Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Estudo do galgamento em estruturas marítimas verticais utilizando o modelo numérico AMAZON

O galgamento oceânico é um fenómeno associado à interação de ondas com estruturas marítimas. Nesta dissertação estudou-se o galgamento em estruturas marítimas verticais através da aplicação do modelo numérico AMAZON, que é um modelo bidimensional, baseado na resolução das equações não lineares de águas pouco profundas. Para validar a sua aplicação a estruturas marítimas verticais impermeáveis, os resultados de caudal médio galgado obtidos com o modelo AMAZON foram comparados com os resultados de ensaios em modelo físico bidimensional realizados anteriormente e disponíveis na base de dados do projeto europeu CLASH. Os resultados foram ainda comparados com os das fórmulas empíricas e semi-empíricas de Van der Meer & Bruce de 2014, de Goda de 2008 e de Hedges & Reis de 2015. Antes da sua validação, o modelo foi calibrado utilizando um dos ensaios do CLASH. Para tal, realizou-se uma análise de sensibilidade a alguns parâmetros do modelo, concretamente da dimensão mínima da malha, da escala de aplicação do AMAZOM (à escala reduzida ou à do protótipo), da forma de modelação da parede vertical, já que, dado este modelo ser integrado na vertical, não é possível considerar paredes verticais sem utilizar um artifício de cálculo, dos valores de alguns dos parâmetros de entrada do modelo (profundidade mínima molhada e a profundidade mínima de fricção) e da dimensão do domínio de cálculo. Os resultados da calibração do AMAZON mostram que, por causa da precisão do modelo para os menores caudais médios de galgamento, se deve aplicar o modelo à escala do protótipo. Para estruturas verticais, a aproximação que o modelo faz, por defeito, para a secção vertical da estrutura através de um talude fictício a 10:1 apresenta melhores resultados que a utilização de um talude a 20:1. Contudo, ambos requerem a adoção de uma dimensão da malha bastante reduzida nesta zona e na sua vizinhança (0.03 m). No caso em estudo, os dois parâmetros de entrada do modelo não têm impacto significativo nos resultados, ao contrário do domínio de cálculo que, tal como recomendado na literatura de referência, se deve iniciar (fronteira de entrada) a aproximadamente um comprimento de onda do pé da estrutura. Os resultados obtidos mostram que, em geral, o AMAZON subestima o valor medido do caudal médio galgado com erros relativos variáveis entre -91% e -49%; apenas para um caso o modelo sobrestima o caudal médio, com um erro relativo de 43%. Quando comparado com as formulações empíricas e semi-empíricas, a fórmula de Van der Meer & Bruce apresenta melhores aproximações aos caudais médios medidos que constam na base de dados do CLASH, seguida da fórmula de Goda e dos resultados do modelo AMAZON, embora estes dois últimos apresentem valores bastante próximos. Assim, uma vez que o modelo está em constante desenvolvimento, espera-se que no futuro seja uma ferramenta mais poderosa. Contudo, enquanto não se fizerem melhorias no modelo e/ou não se estender a outros ensaios a análise feita no âmbito desta dissertação, os resultados sugerem que não há vantagem na sua utilização face às formulações empíricas e semi-empíricas disponíveis, tendo também em conta o significativo tempo computacional exigido para executar o AMAZON com a dimensão da malha escolhida.

The Teggiolo zone: a key to the Helvetic-Penninic connection (stratigraphy and tectonics in the Val Bavona, Ticino, Central Alps)

