Structural and functional analysis of yeast proteins involved in ER-to-Golgi transport: Sec24p family proteins and the GTPase activating protein Gyp5p

Um ihre arbeitsteilige innere Struktur mit spzialisierten Kompartimenten zuerhalten, aber auch, um auf sich verändernde Umweltbedingungen einzustellen, haben eukaryontische Zellen eine ausgefeilte intrazelluläre Transportmaschinerie evolviert, mit deren Hilfe Biomoleküle zu ihren jeweiligen Bestimmungsorten gebracht werde können. In der vorliegenden Arbeit werden einige der Mechanismen beschrieben, die den Transportschritt und die komplizierte Maschinerie auf dem Weg vom Endoplasmatischen Retikulum zum Golgiapparat in der einzelligen Bäckerhefe Saccharomyces cerevisiae regulieren. Ich beschreibe die Hauptmerkmale der beiden Proteine Sfb2p und Sfb3p, die ortholog zu dem COP II-Vesikelhüllprotein Sec24p sind. Diese Proteine sind höchstwahrscheinlich in die Herstellung und die Auswahl der zu verfrachtenden Ladung von COP II-Transportvesikeln eingebunden. Darüberhinaus habe ich Proteine untersucht, die beim Vertäuen, respektive Andocken von Vesikeln an die Zielmembran, in besonderen ein neues Mitglied der Gyp-Familie GTPase-aktivierender Proteine, das von uns Gyp5p genannt worden ist und welches eine bemerkenswerte Spezifität für die GTPase Ypt1p zeigt. In diesem Zusammenhang wurde von mir auch die biologische Funktion der GTP-Hydrolyse an Ypt1p in Zellen untersucht.To maintain their intricate, internal structure of specialized compartments as well as meeting the necessity to react to changes of environmental conditions, eukaryotic cells have evolved an elaborate transport machinery to selectively deliver biochemical components to their various destinations. In this thesis some of the mechanisms that regulate the complex machinery of vesicular transport from the endoplasmic reticulum to the Golgi apparatus in the single-celled eukaryote Saccharomyces cerevisiae are described. I described the main characteristics of two close orthologues of the COPII component Sec24p, named Sfb2p and Sfb3p. These proteins are most likely actively involved in COPII vesicle formation and in cargo selection. In addition, I studied proteins involved in tethering/docking processes, in particular a new member of the Gyp family of GTPase activating proteins (GAPs), named Gyp5p, which shows remarkable specificity for the small Ras-like Ypt1 protein. Furthermore, I investigated the biological function of Ypt1p GTP hydrolysis in the cells

A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B

Homens trans: da invisibilidade à rede de atenção em saúde

A saúde é um direito que deve ser assegurado e promovido pelo Estado. É preciso pensá-la de forma ampliada, principalmente quando se pensa em populações específicas. Esse é o caso dos homens trans, que são pessoas que se percebem e se constroem diferente do sexo assignado ao nascimento. Este estudo, qualitativo/exploratório, teve como objetivo conhecer as principais necessidades em saúde dos homens trans, o acesso e as suas percepções sobre o Sistema Único de Saúde (SUS) e assim, como essa vivencia influencia no seu bem-estar. Participaram de entrevistas individuais oito homens entre 20 e 50 anos que se identificaram enquanto trans. Utilizou-a a Análise Temática para compreensão dos dados e os principais temas emergentes foram: o desrespeito e/ou o não uso do nome social, a falta de qualificação e o preconceito dos/as profissionais da rede de atenção em saúde e, por fim, a dificuldade de acesso aos serviços e procedimentos específicos em hormonoterapia e cirurgias. Mesmo que respaldados por marcos/documentos regulatórios, algumas práticas fogem à promoção dos direitos em saúde para essas pessoas

Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution

Electrophysiological correlates of feedback processing in subarachnoid hemorrhage patients

