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Pcr-Ddge Comparison Of Fecal Bacteria From Captive And Wild Populations Of The Endangered San Esteban Island Chuckwalla (Sauromalus Varius), Tina Kelley

Masters Theses & Specialist Projects

Most modern reptiles are carnivorous or insectivorous, whereas less than 2% of the >7,800 currently recognized species are considered to be herbivorous. Of these, herbivory is confined entirely to lizards; less than 3% of known lizard species eat significant amounts of plants. Most, if not all, herbivorous animals utilize a population of symbiotic microorganisms which can break down plant structural polymers. This study analyzes the bacterial populations from fecal/uric acid deposits of wild and captive samples of the endangered, herbivorous San Esteban chuckwalla (Sauromalus varius: Iguanidae). Substrate and diet samples were also analyzed. Most studies of digestive symbionts use indirect …

Quality Assurance Of Radiation Therapy Planning Systems: Current Status And Remaining Challenges., Jacob Van Dyk

Oncology Publications

Computerized radiation therapy planning systems (RTPSs) are pivotal for treatment planning. The acceptance, commissioning, and quality control of RTPSs are uniquely complex and are described in the American Association of Physicists in Medicine Task Group Report 53 (1998) and International Atomic Energy Agency Technical Report Series No. 430 (2004). The International Atomic Energy Agency also developed a document and data package for use by vendors and purchasers to aid with acceptance testing of RTPSs. This document is based on International Electrotechnical Commission standard 62083 (2000) and describes both "type" tests to be performed in the factory and "site" tests to …

Preparation Of Halogenated Derivatives Of Thiazolo[5,4-D]Thiazole Via Direct Electrophilic Aromatic Substitution, Vladimir Benin, Alan T. Yeates, Douglas Dudis

Chemistry Faculty Publications

Chlorination and bromination reactions of thiazolo[5,4-d]thiazole led to the generation of its mono- and dihalogenated derivatives. These are the first instances of successful direct electrophilic aromatic substitution in the thiazolo[5,4-d]thiazole ring system. X-ray analysis demonstrates that both 2-bromothiazolo[5,4-d]-thiazole and 2,5-dibromothiazolo[5,4-d]thiazole are planar structures, with strongly manifested π-stacking in the solid state. Theoretical analysis of the pyridine-catalyzed halogenation (MP2/6-31+G(d) and B3LYP/6-31+G(d)calculations) reveals that introduction of one halogen actually leads to a slightly enhanced reactivity towards further halogenation. Several halogenation mechanisms have been investigated: 1) The direct C-halogenation with N-halopyridine as electrophile; …

Use Of Ultraviolet Light Irradiated Multiple Myeloma Cells As Immunogens To Generate Tumor Specific Cytolytic T Lymphocytes, Charles A. Gullo Phd, William Y.K. Hwang, Chye K. Poh, Melvin Au, Geraline Cow, Gerrard Teoh

Biochemistry and Microbiology

Background:

As the eradication of tumor cells in vivo is most efficiently performed by cytolytic Tlymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described.

Methods:

Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity.

Results: …

A Luteinizing Hormone Receptor Intronic Variant Is Significantly Associated With Decreased Risk Of Alzheimer's Disease In Males Carrying An Apolipoprotein E Epsilon 4 Allele, Ryan J. Haasl, M Reza Ahmadi, Sivan Vadakkadath Meethal, Carey E. Gleason, Sterling C. Johnson, Sanjay Asthana, Richard L. Bowen, Craig S. Atwood

Research outputs pre 2011

Genetic and biochemical studies support the apolipoprotein E (APOE) ε4 allele as a major risk factor for late-onset Alzheimer's disease (AD), though ~50% of AD patients do not carry the allele. APOE transports cholesterol for luteinizing hormone (LH)-regulated steroidogenesis, and both LH and neurosteroids have been implicated in the etiology of AD. Since polymorphisms of LH beta-subunit (LHB) and its receptor (LHCGR) have not been tested for their association with AD, we scored AD and age-matched control samples for APOE genotype and 14 polymorphisms of LHB and LHCGR. Thirteen gene-gene interactions between the …

