Identification of Possible Failures of Machines with Subsequent Verification

Import 02/11/2016Tématem diplomové práce je navrhnout a sestavit demonstrační model jako učební pomůcku, na kterém půjdou simulovat různé poruchy strojů. Dané poruchy strojů lze následně verifikovat metodami vibrační diagnostiky. První část diplomové práce je věnována návrhu a tvorbě demonstračního modelu. V druhé části diplomové práce jsou popsány jednotlivé simulace poruch, které lze na demonstračním modelu provádět. Poslední část diplomové práce se zabývá reálnými příklady poruch strojů, se kterými jsme se ve své dosavadní vibrodiagnostické praxi setkal.The topic of master thesis is designing and constructing a demonstration model as a teaching aid, on which various machine malfunctions can be simulated. Subsequently, it is possible to verify particular malfunctions by the vibrational diagnostic methods. The first part of master thesis is focused on the design and the construction of a demonstrational model. In the second part of the thesis, particular malfunction simulations, which are possible to be performed on the model, are described. The last part deals with real examples of machine breakdowns which I have encountered in my vibro-diagnostic practice so far.340 - Katedra výrobních strojů a konstruovánívýborn

Roots of angiosperm formins: The evolutionary history of plant FH2 domain-containing proteins

<p>Abstract</p> <p>Background</p> <p>Shuffling of modular protein domains is an important source of evolutionary innovation. Formins are a family of actin-organizing proteins that share a conserved FH2 domain but their overall domain architecture differs dramatically between opisthokonts (metazoans and fungi) and plants. We performed a phylogenomic analysis of formins in most eukaryotic kingdoms, aiming to reconstruct an evolutionary scenario that may have produced the current diversity of domain combinations with focus on the origin of the angiosperm formin architectures.</p> <p>Results</p> <p>The Rho GTPase-binding domain (GBD/FH3) reported from opisthokont and <it>Dictyostelium </it>formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I formins and Class II formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm) formins that define a specific branch (Class III) of the formin family.</p> <p>Conclusion</p> <p>We propose a scenario where formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored) Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II formins, allowing later loss of the RhoGAP-like domain-containing formins in angiosperms.</p

Solution of Reduction of Hydraulic Motor Vibrations of OPKS Assembly Line

Import 05/08/2014Bakalářská práce se zabývá technickou diagnostikou. Teoretická část bakalářské práce obsahuje obecný popis technické diagnostiky a dále popisuje vibrodiagnostiku, jako jednu z metod technické diagnostiky. Praktická část práce se zabývá měřením vibrací na lince OPKS. Z naměřených dat se vyhodnotí stav zařízení. Cílem bakalářské práce je navrhnout vhodné řešení, které bude mít za následek zlepšení stavu zařízení.The bachelor thesis deals with the technical diagnostics. The theoretical part of the thesis contains a general description of technical diagnostics and describes vibrodiagnostics as one of the methods of technical diagnostics. The practical part of the thesis deals with the measurement of vibration on the OPKS line. Aim of this work is to propose a suitable solution that will result in enhancing the state of the device.340 - Katedra výrobních strojů a konstruovánívýborn

Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering

EXO70C2 is a key regulatory factor for optimal tip growth of pollen

The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollenspecific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes

Developmental plasticity of Arabidopsis hypocotyl is dependent on exocyst complex function

The collet (root–hypocotyl junction) region is an important plant transition zone between soil and atmospheric environments. Despite its crucial importance for plant development, little is known about how this transition zone is specified. Here we document the involvement of the exocyst complex in this process. The exocyst, an octameric tethering complex, participates in secretion and membrane recycling and is central to numerous cellular and developmental processes, such as growth of root hairs, cell expansion, recycling of PIN auxin efflux carriers and many others. We show that dark-grown Arabidopsis mutants deficient in exocyst subunits can form a hair-bearing ectopic collet-like structure above the true collet, morphologically resembling the true collet but also retaining some characteristics of the hypocotyl. The penetrance of this phenotypic defect is significantly influenced by cultivation temperature and carbon source, and is related to a defect in auxin regulation. These observations provide new insights into the regulation of collet region formation and developmental plasticity of the hypocotyl

Synergy Among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion during Vegetative Growth

Vesicle exocytosis underpins signaling and development in plants and is vital for wall remodeling during cell expansion. Vesicle tethering and fusion are thought to occur sequentially, tethering mediated by the exocyst and fusion driven by assembly of SNARE protein complexes. Interactions between these two protein complexes are known, although insights into their functional consequences are largely unexplored. We now identify a clear hierarchy of interactions leading to secretion in Arabidopsis. Mating-based split-ubiquitin screens and in vivo FRET analyses showed that exocyst EXO70 subunits bind preferentially with cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distributions and suppressed vesicle traffic like the dominant-negative SYP121deltaC, consistent with the epistasis of exo70A1 over the exo70A1syp121 double mutant. However, the exo70A1vamp721 mutant showed a strong synergistic suppression of growth. These data are best explained by a spatiotemporal hierarchy of exocyst recruitment with the R-SNARE to the plasma membrane, plausibly with the VAMP721 longin domain as a nexus for binding

Dissecting a Hidden Gene Duplication: The Arabidopsis thaliana SEC10 Locus

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation

The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant–pathogen interaction

Recently, the octameric vesicle-tethering complex exocyst was found in plants and its importance for Arabidopsis morphogenesis was demonstrated. Exo70 exocyst subunits in plants, unlike in yeasts and mammals, are represented by a multigene family, comprising 23 members in Arabidopsis. For Exo70B2 and Exo70H1 paralogues, transcriptional up-regulation was confirmed on treatment with an elicitor peptide, elf18, derived from the bacterial elongation factor. Their ability to participate in the exocyst complex formation was inferred by the interaction of both the Exo70s with several other exocyst subunits using the yeast two-hybrid system. Arabidopsis plants mutated in these two genes were used to analyse their local reaction upon inoculation with Pseudomonas syringae pv. maculicola and the fungal pathogen Blumeria graminis f. sp. hordei. The Pseudomonas sensitivity test revealed enhanced susceptibility for the two exo70B2 and one H1 mutant lines. After Blumeria inoculation, an increase in the proportion of abnormal papilla formation, with an unusual wide halo made of vesicle-like structures, was found in exo70B2 mutants. Intracellular localization of both Exo70 proteins was studied following a GFP fusion assay and Agrobacterium-mediated transient expression of the constructs in Nicotiana benthamiana leaf epidermis. GFP-Exo70H1 localizes in the vesicle-like structures, while GFP-Exo70B2 is localized mainly in the cytoplasm. It is concluded that both Exo70B2 and Exo70H1 are involved in the response to pathogens, with Exo70B2 having a more important role in cell wall apposition formation related to plant defence