Vortex Shedding Dynamics in Long Aspect-Ratio Aerodynamics Bodies

The focus of the current dissertation is to study qualitatively the underlying physics of vortex-shedding and wake dynamics in long aspect-ratio aerodynamics in incompressible viscous flow through the use of the KLE method. We carried out a long series of numerical experiments in the cases of flow around the cylinder at low Reynolds numbers. The study of flow at low Reynolds numbers provides an insight in the fluid physics and also plays a critical role when applying to stalled turbine rotors. Many of the conclusions about the qualitative nature of the physical mechanisms characterizing vortex formation, shedding and further interaction analyzed here at low Re could be extended to other Re regimes and help to understand the separation of the boundary layers in airfoils and other aerodynamic surfaces. In the long run, it aims to provide a better understanding of the complex multi-physics problems involving fluid-structure-control interaction through improved mathematical computational models of the multi-physics process. Besides the scientific conclusions produced, the research work on streamlined and bluff-body condition will also serve as a valuable guide for the future design of blade aerodynamics and the placement of wind turbines and hydrakinetic turbines, increasing the efficiency in the use of expensive workforce, supplies, and infrastructure. After the introductory section describing the main fields of application of wind power and hydrokinetic turbines, we describe the main features and theoretical background of the numerical method used here. Then, we present the analysis of the numerical experimentation results for the oscillatory regime right before the onset of vortex shedding for circular cylinders. We verified the wake length of the closed near-wake behind the cylinder and analysed the decay of the wake at the wake formation region, and then studied the St-Re relationship at the Reynolds numbers before the wake sheds compared to the experimental data. We found a theoretical model that describes the time evolution of the amplitude of fluctuations in the vorticity field on the twin vortex wake, which accurately matches the numerical results in terms of the frequency of the oscillation and rate of decay. We also proposed a model based on an analog circuit that is able to interpret the concerning flow by reducing the number of degrees of freedom. It follows the idea of the non-linear oscillator and resembles the dynamics mechanism of the closed near-wake with a common configured sine wave oscillator. This low-dimensional circuital model may also help to understand the underlying physical mechanisms, related to vorticity transport, that give origin to those oscillations.

Selective and Specific Activation of Rab5 during Endocytosis of Receptor Tyrosine Kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

Subfilter-scale Stress Modelling for Large Eddy Simulations

A subfilter-scale (SFS) stress model is developed for large-eddy simulations (LES) and is tested on various benchmark problems in both wall-resolved and wall-modelled LES. The basic ingredients of the proposed model are the model length-scale, and the model parameter. The model length-scale is defined as a fraction of the integral scale of the flow, decoupled from the grid. The portion of the resolved scales (LES resolution) appears as a user-defined model parameter, an advantage that the user decides the LES resolution. The model parameter is determined based on a measure of LES resolution, the SFS activity. The user decides a value for the SFS activity (based on the affordable computational budget and expected accuracy), and the model parameter is calculated dynamically. Depending on how the SFS activity is enforced, two SFS models are proposed. In one approach the user assigns the global (volume averaged) contribution of SFS to the transport (global model), while in the second model (local model), SFS activity is decided locally (locally averaged). The models are tested on isotropic turbulence, channel flow, backward-facing step and separating boundary layer. In wall-resolved LES, both global and local models perform quite accurately. Due to their near-wall behaviour, they result in accurate prediction of the flow on coarse grids. The backward-facing step also highlights the advantage of decoupling the model length-scale from the mesh. Despite the sharply refined grid near the step, the proposed SFS models yield a smooth, while physically consistent filter-width distribution, which minimizes errors when grid discontinuity is present. Finally the model application is extended to wall-modelled LES and is tested on channel flow and separating boundary layer. Given the coarse resolution used in wall-modelled LES, near the wall most of the eddies become SFS and SFS activity is required to be locally increased. The results are in very good agreement with the data for the channel. Errors in the prediction of separation and reattachment are observed in the separated flow, that are somewhat improved with some modifications to the wall-layer model.

