98 research outputs found

    Synthesis And Reactions Of Methyl(aryl)metal Complexes

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    This thesis describes the synthesis of methyl(aryl)platinum(II), -gold(III) and -titanium(IV) complexes and their reactions with electrophiles and with unsaturated reagents. Methyl or phenyl for halogen exchange reactions of dimethyl- and diphenyltitanium complexes are also discussed.;The electrophilic reagents HCl and HgCl(,2) selectively cleave the methyl-platinum bond in the complexes cis- PtMeAr(PMePh(,2))(,2) (Ar = Ph, 4-MeOC(,6)H(,4)), whereas in the complexes cis- AuMe(,2)PhL (L = PMe(,3), PPh(,3)) the phenyl-gold bond is preferentially cleaved. It is argued that the difference in reactivity of the gold and platinum complexes indicates the operation of different mechanisms of electrophilic cleavage.;SO(,2) inserts selectively into the metal-methyl bond of cis- PtMeAr(PMePh(,2))(,2) (Ar = Ph, 4-MeOC(,6)H(,4)) and cis- AuMe(,2)PhL (L = PMe(,3), PPh(,3)), giving the S-sulfinate complexes cis- Pt(SO(,2)Me)Ar(PMePh(,2))(,2) and AuMe(SO(,2)Me)PhL . It is argued that SO(,2) does not behave as a classic electrophile, and the mechanism may involve coordination of SO(,2) to the metal prior to insertion.;Reaction of hexafluorobut-2-yne with cis- PtMeAr(PMePh(,2))(,2) (Ar = Ph, 4-MeOC(,6)H(,4)) gives cis- PtMe C(CF(,3))=C(CF(,3))Ar (PMePh(,2))(,2) .;The synthesis and reactions with electrophiles of a series of methyl(aryl)titanium(IV) complexes TiMeAr((eta)(\u275)-C(,5)H(,5))(,2) are described. The electrophiles HCl, HOAc, HgCl(,2) and HgClMe show little selectivity for cleavage of a titanium-aryl vs. a titanium-methyl bond, except in the case where Ar = 4-MeOC(,6)H(,4). Methyl or phenyl for halogen exchange reactions between TiR(,2)((eta)(\u275)-C(,5)H(,5))(,2) (R = Me, Ph) and TiX(,2)((eta)(\u275)-C(,5)H(,5))(,2) (X = F, Cl, Br, I) are described. In general, TiMe(,2)((eta)(\u275)-C(,5)H(,5))(,2) and TiPh(,2)((eta)(\u275)-C(,5)H(,5))(,2) react at similar rates. Possible mechanisms for the reactions of the organotitanium complexes are discussed

    Using surveys of Affymetrix GeneChips to study antisense expression.

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    We have used large surveys of Affymetrix GeneChip data in the public domain to conduct a study of antisense expression across diverse conditions. We derive correlations between groups of probes which map uniquely to the same exon in the antisense direction. When there are no probes assigned to an exon in the sense direction we find that many of the antisense groups fail to detect a coherent block of transcription. We find that only a minority of these groups contain coherent blocks of antisense expression suggesting transcription. We also derive correlations between groups of probes which map uniquely to the same exon in both sense and antisense direction. In some of these cases the locations of sense probes overlap with the antisense probes, and the sense and antisense probe intensities are correlated with each other. This configuration suggests the existence of a Natural Antisense Transcript (NAT) pair. We find the majority of such NAT pairs detected by GeneChips are formed by a transcript of an established gene and either an EST or an mRNA. In order to determine the exact antisense regulatory mechanism indicated by the correlation of sense probes with antisense probes, a further investigation is necessary for every particular case of interest. However, the analysis of microarray data has proved to be a good method to reconfirm known NATs, discover new ones, as well as to notice possible problems in the annotation of antisense transcripts

    Uncovering the expression patterns of chimeric transcripts using surveys of affymetrix GeneChips.

