Investigating the role of glycine in astrocyte signalling using calcium imaging of rat primary cultures of cortical astrocytes

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Hoxb6 can interfere with somitogenesis in the posterior embryo through a mechanism independent of its rib-promoting activity

Formation of the vertebrate axial skeleton requires coordinated Hox gene activity. Hox group 6 genes are involved in the formation of the thoracic area owing to their unique rib-promoting properties. Here we show that the linker region (LR) connecting the homeodomain and the hexapeptide is essential for Hoxb6 rib-promoting activity in mice. The LR-defective Hoxb6 protein was still able to bind a target enhancer together with Pax3, producing a dominant-negative effect, indicating that the LR brings additional regulatory factors to target DNA elements. We also found an unexpected association between Hoxb6 and segmentation in the paraxial mesoderm. In particular, Hoxb6 can disturb somitogenesis and anterior-posterior somite patterning by dysregulation of Lfng expression. Interestingly, this interaction occurred differently in thoracic versus more caudal embryonic areas, indicating functional differences in somitogenesis before and after the trunk-to-tail transition. Our results suggest the requirement of precisely regulated Hoxb6 expression for proper segmentation at tailbud stages.

Caracterização experimental de uma bomba ejetora

A opção de futuro incide sobre os processos de combustão em regime pobre e com baixos índices de formação de poluentes. Neste contexto, o presente trabalho estuda um ejetor de um queimador de gás propano, que equipa uma marmita utilizada para confeção de alimentos no Exército Português, para compreender o mecanismo de arrastamento de ar, com vista a identificar as causas que possam contribuir para uma diminuição da razão de equivalência da mistura ar/combustível. Estes queimadores são dispositivos de funcionamento estritamente mecânico e de conceção simples que permitem uma gama de potências caloríficas compreendidas entre 8 e 22 kW. Permitem uma boa estabilidade de chama que é um requisito fundamental de segurança. A regulação de potência é executada através de uma válvula de ajuste, contendo três posições, uma em que a alimentação se encontra cortada e as outras duas permitindo funcionar na potência mínima e máxima. De modo a efetuar o estudo do ejetor, numa primeira fase, submeteu-se o sistema a uma caracterização experimental do campo de escoamentos à entrada de ar do ejetor, a partir da técnica de diagnóstico Particle Image Velocimetry (PIV). Assim avalia-se de que forma o escoamento de ar exterior influencia o comportamento do escoamento da mistura de ar/combustível à saída do queimador. Numa segunda fase, recorre-se à técnica de diagnóstico Quimiluminescência para medição de espécies químicas, para avaliar o comportamento do ejetor a alterações de vários parâmetros, tais como caudal de combustível, área de entrada de ar e área de saída da mistura do difusor. Conclui-se que o ejetor mostrase "insensível" a alterações da área de saída e de entrada de ar, em relação ao valor médio da razão de equivalência. Porém observa-se que as misturas de ar/combustível são mais homogéneas quando as áreas de entrada, da saída e da garganta têm valores próximos.

Sistemas urbanos de drenagem sustentáveis

O desenvolvimento e a expansão dos núcleos urbanos conduziram a um aumento das taxas de impermeabilização do solo. Em termos de drenagem urbana, este processo é bastante nefasto, visto que reduz a capacidade de infiltração da água pluvial no solo, aumentando o volume de escoamento superficial, e reduzindo os tempos de concentração, provocando um agravamento nos caudais de ponta. A consequência direta dos fenómenos apontados é a ocorrência de inundações, causadas pela falta de capacidade dos sistemas de drenagem face aos novos caudais de ponta decorrentes das novas urbanizações. Para prevenir estas situações podem implementar-se duas soluções distintas: reforçar a capacidade dos sistemas de drenagem existentes (solução bastante dispendiosa e nem sempre exequível) ou implementar soluções de sistemas de drenagem de águas pluviais alternativos. Esta dissertação apresenta uma forma alternativa de gestão das águas pluviais designada de Sistemas Urbanos de Drenagem Sustentáveis (SUDS), que através de técnicas de controlo na origem (soluções que favorecem a infiltração da água pluvial no solo ou aumentam a capacidade de retenção/armazenamento da água pluvial), reduzem o caudal de ponta e, consequentemente, o risco de inundação.

