Attachment, Physiological and Familial Vulnerability in Childhood Obesity: an Interactive Multisystem Approach

The aims of the present study were to test the association between insecure attachment and basal cortisol and catecholamines levels in a sample of obese children. The role of familial vulnerability and gender was also investigated. Methods: Cortisol and catecholamines levels of 8- to 13-year olds obese children were measured. Self-report questionnaires were used to assess attachment pattern and current anxiety and depression, and parent-report questionnaires were used to assess attachment, current anxiety and depression and familial vulnerability. Linear regression analyses were performed for individuals that scored low versus high on parental internalizing problems, and for boys and girls, separately. Results: In the group with high parental internalizing problems, insecure attachment was significantly associated with reduced basal levels of cortisol, in boys (p=0.007, b= -0.861, R2= 73.0%). In the group with low parental internalizing problems, the association between insecure attachment and cortisol was not significant in either boys or girls, and it was negative in boys (p=0.075, b= -0.606, R2= 36.7%) and positive in girls (p=0.677, b= 0.176, R2= 3.1%) . Conclusions: Apparently, physiological risk factors for psicopathology in obesity are more evident in individuals with a high familial vulnerability. In addition, patterns of physiological risk for psicopathology in obesity are different in boys and girls. Therefore, it is important to take into account familial vulnerability and gender when investigating physiological risk factors for psycopathology in obesity. Insecure attachment in childhood may be a risk factor for obesity. Interventions to increase children's attachment security should examine the effects on children's weight.

Antiretroviral activity of protease inhibitors against Toxoplasma gondii

The introduction of highly active antiretroviral therapy (HAART) has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE). These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API) on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

Focus on: II – physiological principles of acid-base balance: an integrative perspective

Normal human metabolism leads to the daily production of large amounts of volatile and non-volatile acids. The maintenance of the pH within physiological limits is a demanding task in which several mechanisms are involved. The most immediate answer comes from several physiological buffers that quickly neutralize pH deviations caused by the addition of strong acids or bases to the body. Bicarbonate/carbonic acid is the most important buffer pair of the extracellular milieu, but is chemically inefficient and depends on the continuous activity of the lung and kidney. Other physiological buffers have higher efficacy and are very important in the intracellular environment and renal tubules. The capacity of the various chemical buffers is kept by operating in an open system and by several controlling mechanisms. The lung is responsible for the elimination of the carbon dioxide (CO2) produced in the body. In metabolic disorders, respiratory adjustment of the elimination of CO2 prolongs the effect of the bicarbonate/carbonic acid buffer, but this process consumes bicarbonate. The kidney contributes to acid-base balance through several mechanisms: 1) controls the reabsorption of filtered bicarbonate; 2) regenerates bicarbonate consumed in buffer reactions; 3) eliminates non-volatile acids. Renal elimination of acid and bicarbonate regeneration is only possible due to the existence of several urinary buffers and to the ability of the kidneys to produce ammonia

Prophylaxis in Hemophilia A Patients with Inhibitors

The development of antibodies to factor VIII is one of the most serious complications of haemophilia treatment. Approximately 30% of patients with severe haemophilia develop neutralizing inhibitors to replacement FVIII. Although most patients with inhibitors do not bleed more frequently than patients without inhibitors, bleeding is more difficult to control and this patients suffer more severe bleeding and have greater morbidity and mortality. Patients with persistent high-titer inhibitor who are not candidates or fail ITI, pose a great challenge to haemophilia management. The efficacy and safety of prophylaxis with bypassing agents in reducing bleeding tendency, has been described in numerous studies. Patients and methods: We report tree adult severe haemophilia A patients, two with persistent high-titre inhibitors and one who failed ITI, on prophylactic treatment after several significant musculoskeletal and life-threatening haemorrhagic episodes (intrabdominal/intramuscular) and pseudotumor haemorrhage. Treatment regimens consisted of APCC (Feiba®) in doses of 60-70UKg-1, 2-3 times per week, according underlying bleeding phenotype. Breakthrough bleeds were treated with either APCC (Feiba®) or rFVIIa (NovoSeven®). Results and Conclusion: There was reduction in total bleeding episodes in two patients (43% to 80%) and one patient remained stable, while receiving prophylaxis. Absence of severe and life threatening bleeding episodes, as well as inpatient stays, contributing to a better quality of life in those patients, was observed. APCC (Feiba®) was well tolerated and no thrombotic events were observed.