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    BACKGROUND: A chimeric transcript is a single RNA sequence which results from the transcription of two adjacent genes. Recent studies estimate that at least 4% of tandem human gene pairs may form chimeric transcripts. Affymetrix GeneChip data are used to study the expression patterns of tens of thousands of genes and the probe sequences used in these microarrays can potentially map to exotic RNA sequences such as chimeras. RESULTS: We have studied human chimeras and investigated their expression patterns using large surveys of Affymetrix microarray data obtained from the Gene Expression Omnibus. We show that for six probe sets, a unique probe mapping to a transcript produced by one of the adjacent genes can be used to identify the expression patterns of readthrough transcripts. Furthermore, unique probes mapping to an intergenic exon present only in the MASK-BP3 chimera can be used directly to study the expression levels of this transcript. CONCLUSIONS: We have attempted to implement a new method for identifying tandem chimerism. In this analysis unambiguous probes are needed to measure run-off transcription and probes that map to intergenic exons are particularly valuable for identifying the expression of chimeras

    Simkania negevensis nel paziente in dialisi e nel trapiantato di rene. Ricerche cliniche e sperimentali

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    Simkania negevensis is a bacterium belonging to the order Chlamydiales but with certain biological characteristics different from those of chlamydia, according to which it was classified in the family Simkaniaceae. It is widespread in the environment, due to its ability to survive in amoebae also in phase cystic, for which it was hypothesized a possible transmission after contact with water in which they are present amoebae. So far it is known its role in diseases of the lower respiratory tract, such as childhood bronchiolitis and pneumonia in adults of the community, following its transmission through infected aerosols. A recent American study showed, by PCR, a high prevalence of S. negevensis in patients with lung transplant than other transplant recipients, assuming an association between the presence of the bacterium in these patients, and transplant rejection, were more frequent in lung transplant recipients infected compared to uninfected. There are no data so far analyzed in Italy relative to the population of dialysis and kidney transplant recipients relative to simkania negevensis why this study was undertaken in order to start a specific location and evaluate the scientific implications. Because its ability to assume persistent forms of infection, which may lead to a prolonged inflammatory response, Simkania negevensis, similar to other persistent bacteria or viruses, may be ivolved in pathologic complication. Sn may be a factor in graft rejection in mmunesuppressed lung transplant recipients, and further studies are planned to explore the posible association of Sn infections with various in vivo pathologies

    Normalized Affymetrix expression data are biased by G-quadruplex formation

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    Probes with runs of four or more guanines (G-stacks) in their sequences can exhibit a level of hybridization that is unrelated to the expression levels of the mRNA that they are intended to measure. This is most likely caused by the formation of G-quadruplexes, where inter-probe guanines form Hoogsteen hydrogen bonds, which probes with G-stacks are capable of forming. We demonstrate that for a specific microarray data set using the Human HG-U133A Affymetrix GeneChip and RMA normalization there is significant bias in the expression levels, the fold change and the correlations between expression levels. These effects grow more pronounced as the number of G-stack probes in a probe set increases. Approximately 14 of the probe sets are directly affected. The analysis was repeated for a number of other normalization pipelines and two, FARMS and PLIER, minimized the bias to some extent. We estimate that ∼15 of the data sets deposited in the GEO database are susceptible to the effect. The inclusion of G-stack probes in the affected data sets can bias key parameters used in the selection and clustering of genes. The elimination of these probes from any analysis in such affected data sets outweighs the increase of noise in the signal. © 2011 The Author(s)

    Establishing a major cause of discrepancy in the calibration of Affymetrix GeneChips

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    <p>Abstract</p> <p>Background</p> <p>Affymetrix GeneChips are a popular platform for performing whole-genome experiments on the transcriptome. There are a range of different calibration steps, and users are presented with choices of different background subtractions, normalisations and expression measures. We wished to establish which of the calibration steps resulted in the biggest uncertainty in the sets of genes reported to be differentially expressed.</p> <p>Results</p> <p>Our results indicate that the sets of genes identified as being most significantly differentially expressed, as estimated by the z-score of fold change, is relatively insensitive to the choice of background subtraction and normalisation. However, the contents of the gene list are most sensitive to the choice of expression measure. This is irrespective of whether the experiment uses a rat, mouse or human chip and whether the chip definition is made using probe mappings from Unigene, RefSeq, Entrez Gene or the original Affymetrix definitions. It is also irrespective of whether both Present and Absent, or just Present, Calls from the MAS5 algorithm are used to filter genelists, and this conclusion holds for genes of differing intensities. We also reach the same conclusion after assigning genes to be differentially expressed using t-statistics, although this approach results in a large amount of false positives in the sets of genes identified due to the small numbers of replicates typically used in microarray experiments.</p> <p>Conclusion</p> <p>The major calibration uncertainty that biologists need to consider when analysing Affymetrix data is how their multiple probe values are condensed into one expression measure.</p