A Central Function For Perlecan In Skeletal Muscle And Cardiovascular Development, Jason J. Zoeller, Angela Mcquillan, John Whitelock, Shiu-Ying Ho, Renato V. Iozzo

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Perlecan's developmental functions are difficult to dissect in placental animals because perlecan disruption is embryonic lethal. In contrast to mammals, cardiovascular function is not essential for early zebrafish development because the embryos obtain adequate oxygen by diffusion. In this study, we use targeted protein depletion coupled with protein-based rescue experiments to investigate the involvement of perlecan and its C-terminal domain V/endorepellin in zebrafish development. The perlecan morphants show a severe myopathy characterized by abnormal actin filament orientation and disorganized sarcomeres, suggesting an involvement of perlecan in myopathies. In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail …

Drug Resistance In Malaria: A Peruvian Research Experience, Ryan Haley

Inquiry Journal 2008

No abstract provided.

Using Confirmatory Factor Analysis To Understand Executive Control In Preschool Children: I. Latent Structure, Sandra A. Wiebe, Kimberly A. Espy, David Charak

Developmental Cognitive Neuroscience Laboratory: Faculty Publications

*This article may not exactly replicate the final version published in the APA journal. It is not the copy of record. http://content.apa.org/journals/0012-1649 Although many tasks have been developed recently to study executive control in the preschool years, the constructs that underlie performance on these tasks are poorly understood. In particular, it is unclear whether executive control is comprised of multiple, separable cognitive abilities (e.g., inhibition and working memory) or whether it is unitary in nature. A sample of 243 normally developing children between 2.25 and 6 years of age completed a battery of ageappropriate executive control tasks. Confirmatory factor analysis …

Hpr6 Mutants And Uses Thereof, Rolf Joseph Craven

Pharmacology and Nutritional Sciences Faculty Patents

Methods and agents that interfere with Hpr6 function in non-sarcoma tumor cells are disclosed. Anti-Hpr6 agents are used to enhance the killing effect of anti-cancer agents in non-sarcoma tumor cells and to teat non-sarcoma tumors.

Renin Inhibition Reduces Hypercholesterolemia-Induced Atherosclerosis In Mice, Hong Lu, Debra L. Rateri, David L. Feldman, Richard Charnigo, Akiyoshi Fukamizu, Junji Ishida, Elizabeth Grace Oesterling, Lisa A. Cassis, Alan Daugherty

Saha Cardiovascular Research Center Faculty Publications

The role of the renin angiotensin system (RAS) in atherosclerosis is complex because of the involvement of multiple peptides and receptors. Renin is the rate-limiting enzyme in the production of all angiotensin peptides. To determine the effects of renin inhibition on atherosclerosis, we administered the novel renin inhibitor aliskiren over a broad dose range to fat-fed LDL receptor-deficient (Ldlr^-/-) mice. Renin inhibition resulted in striking reductions of atherosclerotic lesion size in both the aortic arch and the root. Subsequent studies demonstrated that cultured macrophages expressed all components of the RAS. To determine the role of macrophage-derived angiotensin in …

Clinical And Microbiological Aspects Of Linezolid Resistance Mediated By The Cfr Gene Encoding A 23s Rrna Methyltransferase, Cesar A Arias, Martha Vallejo, Jinnethe Reyes, Diana Panesso, Jaime Moreno, Elizabeth Castañeda, Maria V Villegas, Barbara E Murray, John P Quinn

Faculty, Staff and Student Publications

The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), …

Insights Into Morphological Nature Of Precipitation Of Cholesterol, Vuk Uskoković