O2/CO2-sensitive cyclic AMP-signalling pathway in peripheral chemoreceptors

RESUMO: O corpo carotídeo (CB) é um pequeno órgão sensível a variações na PaO2, PaCO2 e pH. As células tipo I (células glómicas) do corpo carotídeo, as unidades sensoriais deste órgão, libertam neurotransmissores em resposta às variações dos gases arteriais. Estes neurotransmissores atuam quer em recetores pré-sinápticos, localizados nas células tipo I, quer em recetores póssinápticos, localizados nas terminações do nervo do seio carotídeo, ou em ambos. A activação dos recetores pré-sinápticos modula a atividade do corpo carotídeo, enquanto que, a activação dos recetores pós-sinápticos, de carater excitatório, desencadeia um aumento da frequência de descarga das fibras do CSN, com subsequente despolarização dos neurónios do gânglio petroso, e posterior despolarização de um grupo específico de neurónios do centro respiratório central, desencadeando, como resposta final, hiperventilação. Estes recetores pré- e pós-sinápticos podem ser classificados em ionotrópicos ou metabotrópicos, estando os últimos acoplados a adenilatos ciclases transmembranares (tmAC). O mecanismo exato pelo qual as variações dos gases arteriais são detetadas pelo CB não se encontra ainda completamente elucidado, mas tem sido sugerido que alterações nos níveis de cAMP estejam associadas ao mecanismo de deteção de variações de O2 e CO2. Os níveis de cAMP podem ser regulados através da sua via de síntese, mediada por dois tipos de adenilatos ciclases: tmAC sensível aos eurotransmissores e adenilato ciclase solúvel (sAC)sensível a variações de HCO3/CO2, e pela sua via de degradação mediada por fosfodiesterases. A via de degradação do cAMP pode ser manipulada farmacologicamente, funcionando enquanto alvo terapêutico para o tratamento de patologias do foro respiratório (e.g. asma, hipertensão pulmonar, doença pulmonar obstructiva crónica e apneia do sono), que induzem um aumento da actividade do CB.O trabalho descrito nesta dissertação partiu da hipótese de que a actividade do CB é manipulada por fármacos, que interferem com a via de sinalização do cAMP, tendo sido nosso objectivo geral, investigar o papel do cAMP na quimiotransdução do CB de rato, e determinar se a actividade dos enzimas responsáveis pela via de sinalização do cAMP é ou não regulada por variações de O2/CO2. Assim, a relevância deste trabalho é a de estudar e identificar possíveis alvos moleculares (sAC, isoformas de tmAC e PDE) com potencial para serem usados no tratamento de patologias relacionadas com o controlo respiratório. A primeira parte do presente trabalho, centrou-se na caracterização farmacológica da PDE4 no CB e em tecidos não quimiorecetores (e.g. gânglio cervical superior e artérias carótidas), e na observação do efeito de hipóxia aguda na acumulação dos níveis de cAMP, induzidos pelos inibidores de PDE, nestes tecidos. A quantificação de cAMP foi efectuada por técnica imunoenzimática (EIA), tendo sido elaboradas curvas de dose-resposta para os efeitos de inibidores, não específicos (IBMX) e específicos para a PDE2 e PDE4 (EHNA, Rolipram e Ro 20-1724), nos níveis de cAMP acumulados, em situações de normóxia (20%O2/5%CO2) e hipóxia (5%O2/5%CO2). A caracterização das PDE no gânglio cervical superior foi aprofundada, utilizando-se a técnica de transferência de energia de ressonância por fluorescência (FRET) em culturas primárias de neurónios, na presença de inibidores não específicos (IBMX) e específicos para a PDE3 e PDE4 (milrinone e rolipram, respetivamente). Foram igualmente estudadas, através de RT-qPCR, as alterações na expressão