Dor lombar em crianças

Projeto de Graduação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciada em Fisioterapia

Optimisation de l’extraction de lumière de scintillation dans les matrices de détecteurs pixellisés pour des applications en tomographie d’émission par positrons (TEP) et en tomodensitométrie (TDM)

Résumé : Les performances de détecteurs à scintillation, composés d’un cristal scintillateur couplé à un photodétecteur, dépendent de façon critique de l’efficacité de la collecte et de l’extraction des photons de scintillation du cristal vers le capteur. Dans les systèmes d’imagerie hautement pixellisés (e.g. TEP, TDM), les scintillateurs doivent être arrangés en matrices compactes avec des facteurs de forme défavorables pour le transport des photons, au détriment des performances du détecteur. Le but du projet est d’optimiser les performances de ces détecteurs pixels par l'identification des sources de pertes de lumière liées aux caractéristiques spectrales, spatiales et angulaires des photons de scintillation incidents sur les faces des scintillateurs. De telles informations acquises par simulation Monte Carlo permettent une pondération adéquate pour l'évaluation de gains atteignables par des méthodes de structuration du scintillateur visant à une extraction de lumière améliorée vers le photodétecteur. Un plan factoriel a permis d'évaluer la magnitude de paramètres affectant la collecte de lumière, notamment l'absorption des matériaux adhésifs assurant l'intégrité matricielle des cristaux ainsi que la performance optique de réflecteurs, tous deux ayant un impact considérable sur le rendement lumineux. D'ailleurs, un réflecteur abondamment utilisé en raison de ses performances optiques exceptionnelles a été caractérisé dans des conditions davantage réalistes par rapport à une immersion dans l'air, où sa réflectivité est toujours rapportée. Une importante perte de réflectivité lorsqu'il est inséré au sein de matrices de scintillateurs a été mise en évidence par simulations puis confirmée expérimentalement. Ceci explique donc les hauts taux de diaphonie observés en plus d'ouvrir la voie à des méthodes d'assemblage en matrices limitant ou tirant profit, selon les applications, de cette transparence insoupçonnée.

Hall Effect and transient surface photovoltage (SPV) study of Cu3BiS3 thin films

Se presentan las propiedades eléctricas del compuesto Cu3BiS3 depositado por co-evaporación. Este es un nuevo compuesto que puede tener propiedades adecuadas para ser utilizado como capa absorbente en celdas solares. Las muestras fueron caracterizadas a través de medidas de efecto Hall y fotovoltaje superficial transiente (SPV). A través de medidas de efecto Hall se encontró que la concentración de portadores de carga n es del orden de 1016 cm-3 independiente de la relación de masas de Cu/Bi. También se encontró que la movilidad de este compuesto (μ del orden de 4 cm2V -1s-1) varía de acuerdo con los mecanismos de transporte que la gobiernan en dependencia con la temperatura. A partir de las medidas de SPV se encontró alta densidad de defectos superficiales, defectos que son pasivados al superponer una capa buffer sobre el compuesto Cu3BiS3.

Nocturnal surface ozone enhancement over Portugal during winter: Influence of different atmospheric conditions

our distinct nocturnal surface ozone (NSO) enhancement events were observed, with NSO concentration exceeding 80μg/m3, at multiple ozone (O3) monitoring stations (32 sites) in January, November and December between year 2000–2010, in Portugal. The reasonable explanation for the observed bimodal pattern of surface ozone with enhanced NSO concentration during nighttime has to be transport processes, as the surface ozone production ceases at nighttime. Simultaneous measurements of O3 at multiple stations during the study period in Portugal suggest that horizontal advection alone cannot explain the observed NSO enhancement. Thus, detailed analysis of the atmospheric conditions, simulated with the Weather Research and Forecasting (WRF) model, were performed to evaluate the atmospheric mechanisms responsible for NSO enhancement in the region. Simulations revealed that each event occurred as a result of one or the combination of different atmospheric processes such as, passage of a cold front followed by a subsidence zone; passage of a moving surface trough, with associated strong horizontal wind speed and vertical shear; combination of vertical and horizontal transport at the synoptic scale; formation of a low level jet with associated vertical mixing below the jet stream. The study confirmed that large-scale flow pattern resulting in enhanced vertical mixing in the nocturnal boundary layer, plays a key role in the NSO enhancement events, which frequently occur over Portugal during winter months.

Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.

