Genetic and functional analyses of cell division proteins FtsZ and FtsA in Escherichia coli

In the current model for bacterial cell division, the FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other essential division proteins such as FtsA and ZipA. The putative protein complex ultimately generates the division septum. The essential cell division protein FtsZ is a functional and structural homolog of eukaryotic tubulin, and like tubulin, FtsZ hydrolyzes GTP and self-assembles into protein filaments in a strictly GTP-dependent manner. FtsA shares sequence similarity with members of the ATPase superfamily that include actin, but its actual function remains unknown. To test the division model and elucidate functions of the division proteins, this dissertation primarily focuses on the analysis of FtsZ and FtsA in Escherichia coli. ^ By tagging with green fluorescent protein, we first demonstrated that FtsA also exhibits a ring-like structure at the potential division site. The localization of FtsA was dependent on functional FtsZ, suggesting that FtsA is recruited to the septum by the FtsZ ring. In support of this idea, we showed that FtsA and FtsZ directly interact. Using a novel E. coli in situ assay, we found that the FtsA-FtsZ interaction appears to be species-specific, although an interspecies interaction could occur between FtsA and FtsZ proteins from two closely related organisms. In addition, mutagenesis of FtsA revealed that no single domain is solely responsible for its septal localization or interaction with FtsZ. To explore the function of FtsA, we purified FtsA protein and demonstrated that it has ATPase activity. Furthermore, purified FtsA stimulates disassembly of FtsZ polymers in a sedimentation assay but does not affect GTP hydrolysis of FtsZ. This result suggests that in the cell, FtsA may function similarly in regulating dynamic instability of the FtsZ ring during the cell division process. ^ To elucidate the structure-function relationship of FtsZ, we carried out thorough genetic and functional analyses of the mutagenized FtsZ derivatives. Our results indicate that the conserved N-terminal domain of FtsZ is necessary and sufficient for FtsZ self-assembly and localization. Moreover, we discovered a critical role for an extreme C-terminal domain of FtsZ that consists of only 12 residues. Truncated FtsZ derivatives lacking this domain, though able to polymerize and localize, are defective in ring formation in vivo as well as interaction with FtsA and ZipA. Alanine scanning mutagenesis of this region pinpointed at least five residues necessary for the function of FtsZ. Studies of protein levels and protein-protein interactions suggested that these residues may be involved in regulating protein stability and/or FtsZ-FtsA interactions. Interestingly, two of the point mutants exhibited dominant-negative phenotypes. ^ In summary, results from this thesis work have provided additional support for the division machinery model and will contribute to a better understanding of the coordinate functions of FtsA and FtsZ in the cell division process. ^

A comprehensive assessment of environmental ultraviolet radiation induced DNA damage in marine microorganisms

There is evidence that ultraviolet radiation (UVR) is increasing over certain locations on the Earth's surface. Of primary concern is the annual pattern of ozone depletion over Antarctica and the Southern Ocean. Reduction of ozone concentration selectively limits absorption of solar UV-B (290–320 nm), resulting in higher irradiance at the Earth's surface. The effects of ozone depletion on the human population and natural ecosystems, particularly the marine environment, are a matter of considerable concern. Indeed, marine plankton may serve as sensitive indicators of ozone depletion and UV-B fluctuations. Direct biological effects of UVR result from absorption of UV-B by DNA. Once absorbed, energy is dissipated by a variety of pathways, including covalent chemical reactions leading to the formation of photoproducts. The major types of photoproduct formed are cyclobutyl pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. Marine plankton repair these photoproducts using light-dependent photoenzymatic repair or nucleotide excision repair. The studies here show that fluctuations in CPD concentrations in the marine environment at Palmer Station, Antarctica correlate well with ozone concentration and UV-B irradiance at the Earth's surface. A comparison of photoproduct levels in marine plankton and DNA dosimeters show that bacterioplankton display higher resistance to solar UVR than phytoplankton in an ozone depleted environment. DNA damage in marine microorganisms was investigated during two separate latitudinal transects which covered a total range of 140°. We observed the same pattern of change in DNA damage levels in dosimeters and marine plankton as measured using two distinct quantitative techniques. Results from the transects show that differences in photosensitivity exist in marine plankton collected under varying UVR environments. Laboratory studies of Antarctic bacterial isolates confirm that marine bacterioplankton possess differences in survival, DNA damage induction, and repair following exposure to UVR. Results from DNA damage measurements during ozone season, along a latitudinal gradient, and in marine bacterial isolates suggest that changes in environmental UVR correlate with changes in UV-B induced DNA damage in marine microorganisms. Differences in the ability to tolerate UVR stress under different environmental conditions may determine the composition of the microbial communities inhabiting those environments. ^

Characterization of cytochrome P450 4F subfamily: Response in traumatic brain injury and gene regulation