de PDE3A-B e PDE4A-D, no gânglio cervical superior, em resposta a diferentes percentagens de oxigénio. Na segunda parte do trabalho investigou-se a via de síntese do cAMP no CB em resposta a variações na concentração de HCO3/CO2. Em concreto, o protocolo experimental centrou-se na caracterização da sAC, dado que a sua actividade é regulada por variações de HCO3/CO2. A caracterização da expressão e regulação da sAC, em resposta a variações de HCO3/CO2 ,foi efectuada no CB e em tecidos não quimioreceptores periféricos (e.g. gânglio cervical superior, petroso e nodoso) por qRT-PCR. A actividade deste enzima foi caracterizada indirectamente através da quantificação dos níveis de cAMP (quantificação por EIA), induzidos por diferentes concentrações de HCO3/CO2, na presença de MDL-12,33-A, um inibidore da tmAC. A expressão das isoformas da tmAC no CB e gânglio petroso foi determinada por RT-qPCR. Adicionalmente, estudámos a contribuição relativa da tmAC e sAC no mecanismo de sensibilidade ao CO2 no CB. Para o efeito foram estudadas as alterações: 1) nos níveis de cAMP (quantificado por EIA) na presença de diferentes concentrações de HCO3/CO2 e ao longo do tempo (5-30 min); 2) na ativação da proteína cinase A (PKA, FRET baseado em sensores) em células tipo I do CB; e 3) na frequência de descarga do CSN (registos) na presença e ausência de ativadores e inibidores da sAC,tmAC e PKA. Por último, foi caracterizada a expressão e actividade da sAC nos quimioreceptors centrais (locus ceruleus, rafe e medula ventro-lateral) através de técnicas de RT-qPCR e EIA. A expressão das isoformas da tmAC foi aprofundada no locus coeruleus através de RT-qPCR. Por fim, comparámos a contribuição da tmAC e sAC nos níveis de cAMP no locus coeruleus em condições de normocapnia e hipercapnia.O nosso trabalho teve os seguintes resultados principais: 1) PDE4 está funcional no corpo carotídeo, artérias carótidas e gânglio cervical superior de rato, embora a PDE2 só se encontre funcional neste último; 2) Os efeitos dos inibidores de PDE nos níveis de acumulação de cAMP foram exacerbados em situações de hipóxia aguda no CB e artérias carótidas, mas foram atenuados no gânglio cervical superior; 3) No gânglio cervical superior, diferentes tipos de células apresentaram uma caracterização específica de PDEs, sugerindo uma subpopulação de células neste gânglio com funções fisiológicas distintas; 4) Embora todas as isoformas de PDE4 e PDE3 estivessem presentes no gânglio, a PDE3a, PDE4b e a PDE4d foram as isoformas mais expressas. Por outro lado, incubações de gânglio cervical superior, em diferentes percentagens de oxigénio, não alteraram (não regularam) significativamente a expressão das diferentes isoformas de PDE neste órgão; 5) a sAC encontra-se expressa e funcional no CB e nos quimiorecetores centrais estudados (locus coeruleus, rafe e medula ventrolateral). A sAC apresenta maior expressão no CB comparativamente aos restantes orgãos estudados, exceptuando os testículos, orgão controlo. Variações de HCO3/CO2 de 0/0 para 24/5 aumentaram os níveis de cAMP no CB e quimiorecetores centrais, tendo sido o aumento mais significativo observado no CB. Concentrações acima dos 24mM HCO3/5%CO2 não induziram alterações nos níveis de cAMP, sugerindo que a actividade da sAC se encontra saturada em condições fisiológicas (normocapnia) e que este enzima não desempenha qualquer papel na deteção de situações de hipercapnia; 6) No CB, a expressão das isoformas tmAC1, tmAC4, tmAC6 e tmAC9 é mais elevada comparativamente à expressão da sAC; 7) Utilizamos diferentes inibidores da tmAC (MDL 12-330A, 500μM, 