Contributions à la Paléontologie du Miocène moyen continental du Bassin du Tage. III - Mammifères - Póvoa de Santarém, Pêro Filho et Chões (Secorio). Conclusions générales

This study on middle Miocene mammalian faunas from Tagus'basin deals particularly with some small mammals hitherto undescribed from Portugal, including a new Glirid (Paraglirulus scalabicensis nov. sp.); it allows an accurate datation by biostratigraphical standards, Megacricetodon crusafonti, Fahlbuschia darocensis, Cricetodon jotae being characteristic of mammalian MN6 unit, thus their age is nearly that of Sansan and Manchones (however the presence of Peridyromys hamadryas and Lagopsis verus do suggest, amidst this biozone, a somewhat later age than Sansan's); it contributes with indirect correlation data with marine formations, as underlying oyster-bearing beds most probably are in correspondance to the apogee of the same transgression that deposed near Lisbon ”schlier" facies from VI-a division (Serravalian, Blow's zones 10-13, Globorotalia meyeri zone); the diversity of mammalian assemblages is surely related to an environement with varied biotopes, whose characterisation becomes easier if account is taken of the preceding papers on mollusks (G. Truc) and Cyprinid fishes (J. Gaudant), and also according to some unpublished paleobotanical data (J. Pais). A table with a synthesis of all paleontological data so far known is presented.

Vertébrés du Miocène moyen de Amor (Leiria) - importance stratigraphique

Some outcrops in the western part of Leiria's diapir yielded continental fossils in five points near Amor village (mammals, other vertebrata, and gastropoda). This is most significant as it is the first locality where miocene age could undoubtedly been ascribed to formations northwards the Iberian Central Chain and Nazare's accident. Mammalian fauna comprises 18 taxa. A new cricetid species, Fahlbuschia freudenthali n. sp. is described. This fauna allows to date fossil-bearing units from Upper Orleanian, MN5 mammal zone, that may be correlated to Upper Langhian marine stage. As the fauna is quite varied, it is possible to recognize the main characters of environment and of climatic conditions. It may probably be assumed that at the time the climate was of mediterranean type, generally warmer than today.

Fucosyltransferase IX: characterization and biological role

α3/4-Fucosyltransferases (α3/4-FUTs) are glycosyltransferases (GTs) that catalyze the transfer of fucose in an α3/4-linkage onto the N-acetylglucosamine residue from acceptors containing the type II or type I (Galβ4/3GlcNAc, respectively) structures, thus synthesizing the fucosylated Lewis (Le) carbohydrate determinants. Fucosyltransferase IX (FUT9), the most recently identified member of the family, presents the higher divergence from the other FUTs and its sequence is the only highly conserved among species. FUT9 synthesizes the Lewisx (Lex) epitope (Galβ4(Fucα3)GlcNAc). Recent evidence has suggested that it is the enzyme responsible for the synthesis of Lex in the mouse brain. Lex expression has been described in glycoproteins, proteoglycans and glycolipids from the central nervous system (CNS) of diverse species, including rodents and humans.

Les plus anciens mammifères terrestres du Miocène marin de Lisbonne - le gisement du Km 10