Bioaumento aplicado ao controlo dos níveis de amoníaco em aviários

Este projecto teve como principal objectivo desenvolver um estudo à escala real e laboratorial com o intuito de testar a viabilidade da suspensão biológica comercial JASS para minorar o problema da ocorrência de elevadas concentrações de amoníaco atmosférico no interior dos aviários. Pretendeu-se, assim, estudar a vantagem da utilização de microrganismos com capacidade de degradar o amoníaco ou inibir a sua formação, fixando os compostos azotados na cama de aviário e reduzindo a sua emissão atmosférica. Para o efeito, realizaram-se diversos ensaios à escala laboratorial e real numa instalação industrial, de forma a testar a eficácia da suspensão biológica. Os ensaios laboratoriais consistiram na determinação da influência da aplicação da suspensão biológica na libertação de amoníaco em cama de aviário colocada em reactor fechado ou aberto e numa coluna que permitiu a quantificação do fluxo de amoníaco libertado a diferentes cotas. Noutro conjunto de experiências, procurou-se estudar a caracterização da suspensão biológica essencialmente por técnicas de coloração e de ensaios bioquímicos. Finalmente, avaliou-se a eficácia da suspensão na inibição da transformação da ureia pela enzima urease produzida pelo microrganismo Proteus vulgaris. Os ensaios à escala real decorreram nas instalações de Leiria da Ovopor - Agro-Pecuária dos Milagres, S.A., com uma área de 12500m2. Realizaram-se ensaios em dois ciclos de criação, um com 30970 pintos em que não foi aplicada a suspensão em estudo e outro com 28470 pintos onde foi feita a aplicação da suspensão, como forma de validar as vantagens de aplicação do produto desenvolvido. Durante estes ensaios foi feita a monitorização das condições ambientais, da concentração e do caudal mássico de amoníaco amostrado, bem como de alguns parâmetros caracterizadores da produtividade, como sejam o peso dos animais e a mortalidade. Apesar de não ter sido possível tirar conclusões esclarecedoras quanto à eficiência da suspensão nos estudos comparativos à escala laboratorial, quer nos ensaios em reactor fechado, quer com circulação de ar, observou-se uma redução da emissão de amoníaco de cerca de 13% no estudo da determinação do fluxo de amoníaco a diferentes cotas nas experiências em que se usou a mesma cama de aviário (redução do fluxo mássico total de 32,67 mgNH3/ (m2.h) para 30,68 mgNH3/ (m2.h). Relativamente ao ensaio de campo na recria da Ovopor verificou-se uma clara redução na emissão de amoníaco na segunda campanha, redução essa que foi particularmente relevante nas primeiras quatro semanas de criação, que correspondem ao período em que as condições de ambas as campanhas são praticamente iguais, uma vez que não se promove a ventilação da sala de recria. Constatou-se ainda que os valores mais elevados de amoníaco atmosférico registados foram de 8,2 e 4,8 ppm na 1ª e 2ª campanhas, respectivamente, que correspondem a valores claramente abaixo do valor máximo admissível previsto na legislação nacional (20ppm). Quanto à composição da suspensão biológica JASS verificou-se que é essencialmente composta por esporos de Bacillus sp. e que a germinação destes esporos é favorecida quando a suspensão é diluída. Por fim, através do estudo em meio de cultura com composição controlada, em que foi feita a avaliação da capacidade da suspensão biológica estudada inibir o processo de formação de amoníaco, confirmou-se que esta inibiu a actividade da enzima urease produzida pelo microorganismo Proteus vulgaris e, consequentemente, houve uma redução de cerca de 47% na capacidade de formação de amoníaco comparando o desempenho dos dois reactores aos quais foram adicionados este microrganismo. Em suma, pode-se pois concluir que, com excepção dos resultados dos estudos de determinação do amoníaco libertado realizados à escala laboratorial, todas as restantes experiências indiciam que a suspensão JASS estudada tem capacidade para inibir a formação de amoníaco e, consequentemente, a sua libertação, provando a utilidade do produto na melhoria das condições ambientais dos aviários.

