Doctoral thesis assessing the effects of one-week of escitalopram administration on healthy humans during sequence motor learning training. Published in 3 research articles.

<p>The brain serotonergic and dopaminergic systems broadly influence our internal experience and the ways in which we interact with the outside environment, with crucial regulatory roles in mood, sleep, appetite and the control of voluntary movement. Serotonin and dopamine neurons are themselves influenced by a wide variety of internal and external factors, many of which remain poorly understood. The central aim of this thesis was to better characterize several of these modulatory influences via exploratory investigations involving pharmaceutical agents or environmental modification. Specifically, I examined the modulatory effects of selective serotonin reuptake inhibitors (SSRIs), atypical neuroleptics and environmental enrichment with exercise on the regulation of brain serotonin and dopamine neurons.</p> <p>This thesis documents, for the first time, that (1) inhibition of the serotonin transporter (SERT) by SSRIs induces a rapid and region-selective reduction of tryptophan hydroxylase (TPH)-immunoreactive neurons in serotonergic brainstem nuclei that persists over a prolonged treatment course; that (2) selective blockade of SERT by SSRIs can rapidly induce a reduction of tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra (SN) and the ventral tegmental area (VTA) that, again, persists over a lengthy treatment course; that (3) environmental enrichment with exercise can potentiate the effect of SERT inhibition on SN dopaminergic neurons, but not the dorsal raphe nucleus (DRN) serotonergic neurons; that (4) that SSRI fluoxetine triggers a significant upregulation of microglia in the SN; that (5) environmental enrichment with exercise can reduce TPH immunoreactivity in the DRN and TH immunoreactivity in the SN and VTA, even in the absence of any pharmacological intervention, and finally, that (6) the atypical neuroleptic risperidone significantly reduces TPH in the DRN of both young and aged animals and reduces DRN Nissl counts in aged animals. Taken together, the body of work included in this thesis suggests that SSRIs, atypical neuroleptics and environmental enrichment with exercise can have profound effects on brain serotonergic and dopaminergic neurons, possibly accounting for some of the side effects and therapeutic benefits associated with these interventions.</p> / Doctor of Philosophy (PhD)

Osteoporosis and depression are prevalent among older postmenopausal women 65 years or older. Bisphosphonates (BPs) and selective serotonin reuptake inhibitors (SSRIs) or serotonin norepinephrine reuptake inhibitors (SNRIs) are commonly used medications to treat these conditions. Inhibitory effects of BPs on osteoclasts are responsible for the reduction in fracture risk. SSRIs, however, are associated with increased fracture risk through decreasing osteoblasts and increasing osteoclastic activity. These effects of SSRIs could attenuate the beneficial effects of BPs. This dissertation describes the concomitant use of BPs and SSRIs among postmeopausa women and reports findings from examining the association between concomitant use of BPs and SSRIs and fracture risk. Separate cross-sectional analyses were performed using data from the 2004-2008 Medical Expenditure Panel Survey (MEPS) and Medicare Part D prescriptions claims data (2008-2010) to examine usage patterns of BPs and SSRIs/SNRIs for women aged ≥45 years and ≥65 years, respectively. For our second objective, a nested-case control was conducted using Medicare claims data (2008-2010). Data from Medicare inpatient claims were linked to Medicare Part D data for all female BP users 65 years or older. We used Cox proportional hazards model to assess the increased risk of osteoporotic-related fractures among propensity score matched (1:1 ratio) cohorts of concomitant users of BPs and SSRIs and BP alone users. Concomitant use of BPs and SSRIs was prevalent and increased with age for each timeframe examined. Findings showed that approximately 12% (using MEPS) and 28% (using Medicare data) of women on BPs were also on SSRIs. For the second objective, 4,214 propensity score matched pairs (average age=80.4 years) of subjects were analyzed. Findings showed that concomitant use of BPs and SSRIs was associated with statistically significant increased risk for any fracture (HR=1.29, 95% CI, 1.07-1.57), but statistically non-significant increased risk for hip (HR=1.16, 95% CI, 0.92-1.47) and vertebral fractures (HR=1.55, 95% CI, 0.97-2.48). Current findings indicate that concomitant use of BPs and SSRIs is not uncommon among postmenopausal women and suggest potential attenuation of antifracture efficacy of BPs by SSRIs. Further studies are needed to understand the clinical impact of concomitant use of these medications among older postmenopausal women.

