DBDs compared to ADHD without DBDs. Our results suggest an increased load of common risk variants in ADHD + DBDs compared to ADHD without DBDs, which in part can be explained by variants associated with aggressive behavior.
    • Risk variants and polygenic architecture of disruptive behavior disorders in the context of attention-deficit/hyperactivity disorder

      Demontis, Ditte; Walters, Raymond K.; Rajagopal, Veera M.; Waldman, Irwin D.; Grove, Jakob; Als, Thomas D.; Dalsgaard, Søren; Ribasés, Marta; Bybjerg-Grauholm, Jonas; Bækvad-Hansen, Maria; et al. (Springer Science and Business Media LLC, 2021-01-25)
      Attention-Deficit/Hyperactivity Disorder (ADHD) is a childhood psychiatric disorder often comorbid with disruptive behavior disorders (DBDs). Here, we report a GWAS meta-analysis of ADHD comorbid with DBDs (ADHD + DBDs) including 3802 cases and 31,305 controls. We identify three genome-wide significant loci on chromosomes 1, 7, and 11. A meta-analysis including a Chinese cohort supports that the locus on chromosome 11 is a strong risk locus for ADHD + DBDs across European and Chinese ancestries (rs7118422, P = 3.15×10-10, OR = 1.17). We find a higher SNP heritability for ADHD + DBDs (h2SNP = 0.34) when compared to ADHD without DBDs (h2SNP = 0.20), high genetic correlations between ADHD + DBDs and aggressive (rg = 0.81) and anti-social behaviors (rg = 0.82), and an increased burden (polygenic score) of variants associated with ADHD and aggression in ADHD + DBDs compared to ADHD without DBDs. Our results suggest an increased load of common risk variants in ADHD + DBDs compared to ADHD without DBDs, which in part can be explained by variants associated with aggressive behavior.
    • ROLE OF BMP AND ACTIVIN/NODALSIGNALING REPRESSION IN RETINALPROGENITOR CELL SPECIFICATION ANDCONE PHOTORECEPTOR GENESIS

      Viczian, Andrea; Wong, Kimberly (2017)
      Eye formation begins with the specification of the eye field from the anteriorneural plate during the transition from gastrulation to neuralization. The morphogeneticgradients formed by BMP and Activin/Nodal ligands organize the body axis andregulate intracellular signals to bias the cell lineages.Therefore, to understand theextracellular signaling mechanisms governing eye field specification, we looked closerat the mechanisms responsible for neuralization. This project takes advantage of thedevelopmental accessibility of theXenopus laevisembryo to investigate the role ofBMP and Activin/Nodal signaling pathways during early eye development.Nogginis a secreted BMP antagonist and is sufficient to induce formation of ectopic retinaltissue. My results presented in Chapter 2 show that intracellular repression of BMP-Smad1/5/8 and Activin/Nodal-Smad2/3 signaling was sufficient to replicate the retina-promoting activity of Noggin and efficiently drive pluripotentXenopusprimitive ectodermto a retinal progenitor cell fate. Furthermore, my work presented in Chapter 3 suggeststhat repression of BMP and Activin/Nodal in the anterior neural plate allows the anteriorneural patterning transcription factor, Otx2, to directly activate expression of the earliesteye field transcription factor,tbx3. This study elucidates the initial step by which theanterior neural plate is patterned to specify the eye field. We next translated our findings to mouse embryonic stem cell culture (Chapter 4). We observed that mouse embryonic stemcells can be efficiently directed towards retinal progenitor cells by first converting themto an early primitive ectoderm-like state, followed by simultaneous repression of BMPand Activin/Nodal and activation of FGF signaling. After differentiation, we observedthat photoreceptor genesis is synchronized, resulting in 96% of aggregates expressingmarkers for cone photoreceptors after 9 days. This differentiation protocol is more efficientand quicker than previously published mouse stem cell protocols. Optimizing treatmentconditions resulted in up to 62% of cells expressing the cone/rod photoreceptor marker,CRX. This study was the first to show that photoreceptor-like cells can be generated frommouse pluripotent stem cells as efficiently as published human pluripotent stem cell reports,but with a shorter culture period. These studies will provide the basis of future work todetermine the underlying molecular mechanism driving retinal differentiation.