For the first time, small mammals were found at the earliest marine level in the northeastern part of the lower Tagus basin, to the NE of Lisbon. At this new locality, at the 10 kilometer of the Lisbon-Oporto A1-IP1 highway,conglomerates yielded, along with marine fossils, more or less abraded teeth and bones from insectivores,lagomorphs, rodents and small artiodactyls (sec Tableau 1). Age may he ascribed to the lower Miocene, MN 2b Neogene mammal unit (about 22 My), but an early MN 3 age cannot be entirely excluded. That corresponds to latest Aquitanian (or less probably earliest Burdigalian) (sec Tableau 2). This is the first hitherto found locality with small mammals of this age as far as Portugal is concerned, as well as the oldest locality so far known in the Tagus basin. Km 10 is somewhat older than the localities of Universidade Católica and Avenida do Uruguay in Lisbon (ANTUNES & MEIN, 1986). Hence we can rather accurately date the age of the first marine transgression in the northeastern part of the lower Tagus basin. This shows that in this region there are no marine equivalents of the "Venus ribeiroi beds" (Aquitanian,Division 1 of the Lisbon Miocene series). Correlation between this unit and the uppermost levels of the essentially paleogene "Complexo de Benfica" may be possible. Fossils at km 10 point out to shallow, coastal, highenergy marine environments. Sedimentological features are compatible with this model. Dry land and swamps with brackish (or ev en fresh) waters were present nearby. From those areas came remains of mammals, crocodylians, as well as oysters and charophytes that were later transported to the sea. Sea was warmer than the extant Atlantic at the same latitudes, even if conditions were not strictly tropical then. These conditions surely influenced climate in the nearby regions. Ecological data concerning mammalian faunas distinctly point out to nearby forest-rich environments, much more so than for Universidade Católica and Avenida do Uruguay localities, from where drier, even steppe environment forms largely prevail.

La biochronologie des Mammifères néogènes d'Europe. L'échelle MN, son application à la succession des faunes du Portugal

Similarly to other organisms, Vertebrates changed during the Cenozoic Era. Mammals are the only ones to change quickly; their well mineralised bones and teeth are often fossilised. They are highly diverse and even isolated teeth can be identified. They are thus a good tool for establishing a biochronological framewoork. Among Mammals, Rodents with a short lifetime evolve more quickly than the large Mammals. In Europe, the first elaborated zonation was investigated by the Regional Committee on Neogene Stratigraphy and issued as the MN Zonation (Mammals Neogene) by Mein, 1976. During the following years, progress in knowledge lead to new charts. The latest one resulted from collective work (de Bruijn et al., 1992). Bîochronology gives relative data; if we desire to have numerical age estimates, we must correlate these results with radiometrie data, marine biostratigraphîcal units or the Geomagnetic Polarity Time Scale. For Europe, these results are summarised by Steininger et al. (1989-1996) and Steininger (1999). After some recents developments on MN Zonation, here is discussed the succession of Neogene small mammals Portuguese localities. Fortunately these localities are in majority inbedded in marine context. Their assigment to MN Zones is proposed and correlations with the spanish Aragonian Scale (Daams el al., 1999) are also suggested. In fact, some differences appear between Portugal and Aragonian assemblages, probably for ecological reasons. Therefore, the MN zonation is always useful for short and long distance biochronological correlations.

Nanostructuring silicon probes via electrodeposition: characterization of electrode coatings for acute in vivo neural recordings

Understanding how the brain works will require tools capable of measuring neuron elec-trical activity at a network scale. However, considerable progress is still necessary to reliably increase the number of neurons that are recorded and identified simultaneously with existing mi-croelectrode arrays. This project aims to evaluate how different materials can modify the effi-ciency of signal transfer from the neural tissue to the electrode. Therefore, various coating materials (gold, PEDOT, tungsten oxide and carbon nano-tubes) are characterized in terms of their underlying electrochemical processes and recording ef-ficacy. Iridium electrodes (177-706 μm2) are coated using galvanostatic deposition under different charge densities. By performing electrochemical impedance spectroscopy in phosphate buffered saline it is determined that the impedance modulus at 1 kHz depends on the coating material and decreased up to a maximum of two orders of magnitude for PEDOT (from 1 MΩ to 25 kΩ). The electrodes are furthermore characterized by cyclic voltammetry showing that charge storage capacity is im-proved by one order of magnitude reaching a maximum of 84.1 mC/cm2 for the PEDOT: gold nanoparticles composite (38 times the capacity of the pristine). Neural recording of spontaneous activity within the cortex was performed in anesthetized rodents to evaluate electrode coating performance.

