Genomic screen for genes involved in mammalian craniofacial development

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes. (C) 2003 Wiley-Liss, Inc.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs. (C) 2002 Wiley-Liss, Inc.

Dissecting the role of CLASP1 in the mammalian development

A mitose é o evento celular, através do qual uma células transmite uma cópias do seu DNA às células filhas. Este processo é mediado pelo fuso mitótico, o qual consiste numa rede bipolar microtubulos. A dinâmica dos microtubulos é regulada por proteínas associadas a estes (MAPs – Microtubule-Associated Proteins), tais como as proteínas associadas às extremidades positivas dos microtubulos (+TIPs – Plus-ends Tracking proteins). As proteínas associadas às CLIPs (CLASPs – CLIP-associated proteins) pertencem a esta família e estão altamente conservadas nos eucariotas. Estas interagem com os microtubulos regulando o fuso mitótico, a segregação dos cromossomas e o comportamento dos microtubulos ao nível do cinetocoro. Assim, as CLASPs têm sido descritas como essenciais à manutenção da integridade genética durante a divisão celular. Um modelo animal knockout para o gene Clasp1 é uma ferramenta indispensável à descoberta do papel da CLASP1 a nível fisiológico. Nos animais knockout foi observado um fenótipo letal, no qual 100% dos recém-nascidos morreram poucos minutos após o nascimento, no decurso de falência respiratória. Após análise histopatológica, observamos que os pulmões dos animais knockout apresentam um atraso no desenvolvimento. Porém, a análise da expressão de marcadores de diferenciação celular, mostrou que os pneumócitos tipo I e II estão presente e diferenciados nos animais knockout aquando do seu nascimento. No entanto, um defeito primário a nível pulmonar ainda não pode ser excluído. Níveis elevados de glicogénio no parênquima alveolar dos animais knockout sugerem imaturidade pulmonar ou deficiente produção do líquido surfactante. Adicionalmente, ainda não está esclarecido de que forma pode este atraso explicar a letalidade observada nos recémnascidos knockout. Verificamos também que expressão de CLASP1 é transiente ao longo do desenvolvimento, sendo particularmente elevada no cérebro, o que pode explicar o seu papel já descrito na biologia dos neurónios. A CLASP1 é ubiquamente expressa em mamíferos adultos, o que sugere que esta proteína é também importante em tecidos diferenciados. Nesta fase, o significado biológico da CLASP1 em mamíferos ainda não foi descortinado. No entanto, nenhum animal knockout para Clasp1 foi capaz de sobreviver ex uterus, o que sugere um papel fundamental desta proteína na fase final do desenvolvimento dos mamíferos.

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Mismatch negativity in childhood temporal lobe epilepsy: a proposed paradigm for testing central auditory processing

Background: Temporal lobe epilepsy (TLE) is a neurological disorder that directly affects cortical areas responsible for auditory processing. The resulting abnormalities can be assessed using event-related potentials (ERP), which have high temporal resolution. However, little is known about TLE in terms of dysfunction of early sensory memory encoding or possible correlations between EEGs, linguistic deficits, and seizures. Mismatch negativity (MMN) is an ERP component – elicited by introducing a deviant stimulus while the subject is attending to a repetitive behavioural task – which reflects pre-attentive sensory memory function and reflects neuronal auditory discrimination and perceptional accuracy. Hypothesis: We propose an MMN protocol for future clinical application and research based on the hypothesis that children with TLE may have abnormal MMN for speech and non-speech stimuli. The MMN can be elicited with a passive auditory oddball paradigm, and the abnormalities might be associated with the location and frequency of epileptic seizures. Significance: The suggested protocol might contribute to a better understanding of the neuropsychophysiological basis of MMN. We suggest that in TLE central sound representation may be decreased for speech and non-speech stimuli. Discussion: MMN arises from a difference to speech and non-speech stimuli across electrode sites. TLE in childhood might be a good model for studying topographic and functional auditory processing and its neurodevelopment, pointing to MMN as a possible clinical tool for prognosis, evaluation, follow-up, and rehabilitation for TLE.

Temporal lobe epilepsy in childhood – a study model of auditory processing

TLE in infancy has been the subject of varied research. Topographical and structural evidence is coincident with the neuronal systems responsible for auditory processing of the highest specialization and complexity. Recent studies have been showing the need of a hemispheric asymmetry for an optimization in central auditory processing (CAP) and acquisition and learning of a language system. A new functional research paradigm is required to study mental processes that require methods of cognitive-sensory information analysis processed in very short periods of time (msec), such as the ERPs. Thus, in this article, we hypothesize that the TLE in infancy could be a good model for topographic and functional study of CAP and its development process, contributing to a better understanding of the learning difficulties that children with this neurological disorder have.

Contributions to the Eocene palaeontology and stratigraphy of Beira Alta, Portugal II - New Late Eocene mammalian remnants from Coja (Portugal) and the presence of Palaeotherium magnum Cuvier

The presence of Palaeotherium magnum in the fauna from Coja is recorded. It is well in agreement with the earlier reporting to the Montmartre level from the Ludian stage. Field data as well as compatibility with the remaining taxa and the identical fossilization of all the specimens indicate that all the vertebrate fossils come from the same horizon in the lithostratigraphic unit "Arcoses de Côja".

