Cell-Based Screening to Identify Cytoprotective Compounds

Prevention of cellular injury and consequent cell death is expected to provide therapeutic benefit in various diseases, but with the complexity of cell damaging pathways involved, identification and validation of novel potential drug targets is not a trivial task. New drug targets are expected to take part in complex responses with wide-ranging effects on gene expression and cellular function and drug candidates rather modify these effects than act as simple agonists or antagonists to ultimately protect the cells from an injury. Phenotypic screening may help identify cytoprotective compounds in diseases, in which the lack of drug targets makes target-based approaches unfeasible. This chapter gives an overview of the strategy of cell-based assay development, primary screening, hit selection and confirmation. Considerations about the choice of small molecule compound libraries utilized in cell-based models are discussed as well as the use of clinical drugs for drug repurposing or repositioning. The choice of cell types and issues associated with cell culture techniques are overviewed and the most common assays and readouts are briefly described. Finally, the potential pitfalls of data analysis and hit selection are discussed

Toward a Rhetoric of DNA: The Advent of CRISPR

The nucleic acid DNA, which contains an organism’s genetic information, consists of a four-letter alphabet that has until recently been characterized as a read-only text. The development of a quick, inexpensive DNA targeting and manipulation technique called CRISPR, pronounced “crisper,” though, has changed DNA from this arhetorical, read-only data set, as it has been characterized in the rhetoric literature to date, to a fully rhetorical text—one that can be not only read but created, interpreted, copied, altered, and stored as well. The Book of Nature, an idea with roots in antiquity but popularized during the nineteenth century, provides proof of concept in the form of an historical and theoretical context in which DNA can be viewed in this light. Once ensconced in the Book of Nature, DNA can longer be considered a code; rather, it is a text. DNA text has structural components that are similar to those of traditional text, and now, with CRISPR, it also has purposes, audiences, and stakeholders. Given the enormous potential of DNA text for both good and ill, rhetoricians of science and medicine must participate in discussions of the complex literacy, policy, and ethics issues this new form of text brings about

Green Chemistry Oxidative Modification of Peptoids Utilizing Bleach and TEMPO

Biotherapeutic drugs, derived from biological molecules such as proteins and DNA, are becoming an integral and exceptionally critical aspect of modern medicine. Compared to common pharmaceutical drugs, biotherapeutics are much larger in size and have greater target specificity, allowing them to treat many chronic diseases ranging from cancer to rheumatoid arthritis. The major issue with protein based therapeutics is that they readily undergo proteolysis, or enzymatic degradation, when administered through subcutaneous injections. Traditionally, biotherapeutic modification procedures have centered on the use of PEG derivatives. This process, called PEGylation, is unfavorable due to the increases in molecular weights of the proteins and the heterogeneous mixture of products formed. Instead of PEG derivatives, we propose peptoids with N- methoxyethylglycine (NMEG) side chains to decrease proteolysis. NMEG groups are more advantageous than PEG derivatives due to their low molecular weight and ability to form homogeneous products. Our work focuses on increasing the protease resistance of target biotherapeutic proteins by cross-linking a NMEG-5 peptoid to a cytochrome c via reductive amination. In the presence of a reducing agent, an imine bond is formed through the reduction of the peptoid’s aldehyde group and cytochrome c’s primary amine groups. Due to the expensive and unstable nature of commercially available aldehyde side chains, a green chemistry method, using only sodium hypochlorite (bleach) and 2,6,6-Tetramethylpiperidinoxy (TEMPO, free radical), oxidized the peptoid’s hydroxyl group into the desired aldehyde for cross linkage

Untersuchungen zum Einfluss intronischer miRNAs auf Expression und Funktion ihrer Host-Gene an den Beispielen miR-641/AKT2 und miR-744/MAP2K4 in malignen Gliomen

Genes that regulate essential cellular functions, such as apoptosis or proliferation, must be subject to tight transcriptional control. MiRNAs located in introns of these genes could fulfill important regulatory functions by directly or indirectly controlling the expression of their host genes. The present work dealt with the question of whether intronically localized miRNAs can exert a regulatory influence on their host genes and whether these control mechanisms are impaired or even inactivated in tumors. This was investigated in two known tumorigenic kinases, AKT2 and MAP2K4, and the intronically located miRNA-641 and miRNA-744 by real-time PCR, RNA interference experiments, protein analysis, immunohistochemistry, reporter gene assays, established long-term cell cultures, primary cell lines from glioblastoma tissue and glioblastoma biopsies. It could be shown that both kinases -indeed- are inhibited by their intronic miRNAs via indirect negative feedback: miR-641 inhibits phosphorylation via the targets PIK3R3, MAPKAP1 and NFAT5 and thus the activity of its tumor host gene AKT2; miRNA-744 inhibits the activity of the SMAD and MAPK signaling pathway by targeting TGFB1. We further found that both miRNAs are strongly repressed in glioblastomas as compared to normal brain tissue. In contrast, the miRNA target genes PIK3R3, NFAT5 and TGFB1 were induced in tumor tissue. The present doctoral thesis was thus able to demonstrate an important principle of expressional control: intronic miRNAs can regulate their host genes via complex functional networks. The disruption of these negative feedback mechanisms can significantly influence intracellular signalling promote tumour progression. These results may contribute to the development of miRNA-based future therapeutic strategies