Evaluation of antihypertensive drugs in patients with obstructive sleep apnea and in rats submitted to chronic intermittent hypoxia

RESUMO: A hipertensão arterial (HA) é uma patologia altamente prevalente, embora claramente subdiagnosticada, em doentes com síndrome de apneia obstrutiva do sono (SAOS). Estas duas patologias apresentam uma estreita relação e a monitorização ambulatória da pressão arterial (MAPA), por um período de 24 horas, parece ser o método mais preciso para o diagnóstico de hipertensão em doentes com SAOS. No entanto, esta ferramenta de diagnóstico para além de ser dispendiosa e envolver um número acrescido de meios técnicos e humanos, é mais morosa e, por conseguinte, não é utilizada por rotina no contexto do diagnóstico da SAOS. Por outro lado, apesar da aplicação de pressão positiva contínua nas vias aéreas (CPAP – Continous Positive Airway Pressure) ser considerada a terapêutica de eleição para os doentes com SAOS, o seu efeito no abaixamento da pressão arterial (PA) parece ser modesto, exigindo, por conseguinte, a implementação concomitante de terapêutica anti-hipertensora. Acontece que são escassos os dados relativos aos regimes de fármacos anti-hipertensores utilizados em doentes com SAOS e, acresce ainda que, as guidelines terapêuticas para o tratamento farmacológico da HA, neste grupo particular de doentes, permanecem, até ao momento, inexistentes. A utilização de modelos animais de hipóxia crónica intermitente (CIH), que mimetizam a HA observada em doentes com SAOS, revela-se extremamente importante, uma vez que se torna imperativo identificar fármacos que promovam um controle adequado da PA neste grupo de doentes. No entanto, estudos concebidos com o intuito de investigar o efeito anti-hipertensor dos fármacos neste modelo animal revelam-se insuficientes e, por outro lado, os escassos estudos que testaram fármacos anti-hipertensores neste modelo não foram desenhados para responder a questões de natureza farmacológica. Acresce ainda que se torna imprescindível garantir a escolha de um método para administração destes fármacos que seja não invasivo e que minimize o stress do animal. Embora a gavagem seja uma técnica indiscutivelmente eficaz e amplamente utilizada para a administração diária de fármacos a animais de laboratório, ela compreende uma sequência de procedimentos geradores de stress para os animais e, que podem por conseguinte, constituir um viés na interpretação dos resultados obtidos. O objectivo global da presente investigação translacional foi contribuir para a identificação de fármacos anti-hipertensores mais efectivos para o tratamento da HT nos indivíduos com SAOS e investigar mecanismos subjacentes aos efeitos sistémicos associadas à SAOS bem como a sua modulação por fármacos anti-hipertensores. Os objectivos específicos foram: em primeiro lugar,encontrar novos critérios, baseados nas medidas antropométricas, que permitam a identificação de doentes com suspeita de SAOS, que erroneamente se auto-classifiquem como nãohipertensos, e desta forma promover um uso mais criterioso do MAPA; em segundo lugar, investigar a existência de uma hipotética associação entre os esquemas de fármacos antihipertensores e o controle da PA (antes e após a adaptação de CPAP) em doentes com SAOS em terceiro lugar, avaliar a eficácia do carvedilol (CVD), um fármaco bloqueador β-adrenérgico não selectivo com actividade antagonista α1 intrínseca e propriedades anti-oxidantes num modelo animal de hipertensão induzida pela CIH; em quarto lugar, explorar os efeitos da CIH sobre o perfil farmacocinético do CVD; e, em quinto lugar, investigar um método alternativo à gavagem para a administração crónica de fármacos anti-hipertensores a animais de laboratório. Com este intuito, na primeira fase deste projecto, fizemos uso de uma amostra com um número apreciável de doentes com SAOS (n=369), que acorreram, pela primeira vez, à consulta de Patologia do Sono do CHLN e que foram submetidos a um estudo polissonográfico do sono, à MAPA e que preencheram um questionário que contemplava a obtenção de informação relativa ao perfil da medicação anti-hipertensora em curso. Numa segunda fase, utilizámos um modelo experimental de HT no rato induzida por um paradigma de CIH. Do nosso trabalho resultaram os seguintes resultados principais: em primeiro lugar, o índice de massa corporal (IMC) e o perímetro do pescoço (PP) foram