Techniques and Strategies 2017 – Abstracts

DAY 1

Next-generation Sequencing
Dr Elaine Kenny (TCD)

Next-generation sequencing (NGS) is technology that can generate DNA sequence data in a manner that is fast and accurate. This talk will provide an introduction to Next-Generation Sequencing technology and its many applications.


EPIGENETICS & EPIGENOMICS: Experimental Design, Execution & Analysis
Dr Antoinette Perry (UCD)

“Epigenetics” is defined as the study of heritable changes in gene expression that are not accompanied by changes in DNA sequence. Epigenetic modifications (DNA methylation, changes in the expression and behaviour of chromatin modifiers and non-coding RNAs) are integrally involved in developmental processes as well as many human diseases. This lecture will provide an introductory overview to this field and give practical direction on how to plan, execute and analyse experiments, considering both individual regions as well as -omics technologies.


Research and Clinical Applications of NGS
Dr Denise harold

Next-generation sequencing (NGS) applications have progressed far beyond sequencing (or re-sequencing) genomes. This talk will describe some of the techniques currently employed to identify functional elements within the human genome, particularly those impacting gene regulation, and their relevance to disease. Applications of NGS in the clinic will also be discussed.

Useful Literature

GTEx Consortium. Genetic effects on gene expression across human tissues. Nature, 2017, 550(7675):204-213. PMID: 29022597

Roadmap Epigenomics Consortium. Integrative analysis of 111 reference human epigenomes. Nature, 2015, 518(7539):317-30. PMID: 25693563

ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature, 2012, 489(7414):57-74. PMID: 22955616


Stem Cells – Use, Isolation and Analysis
Dr Cynthia Coleman (NUI Galway)

Stem cells are relatively unspecialised cells lacking tissue-specific characteristics. Under appropriate conditions they can generate one or multiple specialised cell types in a process called ‘differentiation’. Stem cells show enormous therapeutic potential based on their ability to generate cells for repair or regeneration of damaged tissues and organs. Stem cells can also be used as a source of ‘normal’ human cells to study processes such as development and wound healing as well as investigating human-specific toxicity testing of drugs.
This lecture will describe stem cells sources, characteristics and analysis. Hopefully it will give you some ideas about how stem cells could contribute to your research.

Useful Literature

Modeling Development and Disease with Organoids
Hans Clevers1,
1Hubrecht Institute/Royal Netherlands Academy of Arts and Sciences, Princess Maxima Centre and University Medical Centre Utrecht,
3584CT Utrecht, The Netherlands   http://dx.doi.org/10.1016/j.cell.2016.05.082

The Molecular and Cellular Choreography of Appendage Regeneration
Elly M. Tanaka1,
1DFG Research Center for Regenerative Therapies, Technische Universita¨ t Dresden Fetscherstrasse 105, 01307 Dresden, GERMANY
http://dx.doi.org/10.1016/j.cell.2016.05.038

Stem Cells: A Renaissance in Human Biology Research
Jun Wu1,2 and Juan Carlos Izpisua Belmonte1,
1Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA
2Universidad Cato´ lica San Antonio de Murcia (UCAM) Campus de los Jero´ nimos, 135, Guadalupe 30107, Murcia, Spain
http://dx.doi.org/10.1016/j.cell.2016.05.043

Pluripotent stem cells in disease modelling and drug discovery.
Avior Y1, Sagi I1, Benvenisty N1.
Nat Rev Mol Cell Biol. 2016 Mar;17(3):170-82. doi: 10.1038/nrm.2015.27. Epub 2016 Jan 28.


Model Organisms
Dr Alison Reynolds (UCD)

The aim of this lecture is to give an overview of the advantages and disadvantages of the various model organisms which are routinely used in biomedical research. We will discuss the use of vertebrate and mammalian models and their application to the study of development and disease research. We will cover the generation of transgenic models of disease using both targeted methods of genetic manipulation and random screens. These methods have greatly improved our understanding of gene function and drug action. The various models used in vision research will be used as case studies.


Manipulating the Rodent Genome Using Transgenic and Gene Targeting Techniques
Dr Tom Moore (UCC)

Manipulation of gene expression and function in living rodents is virtually a prerequisite for studying developmental genetics and pathology and the creation of rational models of human genetic diseases. However, the available techniques remain challenging and expensive. I will provide an overview of the underlying principles and practical applications of these techniques, which are usually applied to mice, but with increasing frequency to rats and, to a lesser extent, other mammalian species. Techniques covered in this lecture will include electroporation, oocyte microinjection and lentivirus mediated transgenesis, gene targeting in embryonic stem cells, gene trap libraries, inducible vectors, shRNA, zinc finger nucleases, TALENs, and CRISPR. I will provide an overview of national and international centres engaged in large-scale production of mutants and suggestions for additional reading.