Pharmacy Faculty Articles and Research

Additional effects on the previously reported procedure of precipitation of narrowly dispersed and well-defined, brick-shaped cholesterol particles, including non-solvent addition rate, temperature, solvent purity, aging treatments, ultrasound agitation and fine mechanical effects were investigated. Based on the presented results, significant morphological sensitivity of cholesterol precipitation processes upon variations from the standard established procedure of crystallization is induced. However, the tendency of cholesterol to crystallize in the form of biaxially grown particles was evidenced as dominating the precipitation processes, irrespective of any modifications of experimental parameters involved in the preparation procedure investigated hereby. Prolonged aging time and temperature effects lead to …

Functional Interactions Of Alcohol-Sensitive Sites In The N-Methyl-D-Aspartate Receptor M3 And M4 Domains, Hong Ren, Abdelghaffar K. Salous, Jaclyn M. Paul, Kaitlin A. Lamb, Donard S. Dwyer, Robert W. Peoples

Biomedical Sciences Faculty Research and Publications

The N-methyl-d-aspartate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Previous studies have demonstrated sites in the third and fourth membrane-associated (M) domains of the N-methyl-d-aspartate receptor NR2A subunit that influence alcohol sensitivity and ion channel gating. We investigated whether two of these sites, Phe-637 in M3 and Met-823 in M4, interactively regulate the ethanol sensitivity of the receptor by testing dual substitution mutants at these positions. A majority of the mutations decreased steady-state glutamate EC₅₀ values and maximal steady-state to peak current ratios (I_ss/I …

Gating Charge Immobilization In Kv4.2 Channels: The Basis Of Closed-State Inactivation., Kevin Dougherty, Jose A De Santiago-Castillo, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Kv4 channels mediate the somatodendritic A-type K+ current (I(SA)) in neurons. The availability of functional Kv4 channels is dynamically regulated by the membrane potential such that subthreshold depolarizations render Kv4 channels unavailable. The underlying process involves inactivation from closed states along the main activation pathway. Although classical inactivation mechanisms such as N- and P/C-type inactivation have been excluded, a clear understanding of closed-state inactivation in Kv4 channels has remained elusive. This is in part due to the lack of crucial information about the interactions between gating charge (Q) movement, activation, and inactivation. To overcome this limitation, we engineered a charybdotoxin …

Method For Treating A Viral Infection Related Or A Chemical Toxin Related Hepatic Injury With Deltorphin D, Peter R. Oeltgen, Paul D. Bishop, Craig J. Mcclain, Shirish Barve

Pathology and Laboratory Medicine Faculty Patents

A method of modulating cytokine mediated hepatic injury by administering compound-D SEQ ID NO:1 to a mammal. A concentration of the compound in the range of about 0.5 mg/kg to about 20 mg/kg in a physiologically acceptable formulation blocks a cytokine cascade. A therapeutic method of modulating cytokine mediated acute inflammatory, trauma induced and toxin induced hepatic injury, particularly via tumor necrosis factor modulation, is thus disclosed.

Retinoic Acid Decreases Atf-2 Phosphorylation And Sensitizes Melanoma Cells To Taxol-Mediated Growth Inhibition, Ying Huang, Jennifer Minigh, Sarah Miles, Richard N. Niles

Biochemistry and Microbiology

Cutaneous melanoma is often resistant to chemo- and radiotherapy. This resistance has recently been demonstrated to be due, at least in part, to high activating transcription factor 2 (ATF-2) activity in these tumors. In concordance with these reports, we found that B16 mouse melanoma cells had higher levels of ATF-2 than immortalized, but non-malignant mouse melanocytes. In addition, the melanoma cells had a much higher amount of phosphorylated (active) ATF-2 than the immortalized melanocytes. In the course of determining how retinoic acid (RA) stimulates activating protein-1 (AP-1) activity in B16 melanoma, we discovered that this retinoid decreased the phosphorylation of …