CYP4F enzymes metabolize endogenous molecules including arachidonic acid, leukotrienes and prostaglandins. The involvement of these eisosanoids in inflammation has led to the hypothesis that CYP4Fs may modulate inflammatory conditions after traumatic brain injury (TBI). In rat, TBI elicited changes in mRNA expression of CYP4Fs as a function of time in the cerebrum region. These changes in CYP4F mRNA levels inversely correlated with the cerebral leukotriene B4 (LTB4) level following injury at the same time points. TBI also resulted in changes in CYP4F protein expression and localization around the injury site, where CYP4F1 and CYP4F6 immunoreactivity increased in surrounding astrocytes and CYP4F4 immunoreactivity shifted from endothelia of cerebral vessels to astrocytes. The study with rat primary astrocytes indicated that pro-inflammatory cytokines TNFα and IL-1β could affect the transcription of CYP4Fs to a certain degree, whereas the changing pattern in the primary astrocytes appeared to be different from that in the in vivo TBI model.^ In addition, the regulation of CYP4F genes has been an unsolved issue although factors including cytokines and fatty acids appear to affect CYP4Fs expression in multiple models. In this project, HaCaT cells were used as an in vitro cellular model to define signaling pathways involved in the regulation of human CYP4F genes. Retinoic acids inhibited CYP4F11 expression, whereas cytokines TNFα and IL-1β induced transcription of CYP4F11 in HaCaT cells. The induction of CYP4F11 by both cytokines could be blocked by a JNK specific inhibitor, indicating the involvement of the JNK pathway in the up-regulation of CYP4F11. Retinoic acids are known to function in gene regulation through nuclear receptors RARs and RXRs. The RXR agonist LG268 greatly induced transcription of CYP4F11, whereas RAR agonist TTNPB obviously inhibited CYP4F11 transcription, indicating that the down-regulation of CYP4F11 by retinoic acid was mediated by RARs, and that inhibition of CYP4F11 by retinoic acid may also be related to the competition for RXR receptors. Thus, the CYP4F11 gene is regulated by signaling pathways including the RXR pathway and the JNK pathway. In contrast, the regulation mechanism of other CYP4Fs by retinoic acids appears to be different from that of CYP4F11.^

Molecular mechanisms involved in the regulation of cell polarity and genome stability by the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The essential p21-activated kinase (PAK), Shk1, is a critical component of a Ras/Cdc42/PAK complex required for cell viability, normal cell polarity, proper regulation of cytoskeletal dynamics, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. While cellular functions of PAKs have been described in eukaryotes from yeasts to mammals, the molecular mechanisms of PAK regulation and function are poorly understood. This study has characterized a novel Shk1 inhibitor, Skb15, and, in addition, identified the cell polarity regulator, Tea1, as a potential biological substrate of Shk1 in S. pombe. Skb15 is a highly conserved WD repeat protein that was discovered from a two-hybrid screen for proteins that interact with the catalytic domain of Shk1. Molecular data indicate that Skb15 negatively regulates Shk1 kinase activity in S. pombe cells. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution. ^ Our laboratory has recently demonstrated that Shk1, in addition to regulating actin cytoskeletal organization, is required for proper regulation of microtubule dynamics in S. pombe cells. The Shk1 protein localizes to interphase and mitotic microtubules, the septum-forming region, and cell ends. This pattern of localization overlaps with that of the cell polarity regulator, Tea1, in S. pombe cells. The tea1 gene was identified by Paul Nurse's laboratory from a screen for genes involved in the control of cell morphogenesis in S. pombe. In contrast to wild type S. pombe cells, which are rod shaped, tea1 null cells are often bent and/or branched in shape. The Tea1 protein localizes to the cell ends, like Shk1, and the growing tips of interphase microtubules. Thus, experiments were performed to investigate whether Tea1 interacts with Shk1. The tea1 null mutation strongly suppresses the loss of function of Skb15, an essential inhibitor of Shk1 function. All defects associated with the skb15 mutation, including defects in F-actin organization, septation, spindle elongation, and chromosome segregation, are suppressed by tea1Δ, suggesting that Tea1 may function in these diverse processes. Consistent with a role for Tea1 in cytokinesis, tea1Δ cells have a modest cell separation defect that is greatly exacerbated by a shk1 mutation and, like Shk1, Tea1 localizes to the septation site. Molecular analyses showed that Tea1 phosphorylation is significantly dependent on Shk1 function in vivo and that bacterially expressed Tea1 protein is directly phosphorylated by recombinant Shk1 kinase in vitro. Taken together, these results identify Tea1 as a potential biological substrate of Shk1 in S. pombe. ^ In summary, this study provides new insights into a conserved regulatory mechanism for PAKs, and also begins to uncover the molecular mechanisms by which the Ras/Cdc42/PAK complex regulates the microtubule and actin cytoskeletons and cell growth polarization in fission yeast. ^