Useful Literature

Selected publications

Kumar et al. 2009; doi:10.1007/978-1-60327-378-7_22
Schaefer et al. 2017; doi:10.1038/nmeth.4293
Cohen 2016; doi:10.1126/science.aal0334
Quadros et al. 2017; DOI 10.1186/s13059-017-1220-4

Some service centres and companies

http://www.transgenics.ie
http://www.sanger.ac.uk/science/programmes/archive-mouse-and-zebrafish-genetics-programme
https://www.jax.org
https://www.cyagen.com/us/en/service/transgenic-mice.html
https://www.genoway.com/service/transgenic-mouse-models.htm
https://www.ozgene.com


Protein Expression and Purification
Dr Henry Windle (TCD)

There is a growing need to generate and purify multiple types of recombinant proteins for use in diverse areas such as structural studies, diagnostic/pharmaceutical use, biochemical studies, industrial use and for use as research reagents and antibody production. It is relatively easy to clone a gene and express the gene product in a suitable host by making use of the many commercially available versatile expression systems and hosts. Problems are generally encountered at the protein purification stage however. This presentation will cover the basics of protein expression and purification. Emphasis will be placed on alternative strategies and issues that should be considered prior to selection of specific expression systems and purification strategies.

Useful literature

Methods in Enzymology 559, 2-148 (2015): Laboratory methods in Enzymology: Protein part D – This collection represents a very comprehensive overview of protein expression and purification strategies
Burgess R. (2012) Fusion tags: A collection of papers. Protein Express. Purif. 81.
GE Healthcare Life Sciences Handbook Collection: ‘Recombinant Protein Purification: principles and Methods.


Immunodetection Methods on Cell and Tissue Extracts
Dr Ann Hopkins (RCSI)

Antibodies have been used as tools to detect and characterise proteins for decades. With the ongoing development of high throughput protein detection techniques such as tissue microarrays and reverse phase protein arrays, a real challenge now exists to contextualise the cellular locations, levels of expression and the functions of identified proteins. In this lecture, the fundamental immunological principles underlying protein detection in cells and tissues will first be outlined. This will set the scene for discussing experimental approaches to detect either protein expression levels, protein localization or protein-protein interactions. Accordingly the applications of immunodetection in a modern molecular context will be illustrated, including western blotting, ELISA, immunohistochemistry, immunofluorescence, tissue microarrays, co-immunprecipitation, electromobility shift assays, chromatin immunoprecipitation (ChIP) and antibody arrays. Common experimental pitfalls and interpretational challenges will be discussed. After this lecture, the participants should have an appreciation of how to design and optimise experiments to detect proteins in a variety of contexts and models.

Useful Literature

Protein Microarrays for Personalized Medicine. Xiaobo Yu, Nicole Schneiderhan-Marra, Thomas O. Joos
DOI: 10.1373/clinchem.2009.137158 Published February 2010

Protein Analytical Assays for Diagnosing, Monitoring, and Choosing Treatment for Cancer Patients.
Alicia D. Powers and Sean P. Palecek. Department of Chemical and Biological Engineering, University of Wisconsin-Madison, USA. Journal of Healthcare Engineering. Volume 3 (2012), Issue 4, Pages 503-534. http://dx.doi.org/10.1260/2040-2295.3.4.503

Protein expression profiling arrays: tools for the multiplexed high-throughput analysis of proteins. Jens R Sydor and Steffen NockEmail author. Proteome Science20031:3. https://doi.org/10.1186/1477-5956-1-3

 


 

Fluorescence microscopy based high-content screening: Turning pixels into quantitative data
Dr Eugene Dempsey (UCD)

High-content screening (HCS) combines automated microscopy and computer based image analysis to generate quantitative data which can be used to accurately describe phenotypic changes in cells. HCS typically utilises fluorescence based microscopy, thereby, allowing multiple markers of different cellular components to be imaged in parallel. Quantitative data on each cellular component is then extracted using sophisticated image analysis pipelines for further downstream analysis. To date, HCS has been successfully employed in a wide number of fields from novel drug discovery and toxicology to the mapping of biological pathways. This seminar will aim to provide an overview of fluorescence based microscopy, current HCS technology and typical HCS workflows. Example data will be used to demonstrate the power of combining RNA interference with HCS. Finally, looking to the state of the art, we will discuss 3-dimensional in vitro models and HCS.