Compression Loading Induced Cellular Stress Response of Intervertebral Disc Cells in Organ Culture

Introduction: Mechanical stress is often associated to interverterbal disc (IVD) degeneration and the effect of mechanical loading on IVD has been studied and reviewed.1,2 Previously, expression of heat shock proteins, HSP70 and HSP27 has been found in pathological discs.3 However, there is no direct evidence on whether IVD cells respond to the mechanical loading by expression of HSPs. The objective of this study is to investigate the stress response of IVD cells during compressive loading in an organ culture. Materials and Methods: Fresh adult bovine caudal discs were cultured with compressive loading applied at physiological range. Effect of loading type (static and dynamic) and repeated loading (2 hours per day for 2 days) were studied. Nucleus pulposus (NP) and annulus fibrosus (AF) of the IVD were retrieved at different time points: right after loading and right after resting. Positive control discs were heat shocked (43°C). Cell activity was assessed and expression of stress response genes (HSP70 and HSF1) and matrix remodeling genes (ACAN, COL2, COL1, ADAMTS4, MMP3 and MMP13) were studied. Results: Cell activity was maintained in all groups. Both NP and AF expressed high level of HSP70 in heat shock groups, confirming their expression in response to stress. In NP, expression of HSP70 was up-regulated after static loading and dynamic loading with higher fold change was observed after static loading. During repeated loading, HSP70 appeared to be upregulated right after loading and decreased after resting. Such trend was not observed in AF and HSF1 levels. Expressions of matrix remodeling genes did not change significantly with loading except ADAMTS4 decreased in AF during static loading. Conclusion: This study demonstrated that NP cells upregulate expression of HSP70 in response to loading induced stress without changing cell activity and matrix remodeling significantly. Acknowledgments: This project was funded by AO Spine (AOSPN) (grant number: SRN_2011_14) and a fellowship exchange award by AO Spine Scientific Research Network (SRN).

Apoptosis in experimental cerebral malaria: spatial profile of cleaved caspase-3 and ultrastructural alterations in different disease stages

Cerebral malaria (CM) is associated with high mortality and morbidity as a certain percentage of survivors suffers from persistent neurological sequelae. The mechanisms leading to death and functional impairments are yet not fully understood. This study investigated biochemical and morphological markers of apoptosis in the brains of mice infected with Plasmodium berghei ANKA. Cleaved caspase-3 was detected in the brains of animals with clinical signs of CM and immunoreactivity directly correlated with the clinical severity of the disease. Caudal parts of the brain showed more intense immunoreactivity for cleaved caspase-3. Double-labelling experiments revealed processing of caspase-3 primarily in neurons and oligodendrocytes. These cells also exhibited apoptotic-like morphological profiles in ultrastructural analysis. Further, cleavage of caspase-3 was found in endothelial cells. In contrast to neurons and oligodendrocytes, apoptosis of endothelial cells already occurred in early stages of the disease. Our results are the first to demonstrate processing of caspase-3 in different central nervous system cells of animals with CM. Apoptosis of endothelial cells may represent a critical issue for the development of the disease in the mouse model. Neurological signs and symptoms might be attributable, at least in part, to apoptotic degeneration of neurons and glia in advanced stages of murine CM.

Guía de campo para la determinación de tiburones en la pesca artesanal del Perú

Describe 32 especies de tiburones que son capturados por la flota artesanal y comercializados a lo largo de todo nuestro litoral. Además contiene, una sección de términos técnicos usados, caracteres morfológicos externos diferenciales más relevantes, esquemas de la parte ventral de la cabeza, esquemas de dientes y fotos de cada una de las diferentes especies mencionadas. Para la veri cación de algunas características morfológicas como la forma del hocico, posición de la aleta dorsal con respecto a las pectorales, presencia de cresta interdorsal, forma de la aleta caudal, entre otras; se revisaron muestras biológicas de tiburones que se encuentran depositadas en la Colección Cientí ca Ictiológica del IMARPE.