Novel Role Of Antioxidant-1 (Atox1) As A Copper-Dependent Transcription Factor Involved In Cell Proliferation, S. Itoh, H. W. Kim, O. Nakagawa, K. Ozumi, Susan M. Lessner, H. Aoki, K. Akram, R. D. Mckinney, M. Ushio-Fukai, T. Fukai

Faculty Publications

Copper plays a fundamental role in regulating cell growth. Many types of human cancer tissues have higher copper levels than normal tissues. Copper can also induce gene expression. However, transcription factors that mediate copper-induced cell proliferation have not been identified in mammals. Here we show that antioxidant-1 (Atox1), previously appreciated as a copper chaperone, represents a novel copper-dependent transcription factor that mediates copper-induced cell proliferation. Stimulation of mouse embryonic fibroblasts (MEFs) with copper markedly increased cell proliferation, cyclin D1 expression, and entry into S phase, which were completely abolished in Atox1^-/- MEFs. Promoter analysis and EMSA revealed that copper …

Diabetes Alters Contraction-Induced Mitogen Activated Protein Kinase Activation In The Rat Soleus And Plantaris, Anjaiah Katta, Deborah L. Preston, Sunil K. Kakarla, Shinichi Asano, Sarath Meduru, Sriram P. Mupparaju, Ellie Yokochi, Kevin M. Rice, Devashish H. Desai, Eric R. Blough

MIIR Faculty Research

The prescription of anaerobic exercise has recently been advocated for the management of diabetes; however exercise-induced signaling in diabetic muscle remains largely unexplored. Evidence from exercise studies in nondiabetics suggests that the extracellular-signal-regulated kinases (Erk1/2), p38, and c-JUN NH2-terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs) are important regulators of muscle adaptation. Here, we compare the basal and the in situ contraction-induced phosphorylation of Erk1/2- p38- and Jnk-MAPK and their downstream targets (p90rsk and MAPKAP-K2) in the plantaris and soleus muscles of normal and obese (fa/fa) Zucker rats. Compared to lean animals, the time course and magnitude of Erk1/2, p90rsk and …

The Mind-Body Connection: The Association Between Adolescent Locus Of Control And Indicators Of Physical Health, C. Jayne Brahler, James Cropper

Physical Therapy Faculty Publications

Locus of control (LOC) describes an individual’s generalized beliefs or expectancies that their reinforcements are under internal versus external control (1). An individual exhibits either an internal or external LOC. This study examines the link between LOC and selected health risk factors in adolescents. A convenience sample of 167 high school physical education students completed a 13-item LOC questionnaire based on Rotter’s 1966 instrument.

Various anthropometric measurements, blood pressure, body mass index (BMI), and body fat were recorded on all subjects. A subsample of 61 female students received blood chemistry analysis that included a lipid profile, hemoglobin A1c (HbA1c), Apo …

N-Acetyl-B-D-Glucosaminidase And Inflammatory Response After Cardiopulmonary Bypass, Perwaiz Iqbal, Hasanat M Sharif, Naseema Mehboobali, Farzana A Yousuf, Abrar H Khan, Frank W Sellke

Department of Biological & Biomedical Sciences

OBJECTIVE: To determine the changes in activity of plasma N-acetyl-beta-D-glucosaminidase, a marker for inflammation as well as renal, pulmonary and cardiac damage and proinflammatory cytokines in patients undergoing coronary artery bypass grafting and find out the relationship between their plasma levels with clinical outcome of patients.

STUDY DESIGN: Cross-sectional study.

PLACE AND DURATION OF STUDY: The Aga Khan University, Karachi, from January to June 2003.

