Predicting enhancer regions and transcription factor binding sites in D. melanogaster

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 71-75).Identifying regions in the genome that have regulatory function is important to the fundamental biological problem of understanding the mechanisms through which a regulatory sequence drives specific spatial and temporal patterns of gene expression in early development. The modENCODE project aims to comprehensively identify functional elements in the C. elegans and D. melanogaster genomes. The genome- wide binding locations of all known transcription factors as well as of other DNA- binding proteins are currently being mapped within the context of this project [8]. The large quantity of new data that is becoming available through the modENCODE project and other experimental efforts offers the potential for gaining insight into the mechanisms of gene regulation. Developing improved approaches to identify functional regions and understand their architecture based on available experimental data represents a critical part of the modENCODE effort. Towards this goal, I use a machine learning approach to study the predictive power of experimental and sequence-based combinations of features for predicting enhancers and transcription factor binding sites.by Rachel Sealfon.S.M

Elucidation of molecular kinetic schemes from macroscopic traces using system identification

Overall cellular responses to biologically-relevant stimuli are mediated by networks of simpler lower-level processes. Although information about some of these processes can now be obtained by visualizing and recording events at the molecular level, this is still possible only in especially favorable cases. Therefore the development of methods to extract the dynamics and relationships between the different lower-level (microscopic) processes from the overall (macroscopic) response remains a crucial challenge in the understanding of many aspects of physiology. Here we have devised a hybrid computational-analytical method to accomplish this task, the SYStems-based MOLecular kinetic scheme Extractor (SYSMOLE). SYSMOLE utilizes system-identification input-output analysis to obtain a transfer function between the stimulus and the overall cellular response in the Laplace-transformed domain. It then derives a Markov-chain state molecular kinetic scheme uniquely associated with the transfer function by means of a classification procedure and an analytical step that imposes general biological constraints. We first tested SYSMOLE with synthetic data and evaluated its performance in terms of its rate of convergence to the correct molecular kinetic scheme and its robustness to noise. We then examined its performance on real experimental traces by analyzing macroscopic calcium-current traces elicited by membrane depolarization. SYSMOLE derived the correct, previously known molecular kinetic scheme describing the activation and inactivation of the underlying calcium channels and correctly identified the accepted mechanism of action of nifedipine, a calcium-channel blocker clinically used in patients with cardiovascular disease. Finally, we applied SYSMOLE to study the pharmacology of a new class of glutamate antipsychotic drugs and their crosstalk mechanism through a heteromeric complex of G protein-coupled receptors. Our results indicate that our methodology can be successfully applied to accurately derive molecular kinetic schemes from experimental macroscopic traces, and we anticipate that it may be useful in the study of a wide variety of biological systems

BioPP: a tool for web-publication of biological networks

<p>Abstract</p> <p>Background</p> <p>Cellular processes depend on the function of intracellular molecular networks. The curation of the literature relevant to specific biological pathways is important for many theoretical and experimental research teams and communities. No current tool supports web publication or hosting of user-developed large scale annotated pathway diagrams. Sharing via web publication is needed to allow real-time access to the current literature pathway knowledgebase, both privately within a research team or publicly among the outside research community. Web publication also facilitates team and/or community input into the curation process while allowing centralized control of the curation and validation process. We have developed new tool to address these needs. Biological Pathway Publisher (BioPP) is a software suite for converting CellDesigner Systems Biology Markup Language (CD-SBML) formatted pathways into a web viewable format. The BioPP suite is available for private use and for depositing knowledgebases into a newly created public repository.</p> <p>Results</p> <p>BioPP suite is a web-based application that allows pathway knowledgebases stored in CD-SBML to be web published with an easily navigated user interface. The BioPP suite consists of four interrelated elements: a pathway publisher, an upload web-interface, a pathway repository for user-deposited knowledgebases and a pathway navigator. Users have the option to convert their CD-SBML files to HTML for restricted use or to allow their knowledgebase to be web-accessible to the scientific community. All entities in all knowledgebases in the repository are linked to public database entries as well as to a newly created public wiki which provides a discussion forum.</p> <p>Conclusion</p> <p>BioPP tools and the public repository facilitate sharing of pathway knowledgebases and interactive curation for research teams and scientific communities. BioPP suite is accessible at <url>http://tsb.mssm.edu/pathwayPublisher/broadcast/</url></p

Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization

The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section

Stacking weak lensing signals of SZ clusters to constrain cluster physics

We show how to place constraints on cluster physics by stacking the weak lensing signals from multiple clusters found through the Sunyaev-Zeldovich (SZ) effect. For a survey that covers about 200 sq. deg. both in SZ and weak lensing observations, the slope and amplitude of the mass vs. SZ luminosity relation can be measured with few percent error for clusters at z~0.5. This can be used to constrain cluster physics, such as the nature of feedback. For example, we can distinguish a pre-heated model from a model with a decreased accretion rate at more than 5sigma. The power to discriminate among different non-gravitational processes in the ICM becomes even stronger if we use the central Compton parameter y_0, which could allow one to distinguish between models with pre-heating, SN feedback and AGN feedback, for example, at more than 5sigma. Measurement of these scaling relations as a function of redshift makes it possible to directly observe e.g., the evolution of the hot gas in clusters. With this approach the mass-L_SZ relation can be calibrated and its uncertainties can be quantified, leading to a more robust determination of cosmological parameters from clusters surveys. The mass-L_SZ relation calibrated in this way from a small area of the sky can be used to determine masses of SZ clusters from very large SZ-only surveys and is nicely complementary to other techniques proposed in the literature.Comment: Submitted to Ap