2’5’-ddADO, 30-300μM, SQ 22536, 200μM) e da sAC (KH7, 10-100μM) para estudar a contribuição relativa destes enzimas na acumulação do cAMP no CB. Tanto a tmAC como a sAC contribuem para a acumulação dos níveis de cAMP em condições de hipercapnia. Contudo, existe um maior efeito destes inibidores nas condições de 12 mM HCO3/2.5%CO2 do que em condições de normocapnia e hipercapnia, sugerindo um papel relevante destes enzimas na atividade do CB em situações de hipocapnia; 8) Não se observaram variações nos níveis de cAMP em resposta a diferentes concentrações de HCO3/CO2 ao longo do tempo (5-30 min). O efeito inibitório induzido por ddADO e KH7 foi sobreponível após 5 ou 30 minutos de incubação em todas as concentrações de HCO3/CO2 estudadas; 9) Por último, verificou-se um aumento na frequência da descarga do nervo do seio carotídeo entre as condições de normocapnia e hipercapnia acídica. Ao contrário do KH7 (10μM), o 2’5’-ddADO reduziu significativamente a frequência de descarga do nervo, quer em condições de normocapnia quer de hipercapnia acídica. Contudo, não se verificou aumento na frequência de descarga do nervo entre normocapnia e hipercapnia isohídrica, sugerindo que a sensibilidade à hipercapnia no CB é mediada por variações de pH. Em conclusão, os resultados decorrentes deste trabalho permitiram demonstrar que, embora os enzimas que medeiam a via de sinalização do cAMP possam ser bons alvos terapêuticos em condições particulares, a sua actividade não é específica para o CB. Os resultados sugerem ainda que o cAMP não é um mediador específico da transdução à hipercapnia neste orgão. Contudo, os nossos resultados demonstraram que os níveis de cAMP são mais elevados em condições fisiológicas, o que sugere que o cAMP possa ter uma função homeostática neste orgão. Por último, o presente trabalho demonstrou que os aumentos de cAMP descritos por outros em condições de hipercapnia, não são observáveis quando o pH se encontra controlado. ------------------ ABSTRACT: The work presented in this dissertation was aimed to establish how specific is cAMP-signaling pathways in the CB mainly in different CO2 conditions and how O2 concentrations alter/drives the manipulation of cAMP signaling in the CB. The experimental studies included in this thesis sought to investigate the role of cAMP in the rat CB chemotransduction mechanisms and to determine whether the enzymes that participate in cAMP signal transduction in the CB are regulated by O2/CO2. We characterized the enzymes involved in the cAMP-signaling pathway in the CB (sAC, tmAC, PDE) under different O2/CO2 conditions. Our results demonstrated that many of these enzymes are involved in CO2/O2 sensing and while they may be useful in treating conditions with alterations in CO2/O2 sensing,they will not be specific to chemoreception within the CB: 1) PDE4 is ubiquitously expressed in CB and non-chemoreceptor related tissues and their affinity to inhibitors change with O2 tensions in both CB and carotid arteries, and 2) sAC and tmAC are expressed in peripheral and central chemo- and non-chemoreceptor tissues and their effect on cAMP levels do not change between normocapnic and isohydric hypercapnic conditions. Our results provide evidence against a specific role of cAMP as a mediator for O2 and CO2 chemotransduction in the rat CB and emphasized the role of pH in CO2 sensitivity of the CB. Furthermore, our results demonstrate that cAMP levels are maintained higher under physiological conditions, supporting recent finding from our lab, which all together suggests that cAMP has a homeostatic function in this organ.

