Automated data integration for developmental biological research

In an era exploding with genome-scale data, a major challenge for developmental biologists is how to extract significant clues from these publicly available data to benefit our studies of individual genes, and how to use them to improve our understanding of development at a systems level. Several studies have successfully demonstrated new approaches to classic developmental questions by computationally integrating various genome-wide data sets. Such computational approaches have shown great potential for facilitating research: instead of testing 20,000 genes, researchers might test 200 to the same effect. We discuss the nature and state of this art as it applies to developmental research

Programming microbes to treat superbug infection

Superbug infection is one of the greatest public health threat with grave implications across all levels of society. Towards a new solution to combat infection by multi-drug resistant bacteria, this thesis presents an engineering framework and genetic tools applied to repurpose commensal bacteria into “micro-robots” for the treatment of superbug infection. Specifically, a prototype of designer probiotic was developed using the human commensal bacteria Escherichia coli. The engineered commensal was reprogrammed with user-specified functions to sense superbug, produced pathogen-specific killing molecules and released the killing molecules via a lytic mechanism. The engineered commensal was effective in suppressing ~99% of planktonic Pseudomonas and preventing ~ 90% of biofilm formation. To enhance the sensing capabilities of engineered commensal, genetic interfaces comprising orthogonal AND & OR logic devices were developed to mediate the integration and interpretation of binary input signals. Finally, AND, OR and NOT logic gates were networked to generate a myriad of cellular logic operations including half adder and half subtractor. The creation of half adder logic represents a significant advancement of engineering human commensal to be biological equivalent of microprocessor chips in programmable computer with the ability to process input signals into diversified actions. Importantly, this thesis provides exemplary case studies to the attenuation of cellular and genetic context dependent effects through principles elucidated herein, thereby advancing our capability to engineer commensal bacteria.Open Acces

Capsaspora owczarzaki as a unicellular model to study the pre-metazoan integrin adhesome