PATIENTS AND METHODS: N-acetyl-beta-D-glucosaminidase (NAG) activity and concentrations of tumor necrosis factor-alpha of (TNFalpha), interleukin 6 (IL-6), interleukin 8 (IL8) and granulocyte-macrophage colony stimulating factor (GM-CSF) were monitored in plasma samples of 12 angina patients …

Composites Comprising Cholesterol And Carboxymethyl Cellulose, Vuk Uskoković

Pharmacy Faculty Articles and Research

Whereby cholesterol presents one of the major fatty substances in human body, carboxymethyl cellulose is a water-soluble derivative of cellulose, the most abundant dietary fiber. Whereas on one hand in vivo precipitation of cholesterol is the major cause of atherosclerosis, dietary fibers are on the other hand known for their ability to clean the fatty plaque deposited on intestinal pathways, and prevent its build-up in other critical areas within the organism. In this work, a method for the preparation of a composite material comprising cholesterol and carboxymethyl cellulose from 1-hexanol/water biphase mixtures is reported. Specificity of the interaction between the …

C–N Bond Rotation And E–Z Isomerism In Some N-Benzyl-N-Methylcarbamoyl Chlorides: A Dft Study, Michael Horwath, Vladimir Benin

Chemistry Faculty Publications

The current report presents the first theoretical study of the restricted CN bond rotation in carbamoyl chlorides. Several N-benzyl-N-methylcarbamoyl chlorides were investigated, with varying pattern of substitution in the aromatic ring. Optimizations and frequency calculations were conducted employing DFT at the B3LYP/6-31+G(d) level of theory. Each of the studied structures exhibits a pair of rotamers (s-Z and s-E), generated upon rotation around the C(O)N bond. The s-E isomer is the global minimum in every case, but the preference for it is usually less than 1 kcal/mol. Two possible transition state structures were identified …

Dissociation Predicts Later Attention Problems In Sexually Abused Children, Julie B Kaplow, Erin Hall, Karestan C Koenen, Kenneth A Dodge, Lisa Amaya-Jackson

Faculty, Staff and Students Publications

OBJECTIVE: The goals of this research are to develop and test a prospective model of attention problems in sexually abused children that includes fixed variables (e.g., gender), trauma, and disclosure-related pathways.

METHODS: At Time 1, fixed variables, trauma variables, and stress reactions upon disclosure were assessed in 156 children aged 8-13 years. At the Time 2 follow-up (8-36 months following the initial interview), 56 of the children were assessed for attention problems.

RESULTS: A path analysis involving a series of hierarchically nested, ordinary least squares multiple regression analyses indicated two direct paths to attention problems including the child's relationship to …

Medulloblastomas Overexpress The P53-Inactivating Oncogene Wip1/Ppm1d, Robert C. Castellino, Massimiliano De Bortoli, Xiongbin Lu, Sung-Hwan Moon, Thuy-Ai Nguyen, Mark A. Shepard, Pulivarthi H. Rao, Lawrence A. Donehower, John Y H Kim

Faculty, Staff and Students Publications

Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-type p53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens …

Trk: A Neuromodulator Of Age-Specific Behavioral And Neurochemical Responses To Cocaine In Mice., Michelle Niculescu, Shane A Perrine, Jonathan S Miller, Michelle E Ehrlich, Ellen M Unterwald

Farber Institute for Neuroscience Faculty Papers

Responses to psychostimulants vary with age, but the molecular etiologies of these differences are largely unknown. The goal of the present research was to identify age-specific behavioral and molecular adaptations to cocaine and to elucidate the mechanisms involved therein. Postweanling, periadolescent, and adult male CD-1 mice were exposed to cocaine (20 mg/kg) for 7 d. The rewarding effects of cocaine were assessed, as were the response to a Trk antagonist and the regulation of dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32). Cocaine was rewarding in both periadolescent and adult mice using a conditioned place preference procedure. In contrast, postweanling mice …

The Expression Of Microrna Mir-107 Decreases Early In Alzheimer's Disease And May Accelerate Disease Progression Through Regulation Of Β-Site Amyloid Precursor Protein-Cleaving Enzyme 1, Wang-Xia Wang, Bernard W. Rajeev, Arnold J. Stromberg, Na Ren, Guiliang Tang, Qingwei Huang, Isidore Rigoutsos, Peter T. Nelson