    Consistency Analysis of Redundant Probe Sets on Affymetrix Three-Prime Expression Arrays and Applications to Differential mRNA Processing

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    Affymetrix three-prime expression microarrays contain thousands of redundant probe sets that interrogate different regions of the same gene. Differential expression analysis methods rarely consider probe redundancy, which can lead to inaccurate inference about overall gene expression or cause investigators to overlook potentially valuable information about differential regulation of variant mRNA products. We investigated the behaviour and consistency of redundant probe sets in a publicly-available data set containing samples from mouse brain amygdala and hippocampus and asked how applying filtering methods to the data affected consistency of results obtained from redundant probe sets. A genome-based filter that screens and groups probe sets according to their overlapping genomic alignments significantly improved redundant probe set consistency. Screening based on qualitative Present-Absent calls from MAS5 also improved consistency. However, even after applying these filters, many redundant probe sets showed significant fold-change differences relative to each other, suggesting differential regulation of alternative transcript production. Visual inspection of these loci using an interactive genome visualization tool (igb.bioviz.org) exposed thirty putative examples of differential regulation of alternative splicing or polyadenylation across brain regions in mouse. This work demonstrates how P/A-call and genome-based filtering can improve consistency among redundant probe sets while at the same time exposing possible differential regulation of RNA processing pathways across sample types

    Evolving DNA motifs to predict GeneChip probe performance

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    Background: Affymetrix High Density Oligonuclotide Arrays (HDONA) simultaneously measure expression of thousands of genes using millions of probes. We use correlations between measurements for the same gene across 6685 human tissue samples from NCBI's GEO database to indicated the quality of individual HG-U133A probes. Low correlation indicates a poor probe. Results: Regular expressions can be automatically created from a Backus-Naur form (BNF) context-free grammar using strongly typed genetic programming. Conclusion: The automatically produced motif is better at predicting poor DNA sequences than an existing human generated RE, suggesting runs of Cytosine and Guanine and mixtures should all be avoided. © 2009 Langdon and Harrison; licensee BioMed Central Ltd

    Interpretation of multiple probe sets mapping to the same gene in Affymetrix GeneChips

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    BACKGROUND: Affymetrix GeneChip technology enables the parallel observations of tens of thousands of genes. It is important that the probe set annotations are reliable so that biological inferences can be made about genes which undergo differential expression. Probe sets representing the same gene might be expected to show similar fold changes/z-scores, however this is in fact not the case. RESULTS: We have made a case study of the mouse Surf4, chosen because it is a gene that was reported to be represented by the same eight probe sets on the MOE430A array by both Affymetrix and Bioconductor in early 2004. Only five of the probe sets actually detect Surf4 transcripts. Two of the probe sets detect splice variants of Surf2. We have also studied the expression changes of the eight probe sets in a public-domain microarray experiment. The transcripts for Surf4 are correlated in time, and similarly the transcripts for Surf2 are also correlated in time. However, the transcripts for Surf4 and Surf2 are not correlated. This proof of principle shows that observations of expression can be used to confirm, or otherwise, annotation discrepancies. We have also investigated groups of probe sets on the RAE230A array that are assigned to the same LocusID, but which show large variances in differential expression in any one of three different experiments on rat. The probe set groups with high variances are found to represent cases of alternative splicing, use of alternative poly(A) signals, or incorrect annotations. CONCLUSION: Our results indicate that some probe sets should not be considered as unique measures of transcription, because the individual probes map to more than one transcript dependent upon the biological condition. Our results highlight the need for care when assessing whether groups of probe sets all measure the same transcript
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