13 research outputs found

    Developing An Alternative Way to Analyze NanoString Data

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    Nanostring technology provides a new method to measure gene expressions. It\u27s more sensitive than microarrays and able to do more gene measurements than RT-PCR with similar sensitivity. This system produces counts for each target gene and tabulates them. Counts can be normalized by using an Excel macro or nSolver before analysis. Both methods rely on data normalization prior to statistical analysis to identify differentially expressed genes. Alternatively, we propose to model gene expressions as a function of positive controls and reference gene measurements. Simulations and examples are used to compare this model with Nanostring normalization methods. The results show that our model is more stable, efficient, and able to control false positive proportions. In addition, we also derive asymptotic properties of a normalized test of control versus treatment

    Regulation of Innate Immunity in the C. elegans Intestine by Olfactory Neurons

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    The intestinal epithelium represents one of the first lines of defense against pathogenic bacteria. Immune regulation at this critical barrier is necessary to maintain organismal fitness, and mis-regulation here has been linked to numerous debilitating diseases. Functional relationships between the nervous system and immune system have been found to be critical in the proper coordination of immune defenses at barrier surfaces, however the precise mechanisms underlying theses interactions remains unclear. Through conducting a forward genetic screen utilizing the model organism Caenorhabditis elegans, we uncovered a surprising requirement for the olfactory neuron gene olrn-1 in the regulation of intestinal epithelial immunity. During nematode development, olrn-1 is required to program the expression of odorant receptors in the AWC olfactory neuron pair. Here, we show that olrn-1 also functions in AWC neurons in the cell non-autonomous suppression of the canonical p38 MAPK PMK-1 immune pathway in the intestine. Low activity of OLRN-1, which activates the p38 MAPK signaling cassette in AWC neurons during larval development, also de-represses the p38 MAPK PMK-1 pathway in the intestine to promote immune effector transcription, increased clearance of an intestinal pathogen and resistance to bacterial infection. However, derepression of the p38 MAPK PMK-1 pathway also results in severe developmental and reproductive defects, demonstrating the critical function of OLRN-1 to both prime C. elegans intestinal epithelial cells for the induction of anti-pathogen responses, and to limit the deleterious effects of immune hyper-activation. These data reveal an unexpected connection between olfactory receptor development and innate immunity, as well as demonstrate how neuronal regulation of immune responses within the intestinal epithelium is critical for both reproductive and developmental fitness

    Molecular Mechanisms of Chemotherapy Resistance in Oestrogen Receptor Positive Breast Cancer

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    Resistance to chemotherapy is a major obstacle to the successful treatment of breast cancer. In order to optimise treatment regimens and identify novel drug targets, it is important first to understand the molecular mechanisms that can lead to chemoresistance. In this thesis, I have investigated mRNA and miRNA expression profiles of tumour samples taken from patients with oestrogen receptor positive primary breast cancers treated with neoadjuvant chemotherapy (NAC) who displayed only a partial response. Samples were analysed before and after chemotherapy treatment. Gene expression profiles post-NAC suggested that the MAPK and PI3K-AKT pathways were activated. MiRNA expression profiles demonstrated three miRNAs that were consistently deregulated post-NAC. Further in vitro studies revealed that the increased expression of miR-26b and miR-195 contributed to significant increases in chemoresistance (p<0.05). Pulldown assays using mimics of miR-26b and miR-195 as bait, together with RNA-Seq, led to the identification of possible mRNA targets of these two miRNAs. Further in vitro studies confirmed REEP4 and SEMA6D as targets of miR-26b and miR-195 respectively. As targets of these miRNAs, decreased expression of these mRNAs would be expected to contribute to chemoresistance. Chemosensitivity assays suggested a consistent but not significant increase in resistance when REEP4 was silenced, and a significant increase in resistance when SEMA6D was silenced (p<0.05). Investigations were performed to determine whether the expression of either of the corresponding proteins had any prognostic value. Results suggested that REEP4 expression was significantly related to disease free survival, although the precise relationship was unclear. The effect of increased expression of the xenobiotic drug pump BCRP induced by endocrine therapy on chemoresistance was also investigated. Results suggested that increased BCRP expression led to significant increases in chemoresistance (p<0.05), thus suggesting that a treatment regimen of endocrine therapy followed by chemotherapy may not be beneficial. I have identified in this thesis several molecular changes that are induced by chemotherapy or endocrine therapy that contribute to chemoresistance, including changes in mRNA, miRNA and xenobiotic drug pump expression