identificados como preditores independentes de “auto-classificação errónea” da HA em doentes com suspeita de SAOS; em segundo lugar, não encontramos qualquer associação com significado estatístico entre os vários esquemas de fármacos anti-hipertensores bem como o número de fármacos incluídos nesse esquemas, e o controle da PA (antes e depois da adaptação do CPAP); em terceiro lugar, apesar das doses de 10, 30 e 50 mg/kg de carvedilol terem promovido uma redução significativa da frequência cardíaca, não foi observado qualquer decréscimo na PA no nosso modelo animal; em quarto lugar, as razões S/(R+S) dos enantiómeros do CVD nos animais expostos à CIH e a condições de normóxia revelaram-se diferentes; e, em quinto lugar, a administração oral voluntária mostrou ser um método eficaz para a administração diária controlada de fármacos anti-hipertensores e que é independente da manipulação e contenção do animal. Em conclusão, os resultados obtidos através do estudo clínico revelaram que o controle da PA, antes e após a adaptação do CPAP, em doentes com SAOS é independente, quer do esquema de fármacos anti-hipertensores, quer do número de fármacos incluídos num determinado esquema. Os nossos resultados salientam ainda a falta de validade da chamada self-reported hypertension e sugerem que em todos os doentes com suspeita de SAOS, com HA não diagnosticada e com um IMC e um PP acima de 27 kg/m2 e 39 cm, respectivamente, a confirmação do diagnóstico de HA deverá ser realizada através da MAPA, ao invés de outros métodos que com maior frequência são utilizados com este propósito. Os resultados obtidos no modelo animal de HA induzida pela CIH sugerem que o bloqueio do sistema nervoso simpático, juntamente com os supostos efeitos pleiotrópicos do CVD, não parece ser a estratégia mais adequada para reverter este tipo particular de hipertensão e indicam que as alterações farmacocinéticas induzidas pela CIH no ratio S/(R+S) não justificam a falta de eficácia anti-hipertensora do CVD observada neste modelo animal. Por último, os resultados do presente trabalho suportam ainda a viabilidade da utilização da administração oral voluntária, em alternativa à gavagem, para a administração crónica de uma dose fixa de fármacos anti-hipertensores.---------------------------- ABSTRACT: Hypertension (HT) is a highly prevalent condition, although under diagnosed, in patients with obstructive sleep apnea (OSA). These conditions are closely related and 24-hour ambulatory blood pressure monitoring (ABPM) seems to be the most accurate measurement for diagnosing hypertension in OSA. However, this diagnostic tool is expensive and time-consuming and, therefore, not routinely used. On the other hand, although continuous positive airway pressure (CPAP) is considered the gold standard treatment for symptomatic OSA, its lowering effect on blood pressure (BP) seems to be modest and, therefore, concomitant antihypertensive therapy is still required. Data on antihypertensive drug regimens in patients with OSA are scarce and specific therapeutic guidelines for the pharmacological treatment of hypertension in these patients remain absent. The use of animal models of CIH, which mimic the HT observed in patients with OSA, is extremely important since it is imperative to identify preferred compounds for an adequate BP control in this group of patients. However, studies aimed at investigating the antihypertensive effect of antihypertensive drugs in this animal model are insufficient, and most reports on CIH animal models in which drugs have been tested were not designed to respond to pharmacological issues. Moreover, when testing antihypertensive drugs (AHDs) it becomes crucial to ensure the selection of a non-invasive and stress-free method for drug delivery. Although gavage is effective and a widely performed technique for daily dosing in laboratory rodents, it comprises a sequence of potentially stressful procedures for laboratory animals that may constitute bias for the experimental results. The overall goal of the present translational research was to contribute to identify more effective AHDs for the treatment of hypertension in patients with OSA and investigate underlying mechanisms of systemic effects associated with OSA, as well as its modulation by AHDs. The specific aims were: first, to find new predictors based on anthropometric measures to identify patients that