Patients with subarachnoid hemorrhage (SAH) secondary to anterior communicating artery (AComA) aneurysm rupture often experience deficits in executive functioning and decision-making. Effective decision-making is based on the subjects' ability to adjust their performance based on feedback processing, ascribing either positive or negative value to the actions performed reinforcing the most adaptive behavior in an appropriate temporal framework. A crucial brain structure associated to feedback processing is the medial prefrontal cortex (mPFC), a brain region frequently damaged after AComA aneurysm rupture. In the present study, we recorded electrophysiological responses (event-related potentials (ERPs') and oscillatory activity (time frequency analysis) during a gambling task in a series of 15 SAH patients. Previous studies have identified a feedback related negativity (FRN) component associated with an increase on frontal medial theta power in response to negative feedback or monetary losses, which is thought to reflect the degree of negative prediction error. Our findings show a decreased FRN component in response to negative feedback and a delayed increase of theta oscillatory activity in the SAH patient group when compared to the healthy controls, indicating a reduced sensitivity to negative feedback processing and an effortful signaling of cognitive control and monitoring processes lengthened in time, respectively. These results provide us with novel neurophysiological markers regarding feedback processing and performance monitoring patterns in SAH patients, illustrating a dysfunctional reinforcement learning system probably contributing to the maladaptive day-to-day functioning in these patients

Stern twin-propeller effects on harbor infrastructures. Experimental analysis

The growth of marine traffic in harbors, and the subsequent increase in vessel and propulsion system sizes, produces three linked problems at the harbor basin area: (i) higher erosion rates damaging docking structures; (ii) sedimentation areas reducing the total depth; (iii) resuspension of contaminated materials deposited at the seabed. The published literature demonstrates that there are no formulations for twin stern propellers to compute the maximum scouring depth. Another important limitation is the fact that the formulations proposed only use one type of maneuvering during the experimental campaign, assuming that vessels are constantly being undocked. Trying to reproduce the real arrival and departure maneuvers, 24 different tests were conducted at an experimental laboratory in a medium-scale water tank using a twin propeller model to estimate the consequences and the maximum scouring depth produced by stern propellers during the backward/docking and forward/undocking scenarios. Results confirm that the combination of backward and forward scenario differs substantially from the experiments performed so far in the literature using only an accumulative forward scenario, yielding deeper scouring holes at the harbor basin area. The results presented in this paper can be used as guidelines to estimate the effects of regular vessels at their particular docking location.Postprint (published version

Fish pass assessment in the rivers of Catalonia (NE Iberian Peninsula). A case study of weirs associated with hydropower plants and gauging stations