[eng] Animal multicellular structure depends on three general mechanisms: cell adhesion, cell communication and cell differentiation. Some of these mechanisms were thought to be specific to animals. Genome sequencing of unicellular organisms that are closely related to animals has revealed that the unicellular ancestor of all animals already contained homologs of some of the genes that have a role in cell adhesion, cell communication and cell differentiation. Studying the function of these genes in their unicellular context is the way to infer the function they could have had in the ancestor, thus, helping us to understand how animals originated. Capsaspora owczarzaki, a filopodiated amoeba, is the closer unicellular relative of animals that contains key elements of the major cell-matrix adhesion system that animal cells use: the integrin adhesome. Thus, Capsaspora is an ideal model to study the origin of the integrin adhesome. However, the tools that would make feasible the experiments to study the function of these genes are not available. Therefore, this work has focused on developing molecular tools to work with Capsaspora, as well as understanding with other means the function of proteins related to the integrin adhesome. First, the life cycle of Capsaspora has been described at a cellular and molecular level. This revealed that the life stages of Capsaspora are transcriptomically regulated. Characterisation of its epigenome has revealed that these stages are regulated by some mechanisms which are shared with animals. In order to perform genetic studies, a transfection protocol based in calcium-phosphate precipitation has been established for Capsaspora. It was successfully used to label different structures of the cell with fluorescent tags, one of such allowed to visualise with unprecedented detail the filopodia in Capsaspora. As a first approach to the function of the integrin adhesome in Capsaspora, antibodies were designed to localise integrin adhesome proteins in the cell, and an adhesion assay was set up to analyse the adhesive potential of Capsaspora. Localisation of proteins in the filopodia and adhesion to fibronectin suggest that the integrin adhesome proteins in Capsaspora could be involved in adhesive function as in animals. The tools developed during this work are the first steps towards turning Capsaspora into a model organism to study the function of proteins involved in animal multicellularity.[spa] La multicelularidad animal depende de tres mecanismos generales: adhesión, comunicación y diferenciación celular. La secuenciación de los genomas de los organismos unicelulares emparentados con los animales han revelado que el ancestro unicelular de los animales ya contenía homólogos de algunos genes involucrados en estos mecanismos. Si estudiamos la función de estos genes en su contexto unicelular podremos inferir la función que podrían haber tenido en el ancestro de los animales, lo cual nos ayudará a entender cómo se originaron los animales a partir de este ancestro. Capsaspora owczarzaki, una ameba con filopodios, es, entre todos los parientes unicelulares de animales, el más cercano que contiene elementos clave del sistema de adhesión a la matriz extracelular más importante de animales: el adhesoma de integrina. Por lo tanto, Capsaspora es un organismo ideal para estudiar el origen del adhesoma de integrina. Las herramientas necesarias para llevar a cabo estos experimentos, sin embargo, no estaban disponibles. El trabajo realizado durante esta tesis se ha centrado en desarrollar herramientas moleculares para trabajar con Capsaspora, y en estudiar la función de las integrinas y otras proteínas relacionadas con el adhesoma de integrina en Capsaspora, con el fin de acercarnos a la función que podría haber tenido antes de que emergieran los animales. En primer lugar se ha analizado el ciclo celular de Capsaspora en cultivo, a nivel celular y molecular. Además se ha demostrado por primera vez en este organismo la existencia de un estadio agregativo. Comprobamos que las transiciones entre estadios están reguladas transcripcionalmente, y que en el estadio agregativo en concreto, los genes relacionados con la multicelularidad presentan una expresión más alta que en los otros estadios. Esto sugiere que en el ancestro unicelular de los animales, podría haber habido un mecanismo que permitiera la agregación celular y que fue reutilizado para dar lugar al actual sistema que emplean los animales durante su desarrollo. El epigenoma de Capsaspora también se ha analizado. Descubrimos que Capsaspora posee algunos mecanismos de regulación del genoma que comparte con animales, como modificaciones postranscripcionales de histonas y secuencias cis-reguladoras próximas a los genes. Los resultados también sugieren que los genes regulados en Capsaspora forman una red gobernada por factores de transcripción, como en animales. Sin embargo Capsaspora sólo posee un tipo de promotor, mientras que los animales tienen diferentes tipos de promotores (definidos por distintas marcas de histonas), y a su vez, carece de secuencias reguladoras distales, a diferencia de animales. Para poder estudiar la función de genes relacionados con multicelularidad en Capsaspora es necesario establecer herramientas genéticas. Con este fin, se ha desarrollado un protocolo de transfección reproducible y se han construido una serie de plásmidos que nos han permitido visualizar estructuras celulares de Capsaspora con fluorescencia. Por otro lado, y con el objetivo de estudiar la función del adhesoma de integrina en Capsaspora, se ha diseñado un ensayo de adhesión que nos ha permitido estudiar su capacidad de adhesión a distintas proteínas. También se han producido anticuerpos contra proteínas del adhesoma de integrina de Capsaspora para localizarlas en la célula. Estos últimos experimentos realizados nos aportan los primeros datos sobre la función de integrinas en un organismo unicelular, los cuales sugieren que las proteínas del adhesoma de integrina de Capsaspora están involucradas en adhesión, al igual que las de animales. En resumen, en este trabajo se han desarrollado herramientas genéticas y moleculares en un organismo no modelo que permitirá a la comunidad científica plantearse nuevas preguntas sobre el origen de animales

Characterization of post-transcriptional regulatory network of RNA-binding proteins using computational predictions and deep sequencing data