Auditory event-related potentials in children with benign epilepsy with centro-temporal spikes

Benign focal epilepsy in childhood with centro-temporal spikes (BECTS) is one of the most common forms of idiopathic epilepsy, with onset from age 3 to 14 years. Although the prognosis for children with BECTS is excellent, some studies have revealed neuropsychological deficits in many domains, including language. Auditory event-related potentials (AERPs) reflect activation of different neuronal populations and are suggested to contribute to the evaluation of auditory discrimination (N1), attention allocation and phonological categorization (N2), and echoic memory (mismatch negativity – MMN). The scarce existing literature about this theme motivated the present study, which aims to investigate and document the existing AERP changes in a group of children with BECTS. AERPs were recorded, during the day, to pure and vocal tones and in a conventional auditory oddball paradigm in five children with BECTS (aged 8–12; mean = 10 years; male = 5) and in six gender and age-matched controls. Results revealed high amplitude of AERPs for the group of children with BECTS with a slight latency delay more pronounced in fronto-central electrodes. Children with BECTS may have abnormal central auditory processing, reflected by electrophysiological measures such as AERPs. In advance, AERPs seem a good tool to detect and reliably reveal cortical excitability in children with typical BECTS.

The development of the N1 and N2 components in auditory oddball paradigms: a systematic review with narrative analysis and suggested normative values

Auditory event-related potentials (AERPs) are widely used in diverse fields of today’s neuroscience, concerning auditory processing, speech perception, language acquisition, neurodevelopment, attention and cognition in normal aging, gender, developmental, neurologic and psychiatric disorders. However, its transposition to clinical practice has remained minimal. Mainly due to scarce literature on normative data across age, wide spectrumof results, variety of auditory stimuli used and to different neuropsychological meanings of AERPs components between authors. One of the most prominent AERP components studied in last decades was N1, which reflects auditory detection and discrimination. Subsequently, N2 indicates attention allocation and phonological analysis. The simultaneous analysis of N1 and N2 elicited by feasible novelty experimental paradigms, such as auditory oddball, seems an objective method to assess central auditory processing. The aim of this systematic review was to bring forward normative values for auditory oddball N1 and N2 components across age. EBSCO, PubMed, Web of Knowledge and Google Scholarwere systematically searched for studies that elicited N1 and/or N2 by auditory oddball paradigm. A total of 2,764 papers were initially identified in the database, of which 19 resulted from hand search and additional references, between 1988 and 2013, last 25 years. A final total of 68 studiesmet the eligibility criteria with a total of 2,406 participants from control groups for N1 (age range 6.6–85 years; mean 34.42) and 1,507 for N2 (age range 9–85 years; mean 36.13). Polynomial regression analysis revealed thatN1latency decreases with aging at Fz and Cz,N1 amplitude at Cz decreases from childhood to adolescence and stabilizes after 30–40 years and at Fz the decrement finishes by 60 years and highly increases after this age. Regarding N2, latency did not covary with age but amplitude showed a significant decrement for both Cz and Fz. Results suggested reliable normative values for Cz and Fz electrode locations; however, changes in brain development and components topography over age should be considered in clinical practice.

In vitro studies of gum arabic-coated magnetic nanoparticles with mammalian cell cultures

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Neural mechanisms of stimulus generalization in auditory fear conditioning

Dissertation presented to obtain the Ph.D degree in Neuroscience Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

Functional MRI of stimulus interference on auditory processing in normal listeners and tinnitus patients

We are constantly immersed in stimuli. Upon reaching our senses, stimuli are processed within various brain systems along various pathways into the brain, and eventually turned into a percept. However, there are percepts that do not result from responses to external source stimuli. A particular case of this situation is the auditory percept known as tinnitus. Tinnitus can be seen as a task-irrelevant auditory percept, commonly reported to interfere with normal daily tasks. This is known from reports made by tinnitus sufferers that refer to their phantom percept as distracting, and that it diverts their focus from the task-relevant stimuli.(...)

Haematophagic behavior in laboratory of Lutzomyia cruzi (Mangabeira) (Diptera: Psychodidae) in relation to three mammalian blood sources in Manaus, Brazil

The sand fly Lutzomyia cruzi is considered as one of vectors of visceral leishmaniasis in Brazil. This work examined optimum feeding age, feeding time, host preference, fecundity rates, and female blood meal volume taken by single females from a closed colony of L. cruzi. Mean feeding time was longer on hamsters, 6.6 minutes, than on humans, 5.7 minutes. 49.1% of the 48h-old flies fed on humans and 43.3% of 72h-old flies fed on hamsters. Of a total of 120 females, 61% fed on humans and 25% fed on hamsters. Total fecundity was significantly higher in females fed on hamster than on human or opossum. Laboratory-reared L. cruzi females fed earlier, more promptly, and preferably on humans than on hamsters when offered these blood-meal sources simultaneously. The blood-meal volume is higher in females fed on hamsters than other hosts (human and opossum).