Depression and post-traumatic stress disorder are common psychiatric comorbidities related to stress. These conditions are frequently treated with antidepressants such as selective serotonin reuptake inhibitors (SSRI’s). However, there are individual differences in susceptibility to stress-induced psychopathologies and response to antidepressants. Therefore, there is a need to identify biologic factors that predict vulnerability to stress and response to treatment. Furthermore, few studies have examined the neural correlates of antidepressant treatment response in a stress-susceptible animal model. This dissertation had three specific aims: 1) to characterize behavioral predictors of stress vulnerability by studying three dimensions of temperament (reward dependence, novelty-specific activity and harm avoidance) before stress exposure using a stress-susceptible rat strain, 2) to identify the neural network effects of response and non-response to SSRI treatment using a stress-susceptible animal model, and 3) to determine the neurophysiologic correlates of helplessness susceptibility. This was examined via measurement of regional brain metabolic capacity and functional connectivity within relevant neural circuits, and measurements of corticosterone and heart rate. These effects were studied in rats that underwent inescapable shock exposure followed by escape testing. Holtzman rats showed greater predisposition to helpless behavior following inescapable shock compared to Sprague Dawley and Long-Evans strains. Also, increased activity in a novel environment and low heart rate appeared to be markers of helplessness susceptibility in Holtzman rats. Limbic-cortical network effects were identified that distinguished between responders and non-responders to antidepressant treatment in the Holtzman strain. Finally, hypermetabolism of the lateral habenula and a less interactive prefrontal-limbic cortex were identified in subjects with higher susceptibility towards helplessness within the Holtzman strain. Similar findings have been reported with other depression animal models and human neuroimaging studies. These findings support that the helpless dimension of mood disorders can be accurately modeled with the Holtzman rat strain and confirm that the lateral habenula and prefrontal cortex are key regions mediating the helpless phenotype and response to SSRI treatment. / text

Parkinson`s Disease is the second most common motor disorder and is also considered a progressive multisystemic disease associated to several nom motor symptoms (NMS), such as depression, with a prevalence of about 50% among PD patients. Selective serotonin reuptake inhibitors (SSIR) are the main treatment for this NMS, although researches with acutely induced parkinsonism has related fluoxetine to increased motor impairment. The aim of the present research is to evaluate the effect of the fluoxetine hydrochloride on a model of parkinsonism induced by low doses of reserpine. Sixty-four male 7-9-month-old Wistar rats were used, and were obtained from vivarium of the Department of Physiology – Federal University of Sergipe. Animals were divided into four groups: fluoxetine vehicle + reserpine vehicle (CTR); fluoxetine 10 mg/kg + reserpine vehicle (F); fluoxetine 10 mg/kg + reserpine 0,1 mg/kg (F + R); and fluoxetine vehicle + reserpine 0,1 mg/kg (R). During the treatment, the animals were submitted to open field test, catalepsy test and tremoulous jaw movement evaluation. It was shown that animals treated with fluoxetine and reserpine spent more time at the catalepsy test, decreased distance travelled, lower number of rearing at the open field test, increased tremulous jaw movements and increased weight loss. The treatment only with fluoxetine caused immunohistochemistry changes, such as decrease of TH expression in the dorsal striatum and increased staining of the dorsal raphe nucleus, with no correlation with MS for this group. The F + R group showed different immunohistochemistry results for both acute and continued administrations. / A Doença de Parkinson (DP) é a segunda desordem motora mais comum e também é considerada uma doença progressiva multissistêmica ligada a vários sintomas não motores (SNM), como a depressão, que acomete cerca de 50% dos pacientes. Os inibidores seletivos da recaptação de serotonina (ISRS) são considerados os principais medicamentos para o tratamento desse SNM, embora pesquisas que utilizaram indução aguda de parkinsonismo tenham relacionado a fluoxetina ao agravamento dos sintomas motores. Diante disso, este estudo objetivou avaliar o