The Teggiolo zone is the sedimentary cover of the Antigorio nappe, one of the lowest tectonic units of the Penninic Central Alps. Detailed mapping, stratigraphic and structural analyses, and comparisons with less metamorphic series in several well-studied domains of the Alps, provide a new stratigraphic interpretation. The Teggiolo zone is comprised of several sedimentary cycles, separated by erosive surfaces and large stratigraphic gaps, which cover the time span from Triassic to Eocene. At Mid-Jurassic times it appears as an uplifted, partially emergent block, marking the southern limit of the main Helvetic basin (the Limiting South-Helvetic Rise LSHR). The main mass of the Teggiolo calcschists, whose base truncates the Triassic-Jurassic cycles and can erode the Antigorio basement, consists of fine-grained clastic sediments analogous to the deep-water flyschoid deposits of Late Cretaceous to Eocene age in the North-Penninic (or Valais s.l.) basins. Thus the Antigorio-Teggiolo domain occupies a crucial paleogeographic position, on the boundary between the Helvetic and Penninic realms: from Triassic to Early Cretaceous its affinity is with the Helvetic; at the end of Cretaceous it is incorporated into the North-Penninic basins. An unexpected result is the discovery of the important role played by complex formations of wildflysch type at the top of the Teggiolo zone. They contain blocks of various sizes. According to their nature, three different associations are distinguished that have specific vertical and lateral distributions. These blocks give clues to the existence of territories that have disappeared from the present-day level of observation and impose constraints on the kinematics of early folding and embryonic nappe emplacement. Tectonics produced several phases of superimposed folds and schistosities, more in the metasediments than in the gneissic basement. Older deformations that predate the amplification of the frontal hinge of the nappe generated the dominant schistosity and the km-wide Vanzèla isoclinal fold.

New Sorex araneus karyotypes from Germany and the postglacial recolonization of central Europe

New G-banded karyotypes from populations of the common shrew Sorex araneus Linnaeus, 1758 provide a clearer picture of the distribution of chromosome races in central Europe. As expected according to their occurrence in neighbouring countries, the Jutland (kq, no), Laska (k/o) and Drnholec (ko, nr) races are also found in Germany. A new chromosome race "Rugen" (kq) is described from this Baltic Island. Together with the previously recorded races Ulm and Mooswald (kr), six chromosome races are now known from Germany. The resulting distribution pattern is characterized by high frequencies of different race-specific metacentrics at the periphery of the country and clines with decreasing frequencies towards the centre which is occupied by the Ulm race. This race is acrocentric for all chromosome arms involved in the observed race-specific fusions and represents a buffer between the surrounding, more metacentric races. According to the present distribution of these metacentrics, a scenario for the postglacial recolonization of central Europe by S. araneus populations on three different routes is proposed: from the east along the northern slopes of the Carpathian Arc, from the south-east along the Danube Valley and from the south-west through the Upper Rhine Valley.

Características generales de las embarcaciones pesqueras de la zona central del litoral peruano: Puerto Chico (10º45' L.S.) Laguna Grande (14º 08' L.S.)

Informe técnico sobre embarcaciones pesqueras, el cual abarca la zona centro del litoral peruano, entre las localidades pesqueras de río Chico - laguna Grande, cumpliendo así con el desarrollo de la primera línea de investigación

Biología reproductiva del Potoyunco peruano Pelecanoides Garnotii en isla La Vieja, Costa Central del Perú

Se encontraron adultos reproductivos durante todo el año; sin embargo, se identificaron dos picos reproductivos. El primero, más sincrónico, ocurre en los meses de verano entre diciembre y marzo; el segundo, menos marcado, ocurre en invierno entre mayo y setiembre. La duración del período de incubación se ha estimado en 50 a 60 días. Los pichones que nacen requieren de 70 a 80 días para completar el desarrollo. Se observaron tasas de crecimiento mayores durante los meses de invierno, debido probablemente, a la calidad diferencial en el alimento. En invierno, los potoyuncos consumen un porcentaje elevado de larvas de peces, en tanto que en verano se presenta un alto consumo de crustáceos planctónicos. Durante 1997, año anormal donde se inició un evento El Niño de fuerte intensidad, el primer pico reproductivo se realizó de manera normal, observándose una clara disminución en lo que respecta al segundo pico reproductivo. La proporción de adultos con huevo en mayo de 1997, bajó de 45,25% a 17,5% en julio y la proporción de pichones disminuyó de 15% en mayo y julio a menos del 5% en setiembre, sugiriendo un bajo éxito de eclosión en los huevos y una elevada mortalidad de pichones. Un censo realizado en noviembre de 1997 permitió estimar una población de 12.800 parejas y no difiere de las estimaciones de años anteriores. Durante el desarrollo de este censo el 38,41% de los nidos monitoreados contenían adultos con huevos, de los cuales el 66,27% eran adultos previamente anillados.

