The mapping of velocity profiles in a three generation lung model using Particle Image Velocimetry flow analysis techniques

The intent of this thesis was to develop an understanding for the particle flow characteristics in the human lung by examining particle flow profiles in a three generation lung model. To develop this understanding, experimentally derived flow profiles were compared to analytical solutions, where applicable, and Computational Fluid Dynamics (CFD) generated models for validation of both the CFD model and the experimental set-up. Validation of flow velocities in a three generation model could contribute significantly to the medical industry as a better understanding of particle behavior in the lung could lead to more accurate treatment of certain diseases and better prediction of health effects of inhaled contaminants. Particle flow in a three generation lung model was studied using a technique known as Particle Image Velocimetry (PIV). PIV involves passing a widely dispersed laser beam through a flow field of specifically sized and fluorescently colored particles. These fluorescent particles are photographed by a high-speed camera under high magnification. These images are then digitally sent to VisiFlow analysis software where each particle\u27s flow path is mapped and converted, through a cross-correlation technique, into a vector field, from which the velocity profiles can be derived. Following successful interpretation of the experimentally derived velocity profiles, a comparison was drawn between the experimentally collected data and the anticipated result based on an existing CFD model. This comparison served to not only validate the experimental test set-up but also to validate the CFD model. A favorable correlation between the experimental results and the CFD results provided confidence that a valid solution for flow profiles was achieved. Achieving a valid experimental result was dependent on many factors. The ability of the test setup to accurately produce flow profiles was measured using less complicated and easily calculated models. In doing so, confidence was developed that current scientific, analytical and experimental practices and procedures were accurately eliminating or minimizing sources of error and ultimately providing an accurate solution. As a result, not only was a valid experimental model derived but a thorough understanding of proper laboratory techniques was achieved

Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1–5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways

FHOD‐1 is the only formin in Caenorhabditis elegans that promotes striated muscle growth and Z‐line organization in a cell autonomous manner

The striated body wall muscles of Caenorhabditis elegans are a simple model for sarcomere assembly. Previously, we observed deletion mutants for two formin genes, fhod‐1 and cyk‐1, develop thin muscles with abnormal dense bodies (the sarcomere Z‐line analogs). However, this work left in question whether these formins work in a muscle cell autonomous manner, particularly since cyk‐1(Δ) deletion has pleiotropic effects on development. Using a fast acting temperature‐sensitive cyk‐1(ts) mutant, we show here that neither post‐embryonic loss nor acute loss of CYK‐1 during embryonic sarcomerogenesis cause lasting muscle defects. Furthermore, mosaic expression of CYK‐1 in cyk‐1(Δ) mutants is unable to rescue muscle defects in a cell autonomous manner, suggesting muscle phenotypes caused by cyk‐1(Δ) are likely indirect. Conversely, mosaic expression of FHOD‐1 in fhod‐1(Δ) mutants promotes muscle cell growth and proper dense body organization in a muscle cell autonomous manner. As we observe no effect of loss of any other formin on muscle development, we conclude FHOD‐1 is the only worm formin that directly promotes striated muscle development, and the effects on formin loss in C. elegans are surprisingly modest compared to other systems

Polarized Growth in the Absence of F-Actin in Saccharomyces cerevisiae Exiting Quiescence

Polarity establishment and maintenance are crucial for morphogenesis and development. In budding yeast, these two intricate processes involve the superposition of regulatory loops between polarity landmarks, RHO GTPases, actin-mediated vesicles transport and endocytosis. Deciphering the chronology and the significance of each molecular step of polarized growth is therefore very challenging.We have taken advantage of the fact that yeast quiescent cells display actin bodies, a non polarized actin structure, to evaluate the role of F-actin in bud emergence. Here we show that upon exit from quiescence, actin cables are not required for the first steps of polarized growth. We further show that polarized growth can occur in the absence of actin patch-mediated endocytosis. We finally establish, using latrunculin-A, that the first steps of polarized growth do not require any F-actin containing structures. Yet, these structures are required for the formation of a bona fide daughter cell and cell cycle completion. We propose that upon exit from quiescence in the absence of F-actin, secretory vesicles randomly reach the plasma membrane but preferentially dock and fuse where polarity cues are localized, this being sufficient to trigger polarized growth

Intrinsic Capability of Budding Yeast Cofilin to Promote Turnover of Tropomyosin-Bound Actin Filaments

The ability of actin filaments to function in cell morphogenesis and motility is closely coupled to their dynamic properties. Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled. Although genetic studies have shown that rapid actin turnover in patches and cables depends on cofilin, how cofilin might control cable disassembly remains unclear, because tropomyosin, a component of actin cables, is thought to protect actin filaments against the depolymerizing activity of ADF/cofilin. We have identified cofilin as a yeast tropomyosin (Tpm1) binding protein through Tpm1 affinity column and mass spectrometry. Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4. Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1

Globus Data Publication as a Service: Lowering Barriers to Reproducible Science

Abstract-Broad access to the data on which scientific results are based is essential for verification, reproducibility, and extension. Scholarly publication has long been the means to this end. But as data volumes grow, new methods beyond traditional publications are needed for communicating, discovering, and accessing scientific data. We describe data publication capabilities within the Globus research data management service, which supports publication of large datasets, with customizable policies for different institutions and researchers; the ability to publish data directly from both locally owned storage and cloud storage; extensible metadata that can be customized to describe specific attributes of different research domains; flexible publication and curation workflows that can be easily tailored to meet institutional requirements; and public and restricted collections that give complete control over who may access published data. We describe the architecture and implementation of these new capabilities and review early results from pilot projects involving nine research communities that span a range of data sizes, data types, disciplines, and publication policies

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin-based motility

Vaccinia virus dissemination relies on the N-WASP– ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP– ARP2/3 and Rac1–FHOD1 pathways.Fil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Yale. School of Medicine; Estados UnidosFil: Agaisse, Herve. University of Yale. School of Medicine; Estados Unido

The mating-specific Gα interacts with a kinesin-14 and regulates pheromone-induced nuclear migration in budding yeast

As a budding yeast cell elongates toward its mating partner, cytoplasmic microtubules connect the nucleus to the cell cortex at the growth tip. The Kar3 kinesin-like motor protein is then thought to stimulate plus-end depolymerization of these microtubules, thus drawing the nucleus closer to the site where cell fusion and karyogamy will occur. Here, we show that pheromone stimulates a microtubule-independent interaction between Kar3 and the mating-specific Gα protein Gpa1 and that Gpa1 affects both microtubule orientation and cortical contact. The membrane localization of Gpa1 was found to polarize early in the mating response, at about the same time that the microtubules begin to attach to the incipient growth site. In the absence of Gpa1, microtubules lose contact with the cortex upon shrinking and Kar3 is improperly localized, suggesting that Gpa1 is a cortical anchor for Kar3. We infer that Gpa1 serves as a positional determinant for Kar3-bound microtubule plus ends during mating. © 2009 by The American Society for Cell Biology

A mother's sacrifice: what is she keeping for herself?

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited life span and undergo a form of senescence termed replicative aging. Replicative life span is defined as the number of daughter cells produced by a yeast mother cell before she ceases dividing. Replicative aging is asymmetric: a mother cell ages but the age of her daughter cells is 'reset' to zero. Thus, one or more senescence factors have been proposed to accumulate asymmetrically between mother and daughter yeast cells and lead to mother-specific replicative senescence once a crucial threshold has been reached. Here we evaluate potential candidates for senescence factors and age-associated phenotypes and discuss potential mechanisms underlying the asymmetry of replicative aging in budding yeast