This report is divided into three parts: Data Analysis, Mathematical Modeling and Conclusion and future directions. In the Data Analysis part, various methods and tools for characterizing the post-transcriptional regulatory networks of RNA-binding proteins are discussed and applied. Chapter 2 introduces PAR-CLIP, a method for transcriptomewide identification of RNA binding proteins at nucleotide resolution. PAR-CLIP was successfully applied on RNA binding proteins and their binding specificity was characterized. Partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, Chapter 3 presents CLIPZ, which is a database and analysis environment for various kinds of deep sequencing (and in particular CLIP) data, that aims to provide an open-access repository of information for post-transcriptional regulatory elements. Chapter 4 revisits various CLIP methods. A set of ideas in terms of both experimental protocols and data analysis are presented to improve the quality and reproducibility of such experiments. In general, cytoplasmic RNAs are isolated in CLIP experiments. Like many high-throughput experiments, CLIP has a certain amount of isolated RNAs which do not represent regulatory binding sites. To improve the quality of the obtained RNAs, a set of novel methods for data analysis are also suggested. These methods are added as new tools to the CLIPZ analysis platform. Argonaute CLIP data could in principle be beneficial in improving the microRNA target site predictions. However, several questions still remain which cannot be addressed using CLIP methods. For example: • Argonaute CLIP data by default does not reveal which microRNAs are more likely to interact to the mRNA binding site at the time of cross-linking. • As mentioned earlier, biochemical and structural studies of Thermus thermophilus Argonaute protein suggest that the protein-RNA interaction between microRNA and the Argonaute protein forms a physical structure that only some positions in the microRNA become accessible to the target binding site. Having inferred the interacting microRNA, it is also interesting to predict the most plausible secondary structure of the hybridized microRNA-mRNA complex. Mathematical Modeling part of the report contains Chapter 5. This chapter presents a novel mathematical model called MIRZA to address the above mentioned questions. An in-depth introduction to MIRZA is presented and its performance in terms of identifying functionally relevant targets of microRNAs is discussed. Finally, Conclusion and future directions part of the report contains Chapter 6 in which discusses the main findings of the projects and gives an outlook of where future work could be taken up

Population Genomics of a Baboon Hybrid Zone in Zambia

Hybridization is increasingly recognized as a common, important process shaping the evolution of organisms including humans. Across hybrid zones, the genomes of incipient species are mixed and recombined through hybridization and backcrossing, creating conditions ideal for evaluating the actions of natural selection on gene variants in novel genomic contexts. This dissertation aims to increase our understanding of hybridization using a Zambian baboon study system in which two species, Kinda baboons (Papio kindae) and grayfoot baboons (Papio griseipes), hybridize despite exhibiting pronounced differences in body size and behavior. Using genome-wide genotypic data prepared using double-digest RADseq, I scan for genomic regions under selection in these species and in their hybrids.Because a large section of the hybrid zone contains groups unhabituated to human presence, I develop a new method for noninvasive genomic-scale genotyping from feces. I demonstrate that an enrichment procedure using methyl-CpG-binding-domain proteins to preferentially capture densely CpG-methylated mammalian DNA effectively partitions baboon host DNA from contaminating bacterial DNA, yielding efficient sequencing of target genomic DNA. Comparisons of same-animal double-digest RADseq libraries demonstrate high concordance between feces-derived and blood-derived genotypes.By scanning genome-wide data for regions with significant levels of differentiation between Kinda and grayfoot baboon populations, I identify candidate genes under selection in the two species. I find evidence for selection on genes and biological pathways that underlie differences in body size between the parental species. One pathway exhibiting significantly elevated differentiation was the JAK/STAT signaling pathway, which notably serves an important role in mediating the effects of cytokine signals on processes including epiphyseal chondrocyte proliferation essential for bone growth.Analysis of hybrids reveals that Kinda and grayfoot baboons form a relatively wide cline in the Kafue river valley in central Zambia. Comparison of autosomal ancestry patterns to mitochondrial-DNA and Y-chromosome ancestry patterns reveals that the Kinda baboon Y chromosome has introgressed extensively across the species barrier relative to both the mitochondrial genome and the remainder of the nuclear genome. The JAK/STAT signaling pathway exhibits restricted introgression, suggesting a role in barriers to reproduction possibly due to the unusually high or low body size sexual dimorphism between male grayfoot x female Kinda and male Kinda x female grayfoot baboon mating partners. The toll-like receptor pathway exhibits enhanced introgression, suggesting adaptive introgression of pathogen defenses. Finally, the sperm tail gene ODF2 exhibits enhanced introgression and an advantage of the grayfoot baboon variant. I suggest based on a house mouse analogy that male hybrids may be subjected to reduced sperm quality but that this effect may be mitigated or overcome by the presence of an invading Y chromosome. This effect potentially explains the extreme introgression of the Kinda baboon Y chromosome

COMPARATIVE GENOMICS AND MOLECULAR EVOLUTION: NEW GENOMIC RESOURCES FOR THE HYMENOPTERA AND EVOLUTIONARY STUDIES ON THE GENES OF THE \u3ci\u3eNasonia vitripennis\u3c/i\u3e HOX COMPLEX.