efeito da administração de cloridrato de fluoxetina em um modelo de parkinsonismo induzido por baixas doses de reserpina. Foram utilizados 64 ratos Wistar, machos, com idade de 7 a 9 meses, provenientes do Biotério Setorial do Departamento de Fisiologia da Universidade Federal de Sergipe. Os animais foram divididos aleatoriamente em quatro grupos: veículo fluoxetina + veículo reserpina (CTR); fluoxetina 10 mg/kg + veículo reserpina (F); fluoxetina 10 mg/kg + reserpina 0,1 mg/kg (F + R); e veículo fluoxetina + reserpina 0,1 mg/kg (R). Durante o tratamento, os animais foram submetidos aos testes de campo aberto, catalepsia e avaliação dos movimentos orofaciais. Foi observado aumento da latência na barra, diminuição da distância total percorrida em campo aberto, diminuição do número de rearing, aumento dos movimentos involuntários de mandíbula e maior alteração de peso corporal dos animais do grupo F + R. O tratamento apenas com fluoxetina provocou alterações imunohistoquímicas, como a diminuição da expressão de TH no estriado dorsal e aumento da marcação para 5-HT no núcleo dorsal da rafe, sem correlação com nenhum sintoma motor para esse grupo. O grupo F + R apresentou resultados de imunorreatividade distintos para as administrações breve e continuada. / São Cristóvão, SE

Previous findings in healthy humans suggest that selective serotonin reuptake inhibitors (SSRIs) modulate emotional processing via earlier changes in attention. However, many previous studies have provided inconsistent findings. One possible reason for such inconsistencies is that these studies did not control for the influence of either sex or sex hormone fluctuations. To address this inconsistency, we administered 20 mg escitalopram or placebo for seven consecutive days in a randomized, double-blind, placebocontrolled design to sixty healthy female participants with a minimum of 3 months oral contraceptive (OC) intake. Participants performed a modified version of an emotional flanker task before drug administration, after a single dose, after 1 week of SSRI intake, and after a 1-month wash-out period. Supported by Bayesian analyses, our results do not suggest a modulatory effect of escitalopram on behavioral measures of early attentional-emotional interaction in female individuals with regular OC use. While the specific conditions of our task may be a contributing factor, it is also possible that a practice effect in a healthy sample may mask the effects of escitalopram on the attentional-emotional interplay. Consequently, 1 week of escitalopram administration may not modulate attention toward negative emotional distractors outside the focus of attention in healthy female participants taking OCs. While further research in naturally cycling females and patient samples is needed, our results represent a valuable contribution toward the preclinical investigation of antidepressant treatment.

Pediatric anxiety disorders characterized as Generalized, Separation, and Social Anxiety Disorders, are chronic debilitating conditions that leave children feeling tense and isolated, both physically and emotionally. Selective serotonin reuptake inhibitors (SSRIs) are a classification of antidepressants that can be prescribed to children diagnosed with these disorders. SSRIs have been shown to be effective in treating anxiety disorders in children. The purpose of this literature review was to examine and determine if there are more risks or benefits associated with SSRIs, as well as evaluate teaching and education regarding anxiety disorder medication compliance in both children and parents. A secondary purpose of this research was to provide recommendations in nursing practice to allow children to feel more involved in their medical regimen. The following databases were used for the search: CINAHL, Academic Search Premier, and Web of Science. Key terms used in the search include but are not limited to: child* and anxiety, not autism, and selective serotonin reuptake inhibitors, OR SSRI*, OR adolsecen*, not med*, pediatric*, OR side effects. The results suggest that the benefits of SSRI therapy in children with anxiety disorder, when taken on a regularly scheduled basis, outweigh the risks, however more research aimed at compliance with SSRI therapy in children and parents is necessary. Further research analyzing children with anxiety disorders is needed to assess SSRI usage based specifically on their developmental age, and the inclusion of appropriate teaching and explanation related to their diagnoses to identifying stressors that can include behavioral therapy as well.