misclassify themselves as non-hypertensive, and thereby promote the selective use of ABPM; second, to investigate a hypothetical association between ongoing antihypertensive regimens and BP control rates in patients with OSA, before and after CPAP adaptation; third, to determine, in a rat model of CIH-induced hypertension, the efficacy of carvedilol (CVD), a nonselective beta-blocker with intrinsic anti-α1-adrenergic activity and antioxidant properties; fourth, to explore the effects of CIH on the pharmacokinetics profile of CVD and fifth, to investigate an alternative method to gavage, for chronic administration of AHDs to laboratory rats. For that, in the first phase of this project, we used a sizeable sample of patients with OSA (n=369), that attended a first visit at Centro Hospitalar Lisboa Norte, EPE Sleep Unit, and underwent overnight polysomnography, 24-h ABPM and filled a questionnaire that included ongoing antihypertensive medication profile registration. In the second phase, a rat experimental model of HT induced by a paradigm of CIH that simulates OSA was used. The main findings of this work were: first, body mass index (BMI) and neck circumference (NC) were identified as independent predictors of hypertension misclassification in patients suspected of OSA; second, in patients with OSA, BP control is independent of both the antihypertensive regimen and the number of antihypertensive drugs, either before or after CPAP adaptation; third, although the doses of 10, 30 and 50 mg/Kg of CVD promoted a significant reduction in heart rate, no decrease in mean arterial pressure was observed; fourth, the S/(R+S) ratios of CVD enantiomers, between rats exposed to CIH and normoxic conditions, were different and fifth, voluntary ingestion proved to be an effective method for a controlled daily dose administration, with a define timetable, that is independent of handling and restraint procedures. In conclusion, the clinical study showed that BP control in OSA patients is independent of both the antihypertensive regimen and the number of antihypertensive drugs. Additionally, our results highlight the lack of validity of self-reported hypertension and suggest that all patients suspected of OSA with undiagnosed hypertension and with a BMI and NC above 27 Kg/m2 and 39 cm should be screened for hypertension, through ABPM. The results attained in the rat model of HT related to CIH suggest that the blockade of the sympathetic nervous system together with the putative pleiotropic effects of carvedilol is not able to revert hypertension induced by CIH and point out that the pharmacokinetic changes induced by CIH on S/(R+S) ratio are not apparently responsible for the lack of efficacy of carvedilol in reversing this particular type of hypertension. Finally, the results here presented support the use of voluntary oral administration as a viable alternative to gavage for chronic administration of a fixed dose of AHDs.

Improving silicon probe performance through layer-by-layer coating

Fully comprehending brain function, as the scale of neural networks, will only be possi-ble with the development of tools by micro and nanofabrication. Regarding specifically silicon microelectrodes arrays, a significant improvement in long-term performance of these implants is essential. This project aims to create a silicon microelectrode coating that provides high-quality electrical recordings, while limiting the inflammatory response of chronic implants. To this purpose, a combined chitosan and gold nanoparticles coating was produced allied with electrodes modification by electrodeposition with PEDOT/PSS in order to reduce the im-pedance at 1kHz. Using a dip-coating mechanism, the silicon probe was coated and then charac-terized both morphologically and electrochemically, with focus on the stability of post-surgery performance in anesthetized rodents. Since not only the inflammatory response analysis is vital, the electrodes recording degradation over time was also studied. The produced film presented a thickness of approximately 50 μm that led to an increase of impedance of less than 20 kΩ in average. On a 3 week chronic implant, the impedance in-crease on the coated probe was of 641 kΩ, compared with 2.4 MΩ obtained for the uncoated probe. The inflammatory response was also significantly reduced due to the biocompatible film as proved by histological tests.