1. Bray MA, Carpenter A. Advanced Assay Development Guidelines for Image-Based High Content Screening and Analysis. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
2. Martin S, Buehler G, Ang KL, Feroze F, Ganji G, Li Y. Cell-Based RNAi Assay Development for HTS. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
3. Buchser W, Collins M, Garyantes T, Guha R, Haney S, Lemmon V, et al. Assay Development Guidelines for Image-Based High Content Screening, High Content Analysis and High Content Imaging. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
4. Strovel J, Sittampalam S, Coussens NP, Hughes M, Inglese J, Kurtz A, et al. Early Drug Discovery and Development Guidelines: For Academic Researchers, Collaborators, and Start-up Companies. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, et al., editors. Assay Guidance Manual. Bethesda (MD)2004.
5. Boutros M, Heigwer F, Laufer C. Microscopy-Based High-Content Screening. Cell. 2015;163(6):1314-25.


DAY 2

 

Translational fMRI Functional Connectomics
Dr Clare Kelly (TCD)

Task-independent or “resting state” functional magnetic resonance imaging (fMRI) approaches (“functional connectomics”) have revolutionized our understanding of brain functional organisation and have driven significant advances toward the goal of identifying valid and reliable biomarkers of psychiatric illness. Functional connectomics offers the promise of a truly translational tool; the correspondence between functional circuits identified in the human, macaque, and rodent has been demonstrated. This talk will provide an overview of fMRI-based functional connectomics and will illustrate how translational work has the potential to provide mechanistic insights into how disturbances in typical brain development give rise to psychiatric and neurological disorders.

Useful Literature

Di Martino, A., Fair, D. A., Kelly, C., Satterthwaite, T. D., Castellanos, F. X., Thomason, M. E., et al. (2014). Unraveling the miswired connectome: a developmental perspective. Neuron, 83(6), 1335–1353. http://doi.org/10.1016/j.neuron.2014.08.050

Gorges, M., Roselli, F., Müller, H.-P., Ludolph, A. C., Rasche, V., & Kassubek, J. (2017). Functional Connectivity Mapping in the Animal Model: Principles and Applications of Resting-State fMRI. Frontiers in Neurology, 8, 200. http://doi.org/10.3389/fneur.2017.00200

Hutchison, R. M., & Everling, S. (2012). Monkey in the middle: why non-human primates are needed to bridge the gap in resting-state investigations. Frontiers in Neuroanatomy, 6, 29. http://doi.org/10.3389/fnana.2012.00029

 


Flow Cytometry, Cell Sorting and Flow Imaging
Dr Alfonso Blanco (UCD)

Flow cytometry is a method for qualitative and quantitative analysis of components or structural features of cells, primarily by optical means, but also particles. Although it makes measurements on one cell at a time, it can process thousands of cells per second. Since cell types can be distinguished by quantitating structural and/or physiological features, flow cytometry can be used to count prokaryotic or eukaryotic cells of different types in complex mixtures. Flow cytometry PDF

Useful Literature

Flow Cytometry – A Basic Introduction (by Mike Ormerod)
http://flowbook-wiki.denovosoftware.com/site/introtoflowormerod.shtml

Clinical Flow Wiki

http://wiki.clinicalflow.com/

Compensation by Mario Roederer
http://www.drmr.com/compensation/index.html

How to perform cell counts using a hemocytometer
https://www.youtube.com/watch?v=dLly_DFjXEo&feature=em-uploademail-new

ThermoFisher. Resource Center. Introduction to Flow Cytometry. Analyzing Flow Cytometry Data
http://www.thermofisher.com/ie/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-technical-resources.html?icid=fr-flow-4

ThermoFisher. Fluorescence Tutorials
http://www.thermofisher.com/ie/en/home/support/tutorials.html

ExCyte. Expert Cytometry
https://expertcytometry.com/mastery-class/

Purdue University Cytometry Lab
http://www.cyto.purdue.edu/

BiteSized Immunology
https://www.immunology.org/public-information/bitesized-immunology


Imaging Using Fluorescent/Confocal Microscopy
Dr Gavin McManus (TCD)

Fluorescence microscopy is an important and fundamental tool for biomedical research. Optical microscopy is almost non-invasive and allows highly spatially resolved images of organisms, cells, macromolecular complexes and biomolecules to be obtained. Generally speaking, the architecture of the observed structures is not significantly modified and the environmental conditions can be kept very close to physiological reality. The development of fluorescence microscopy was revolutionised with the invention of Laser Scanning Confocal Microscopy (LSCM). With its unique three-dimensional representation and analysis capabilities, this technology gives us a more real view of the world.