Research on insects, the most successful group from all metazoans on earth, has important societal, as well as scientific benefits. Insects occupy a wide range of roles, which have an effect on human life either because the former pose serious threats to public health and commercial crops as well as in some cases represent the only way to propagate food resources. Despite their tremendous importance, insect genomics remained an uneven territory dominated by studies in the Drosophila group and the mosquitoes. This dissertation attempts to: 1) report on advances in the development and characterization of genomic tools for species of the order Hymenoptera in the hopes of helping to close this gap; and 2) to shed light on the organization, origin and evolution of genes of the Hox cluster in species of the order Hymenoptera through molecular evolution analyses that were possible thanks to the availability of the aforementioned genomic resources

Arabidopsis leaf mutants reveal conserved and unique proteins involved in light and auxin signaling

Growth and development in plants is steered by the meristems which are established on both poles of the embryo and become active after germination. The activity of the shoot apical meristem (SAM) leads to the formation of lateral organs such as leaves which are responsible for photosynthesis. At flowering time the SAM is transformed to the inflorescence meristem which produces flowers as lateral organs. The root apical meristem is responsible for the growth of the primary root and is important in water- and mineral-uptake. Within the research group “Chromatin and Growth Control” several genes are being cloned of which the mutant phenotype shows a defect in leaf growth. This way the genes coding for components of the Elongator complex were identified. In yeast Elongator was defined as a histone acetyltransferase (HAT) complex associated with RNA polymerase II to facilitate transcription elongation. In this thesis the loci of two other leaf mutants ang3 and ron3 belonging to the angusta and rotunda class respectively were cloned. By transcript profiling of the leaf mutants using micro-array data we try to pinpoint in which biological process the gene is involved. Localization studies and tandem affinity purification of the respective protein helps to identify which molecular pathways are likely to be affected. With detailed phenotypical analysis of the mutants we are often able to link the affected processes to observed phenotypes. Depending on the obtained results we perform specific experiments (ChIP, hormone measurements,…) to verify the true function of the protein. This work aimed at investigating the role of the Elongator complex in plants and several objectives were put forward 1. To prove the role of Elongator in transcription 2. To identify target genes of Elongator using chromatin-immuno-precipitation Besides the functional characterization of the Elongator complex, two other leaf mutants ang3 and ron3 belonging to the angusta and rotunda class respectively were cloned and functionally characterized. The genes were cloned by fine-mapping combined with sequence analysis of candidate genes in the genetic interval around the locus of interest. Molecular analysis of the genes was combined with morphological and cellular analyses of the corresponding mutants to determine the cellular basis of the observed growth defects and to gain deeper insight into the function of the genes

Genomic Regulation Of Abiotic Stress Response In The Soil Nematode Oscheius Tipulae

Oscheius tipulae is a species of free-living soil nematode that can be found in ecosystems worldwide. Because of this, individuals must be able to respond to heat, freezing, and desiccation stresses in order to survive. They do this by producing a suite of cellular responses, some of which are necessary to survive multiple stresses, and some are stress-specific. While these cellular responses are well known, the ways in which they are regulated in a genome-wide context are not. In this project, multiple high throughput sequencing and bioinformatics analyses were utilized to answer this question. First, the O. tipulae genome was sequenced via Illumina HiSeq, assembled, and annotated. An RNA-Seq experiment was performed to determine transcription patterns within stress responses. Pooled nematode samples were subjected to heat, freezing, or desiccation stress prior to RNA sequencing and read mapping. Results showed that shared cellular responses were controlled by the upregulation of both shared and stress-specific genes. This suggests that the genome remains efficient by utilizing overlapping response genes and reinforcing them with stress-specific genes. Whole genome bisulfite sequencing and MethylCap-Seq analyses were performed to assess DNA cytosine methylation presence in O. tipulae and the model organism Caenorhabditis elegans and to determine its role in the abiotic stress response process in O. tipulae. Methylated cytosines were found in both O. tipulae and C. elegans, contradicting the historical belief that cytosine methylation is absent in nematodes. Changes in DNA methylation were not associated with the abiotic stress response as very few methylation cites were found within upregulated genes. This project utilized new sequencing technologies and various bioinformatics programs to provide an in-depth look into the genome-wide responses to abiotic stress in O. tipulae