Analysis of epigenomic variability in grapevine and its relation with structural variation

Epigenetics encompasses a series of chromatin modifications that are potentially inheritable and can result into a change of gene expression without involving a change in the underlying DNA sequence. Epigenetics is involved in several fundamental mechanisms that regulate cell cycle in all eukaryotes including X chromosome inactivation, gene silencing, paramutation, parental imprinting, chromatin position effect, plant gametogenesis, flowering time, stress responses and light signaling. Within epigenetic modifications, DNA methylation is predominant and widespread in all eukaryotic kingdoms and consists in a reversible reaction which transfers a methyl group on a cytosine. In mammals methylation occurs in CG-rich regions known as CpG islands, whereas in plant methylation may occurs in CG, CHG and CHH contexts, where H may be A,C or T, with different mechanisms. Depending on the location, DNA methylation may have opposite effects: in heterochromatin it is generally associated to transcriptional inactivity but in the transcribed region of genes, methylation in the CG context is associated to medium-to-high transcriptional level. The silencing effect of DNA methylation represents a useful defense weapon against both retrovirus infection and transposable element (TE) insertions: indeed such sequence elements generally become highly methylated as a plant response to prevent further mobilization. Despite this mechanism, during evolution TEs colonized eukaryotic genomes, up to represent 75% of the total genome in some plant species. . TEs are a major factor underlying the tremendous intra-species genome variability that has been revealed thanks to the introduction of next generation sequencing (NGS) technology and the resequencing of several individuals of the same species. This led scientists to introduce, initially only for bacteria then for any organism, the concept of pan-genome, composed by a common genome shared by all the individuals of the species and a dispensable genome which is not essential for survival, but is the foundation for phenotypic variability. The dispensable genome consists of the entire set of structural variations (SVs) observed among individuals and is mainly represented by TEs. TE sequences are generally methylated and their methylation may spread in their flanking regions.Thus, when TEs accidentally insert nearby genes or regulating sequences, they may alter their epigenetic status creating epigenetic variants called epialleles. 4 Specific protocols of NGS, involving the use of bisulfite, which in the overall process converts unmethylated cytosines to thymines, allow to map all methylcytosines of a genome with single-base resolution. The aim of this work was to analyse the relationship between structural variations, mainly represented by TEs, and epigenetic variations in plants. Grapevine is a suitable model for this study because it is a perennial species and generally, it is vegetatively propagated in agriculture. This technique preserves the genome from recombination, thus it allows maintaining the genotypes stable across clonal generations and focusing on mere epigenetic variation. Moreover for grapevine a highly homozygous reference genome is already available as well as a set of grapevine-specific TEs annotated. Three biological replicates of leaf nuclear DNA of the cultivar Pinot Noir, which shares one haplotype with the sequenced genome reference, have been sequenced and analysed. To evaluate the spreading of internal TE methylation on the flanking regions, we considered hemizygous TEs in order to compare the same regions on homologous chromosome in presence or absence of TEs. Consistently with other species, grapevine TEs show high methylation in their sequence in both CG and CHG context whereas CHH context is extremely low methylated. Internal TE methylation is generally spread on their flanking regions. Within TEs, retrotranspons show a stronger impact on flanking regions compared to DNA-transposons, with different behaviors according to the differential genomic distribution of TE-groups: Ty3-Gypsy usually insert in highly methylated regions of pericentromeric chromatin, LINEs element are frequently found in highly CG methylated gene bodies, Ty1-Copia display more variable locations. Generally, where not saturated, retrotransposon insertions provoke an increase of methylation in both CG and CHG contexts, supported by statistical analyses. DNA methylation is also present in transcribed regions of grapevine genes, in particular in the CG context. A set of about 19000 genes was utilized to analyze gene body methylation (GBM) in grapevine. Similarly to other species, grapevine GBM displays an asymmetrical bell-shape profile, in which the 5\u2019 is much less methylated than 3\u2019. Surprisingly introns appear more methylated than exons, in contrast with other species such as Arabidopsis, humans and honey bee. Grapevine introns occupy a large part of the genome (36.7%) and are quite rich in TEs that represent 12.4% of their sequence. The moderate TE content of introns may partially explain their higher methylation compared to flanking exons. However, when excluding genes carrying TE from the analysis, methylation in both exons and introns is reduced but still present, confirming that GBM methylation is independent from TEs, although their insertion may increase it. 5 Analysis of gene expression showed that genes located in highly methylated regions, especially in the CHG context, show on average a lower expression rate and furthermore their expression tend to be more conserved within varieties. On the contrary, when methylation occurs in gene bodies, transcriptional activity is not reduced and it may be even higher. Gene expression may also be modulated by TEs: when these are located in gene flanking regions, gene expression rate is significantly lower than unaffected genes, whereas genes whose introns are enriched in TEs display significantly higher methylation and expression rates. Lastly, allele-specific expression analyses indicate that hemizygous TEs may affect the contribution of the two alleles to the expression rate of the gene. Taken together these data confirm that DNA methylation occurs in grapevine withpatterns comparable with other plant species, but with the peculiarity of highly methylated introns whose methylation is generally associated to moderate TE content and medium-to-high expression level