This study evaluated the effectiveness of fish passes in Catalan rivers (NE Iberian Peninsula). The aim was to test current functionality to enhance these structures for the purpose of optimising their management. We analysed river connectivity and fish pass facilities to generate information to improve the design, construction, management and assessment of solutions related to fish passage in Catalan rivers according to international best practices. In 2006, a preliminary evaluation of the locations and effectiveness of fish pass facilities in Catalonia was conducted through direct inspection of 78 fishways. Most of them were retro-fitted solutions using broad-spectrum technical structures, mainly pool fishway or pool pass facilities. An analysis of the effectiveness of 7 of the new fish passes (4, 2 and 1 passes in the Ter, Ebro and Tordera rivers, respectively) was performed between 2006 and 2009. The fish passes were located in weirs associated with hydropower plants and gauging stations. Our preliminary assessment of fish connectivity was based on a field visit and collection of information to populate a database of existing obstacles and associated fishways and calculation of the new version of the ICF index (river connectivity index) for all of them. Several methodologies were used to check the effectiveness of a selection of fish pass solutions, most of which were based on passage rates directly or indirectly estimated for each species. Direct estimation techniques involved the installation of fish traps upstream of the facility at the exit of the fish pass and visual counts. Indirect estimation techniques compared the fish population structure on each side of the obstacle in river sections with equivalent hydrology and habitat characteristics using electric fishing or trapping systems, group mark-recapture methods and individual mark-recapture methods (PIT tags). The results showed that (1) existing solutions to improve fish migration in Catalonia are insufficient (only present at approximately 8% of obstacles), and where they do exist, the fish passes are usually inadequate or poorly maintained. (2) Fish passes are generally inadequate or insufficient for the total native fish fauna from each water body. Additionally, with few exceptions, fish passage rates are too low, and in most cases, only fish with a high capacity to overcome obstacles or the largest individuals succeed in migrating. (3) Fish pass effectiveness agrees with the results of the new version of the ICF index.Este estudio evalua la eficacia de dispositivos o soluciones de paso para peces en los ríos de Catalunya (NE de la Península Ibérica). Se realizó con el propósito de comprobar su funcionalidad actual y también para poder mejorar elementos constructivos y/o para optimizar aspectos de gestión. El objetivo del estudio era analizar la conectividad de los ecosistemas fluviales y generar información para mejorar el diseño, construcción, gestión y evaluación de soluciones para el paso de peces en los ríos de Catalunya, de acuerdo con los estándares internacionales. El año 2006 se realizó un estudio de localización y evaluación preliminar de la eficacia de los dispositivos de paso para peces existentes en Catalunya mediante la inspección directa de 78 dispositivos. La mayor'ıa eran soluciones de rehabilitación por medio de estructuras técnicas de amplio espectro, como dispositivos de paso de estanques sucesivos. Entre 2006 y 2009 se efectuó un análisis de la eficacia de 7 de estos elementos (4, 2 y 1 en los ríos Ter, Ebro y Tordera, respectivamente). Los dispositivos de paso para peces estaban localizados en azudes para uso hidroeléctrico y estaciones de aforo. La evaluación preliminar de la connectividad para los peces se basó en una visita de campo y la recolección de información para completar una base de datos sobre los obstáculos existentes y las soluciones de paso para peces associadas. Para cada uno de estos casos analizados se calculó la nueva versión del Índice de Conectividad Fluvial (ICF). Posteriormente, se usaron varias metodologías para comprobar la eficacia de una selección de soluciones de paso para peces, la mayoría basadas en la estimación de la tasa de franqueo de cada especie. Las t'ecnicas de estimación directa consistieron en la instalación de trampas aguas arriba del obstáculo, a la salida del dispositivo de paso para peces, y los recuentos visuales. La estimación indirecta permitió la comparación de las estructuras poblacionales por especie de ambos lados del obstáculo en tramos fluviales de características hidrológicas y de hábitat equivalentes, a partir de muestreos con pesca eléctrica o trampeo, métodos de marcaje y recaptura de grupo, y métodos de marcaje y recaptura individualizados (con marcas PIT). Los resultados obtenidos indicaron (1) Las soluciones existentes en Catalunya para mejorar las migraciones de los peces resultan insuficientes (están presentes sólo alrededor del 8 % de los obstáculos). Cuando los hay, en general, los dispositivos de paso son inadecuados o con un mal mantenimiento. (2) Los dispositivos de paso para peces son generalmente inadecuados o insuficientes para el conjunto de las especies autóctonas de fauna piscícola de cada masa de agua. Las tasas de franqueo de los dispositivos de paso para peces son, con algunas excepciones, demasiado bajas y, en la majoría de los casos, sólo facilitan las migraciones de los peces con gran capacidad de superar obst'aculos o los individuos de mayor talla. (3) La eficacia de los dispositivos de paso para peces coincide con los resultados de la nueva versión del índice ICF

Identification, characterization and expression of novel Sex Hormone Binding Globulin alternative first exons in the human prostate

<p>Abstract</p> <p>Background</p> <p>The human Sex Hormone Binding Globulin (SHBG) gene, located at 17p13.1, comprises, at least, two different transcription units regulated by two different promoters. The first transcription unit begins with the exon 1 sequence and is responsible for the production of plasma SHBG by the hepatocytes, while the second begins with an alternative exon 1 sequence, which replaces the exon 1 present in liver transcripts. Alternative exon 1 transcription and translation has only been demonstrated in the testis of transgenic mice containing an 11-kb human SHBG transgene and in the human testis. Our goal has been to further characterize the 5' end of the SHBG gene and analyze the presence of the SHBG alternative transcripts in human prostate tissue and derived cell lines.</p> <p>Results</p> <p>Using a combination of <it>in silico </it>and <it>in vitro </it>studies, we have demonstrated that the SHBG gene, along with exon 1 and alternative exon 1 (renamed here exon 1A), contains four additional alternative first exons: the novel exons 1B, 1C, and 1E, and a previously identified exon 1N, which has been further characterized and renamed as exon 1D. We have shown that these four alternative first exons are all spliced to the same 3' splice site of SHBG exon 2, and that exon 1A and the novel exon 1B can be spliced to exon 1. We have also demonstrated the presence of SHBG transcripts beginning with exons 1B, 1C and 1D in prostate tissues and cell lines, as well as in several non-prostatic cell lines. Finally, the alignment of the SHBG mammalian sequences revealed that, while exons 1C, 1D and 1E are very well conserved phylogenetically through non-primate mammal species, exon 1B probably aroused in apes due to a single nucleotide change that generated a new 5' splice site in exon 1B.</p> <p>Conclusion</p> <p>The identification of multiple transcription start sites (TSS) upstream of the annotated first exon of human SHBG, and the detection of the alternative transcripts in human prostate, concur with the prediction of the ENCODE (ENCyclopedia of DNA Elements) project, and suggest that the regulation of SHBG is much more complex than previously reported.</p