A monitorização terapêutica permite a individualização do regime de dosagem, assegurando a eficácia clínica e minimizando os efeitos adversos dos fármacos, prescritos na clínica. Os antidepressivos têm sido monitorados, pois, apresentam intervalos terapêuticos bem estabelecidos, ou seja, a maioria dos pacientes, que apresentam concentrações plasmáticas dentro deste intervalo fixo, tem as desordens psiquiátricas mantidas sob controle e efeitos adversos aceitáveis. Os antidepressivos tricíclicos (ADTs): imipramina, amitriptilina, nortriptilina e desipramina, embora eficazes e ainda muito utilizados, apresentam efeitos adversos, não desejáveis. Os antidepressivos, inibidores seletivos da recaptação de serotonina (ISRSs): citalopram e sertralina, apresentam eficácia clínica comparável aos clássicos ADTs, mas destituídos dos efeitos adversos associados aos mesmos. Os métodos convencionais, empregados no tratamento de amostras biológicas, para análises de antidepressivos por técnicas cromatográficas, têm sido a extração líquido-líquido e extração em fase sólida. A extração sortiva em barra de agitação (SBSE), técnica recente de preparo de amostras para a préconcentração de compostos orgânicos presentes em amostras biológicas, baseiase na extração estática, através do polímero polidimetilsiloxano (PDMS), no qual ocorre a dissolução (sorção, partição) do analito. Neste trabalho, as técnicas SBSE e cromatografia líquida de alta eficiência foram avaliadas para a análise simultânea dos antidepressivos em amostras de plasma para fins de monitorização terapêutica. As condições cromatográficas de análise, assim como as variáveis SBSE de extração (tempo, temperatura, força iônica, pH da matriz) e tempo de dessorção, foram otimizadas, visando adequada sensibilidade analítica. A validação analítica foi realizada segundo normas da ANVISA, em diferentes concentrações plasmáticas, as quais contemplam o intervalo terapêutico. O método SBSE/HPLC padronizado apresentou linearidade na faixa de concentração plasmática de 20 a 1000 ng mL-1, precisão interensaio com coeficientes de variação menor que 14% e recuperação relativa de 83 a 110%. Segundo a validação analítica, a metodologia SBSE/HPLC apresentou linearidade, alta sensibilidade, seletividade e precisão analítica adequadas para a análise dos antidepressivos: imipramina, amitriptilina, nortriptilina, desipramina, citalopram e sertralina, em amostra de plasma, para fins de monitorização terapêutica. / Therapeutic drug monitoring allows individualization of drug dosage assuring its clinical efficacy and at the same time minimizing adverse effects of the drugs prescribed in clinics. The antidepressants have been monitored since they present a very well established therapeutic interval. In this sense, most of the patients whose plasmatic concentrations are ranged at that interval present psychiatric disorders under control and drug adverse effects at bearable levels. Despite tricyclic antidepressants (TCAs) such as imipramine, amitriptyline, nortriptyline and desipramine are highly efficient and widely used, they also present undesirable adverse effects. The antidepressants: citalopram and sertraline, which are selective serotonin reuptake inhibitors (SSRIs), present clinical efficacy comparable to the classic TACs, but with no adverse effects associated to the last ones. Liquid-liquid extraction (LLE) and solid phase extraction (SPE) have been usually employed in biological sample pre-treatment for chromatographic analysis. A new technique named sorptive stir bar extraction (SBSE) for sample pre-concentration of organic compounds from biological samples was recently proposed. This technique is based on static extraction through the polymer polidimetilsiloxane (PDMS), in which analyte sorption occurs. In this work, SBSE and HPLC techniques have been evaluated for out antidepressants simultaneous analysis in plasma samples for therapeutic drug monitoring. The chromatographic conditions of analysis, as well as SBSE parameters (time, temperature, ionic strength, matrix pH, desorption time) have been optimized in order to obtain best analytical sensitivity. Analytical validation was carried out according to the norms established by ANVISA, in different plasmatic concentrations, which represent the therapeutic interval. The SBSE/HPLC method developed showed linearity in a concentration plasmatic ranging from 20 to 1000 ng mL-1, inter assay precision with coefficient of the variation lower than 14%, and relative recovery from 83 a 110%. Based on analytical validation, the SBSE/HPLC methodology showed good linearity, high sensitivity, selectivity and suitable repeatability to the analyzed antidepressants: imipramine, amitriptyline, nortriptyline, desipramine, citalopram and sertraline in plasma samples for therapeutic drug monitoring.

