1,721,067 research outputs found
Application of multivariate statistics and machine learning to phenotypic imaging and chemical high-content data
Full text linkImage-based high-content screens (HCS) hold tremendous promise for cell-based
phenotypic screens. Challenges related to HCS include not only storage and
management of data, but critical analysis of the complex image-based data. I
implemented a data storage and screen management framework and developed
approaches for data analysis of a number high-content microscopy screen formats.
I visualized and analysed pilot screens to develop a robust multi-parametric assay
for the identification of genes involved in DNA damage repair in HeLa cells.
Further, I developed and implemented new approaches for image processing and
screen data normalization. My analyses revealed that the ubiquitin ligase RNF8
plays a central role in DNA-damage response and that a related ubiquitin ligase
RNF168 causes the cellular and developmental phenotypes characteristic for the
RIDDLE syndrome. My approaches also uncovered a role for the MMS22LTONSL
complex in DSB repair and its role in the recombination-dependent repair
of stalled or collapsed replication forks.
The discovery of novel bioactive molecules is a challenge because the fraction of active
candidate molecules is usually small and confounded by noise in experimental
readouts. Cheminformatics can improve robustness of chemical high-throughput
screens and functional genomics data sets by taking structure-activity relationships
into account. I applied statistics, machine learning and cheminformatics
to different data sets to discern novel bioactive compounds. I showed that phenothiazines
and apomorphines are regulators for cell differentiation in murine
embryonic stem cells. Further, I pioneered computational methods for the identification of structural features that influence the degradation and retention of
compounds in the nematode C. elegans. I used chemoinformatics to assemble a
comprehensive screening library of previously approved drugs for redeployment
in new bioassays. A combination of chemical genetic interactions, cheminformatics
and machine learning allowed me to predict novel synergistic antifungal small
molecule combinations from sensitized screens with the drug library. In another
study on the biological effects of commonly prescribed psychoactive compounds,
I discovered a strong link between lipophilicity and bioactivity of compounds in
yeast and unexpected off-target effects that could account for unwanted side effects
in humans. I also investigated structure-activity relationships and assessed
the chemical diversity of a compound collection that was used to probe chemical-genetic
interactions in yeast. Finally, I have made these methods and tools available
to the scientific community, including an open source software package called
MolClass that allows researchers to make predictions about bioactivity of small
molecules based on their chemical structure
Approaches to target WD40 proteins and synthesis and evaluation of chemical tools for on-bead screening
Full text linkA database consisting of information on human WD40 domains was compiled from literature
sources. Data collected included information on function, structure, links to disease and
information on molecules known to bind to WD40 domain containing proteins. Curation of
the data collected suggested that 21% of WD40 domain containing proteins are linked to
cancer, and that only 6% had known small molecule binders. From the database a shortlist of
WD40 domain containing proteins that were considered of interest as research targets was
produced. It was determined that WD40 Domain Containing Protein 5 (WDR5) was a
potential cancer target open to several targeting methods.
WDR5 normally plays a structural roll in the formation of a complex containing WDR5,
RbBP5, ASH2L, and DPY-30. This complex is required for methylation of H3K4, when MLL1
joins the complex it is able to methylate H3K4me2. It was also recently determined that WDR5
complexes with MYC, another protein with roles in transcriptional control. Both MLL1 and
MYC are known to be prominent cancer targets.
His-tagged WDR5 was successfully expressed in BL21 (DE3) cell line and purified by a 2 step
method. First the protein was purified via His tag - Ni-NTA agarose affinity chromatography,
the eluted protein was then further purified via Size Exclusion Chromatography.
The first approach targeting WDR5 consisted of a combination of an in-silico approach and a
small molecule binder screen. Two in-silico methods, Q-mol and USRCAT, were used to
determine small molecules that would potentially bind to WDR5. From this suggested set, 81
compounds were screened against WDR5. Thermal denaturation fluorescence (TDF) was
chosen as assay technique. A single compound increased the thermal stability of WDR5 in
repeated experiments. This compound, NCI292249, was further characterised in microdialysis
experiments where it was determined to have a low affinity of 564 μM to WDR5.
In a second approach, in order to target the MYC-WDR5 interaction, a series of truncated
peptides derived from the WDR5 binding motif from MYC were produced testing a variation
of the One-Bead One-Compound (OBOC) synthesis method derived in this work. These
peptide fragments were synthesised using Fmoc solid phase peptide synthesis on TentaGel
micobeads experimenting with the SOBOC technique (Scanning OBOC) adapted to produce
all possible fragments of a peptide in parallel. The peptide fragments were fluorescently
labelled with tetramethylrhodamine and screened against 6XHis-WDR5 isolated on Ni-NTA
functionalised agarose beads. The peptide fragments were ranked based on their affinity to
the WDR5-coated micro-beads assayed by Confocal Scanning (CONA). The highest affinity
peptide found in the CONA screen was further tested for WDR5 binding in solution by
fluorescence anisotropy which resulted in an affinity of 96 μM to WDR5. This 7-mer truncate
of the MYC peptide was used as input for an in-silico method of peptide optimisation named
MorPH. MorPH is a technique developed in the Auer Lab in which amino acids in a peptide
are systematically replaced by all commercially available non-natural amino acids in a
sequential manner. Each of ~ 1000 modified peptidomimetics are docked in-silico to the target
structure. The suggestions from MorPH for the MYC peptide truncate were analysed and the
potential replacements discussed in order to plan a possible future synthesis.
The MorPH technique was tested experimentally in this thesis in a second example, targeting
of Survivin. Survivin is followed as cancer target in the Auer lab and it is found in significantly
high concentrations in cancer cell lines and in stem cells. Increased Survivin expression has
also been linked to a poor prognosis and reduced patient survivability in the clinic. Several
suggestions resulting from the MorPH in-silico screen were synthesised and screened against
Survivin. The best-in-series peptide was shown to have a Kd of 2.5 μM, with significantly
increased plasma stability.
Several chemical tools were developed and characterised for use with on-bead synthesis
methods. A contribution was made to a novel synthetic method for isomerically pure
rhodamine dyes and their functionalisation[1] (Tetramethylrhodamine was azide-functionalised
for use in peptide labelling, based on the azide-alkyne Huisgen cycloaddition
reaction). The Auer lab synthesises many of its compounds and libraries using solid phase
synthesis techniques. Several compounds exist in literature for the linking of chemicals to a
solid-support, all of which are stable to different chemistries and require different conditions
to cleave the reaction product from the solid phase. The use of methionine as a linker is
described in literature as being highly specific in its cleavage conditions. A series of literature
cleavage conditions were tested, the method that offered the highest purity was selected to be
improved through further testing. The improved cleavage method was then characterised by
cleaving the 20 natural amino acids from the methionine linker. In this experiment it was
determined that all amino acids tolerated the new conditions with the exception of
methionine, cysteine and tryptophan, which were expected to react poorly to the harsh
conditions. This verified that methionine was a suitable alternative to current lab standards
for bead linkage
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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