The Saccharomyces cerevisiae v-SNARE Vti1p Is Required for Multiple Membrane Transport Pathways to the Vacuole

The interaction between v-SNAREs on transport vesicles and t-SNAREs on target membranes is required for membrane traffic in eukaryotic cells. Here we identify Vti1p as the first v-SNARE protein found to be required for biosynthetic traffic into the yeast vacuole, the equivalent of the mammalian lysosome. Certain vti1-ts yeast mutants are defective in alkaline phosphatase transport from the Golgi to the vacuole and in targeting of aminopeptidase I from the cytosol to the vacuole. VTI1 interacts genetically with the vacuolar t-SNARE VAM3, which is required for transport of both alkaline phosphatase and aminopeptidase I to the vacuole. The v-SNARE Nyv1p forms a SNARE complex with Vam3p in homotypic vacuolar fusion; however, we find that Nyv1p is not required for any of the three biosynthetic pathways to the vacuole. v-SNAREs were thought to ensure specificity in membrane traffic. However, Vti1p also functions in two additional membrane traffic pathways: Vti1p interacts with the t-SNAREs Pep12p in traffic from the TGN to the prevacuolar compartment and with Sed5p in retrograde traffic to the cis-Golgi. The ability of Vti1p to mediate multiple fusion steps requires additional proteins to ensure specificity in membrane traffic.

Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

Vps10p Transport from the trans-Golgi Network to the Endosome Is Mediated by Clathrin-coated Vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12Δ and vps34Δ, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10CtΔp mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN

The four mammalian golgins, p230/golgin-245, golgin-97, GCC88 and GCC185 are targeted to trans-Golgi network ITGN) membranes by their C-terminal GRIP domain in a G-protein-dependent process. The Arf-like GTPase, Arl1, has been shown to mediate TGN recruitment of p230/golgin245 and golgin-97 by interaction with their GRIP domains; however, it is not known whether all the TGN golgins bind to Arl1 and whether they are all recruited to the same or different TGN domains. Here we demonstrate differences in membrane binding properties and TGN domain recruitment of the mammalian GRIP domain proteins. Overexpression of full-length GCC185 resulted in the appearance of small punctate structures dispersed in the cytoplasm of transfected cells that were identified as membrane tubular structures by immunoelectron microscopy. The cytoplasmic GCC185-labelled structures were enriched for membrane binding determinants of GCC185 GRIP, whereas the three other mammalian GRIP family members did not colocalize with the GCC185-labelled structures. These GCC185-labelled structures included the TGN resident protein alpha2,6 sialyltransferase and excluded the recycling TGN protein, TGN46. The Golgi stack was unaffected by overexpression of GCC185. Overexpression of both full-length GCC185 and GCC88 showed distinct and nonoverlapping structures. We also show that the GRIP domains of GCC185 and GCC88 differ in membrane binding properties from each other and, in contrast to p230/golgin245 and golgin-97, do not interact with Arl1 in vivo. Collectively these results show that GCC88, GCC185 and p230/golgin245 are recruited to functionally distinct domains of the TGN and are likely to be important for the maintenance of TGN subdomain structure, a critical feature for mediating protein sorting and membrane transport.

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

Performance of transport and selective media for swine Bordetella bronchiseptica recovery and it comparison to polymerase chain reaction detection

Three comparative assays were performed seeking to improve the sensitivity of the diagnosis of Bordetella bronchiseptica infection analyzing swine nasal swabs. An initial assay compared the recovery of B. bronchiseptica from swabs simultaneously inoculated with B. bronchiseptica and some interfering bacteria, immersed into three transport formulations (Amies with charcoal, trypticase soy broth and phosphate buffer according to Soerensen supplemented with 5% of bovine fetal serum) and submitted to different temperatures (10ºC and 27ºC) and periods of incubation (24, 72 and 120 hours). A subsequent assay compared three selective media (MacConkey agar, modified selective medium G20G and a ceftiofur medium) for their recovery capabilities from clinical specimens. One last assay compared the polymerase chain reaction to the three selective media. In the first assay, the recovery of B. bronchiseptica from transport systems was better at 27ºC and the three formulations had good performances at this temperature, but the collection of qualitative and quantitative analysis indicated the advantage of Amies medium for nasal swabs transportation. The second assay indicated that MacConkey agar and modified G20G had similar results and were superior to the ceftiofur medium. In the final assay, polymerase chain reaction presented superior capability of B. bronchiseptica detection to culture procedures.