Estudios de la edad y crecimiento de la Sardina Sardinops sagax sagax de la region central del Perú

En el presente trabajo se estima la edad y crecimiento de sardina, sardinops Sagax Sagax en la región central del Perú durante 1980, mediante la lectura directa de otolitos por contaje de anillos de crecimiento y por retrocálculo en las medidas de las mismas estructuras. Para determinar la edad, se ha seguido la evolución de la naturaleza del borde del otolito, así como también se realizaron mediciones de cada anillo de crecimiento y se estableció la proporcionalidad entre el incremento en longitud del otolito (R) y del pez (L), resultando la ecuación siguiente: L = - 1.032 + 15.174 R Las expresiones que describen el crecimiento en longuitud según el modelo de Van Bertalanffy, se señalan a continuación: Por contaje de anillos: L = 39.8 [1 - e-.22 (t+0.84) ] Por retrocálculo: L = 37.49 [1 - e-.22(t+ o.71)] La relación de la edad con las tallas de captura determinan que la pesquería de consumo durante 1980, estuvo constituida mayormente por ejemplares adultos de 4, 5 y 6 años de edad; análogamente en la pesquería industrial predominaron los individuos jóvenes (edades O, 1 y 2).

La recolonización de las comunidades de Mitílidos en la costa central del Perú de El Niño 1982-83

Se estudia la recuperación de los bancos de los mitílidos Aulacomya ater y Semimytilus algosus después de agosto de 1983 en la Bahía de Ancón y después de agosto de 1986 en Bahía Independencia. En la Bahía de Ancón la recolonización de Semimytilus algosus se inició en octubre de 1983, sostenida por una intensiva proliferación de larvas en el plancton. En febrero de 1984 alcanz.aban una máxima longitud de 31mm y los otros constituyentes de la comunidad estaban completando su recuperación. Sin embargo, hasta marzo de 1987 no habían evidencias de una recuperación de A. ater. En Bahía Independencia hubo una recuperación de S. algosus en el nivel intermareal. En agosto de 1986 recién encontramos una recolonización simultánea de ambos mitílidos en los rizoides de las algas pardas (Macrocystis pyrifera y Lessonia spp.). En los meses posteriores las algas pardas extendieron su distribución vertical de los 6 m hasta más de 12 m de profundidad y junto con ellos también A. ater. Se discute los mecanismos y procesos que estarían implicados en la diferente recolonización de las comunidades de mitílidos en las orillas y fondos rocosos de las dos áreas de estudio.

Spurious hyperphosphatemia in a patient with alteplase-locked central venous catheter.

Alteplase has been shown to be effective in preventing central venous access clotting in patients on hemodialysis. Because of a high phosphorus content in its excipient, it can inadvertently contaminate blood samples, leading the physician in care of the patient to erroneously increase dialysis time or change diet in order to control the pseudo-hyperphosphatemia.

Neuromodulation by oxytocin and vasopressin in the central nervous system as a basis for their rapid behavioral effects.

The last several years have seen an increasing number of studies that describe effects of oxytocin and vasopressin on the behavior of animals or humans. Studies in humans have reported behavioral changes and, through fMRI, effects on brain function. These studies are paralleled by a large number of reports, mostly in rodents, that have also demonstrated neuromodulatory effects by oxytocin and vasopressin at the circuit level in specific brain regions. It is the scope of this review to give a summary of the most recent neuromodulatory findings in rodents with the aim of providing a potential neurophysiological basis for their behavioral effects. At the same time, these findings may point to promising areas for further translational research towards human applications.

Sharing data between LSDBs and central repositories.

Several Locus-Specific DataBases (LSDBs) have recently been approached by larger, more general data repositories (including NCBI and UCSC) with the request to share the DNA variant data they have collected. Within the Human Genome Variation Society (HGVS) a document was generated summarizing the issues related to these requests. The document has been circulated in the HGVS/LSDB community and was discussed extensively. Here we summarize these discussions and present the concluded recommendations for LSDB data sharing with central repositories.