Baseline Susceptibility of Primary HIV-2 to Entry Inhibitors

BACKGROUND: The baseline susceptibility of primary HIV-2 to maraviroc (MVC) and other entry inhibitors is currently unknown. METHODS: The susceptibility of 19 HIV-2 isolates obtained from asymptomatic and AIDS patients and seven HIV-1 clinical isolates to the fusion inhibitors enfuvirtide (ENF) and T-1249, and to the coreceptor antagonists AMD3100, TAK-779 and MVC, was measured using a TZM-bl cell-based assay. The 50% inhibitory concentration (IC(50)), 90% inhibitory concentration (IC(90)) and dose-response curve slopes were determined for each drug. RESULTS: ENF and T-1249 were significantly less active on HIV-2 than on HIV-1 (211- and 2-fold, respectively). AMD3100 and TAK-779 inhibited HIV-2 and HIV-1 CXCR4 tropic (X4) and CCR5 tropic (R5) variants with similar IC(50) and IC(90) values. MVC, however, inhibited the replication of R5 HIV-2 variants with significantly higher IC(90) values (42.7 versus 9.7 nM; P<0.0001) and lower slope values (0.7 versus 1.3; P<0.0001) than HIV-1. HIV-2 R5 variants derived from AIDS patients were significantly less sensitive to MVC than variants from asymptomatic patients, this being inversely correlated with the absolute number of CD4(+) T-cells. CONCLUSIONS: T-1249 is a potent inhibitor of HIV-2 replication indicating that new fusion inhibitors might be useful to treat HIV-2 infection. Coreceptor antagonists TAK-779 and AMD3100 are also potent inhibitors of HIV-2 replication. The reduced sensitivity of R5 variants to MVC, especially in severely immunodeficient patients, indicates that the treatment of HIV-2-infected patients with MVC might require higher dosages than those used in HIV-1 patients, and should be adjusted to the disease stage.

Biological and Physiological Markers of Tactile Sensorial Processing in Healthy Newborns

The main objective of this review is to provide a descriptive analysis of the biological and physiological markers of tactile sensorial processing in healthy, full-term newborns. Research articles were selected according to the following study design criteria: (a) tactile stimulation for touch sense as an independent variable; (b) having at least one biological or physiological variable as a dependent variable; and (c) the group of participants were characterized as full-term and healthy newborns; a mixed group of full-term newborns and preterm newborns; or premature newborns with appropriate-weight-for-gestational age and without clinical differences or considered to have a normal, healthy somatosensory system. Studies were then grouped according to the dependent variable type, and only those that met the aforementioned three major criteria were described. Cortisol level, growth measures, and urinary catecholamine, serotonin, and melatonin levels were reported as biological-marker candidates for tactile sensorial processing. Heart rate, body temperature, skin-conductance activity, and vagal reactivity were described as neurovegetative-marker candidates. Somatosensory evoked potentials, somatosensory evoked magnetic fields, and functional neuroimaging data also were included.

Applications of the indole scaffold in medicinal chemistry: development of new antioxidants, COX inhibitors and antitubercular agents

Dissertação para obtenção do Grau de Doutor em Química, especialidade Química Orgânica

Development of novel benzimidazole- based COX Inhibitors new synthetic strategies and screening of heterocyclic structures

Dissertação para obtenção do Grau de Doutor em Química, especialidade Química Orgânica