Longitudinal connectivity in hydromorphological quality assessments of rivers. The ICF index : a river connectivity index and its application to Catalan rivers

The Water Framework Directive urges assessment of river continuity as an input for the evaluation of hydromorphological quality. The existence of transverse obstacles in river beds has serious ecological consequences because it blocks the natural flow of water, sediments and biota, and this is considered one of the major causes of declines in many continental fish species. The index of river connectivity (ICF, from the Catalan name Index de Connectivitat Fluvial) evaluates obstacle effects on fish movement alone and does not take into account other elements affected by the obstacles. However, it can be used as a complementary element in hydromorphological condition assessments. The index is based on comparison of obstacle and fish pass (if any) characteristics with the capabilities of the fishes potentially present in the considered river section to overcome the obstacle. In this study, we present a new version of the ICF improved from its earlier version that was tested by different consultants and research centres for several obstacles from 2006 to 2009. The new version of the ICF is divided into three blocks that encompass assessment of (1) the obstacle and (2) the fish pass (if any) as well as (3) the estimation of certain modulators. Finally, the ICF classifies connectivity into five levels from very good to bad depending on the degree of permeability for different fish groups. This new version of the ICF has been tested for 101 transverse obstacles in rivers in Catalonia (NE Iberian Peninsula) both with and without fish passes, obtaining representation of the five expected quality levels (from very good to bad), and it is considered coherent with the real permeability of the obstacles. Its ease of application compared to in situ measurements of fish movements and the detailed information recorded by the index make it a very useful tool for the diagnosis of the longitudinal connectivity of rivers and for guiding measures for hydromorphological quality improvement.La Directiva Marco del Agua define que para evaluar la calidad hidromorfológica de los ríos hay que valorar, entre otros factores, la continuidad fluvial. La existencia de obstáculos transversales al cauce fluvial tiene importantes consecuencias ecológicas, y se considera una de las causas principales del declive de muchas especies continentales de peces. Por ello se ha diseñado el Índice de Conectividad Fluvial (ICF), que aunque sólo considera los peces y no otros compartimentos afectados (agua y sedimentos), puede ser utilizado como parte integrante del protocolo completo de evaluación de la calidad hidromorfológica de los ríos en Catalunya (protocolo HIDRI). El ICF se basa en la comparación de las características de un determinado obstáculo y de los dispositivos de paso para peces (si dispone de ellos) con las capacidades para superarlo de las distintas especies de peces autóctonos potencialmente presentes en el tramo. En este estudio se presenta una versión mejorada del ICF, que ha sido probada por distintos equipos de consultorías y centros de investigación entre 2006 y 2009. La nueva versión consta de tres grandes bloques que incluyen la evaluación de (1) el obstáculo, (2) el o los dispositivos de paso de peces, en caso que los haya, y (3) la estimación de distintos moduladores. Finalmente, el ICF clasifica el nivel de conectividad en cinco rangos, de muy bueno a malo, en función del grado de franqueabilidad para los distintos grupos de especies potencialmente presentes en el tramo. La nueva versión del ICF ha sido aplicada en 101 obstáculos transversales de distintos ríos de Catalunya (NE de la península Ibérica), con y sin dispositivos de paso para peces. En los resultados se encuentran representados los cinco rangos de calidad posibles (desde muy bueno a malo), resultados que se consideran coherentes con la permeabilidad real del obstáculo. Su fácil aplicación, en comparación con la evaluación in situ de los movimientos de los peces, así como los detalles de la información recogida en las hojas de campo, sugieren que el ICF es una buena herramienta tanto para diagnosticar la conectividad longitudinal como para orientar las medidas a tomar para la mejora de la calidad hidromorfológica