Useful Literature

https://www.ibiology.org/online-biology-courses/microscopy-series/microscopy-series-table-contents/

https://www.microscopyu.com/

https://www.thermofisher.com/ie/en/home/references/molecular-probes-the-handbook.html

https://www.chroma.com/sites/default/files/HandbookofOpticalFilters.pdf


Systems Systems Medicine – what it is and where we are
Dr Manuela Salvucci (RCSI)

Mathematical modelling has provided mechanistic insight into cell signalling and disease processes by identifying network-level control functions that originate from the interplay of multiple proteins. Validated systems models can accurately predict cell responses both kinetically and quantitatively. In translational studies, systems models also show potential for predicting for example tumour responsiveness to therapy. This talk will outline principles and concepts of systems biology research and illustrate the benefits of such approaches in the context of apoptosis signalling.

Useful Literature

Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein
https://www.ncbi.nlm.nih.gov/pubmed/16932741

Mathematical modelling of the mitochondrial apoptosis pathway
https://www.ncbi.nlm.nih.gov/pubmed/20950651

Harnessing system models of cell death signalling for cytotoxic chemotherapy: towards personalised medicine approaches?
https://www.ncbi.nlm.nih.gov/pubmed/24477766


Polymorphisms Associated with Disease
Dr Ricardo Segurado (UCD)

Different strategies are required to identify rare and common genetic variants underlying both rare and common diseases. For common genetic variants, there is now a very rich dataset of identified common single nucleotide polymorphisms (SNPs). These can be investigated in disease groups (compared to controls) in candidate genes, or by whole genome association analysis, using up to millions of SNPs. Analysis of these variants requires careful attention to the patterns of association of SNPs that are chromosomally adjacent (in linkage disequilibrium) and to the population history or genetic structure of the patients and control individuals. Linkage analysis tracks in families the disease co-inheritance with widely spaced gene markers, and is the traditional approach of choice for rare mutations with strong phenotypic effects. High throughput sequencing of whole genomes has greatly accelerated the rate of data accumulation for research, as well as clinical characterisation of rare disorders, and there are many and varied data resources available for dry-bench research.

Useful Literature

Book: An Introduction to Genetic Epidemiology. Palmer, Burton & Davey Smith (Policy Press, 2011). The chapters were also published in the Lancet journal volume 266 (2005), in a series running from issue 9489: http://www.sciencedirect.com/science/article/pii/S0140673605673229 through to issue 9495.

10 Years of GWAS Discovery: Biology, Function, and Translation. Am J Hum Genet. 2017 Jul 6;101(1):5-22. doi: 10.1016/j.ajhg.2017.06.005.  https://www.ncbi.nlm.nih.gov/pubmed/28686856


Mass Spectrometry in Biological and Medical Research
Prof Matthias Wilm (UCD)

With the discovery of the two soft ionisation methods electrospray and matrix assisted laser desorption/ionization mass spectrometry as an analytical technique made its way into biological research. Today this technique is the major tool for protein identification, the characterisation of secondary modifications and the analysis of lipids and small molecules in biological research.

With thousands of proteins identified in each analysis the information of how much of each protein was in the sample became of outermost importance. With new acquisition regimes and software tools the ability to quantify proteins in a reliable and reproducible way just got massively improved. By this mass spectrometry based protein characterisation will be broadly applicable to medical research.