Wnt/β-catenin Signaling Regulates Regeneration in Diverse Tissues of the Zebrafish

Thesis (Ph.D.)--University of Washington, 2016-06

A new crocodilian from the Lower Cretaceous Crato Formation of north-eastern Brazil

A new mesosuchian crocoddian from the Nova Olinda Member of the Crato Formation (Lower Cretaceous, Aptian) of north-eastern Brazil is described. Susisuchus anatoceps gen. et sp. nov. is the first crocodillan to be reported from this formation. It is represented by an incomplete, partially articulated skeleton: the skull and mandible, partial postcranial axial skeleton, forelimbs and portions of the osteodermal skeleton. Preservation of soft tissues includes the skin surrounding both forelimbs and the digits of the right hand. The state of preservation of the specimen suggests that it was incorporated into the basin as a desiccated carcass. Susisuchus anatoceps is one of the oldest crocodilians with a eusuchian-type dorsal shield, comprising a tetraserial paravertebral shield and, either side of this, two sagittal rows of accessory osteoderms. It also possesses amphicoelous thoracic, lumbar and caudal vertebrae. This combination of postcranial features have never before been seen in a crocodilian and warrant the erection of a new family within Mesosuchia: Susisuchidae. Taxonomically, S. anatoceps is similar to a number of Lower Cretaceous mesosuchians previously considered to have given rise to eusuchians, most notably the Glen Rose crocodilian and a new, but as yet undescribed crocodillan from the Lower Cretaceous Winton Formation of western Queensland, Australia. Preliminary preparation of the Winton crocodilian indicates that it may belong to Susisuchidae, supporting the hypotheses of interchange between the vertebrate faunas of South America and Australia during the Lower Cretaceous.

Moravecia australiensis n. g., n. sp (Dracunculoidea : Guyanemidae) from the gills of the green porcupine fish Tragulichthys jaculiferus (Cuvier) in Australia

A new dracunculoid genus and species, Moravecia australiensis, is described from gill-filaments of the green porcupine fish Tragulichthys jaculiferus (Cuvier) (Tetraodontiformes: Diodontidae) from Moreton Bay, Queensland, Australia. Abundant mobile larvae and a few adult males with females occurred in the gill-filament between the epithelial basement membrane and efferent artery. Gills of all 69 fish examined contained larvae. Eleven harboured adult nematodes of a previously undescribed species belonging to the family Guyanemidae. The new species is placed within a newly proposed genus because it differs from the four existing genera in the family in possessing fine cuticular transverse striations, two forward protruding cephalic elevations, a circumoral elevation, a small triangular mouth surrounded by six cephalic papillae arranged in two lateral clusters of three each and a pair of large lateral amphids. Males have two pairs of pedunculate caudal papillae supporting the caudal alae. A key to the genera of the Guyanemidae is presented.

Visual thalamocortical projections in the flying fox: Parallel pathways to striate and extrastriate areas

We studied thalamic projections to the visual cortex in flying foxes, animals that share neural features believed to resemble those present in the brains of early primates. Neurones labeled by injections of fluorescent tracers in striate and extrastriate cortices were charted relative to the architectural boundaries of thalamic nuclei. Three main findings are reported: First, there are parallel lateral geniculate nucleus (LGN) projections to striate and extrastriate cortices. Second, the pulvinar complex is expansive, and contains multiple subdivisions. Third, across the visual thalamus, the location of cells labeled after visual cortex injections changes systematically, with caudal visual areas receiving their strongest projections from the most lateral thalamic nuclei, and rostral areas receiving strong projections from medial nuclei. We identified three architectural layers in the LGN, and three subdivisions of the pulvinar complex. The outer LGN layer contained the largest cells, and had strong projections to the areas V1, V2 and V3. Neurones in the intermediate LGN layer were intermediate in size, and projected to V1 and, less densely, to V2. The layer nearest to the origin of the optic radiation contained the smallest cells, and projected not only to V1, V2 and V3, but also, weakly, to the occipitotemporal area (OT, which is similar to primate middle temporal area) and the occipitoparietal area (OP, a third tier area located near the dorsal midline). V1, V2 and V3 received strong projections from the lateral and intermediate subdivisions of the pulvinar complex, while OP and OT received their main thalamic input from the intermediate and medial subdivisions of the pulvinar complex. These results suggest parallels with the carnivore visual system, and indicate that the restriction of the projections of the large- and intermediatesized LGN layers to V1, observed in present-day primates, evolved from a more generalized mammalian condition. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