RNA contact prediction by data efficient deep learning

On the path to full understanding of the structure-function relationship or even design of RNA, structure prediction would offer an intriguing complement to experimental efforts. Any deep learning on RNA structure, however, is hampered by the sparsity of labeled training data. Utilizing the limited data available, we here focus on predicting spatial adjacencies ("contact maps") as a proxy for 3D structure. Our model, BARNACLE, combines the utilization of unlabeled data through self-supervised pre-training and efficient use of the sparse labeled data through an XGBoost classifier. BARNACLE shows a considerable improvement over both the established classical baseline and a deep neural network. In order to demonstrate that our approach can be applied to tasks with similar data constraints, we show that our findings generalize to the related setting of accessible surface area prediction

Three-dimensional chromatin organisation in human pancreatic islets

Diabetes is a group of metabolic diseases that affects millions of people. Despite this, little is known about the underlying molecular mechanisms. Diabetes is characterised by an impaired blood-glucose regulation that can lead to severe consequences, such as kidney failure, and premature death. Pancreatic islets are one of the major tissues to understand diabetes pathogenesis as they produce insulin, a hormone central for blood-glucose homeostasis. Our previous work showed that studying epigenomic regulation is key to giving insight into the molecular mechanisms underlying diabetes, as risk-associated genomic variants are enriched at transcriptional regulatory regions named enhancers. To give further insight in pancreatic islet transcriptional regulation, I aimed to decipher the 3D chromatin organisation, an aspect of epigenomic regulation in human pancreatic islets that remained largely unexplored until now. As part of my PhD project I have studied high-resolution chromatin interaction maps that characterise 3D chromatin organisation at different levels, from single interactions between specific pair of genomic loci to large genomic topological domains known as TADs. These high-resolution chromatin interaction maps, integrated with a large collection of epigenomic datasets, allowed me to describe several aspects of islet 3D chromatin organisation, such as the identification of islet-selective chromatin structures associated to islet-specific gene expression. Moreover, I identified groups of enhancers that gather in 3D space. These 3D enhancer clusters were frequently found in loci key for islet function and highly enriched in diabetes associated variants. The results of this thesis allow us to have a more accurate picture of the epigenomic regulation in human pancreatic islets and how non-coding diabetes risk variants could be impairing enhancer-promoter communication.Open Acces

Dual RNA-Seq analysis of Mus musculus and Leishmania donovani transcriptomes

Parasitic protozoa of the genus Leishmania cause a spectrum of disease, affecting 12 million people worldwide. This project aimed to investigate the effect of Leishmania donovani infection on the gene expression of healthy/WT (Black 6) and immunocompromised (RAG2KO) mice. Differences in the gene expression of parasites in inoculum and tissue were also elucidated. WT and RAG mice were infected using an L. donovani inoculum, and were euthanised after 28 days. Harvested spleens (and the inoculum) were used to generate RNA samples, from which mRNA was isolated and purified. Transcriptome data was generated using dual RNA-Seq approaches from the mRNA samples. After appropriate pre-processing, data underwent a number of bioinformatic analyses to explore differential gene expression, such as Gene Ontology, Gene Set Enrichment, and KEGG Pathway analysis. Comparison of different mouse spleen transcriptomes revealed that even in uninfected mice, WT mice more highly express genes related to immunoglobulins when compared with their immunocompromised counterparts. Healthy mice were found to react to infection through the induction of inflammatory response, and the production of NOX generating species. RAG mice still upregulated immunoglobulin-related genes in response to infection, despite their inability to generate antibodies, T-cells, and B-cells. However, RAG modulation of haeme and iron metabolism may contribute to defence against the parasites despite a lack of acquired immunity. Differences in the amastin, the key glycoprotein on the surface on intracellular-stage parasites, are apparent between the inoculum and tissue parasites, which may reflect microenvironment adaptation. Additionally, tissue-derived parasites showed significant upregulation of genes related to gene expression control, such as histones and DNA-packing. These experiments are among the first attempts to in vivo transcriptome sequence mice and Leishmania simultaneously, a powerful approach giving insight to action and reaction. However, these techniques are not without challenge, such as low parasite read counts