Aptamers binding to Bradykinin

Bradykinin is a peptide of the kinin group, involved in a number of receptor-mediated physiological actions, including inflammation and vasodilation, as well as neuromodulation, neuroprotection and promotion of neurogenesis. Bradykinin is the main ligand of the B2 receptor- the main kinin receptor- which is involved in the cardiac and renal protective effects of kinins in diseases. Antibodies have been considered for a long time as promising therapeutic agents in various fields, especially cancer-related ones. Aptamers, on the other hand, have proven to be an excellent alterative, since they have similar properties to those of monoclonal antibodies, such a high-specificity of recognition and high-affinity binding. Plus, they are developed using in vitro selection procedures and can be reproduced by enzymatic reactions. SELEX is a powerful tool for the development of both DNA and RNA aptamers. The main goal of this project was to design a method to select aptamers against bradykinin using capillary electrophoresis alongside the SELEX technique. The selection was done by comparing the aptamers’ (ssDNA-target complex) electrophoretic mobility with that of the ssDNA and the target, which allowed us to define an appropriate collection window that took into consideration the analytes’ detection time, thus enabling the collection of the desired oligonucleotides. After two selection rounds, the collected pool was sequenced, the affinity was measured and the aptamers’ secondary structure was predicted. We concluded that with only two selection cycles, the original DNA library’s bulk affinity grew around 0.4%. The structural characterization of the aptamers, performed with the aid of the Mfold software, revealed that there are many repetitive motifs amongst them, indicating that the selection process was successful. We have obtained 16 sequences of candidate aptamers as bradykinin ligands of similar sequences and secondary structures whose biological activity should be analyzed after synthesis; mainly in regard to their role as bradykinin inhibitors.

Estimate of Physiological Age in Females of PASSALUS (Insecta, Coleoptera)

Studies on development of female reproductive system insects have shown its utility in physiological age grading. The irreversible charocter of ovarian alterations correlated with mandibular wear and presence of yellow body, were applied to estimate the physiological age of Passalus convexus and Passalus latifrons females, giving results comparable to other studies and indicating the value of these methods in determination of physiological age.

Photochemical synthesis and functional transformations of bicyclic vinyl aziridines

Aziridines, a class of organic compounds containing a three membered heterocycle with a nitrogen atom, are extremely valuable molecules in organic and medicinal chemistry. They are frequently used as versatile precursors in the synthesis of natural products, and many biologically active molecules possess the aziridine moiety. The reactivity of aziridines has been studied, for example, in ring-opening reactions with thiols. However, not much interest seems to be given to reactions of aziridines in aqueous media, despite the numberless advantages of using water as solvent in organic chemistry. The nucleophilic ring-opening reaction of aziridines in aqueous media was here explored. Following the Kaplan aziridine synthetic methodology, in which pyridinium salts undergo a photochemical transformation to give bicyclic vinyl aziridines, new aziridines were synthetized. Their nucleophilic ring-opening reaction in water under physiological conditions was investigated and a range of sulphur, nitrogen, carbon and oxygen nucleophiles tested. Thiols, anilines and azide proved to be good nucleophiles to react with the aziridines, giving the ring-opening product in moderate to good yields. The best results were obtained with thiols, more specifically with cysteine-derived nucleophiles. Preliminary results show that these bicyclic vinyl aziridines can modify calcitonin, a peptide containing two cysteine amino acids residues, grating them the potential to be used in bioconjugation as ligands to cysteine-containing proteins, or even as enzyme inhibitors of, for example, cysteine proteases. Additionally, exploratory investigations suggest that the separation of both enantiomers of the bicyclic vinyl aziridine can be performed by taking advantage of an enzymatic methodology for the resolution of racemic secondary alcohols. Both enantiomers would be highly valuable as precursors in the synthesis of enantiomerically pure molecules, as no other method is currently reported for their separation.

The role of ascorbate peroxidase, guaiacol peroxidase, and polysaccharides in cassava (Manihot esculenta Crantz) roots under postharvest physiological deterioration

Abstract This study aimed to investigate the role of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), polysaccharides, and protein contents associated with the early events of postharvest physiological deterioration (PPD) in cassava roots. Increases in APX and GPX activity, as well as total protein contents occurred from 3 to 5 days of storage and were correlated with the delay of PPD. Cassava samples stained with periodic acid-Schiff (PAS) highlighted the presence of starch and cellulose. Degradation of starch granules during PPD was also detected. Slight metachromatic reaction with toluidine blue is indicative of increasing of acidic polysaccharides and may play an important role in PPD delay. Principal component analysis (PCA) classified samples according to their levels of enzymatic activity based on the decision tree model which showed GPX and total protein amounts to be correlated with PPD. The Oriental (ORI) cultivar was more susceptible to PPD.