I. IR Photodissociation Spectroscopy of Large van der Waals Clusters. II. UV Laser-Induced Photochemistry of Fe(CO)₅ on Single Crystal Surfaces

Chapter 2 is a summary of vibrational predissociation spectra of weakly bound clusters containing either CF3I or CF3Br. Spectra indicative of ArCF3I, KrCF3I, (CF3I)2, ArCF3Br and (CF3Br)2 clusters are presented with comparison to the matrix-isolation spectra of CF3I and CF3Br. The infrared photodissociation technique can be applied to the study of weakly bonded clusters which do not absorb in the infrared by attaching an IR chromophore; this work is presented in Chapter 3. Vibrational predissociation spectra of large clusters of Ar, Kr, N2 and CH4 containing a single CH3F or C2H4 chromophore are obtained as a function of cluster size. The IR spectra distinguish three regimes of cluster size in the CH3F case. The dissociation profiles yield information on the chromophore environment and the lifetime of the excited vibration. The clusters are gas-phase analogies to the matrix-isolation technique, although differences between the two are consistent with the presumed icosahedral cluster geometry, and "matrix" and "surface" sites are distinguishable. Chapter 4 presents the application of a simple two-level-with-decay model to the photodesorption of weakly bound adsorbates on crystal surfaces. Feasibility estimates are reported and establish that vibrational predissociation of the excited adsorbes can be induced in the infrared, where energy quenching mechanisms on surfaces can be quite fast. The effect of these quenching mechanisms on the lineshapes present the opportunity to probe the processes with the use of the photodesorption technique. Extraction of the phenomenological rate constants is described. Also reported are attempts to observe photodesorption induced by low-power lasers. The photodecomposition of Fe(CO)5 on three single crystal surfaces, sapphire (Al2O3), Si(100) and Ag(110), using UV irradiation is described in Chapter 5. The quenching of excited electronic states of adsorbates is expected to be quite different on the surfaces, yet dissociation of the Fe(CO)5 is observed in all cases. The results allow determination of the dissociation mechanism and also imply that the Fe(CO)5 dissociation rate is ultra-fast (&lt; 10-13). Extensions of this initial investigation are discussed.</p

Bakeout controller for the use of helium closed-cycle refrigerators in ultrahigh vacuum applications

imple control electronics are described which provide automated bakeout of an ultrahigh vacuum (UHV) chamber equipped with a helium closed-cycle refrigerator. By preventing the refrigerator from reaching elevated temperatures (T>50 °C) during bakeout, these devices can be utilized in the ultrahigh vacuum environment

Low Resistance Polycrystalline Diamond Thin Films Deposited by Hot Filament Chemical Vapour Deposition