    Exosomal RNA as a source of urine biomarkers for prostate cancer

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    Introduction In this study we exploited the recent development of methods that have enabled the analysis of RNA present in urine exosomes of prostate cancer patients. We report RNA expression patterns that contain diagnostic and prognostic information for prostate cancer, and association with response to hormone treatment. Methods First catch urine following digital rectal examination were collected from 662 men. 3 groups of patients were used: Low, Intermediate, and High-risk according to NICE stratification criteria, and two control groups: benign and advanced disease. 50-gene transcript expression analysis using NanoString technology was performed on 192 samples. Exosomal RNA Next-Generation Sequencing was performed on 18 samples for novel biomarker discovery. Results Expression analysis showed that PCa-specific transcripts such as TMPRSS2/ERG fusion transcripts were identifiable in exosomes from PCa urine samples. LPD analysis highlighted expression levels of 15 transcripts with diagnostic potential (significantly up-regulated in cancer samples in comparison to benign control) and 17 transcripts with prognostic potential (differentialy expressed in high risk and advanced disease in comparison to lower grade disease). I also report two gene transcripts (SERPINB5/Maspin, HPRT) that were significantly differentially expressed in patients who failed to respond to hormone deprivation therapy for high risk/metastatic disease. Three genes (STEAP4, ARexons4_8 and NAALADL2) were significantly differentially expressed in patients who relapsed within 12 months of hormone treatment initiation. Next-Generation Sequencing of twenty samples identified 45 genes to be significantly differentially expressed between non-cancer and cancer samples (28 were up regulated and 17 down regulated). 33 out of the 45 genes showed a significant linear trend in association with cancer risk. Conclusions Urine Exosomal RNA contains PCa specific transcripts. Gene expression analysis and Next Generation Sequencing identified genes that are significantly differentially expressed between cancer and non-cancer cases as well as prognostic genes and genes that can predict response to hormone treatmen

    Defining The Effect Of Environmental Perturbation On The Male Germline

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    Periconceptional environment, according to the Developmental Origins of Health and Disease (DOHaD) theory, influences offspring phenotype, primarily via epigenetic mechanisms. Although the paternal component in humans is poorly understood, both maternal and paternal peri-conceptional environment are now believed to contribute to this phenomenon. Manipulation of the early embryo for treating human infertility, is suspected of contributing to offspring abnormalities through epigenetic mechanisms. To directly address the effects of common assisted reproductive technology procedures on the offspring epigenome, the DNA methylation profiles of newborns conceived naturally, or through the use of intrauterine insemination (IUI), or in vitro fertilization (IVF) using Fresh or Cryopreserved (Frozen) embryo transfer, were compared. In addition to a reduction of epigenetic aberrations in the IVF conceptions using cryopreservation, metastable epialleles also exhibited altered methylation with fertility status. ART, embryo nutrition, and fertility status are thus suggested to have a lasting epigenetic effect of on the developing embryo. While the paternal contribution to the human embryo is uncertain, sperm deliver a collection of proteins and RNA to the zygote. To identify the entire cadre of intergenic spermatozoal RNAs, RNA Element (RE) discovery algorithm (REDa) was developed and applied to a spectrum of germline, embryonic, and somatic tissues. This highlighted extensive transcription throughout the human genome and yielded previously unidentified human RNAs. Human spermatogenesis was found to exhibit extensive intergenic transcription and pervasive repetitive sequence expression. By analyzing the collection of novel and annotated spermatozoal RNAs in sperm samples from the Mesalamine and Reproductive Health Study (MARS), the effect of endocrine disruptor exposure on human sperm RNA profiles was determined. Sperm RNA profiles among men and their relationship to di-butyl phthalate (DBP) was longitudinally assessed across binary (high or background) DBP crossover exposures. Numerous changes in the composition of sperm RNA elements were detected during the acute and recovery phases, which suggest that exposure to, or removal from high DBP, produces effects that require longer than one spermatogenic cycle to resolve, if at all. Overall, chronic phthalate exposure influences the male germline, and acts on the dynamic RNA expression during human spermiogenesis

    Exploring unknown avenues of intra- and interspecies communication in Drosophila

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    The manuscripts within this dissertation aimed to investigate odor-mediated communication channels for the interactions of Drosophila flies with each other, other Drosophila species, and with microorganisms. Throughout the dissertation, I introduce the frass of adult Drosophila flies as a previously overseen intra- and possibly interspecies communication medium (manuscript I) and demonstrate the sensitivity of hardwired stereotypical behaviors, which are mediated by only a few chemosignals, to manipulation through harmful microbes (manuscript II). Furthermore, this dissertation highlights different factors that may be involved in Drosophila speciation events, such as niche partitioning (manuscript IV) and insect-microbe interactions (manuscript V). Finally, I worked on a previously described method that is used for the identification of ligand-receptor pairs of olfactory systems in mammals and tried to further establish this technique for the high-throughput identification of chemosensory receptor ligands in insects on the example of the vinegar fly D. melanogaster (manuscript III)

    Identification and Characterization of Genetic Components in Autism Spectrum Disorders 2019

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    The Identification of the Genetic Components of Autism Spectrum Disorders 2019 will serve as a resource for laboratory and clinical scientists as well as translational-based researchers, primary healthcare providers or physicians, psychologists/psychiatrists, neurologists, developmental pediatricians, clinical geneticists, and other healthcare providers, teachers, caregivers and students involved in autism spectrum disorders (ASD) with the goal to translate information directly to the clinic, education and home setting. Other professionals, students and families might find this textbook of value based on better awareness, causes and understanding of genetic components leading to autism and open avenues for treatment. Genetics play a role with up to 90% of autism, with over 800 currently recognized genes contributing to causes, clinical presentation, treatment, and counseling of family members. This textbook includes 13 chapters divided into three sections (clinical, genetics, other) written by experts in the field dedicated to research and clinical care, description, treatment and generating relevant reviews for ASD and related disorders impacting gene expression, profiling, and pathways. Identification of potential risk factors will be discussed, including obesity, microbiota, malignancy, and the immune system, as well as their direct or indirect contribution to ASD treatment and causation

    Parallel Genetics of Gene Regulatory Sequences in Caenorhabditis elegans

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    Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren.How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals
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