Body mass, flipper length, oxidative balance indices and telomere length of king penguin chicks (A. patagonicus) on Possession Island

One of the reasons for animals not to grow as fast as they potentially could is that fast growth has been shown to be associated with reduced lifespan. However, we are still lacking a clear description of the reality of growth-dependent modulation of ageing mechanisms in wild animals. Using the particular growth trajectory of small king penguin chicks naturally exhibiting higher-than-normal growth rate to compensate for the winter break, we tested whether oxidative stress and telomere shortening are related to growth trajectories. Plasma antioxidant defences, oxidative damage levels and telomere length were measured at the beginning and at the end of the post-winter growth period in three groups of chicks (small chicks, which either passed away or survived the growth period, and large chicks). Small chicks that died early during the growth period had the highest level of oxidative damage and the shortest telomere lengths prior to death. Here, we show that small chicks that grew faster did it at the detriment of body maintenance mechanisms as shown by (i) higher oxidative damage and (ii) accelerated telomere loss. Our study provides the first evidence for a mechanistic link between growth and ageing rates under natural conditions.

Chemical and isotopic compositions of basaltic glasses and crusts from the Mid-Atlantic Ridge rift valley

Data presented in the paper suggest significant differences between thermodynamic conditions, under which magmatic complexes were formed in MAR at 29°-34°N and 12°-18°N. Melts occurring at 29°-34°N were derived by melting of a mantle source with homogeneous distribution of volatile components and arrived at the surface without significant fractionation, likely, due to their rapid ascent. The MAR segments between 12° and 18°N combine contrasting geodynamic environments of magmatism, which predetermined development of a large plume region with widespread mixing of melting products of geochemically distinct mantle sources. At the same time, this region is characterized by conditions favorable for origin of localized zones of anomalous plume magmatism. These sporadic magmatic sources were spatially restricted to MAR fragments with the Hess crust, whose compositional and mechanical properties were, perhaps, favorable for focusing and localization of plume magmatism. The plume source between 12° and 18°N beneath MAR may be geochemically heterogeneous.

Improving target localization accuracy of wireless visual sensor networks

This paper discusses the target localization problem of wireless visual sensor networks. Specifically, each node with a low-resolution camera extracts multiple feature points to represent the target at the sensor node level. A statistical method of merging the position information of different sensor nodes to select the most correlated feature point pair at the base station is presented. This method releases the influence of the accuracy of target extraction on the accuracy of target localization in universal coordinate system. Simulations show that, compared with other relative approach, our proposed method can generate more desirable target localization's accuracy, and it has a better trade-off between camera node usage and localization accuracy.

Intraply fracture of fiber-reinforced composites: microscopic mechanisms and modeling

The fracture behavior parallel to the fibers of an E-glass/epoxy unidirectional laminate was studied by means of three-point tests on notched beams. Selected tests were carried out within a scanning electron microscope to ascertain the damage and fracture micromechanisms upon loading. The mechanical behavior of the notched beam was simulated within the framework of the embedded cell model, in which the actual composite microstructure was resolved in front of the notch tip. In addition, matrix and interface properties were independently measured in situ using a nanoindentor. The numerical simulations very accurately predicted the macroscopic response of the composite as well as the damage development and crack growth in front of the notch tip, demonstrating the ability of the embedded cell approach to simulate the fracture behavior of heterogeneous materials. Finally, this methodology was exploited to ascertain the influence of matrix and interface properties on the intraply toughness.

A practical approach for outdoors distributed target localization in wireless sensor networks

Wireless sensor networks are posed as the new communication paradigm where the use of small, low-complexity, and low-power devices is preferred over costly centralized systems. The spectra of potential applications of sensor networks is very wide, ranging from monitoring, surveillance, and localization, among others. Localization is a key application in sensor networks and the use of simple, efficient, and distributed algorithms is of paramount practical importance. Combining convex optimization tools with consensus algorithms we propose a distributed localization algorithm for scenarios where received signal strength indicator readings are used. We approach the localization problem by formulating an alternative problem that uses distance estimates locally computed at each node. The formulated problem is solved by a relaxed version using semidefinite relaxation technique. Conditions under which the relaxed problem yields to the same solution as the original problem are given and a distributed consensusbased implementation of the algorithm is proposed based on an augmented Lagrangian approach and primaldual decomposition methods. Although suboptimal, the proposed approach is very suitable for its implementation in real sensor networks, i.e., it is scalable, robust against node failures and requires only local communication among neighboring nodes. Simulation results show that running an additional local search around the found solution can yield performance close to the maximum likelihood estimate.