Useful Literature

The Coming Age of Complete, Accurate, and Ubiquitous Proteomes
Matthias Mann,1 ,2 , Nils A. Kulak,1 Nagarjuna Nagaraj,1 and Ju¨ rgen Cox1
1Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
2The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen,
2200 Copenhagen, Denmark  http://dx.doi.org/10.1016/j.molcel.2013.01.029

Multiplexed peptide analysis using data-independent acquisition and Skyline
Jarrett D Egertson1, Brendan MacLean1, Richard Johnson1, Yue Xuan2 & Michael J MacCoss1
1Department of Genome Sciences, University of Washington, Seattle, Washington, USA. 2Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany. Correspondence
should be addressed to M.J.M. (maccoss@uw.edu).
Published online 21 May 2015; doi:10.1038/nprot.2015.055


Application of Proteomics for new Biomedical Discoveries
Prof Steve Pennington (UCD)

Following the sequencing of the genomes of a large number of organisms including the landmark publications of the human genome it has become increasingly apparent that the study of their encoded proteins – on a genome-wide scale – is required. This is the field of proteomics. When, in 1994, Marc Wilkins coined the term ‘proteome’, defined as the protein complement of a genome, the methods for investigating protein expression on such a scale were in their infancy and today (over two decades later) they are still in evolution.  Data from recent efforts to map the human proteome and several biomedical applications of proteomics will be shown to illustrate how protein-centric strategies can lead to new biomedical ‘discoveries’.

Useful Literature

1: Meyer JG, Schilling B. Clinical applications of quantitative proteomics using targeted and untargeted data-independent acquisition techniques. Expert Rev Proteomics. 2017 May;14(5):419-429. doi: 10.1080/14789450.2017.1322904. PubMed
PMID: 28436239; PubMed Central PMCID: PMC5671767.

2: Jones LH, Neubert H. Clinical chemoproteomics-Opportunities and obstacles. Sci Transl Med. 2017 Apr 19;9(386). pii: eaaf7951. doi: 10.1126/scitranslmed.aaf7951.
Review. PubMed PMID: 28424333.

3: Murray HC, Dun MD, Verrills NM. Harnessing the power of proteomics for identification of oncogenic, druggable signalling pathways in cancer. Expert Opin
Drug Discov. 2017 May;12(5):431-447. doi: 10.1080/17460441.2017.1304377. Epub
2017 Mar 17. Review. PubMed PMID: 28286965.

4: Duarte TT, Spencer CT. Personalized Proteomics: The Future of Precision Medicine. Proteomes. 2016 Oct 1;4(4). pii: E29. doi: 10.3390/proteomes4040029.
Review. PubMed PMID: 28248239.

5: Geyer PE, Holdt LM, Teupser D, Mann M. Revisiting biomarker discovery by plasma proteomics. Mol Syst Biol. 2017 Sep 26;13(9):942. doi:
10.15252/msb.20156297. Review. PubMed PMID: 28951502; PubMed Central PMCID:
PMC5615924.

6: Mardamshina M, Geiger T. Next-Generation Proteomics and Its Application to Clinical Breast Cancer Research. Am J Pathol. 2017 Oct;187(10):2175-2184. doi:
10.1016/j.ajpath.2017.07.003. Epub 2017 Jul 20. Review. PubMed PMID: 28736317.


Cellular Oxygen Consumption as an index of Metabolic Activity
Dr Richard Porter (TCD)

 

Cellular oxygen consumption gives a good measure of cellular metabolic activity. In primary cells, oxygen consumption is primarily due to oxidative phosphorylation by mitochondria. However, in some primary cells and cell lines a more comprehensive account of metabolism can be achieved by also measuring glycolytic flux. The Agilent Seahorse Flux Analyzer and the Oroboros Oxygraph Respirometer are two excellent and popularly used apparati to detect cellular oxygen consumption. In the former cells have to be adherent, whereas in the latter cells have to be in suspension. The lecture will shed light on the value of measuring cellular oxygen consumption.

Useful Literature

Porter RK, Brand MD.(1995) Cellular oxygen consumption depends on body mass, Am J Physiol. 1995 Jul;269(1 Pt 2):R226-8. PMID: 7631898

Geoghegan F, Chadderton N, Farrar GJ, Zisterer DM, Porter RK.(2017) Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells. Oncol Lett. 2017 Nov;14(5):6298-6306. doi: 10.3892/ol.2017.6929.

Martin DS, Leonard S, Devine R, Redondo C, Kinsella GK, Breen CJ, McEneaney V, Rooney MF, Munsey TS, Porter RK, Sivaprasadarao A, Stephens JC, Findlay JB.(2016) Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice. J Mol Endocrinol. 2016 Apr;56(3):261-71. doi: 10.1530/JME-15-0225.

https://www.agilent.com/en/promotions/seahorse-xf-technology

http://www.oroboros.at/index.php?id=high-resolution-respirometry