A monitorização terapêutica permite a individualização do regime de dosagem, assegurando a eficácia clínica e minimizando os efeitos adversos dos fármacos, prescritos na clínica. Os antidepressivos têm sido monitorados, pois, apresentam intervalos terapêuticos bem estabelecidos, ou seja, a maioria dos pacientes, que apresentam concentrações plasmáticas dentro deste intervalo fixo, tem as desordens psiquiátricas mantidas sob controle e efeitos adversos aceitáveis. Os antidepressivos tricíclicos (ADTs): imipramina, amitriptilina, nortriptilina e desipramina, embora eficazes e ainda muito utilizados, apresentam efeitos adversos, não desejáveis. Os antidepressivos, inibidores seletivos da recaptação de serotonina (ISRSs): citalopram e sertralina, apresentam eficácia clínica comparável aos clássicos ADTs, mas destituídos dos efeitos adversos associados aos mesmos. Os métodos convencionais, empregados no tratamento de amostras biológicas, para análises de antidepressivos por técnicas cromatográficas, têm sido a extração líquido-líquido e extração em fase sólida. A extração sortiva em barra de agitação (SBSE), técnica recente de preparo de amostras para a préconcentração de compostos orgânicos presentes em amostras biológicas, baseiase na extração estática, através do polímero polidimetilsiloxano (PDMS), no qual ocorre a dissolução (sorção, partição) do analito. Neste trabalho, as técnicas SBSE e cromatografia líquida de alta eficiência foram avaliadas para a análise simultânea dos antidepressivos em amostras de plasma para fins de monitorização terapêutica. As condições cromatográficas de análise, assim como as variáveis SBSE de extração (tempo, temperatura, força iônica, pH da matriz) e tempo de dessorção, foram otimizadas, visando adequada sensibilidade analítica. A validação analítica foi realizada segundo normas da ANVISA, em diferentes concentrações plasmáticas, as quais contemplam o intervalo terapêutico. O método SBSE/HPLC padronizado apresentou linearidade na faixa de concentração plasmática de 20 a 1000 ng mL-1, precisão interensaio com coeficientes de variação menor que 14% e recuperação relativa de 83 a 110%. Segundo a validação analítica, a metodologia SBSE/HPLC apresentou linearidade, alta sensibilidade, seletividade e precisão analítica adequadas para a análise dos antidepressivos: imipramina, amitriptilina, nortriptilina, desipramina, citalopram e sertralina, em amostra de plasma, para fins de monitorização terapêutica. / Therapeutic drug monitoring allows individualization of drug dosage assuring its clinical efficacy and at the same time minimizing adverse effects of the drugs prescribed in clinics. The antidepressants have been monitored since they present a very well established therapeutic interval. In this sense, most of the patients whose plasmatic concentrations are ranged at that interval present psychiatric disorders under control and drug adverse effects at bearable levels. Despite tricyclic antidepressants (TCAs) such as imipramine, amitriptyline, nortriptyline and desipramine are highly efficient and widely used, they also present undesirable adverse effects. The antidepressants: citalopram and sertraline, which are selective serotonin reuptake inhibitors (SSRIs), present clinical efficacy comparable to the classic TACs, but with no adverse effects associated to the last ones. Liquid-liquid extraction (LLE) and solid phase extraction (SPE) have been usually employed in biological sample pre-treatment for chromatographic analysis. A new technique named sorptive stir bar extraction (SBSE) for sample pre-concentration of organic compounds from biological samples was recently proposed. This technique is based on static extraction through the polymer polidimetilsiloxane (PDMS), in which analyte sorption occurs. In this work, SBSE and HPLC techniques have been evaluated for out antidepressants simultaneous analysis in plasma samples for therapeutic drug monitoring. The chromatographic conditions of analysis, as well as SBSE parameters (time, temperature, ionic strength, matrix pH, desorption time) have been optimized in order to obtain best analytical sensitivity. Analytical validation was carried out according to the norms established by ANVISA, in different plasmatic concentrations, which represent the therapeutic interval. The SBSE/HPLC method developed showed linearity in a concentration plasmatic ranging from 20 to 1000 ng mL-1, inter assay precision with coefficient of the variation lower than 14%, and relative recovery from 83 a 110%. Based on analytical validation, the SBSE/HPLC methodology showed good linearity, high sensitivity, selectivity and suitable repeatability to the analyzed antidepressants: imipramine, amitriptyline, nortriptyline, desipramine, citalopram and sertraline in plasma samples for therapeutic drug monitoring.