UPM Activities on the STLs processing (Task 6.2) and the generation of DPA cross section library up to 150 MeV (Task 4.1)

Generation of a complete damage energy and dpa cross section library up to 150 MeVbased on JEFF- 3.1.1 and suitable approximations (UPM) Postprocessing of photonuclear libraries (by CCFE) and thermal scattering  tables (by UPM) at the backend of the calculational system (CCFE/UPM)

Impact parameters in relation to bruising and other fruit properties.

Impact response in fruits, primarily appl.es and pears (Pomaceae fruits), has been studied during the last five years. Using a laboratory impact testing device and also free-fall tests of instrumented apples, a significant body of results has been established, relative to the parameters which best characterize the impact response of these materials, and to their correlation with bruise damage, variety and ripeness level of the fruits. Bruise damage, measured as the size and/or volume of the affected fruit tissue is related primarily to applied energy (i.e. mainly drop height) for a given variety at a given ripeness stage. The relevant impact response parameters are maximum deformation (DM), permanent deformation (DP), maximum impulse (IM), maximum impact force (FM), maximum value of the force/time slope during impact (F/T) and impact time (T). The effect of ripeness differences was also studied in selected varieties of pears, being the most relevant parameters: maximum force (FM) and F/T slope.

Gear dynamics monitoring using discrete wavelet transformation and multi-layer perceptron neural networks

This paper presents a multi-stage algorithm for the dynamic condition monitoring of a gear. The algorithm provides information referred to the gear status (fault or normal condition) and estimates the mesh stiffness per shaft revolution in case that any abnormality is detected. In the first stage, the analysis of coefficients generated through discrete wavelet transformation (DWT) is proposed as a fault detection and localization tool. The second stage consists in establishing the mesh stiffness reduction associated with local failures by applying a supervised learning mode and coupled with analytical models. To do this, a multi-layer perceptron neural network has been configured using as input features statistical parameters sensitive to torsional stiffness decrease and derived from wavelet transforms of the response signal. The proposed method is applied to the gear condition monitoring and results show that it can update the mesh dynamic properties of the gear on line.

Experimental study on bridge damage identification based on wavelet packet energy curvature difference method

Among those damage identification methods, the Wavelet Packet Energy Curvature Difference (WPECD) Method is an effective one. However, most of the existing methods rely on numerical simulation and are unverified via experiment, and very few of them have been applied to practice. In this paper, the validity of WPECD in structural damage identification is verified by a numerical example. A damage simulation experiment is taken on a real replaced girder at the Ziya River New Bridge in Cangzhou. Two damage cases are applied and the acceleration responses at the measuring points are obtained, based on which the damages are identified with the WPECD Method, and the influence of wavelet function and decomposition level is studied. The results show that the WPECD Method can identify structure damage efficiently and can be put into practice.

Active Optical Sensors for Tree Stem Detection and Classification in Nurseries.

Active optical sensing (LIDAR and light curtain transmission) devices mounted on a mobile platform can correctly detect, localize, and classify trees. To conduct an evaluation and comparison of the different sensors, an optical encoder wheel was used for vehicle odometry and provided a measurement of the linear displacement of the prototype vehicle along a row of tree seedlings as a reference for each recorded sensor measurement. The field trials were conducted in a juvenile tree nursery with one-year-old grafted almond trees at Sierra Gold Nurseries, Yuba City, CA, United States. Through these tests and subsequent data processing, each sensor was individually evaluated to characterize their reliability, as well as their advantages and disadvantages for the proposed task. Test results indicated that 95.7% and 99.48% of the trees were successfully detected with the LIDAR and light curtain sensors, respectively. LIDAR correctly classified, between alive or dead tree states at a 93.75% success rate compared to 94.16% for the light curtain sensor. These results can help system designers select the most reliable sensor for the accurate detection and localization of each tree in a nursery, which might allow labor-intensive tasks, such as weeding, to be automated without damaging crops.

Effects of hydrogen assisted stress corrosion on damage tolerance of a high-strength duplex stainless steel wire for prestressing concrete

The study brings new insights on the hydrogen assisted stress corrosion on damage tolerance of a high-strength duplex stainless steel wire which concerns its potential use as active reinforcement for concrete prestressing. The adopted procedure was to experimentally state the effect of hydrogen on the damage tolerance of cylindrical smooth and precracked wire specimens exposed to stress corrosion cracking using the aggressive medium of the standard test developed by FIP (International Prestressing Federation). Stress corrosion testing, mechanical fracture tests and scanning electron microscopy analysis allowed the damage assessment, and explain the synergy between mechanical loading and environment action on the failure sequence of the wire. In presence of previous damage, hydrogen affects the wire behavior in a qualitative sense, consistently to the fracture anisotropy attributable to cold drawing, but it does not produce quantitative changes since the steel fully preserves its damage tolerance.