Homology of fin lepidotrichia in osteichthyan fishes

Lepidotrichia are dermal elements located at the distal margin of osteichthyan fins. In sarcopterygians and actinopterygians, the term has been used to denote the most distal bony hemisegments and also the more proximal, scale-covered segments which overlie endochondral bones of the fin. In certain sarcopterygian fishes, including the Rhizodontida, these more proximal, basal segments are very long, extending at least half the length of the fin. The basal segments have a subcircular cross section, rather than the crescentic cross section of the distal lepidotrichial hemisegments, which lack a scale cover and comprise short, generally regular, elements. In rhizodonts and other sarcopterygians, e.g. Eusthenopteron, the basal elements are the first to appear during fin development, followed by the endochondral bones and then the distal lepidotrichia. This sequence contradicts the 'clock-face model' of fin development proposed by Thorogood in which the formation of endochondral bones is followed by development of lepidotrichia. However, if elongate basal 'lepidotrichia' are not homologous with more distal, jointed lepidotrichia and if the latter form within a distal fin-fold and the former outside this fold, then Thorogood's 'clock-face' model remains valid. This interpretation might indicate that the fin-fold has been lost in early digited stem-tetrapods such as Acanthostega and Ichthyostega and elongate basal elements, but not true lepidotrichia, occur in the caudal fins of these taxa.

Target tissue influences the peripheral trajectory of mouse primary sensory olfactory axons

Primary olfactory neurons situated in the nasal septum project axons within fascicles along a highly stereotypical trajectory en route to the olfactory bulb. The ventral fascicles make a distinct dorsovental turn at the rear of the septum so as to reach the olfactory bulb. In the present study we have used a brain and nasal septum coculture system to examine the role of target tissue on the peripheral trajectory of olfactory sensory axons. In cultures of isolated embryonic nasal septa, olfactory axons form numerous parallel fascicles that project caudally in the submucosa, as they do in vivo. The ventral axon fascicles in the septum, however, often fail to turn, and do not project dorsally towards the roof of the nasal cavity. The presence of olfactory bulb, cortical, or tectal tissue apposed to the caudal end of the septum rescued this phenotype, causing the ventral fascicles to follow a normal in vivo-like trajectory. Ectopic placements of the explants revealed that brain tissue is not tropic for olfactory axons but appears to maintain the peripheral trajectory of growing axons in the nasal septum. Although primary olfactory axons are able to penetrate into olfactory bulb in vitro, they only superficially enter cortical tissue, whereas they do not grow into tectal explants. The ability of axons to differentially grow into different brain regions was shown to be unrelated to the migratory behavior of olfactory ensheathing cells, indicating that olfactory axons are directly responsive to guidance cues in the brain. (C) 2004 Wiley Periodicals, Inc.

Suspected fibrocartilaginous embolism in a cat

A 12-year-old cat was presented to the University of Queensland's Small Animal Teaching Hospital with a 1-day history of left herniparesis of acute onset, with no evidence of trauma or toxin exposure. Neurological examination findings were consistent with a lesion in the caudal left cervical spinal cord (C6 to C8), which was non-painful and had not progressed since the onset of clinical signs. No other abnormalities were found, although myelography showed a mild swelling involving the caudal cervical and cranial thoracic spinal segments. A diagnosis of suspected fibrocartilaginous embolism was made on the basis of the history, clinical presentation and diagnostic tests results, making this case the first report of a suspected fibrocartilaginous embolism in a cat that returned to normal function.