Sanders-Brown Center on Aging Faculty Publications

MicroRNAs (miRNAs) are small regulatory RNAs that participate in posttranscriptional gene regulation in a sequence-specific manner. However, little is understood about the role(s) of miRNAs in Alzheimer's disease (AD). We used miRNA expression microarrays on RNA extracted from human brain tissue from the University of Kentucky Alzheimer's Disease Center Brain Bank with near-optimal clinicopathological correlation. Cases were separated into four groups: elderly nondemented with negligible AD-type pathology, nondemented with incipient AD pathology, mild cognitive impairment (MCI) with moderate AD pathology, and AD. Among the AD-related miRNA expression changes, miR-107 was exceptional because miR-107 levels decreased significantly even in patients with …

Diverse Antidepressants Increase Cdp-Diacylglycerol Production And Phosphatidylinositide Resynthesis In Depression-Relevant Regions Of The Rat Brain, Kimberly R. Tyeryar, Habiba O.U. Vongtau, Ashiwel S. Undieh

College of Pharmacy Faculty Papers

BACKGROUND: Major depression is a serious mood disorder affecting millions of adults and children worldwide. While the etiopathology of depression remains obscure, antidepressant medications increase synaptic levels of monoamine neurotransmitters in brain regions associated with the disease. Monoamine transmitters activate multiple signaling cascades some of which have been investigated as potential mediators of depression or antidepressant drug action. However, the diacylglycerol arm of phosphoinositide signaling cascades has not been systematically investigated, even though downstream targets of this cascade have been implicated in depression. With the ultimate goal of uncovering the primary postsynaptic actions that may initiate cellular antidepressive signaling, we …

Genome-Wide Expression Profiling Reveals Transcriptomic Variation And Perturbed Gene Networks In Androgen-Dependent And Androgen-Independent Prostate Cancer Cells., Ajay P. Singh, Sangeeta Bafna, Kunal Chaudhary, Ganesh Venkatraman, Lynette Smith, James D. Eudy, Sonny L. Johansson, Ming-Fong Lin, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

Previously, we have developed a unique in vitro LNCaP cell model, which includes androgen-dependent (LNCaP-C33), androgen-independent (LNCaP-C81) and an intermediate phenotype (LNCaP-C51) cell lines resembling the stages of prostate cancer progression to hormone independence. This model is advantageous in overcoming the heterogeneity associated with the prostate cancer up to a certain extent. We characterized and compared the gene expression profiles in LNCaP-C33 (androgen-dependent) and LNCaP-C81 (androgen-independent) cells using Affymetrix GeneChip array analyses. Multiple genes were identified exhibiting differential expression during androgen-independent progression. Among the important genes upregulated in androgen-independent cells were PCDH7, TPTE, TSPY, EPHA3, HGF, MET, EGF, TEM8, etc., …

Long Lasting Synchronization Of Calcium Oscillations By Cholinergic Stimulation In Isolated Pancreatic Islets, Min Zhang, Bernard Fendler, Bradford Peercy, Pranay Goel, Richard Bertram, Arthur Sherman, Leslie Satin

Pharmacology and Toxicology Publications

Abstract

Individual mouse pancreatic islets exhibit oscillations in [Ca²⁺]_i and insulin secretion in response to glucose in vitro, but how the oscillations of a million islets are coordinated within the human pancreas in vivo is unclear. Islet to islet synchronization is necessary, however, for the pancreas to produce regular pulses of insulin. To determine whether neurohormone release within the pancreas might play a role in coordinating islet activity, [Ca²⁺]_i changes in 4–6 isolated mouse islets were simultaneously monitored before and after a transient pulse of a putative synchronizing agent. The degree of synchronicity was …

Fusion, 2008, George Washington University, William H. Beaumont Medical Research Honor Society

Fusion

No abstract provided.