Polycrystalline diamond thin films with outgrowing diamond (OGD) grains were deposited onto silicon wafers using a hydrocarbon gas (CH4) highly diluted with H2 at low pressure in a hot filament chemical vapour deposition (HFCVD) reactor with a range of gas flow rates. X-ray diffraction (XRD) and SEM showed polycrystalline diamond structure with a random orientation. Polycrystalline diamond films with various textures were grown and (111) facets were dominant with sharp grain boundaries. Outgrowth was observed in flowerish character at high gas flow rates. Isolated single crystals with little openings appeared at various stages at low gas flow rates. Thus, changing gas flow rates had a beneficial influence on the grain size, growth rate and electrical resistivity. CVD diamond films gave an excellent performance for medium film thickness with relatively low electrical resistivity and making them potentially useful in many industrial applications

Anisotropic optical response of the diamond (111)-2x1 surface

The optical properties of the 2$\times$1 reconstruction of the diamond (111) surface are investigated. The electronic structure and optical properties of the surface are studied using a microscopic tight-binding approach. We calculate the dielectric response describing the surface region and investigate the origin of the electronic transitions involving surface and bulk states. A large anisotropy in the surface dielectric response appears as a consequence of the asymmetric reconstruction on the surface plane, which gives rise to the zigzag Pandey chains. The results are presented in terms of the reflectance anisotropy and electron energy loss spectra. While our results are in good agreement with available experimental data, additional experiments are proposed in order to unambiguously determine the surface electronic structure of this interesting surface.Comment: REVTEX manuscript with 6 postscript figures, all included in uu file. Also available at http://www.phy.ohiou.edu/~ulloa/ulloa.html Submitted to Phys. Rev.

Dependence of the emission from tris(8-hydroxyquinoline) aluminum based microcavity on device thickness and the emission layer position

In this work, we present a systematic study of the emission from bilayer organic microcavity light emitting diodes with two metal mirrors. The devices consisting of two organic layers, N,NV-di(naphthalene-1-yl)-N,NV-diphenylbenzidine as the hole transport layer and tris (8-hydroxyquinoline) aluminum as the emitting layer, and two metal mirrors were fabricated and characterized by transmittance, reflectance, photoluminescence, and electroluminescence measurements. The effects of layer thickness, interface position, and the choice of anode(bottom mirror) were investigated. The transmittance and reflectance spectra were modeled using a transfer matrix model, and the optical functions for all the materials used were determined by spectroscopic ellipsometry. The dependence of the photoluminescence and electroluminescence spectra on the device thickness and interface position is discussed

Device Optimization of Tris-Aluminum (Alq3) Based Bilayer Organic Light Emitting Diode Structures

In this work we present detailed analysis of the emitted radiation spectrum from tris(8-hydroxyquinoline) aluminum (Alq3) based bilayer OLEDs as a function of: the choice of cathode, the thickness of organic layers, and the position of the hole transport layer/Alq3 interface. The calculations fully take into account dispersion in the glass substrate, the indium tin oxide anode, and in the organic layers, as well as the dispersion in the metal cathode. Influence of the incoherent transparent substrate (1 mm glass substrate) is also fully accounted for. Four cathode structures have been considered: Mg/Ag, Ca/Ag, LiF/Al, and Ag. For the hole transport layer, N,N'-diphenyl-N,N'-(3-methylphenyl)-1,1'-biphenyl-4,4'-diamine (TPD) and N,N'-di(naphthalene-1-yl)-N,N'-diphenylbenzidine (NPB) were considered. As expected, emitted radiation is strongly dependent on the position of the emissive layer inside the cavity and its distance from the metal cathode. Although our optical model for an OLED does not explicitly include exciton quenching in vicinity of the metal cathode, designs placing the emissive layer near the cathode are excluded to avoid unrealistic results. Guidelines for designing devices with optimum emission efficiency are presented. Finally, several different devices were fabricated and characterized and experimental and calculated emission spectra were compared