Irritable bowel syndrome (IBS) is a functional disorder that affects the gastrointestinal tract and especially the large intestine. The pathogenesis of the disease is not fully understood, for that reason there is still no cure and current therapy focuses on symptom relief. The disease manifests itself in the form of abdominal pain, bloating, constipation or diarrhoea. New studies have shown a link between IBS and communication between the central nervous system and the gut. Serotonin and norepinephrine seem to be important for the course of the disease. The purpose of this literature review was to study the efficacy and safety of certain antidepressant preparations belonging to tricyclic antidepressants and selective serotonin reuptake inhibitors in the treatment of IBS. This work focused on the effect of preparations and how tolerable they are with respect to their side effects. Articles presented in this work were found in the PubMed database with the keywords "irritable bowel syndrome", "serotonin reuptake inhibitor", " tricyclic antidepressants ". Amitriptyline 10 mg and imipramine 25 mg provided a statistically significant symptom relief in IBS with diarrhoea (IBS-D) compared to placebo. In addition, tianeptine 12.5 mg 3 times / day, which belongs to the selective serotonin reuptake enhancer (SSRE) group, gave a corresponding symptom relief as amitriptyline 10 mg once / day. The difference between the two was not statistically significant, but the study was open label and non-placebo-controlled, which made it difficult to draw any conclusions. Fluoxetine, which belongs to the selective serotonin reuptake inhibitor (SSRI) group, produced a statistically significant effect compared to placebo in the treatment of constipation IBS (IBS-C). In contrast, paroxetine-CR did not have any statistically significant effect on abdominal pain in IBS-C compared with placebo. However, more patients in the paroxetine group achieved the secondary outcome (clinical global improvement) with scores of 1-2 on the scale Clinical Global Impressions-Improvement (CGI-I). Based on the studies presented in this literature study, it is concluded that a low dose of TCA is an effective and safe treatment for IBS-D while SSRIs are effective and safe in the treatment of IBS-C compared to placebo. / Irritable bowel syndrome (IBS), är funktionella störningar som drabbar gastrointestinalkanalen och framför allt kolon. Patogenesen till sjukdomen är inte helt klarlagd, av den anledningen saknas fortfarande någon botande behandling och dagens terapi fokuserar på symtomlindring. Sjukdomen yttrar sig i form av buksmärta, uppblåsthet, förstoppning eller diarré. Senaste studier har visat en koppling mellan IBS och kommunikation mellan centrala nervsystemet och tarmen. Serotonin och noradrenalin verkar ha betydelse för sjukdomsförlopp. Syftet med detta litteraturarbete var att studera effektivitet och säkerhet för några antidepressiva preparat som tillhör tricykliska antidepressiva och selektiva serotonin återupptagshämmare vid behandling av IBS. Detta arbete fokuserade på effekten av preparaten och hur tolererbara de är med avseende på deras biverkningar. Artiklar som presenteras i detta arbete söktes i databasen Pubmed med sökord ”irritable bowel syndrome”, ”serotonin reuptake inhibitors”, ” tricyclic antidepressants”. Amitriptylin 10mg och imipramin 25mg gav en statistiskt signifikant symtomlindring vid IBS med diarré (IBS-D) jämfört med placebo. Dessutom gav tianeptin 12,5mg 3 gånger/dag, som tillhör läkemedelsgruppen selektiv serotonin återupptagsenhancer (SSRE), en likvärdig symtomlindring som amitriptylin 10mg en gång/dag. Skillnaden mellan de två var inte statistiskt signifikant, däremot var studien openlabel och icke-placebokontrollerad vilket gör det svårt att dra någon slutsats. Fluoxetin, som tillhör läkemedelsgruppen selektiva serotonin återupptagshämmare SSRI, gav en statistiskt signifikant effekt jämfört med placebo vid behandling av IBS med förstoppning (IBS-C). Däremot gav paroxetin-CR ingen statistiskt signifikant effekt på buksmärta vid IBS-C jämfört med placebo, dock uppnådde fler i paroxetingruppen den sekundära utfallsvariabeln (global klinisk förbättring) och fick poäng mellan 1 – 2 på skalan Clinical Global Impressions-Improvement (CGI-I).  Baserat på studierna som presenteras i denna litteraturstudie dras slutsatsen att en låg dos TCA är en effektiv och säker behandling vid IBS-D medan SSRI är effektiva och säkra vid behandling av IBS-C jämfört med placebo.