Recent advances in global gene expression measurement and the development of large- scale public repositories for storage of such data have made a wealth of information available to researchers. While one gene expression study may lack sufficient replicates to make statistically significant pronouncements, the combination of studies through meta-analysis can yield results with a much greater likelihood of accuracy. In order to combine multiple sets of data, one must first address the issue of cross-comparison between global gene expression platforms, as well as resolve the issue of repeated measures (multiple probes representing the same gene) within each platform. In this work, I present computational methods for probe reannotation and scoring and for redundant probe consolidation that together allow for greatly improved access to data for meta-analysis. I also present an example of the application of these methods, in the analysis of the gene expression regulated by estrogen across multiple cell types. Estrogen, a steroid hormone, interacts with its receptors to regulate gene transcription in both direct and indirect manners. Estrogen has the effect of increasing proliferation in some tissues, while inhibiting proliferation or increasing apoptosis in others. How estrogen achieves these highly divergent results remains unclear. Through meta-analysis of gene expression experiments across multiple cell types, I show that patterns of estrogen regulation in many tissues involve the same key genes and pathways, including cell cycle, p53 signaling, and TGFβ signaling pathways. However, regulation in different cell types can result from regulation of different genes, or the same genes regulated in different directions. Many patterns of gene regulation support known physiological consequences of estrogen on these tissues. In particular, genes promoting proliferation are upregulated in uterus and certain breast and ovarian cancer cell lines. One gene, thrombospondin-1, is up-regulated in eleven out of nineteen cell types and may be a key player in regulating proliferation in re- sponse to estrogen. Results in other cell types are unexpected. Most notably, neither genes promoting nor inhibiting proliferation are differentially regulated upon estrogen treatment in vascular smooth muscle cells, despite estrogen inhibiting proliferation of these cells.

The quality of RNA is of great importance in gene expression studies. It is mostly measured using the RNA integrity number (RIN). Lately it has been shown that samples with low RIN and different fragmentation patterns could affect quality of sequencing data. For such low RIN samples a new approach has been developed by Illumina called the DV200 metric, which is the percentage of fragments >200 nucleotides. For samples with low RIN, DV200 has proved to be a better method to predict if good quality data from RNA sequencing can be generated. However, neither RIN nor DV200 provide spatial infromation on the RNA integrity. Thus, tissues with areas of heterogeneous RNA integrity, where regions of good quality RNA sequencing data could be generated from are missed. We have designed a method to spatially evaluate the RNA integrity in tissue, which we named in situ RNA QC. The method uses three probes with three different fluorophores, each bound to three specific cDNA regions synthesized from the high abundant and well conserved 18S rRNA. With the help of in-house technology from the Spatial Transcriptomics (ST) group at SciLifeLab, we enable creation of heat maps over the RNA integrity to show spatial fragmentation patterns of RNA in tissue. This could reveal the regional quality of transcripts in situ, which is crucial knowledge when selecting samples for further RNA sequencing. The assay has been tried using different tissue fixation methods in order to show a proof of concept that formalin gives shorter cDNA fragments than acetone. The generated heat-map provides a visual overview of RNA integrity in situ; hence this method could be used to select samples for sequencing by evaluation the spatial quality of RNA. For instance from fresh frozen and formalin fixated paraffin embedded (FFPE) tissue (biobanks contain large number of longterm storage FFPE samples). With this assay we will be able to determine which samples are suitable for sequencing.

Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents. / QC 20111117 / Development and applications of protein microarrays / The Swedish Human Proteome Resource (HPR) program

Pathogenic bacterial infections are a serious threat to public health, claiming millions of lives every year. In order to contain the spread of infectious diseases sensitive and timely diagnosis of pathogenic bacteria is of significant importance. The rapid detection of low abundance analytes is still challenging in the most common bacteria detection techniques including, culture and colony counting, Enzyme-linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR). Conventional bacteria detection techniques suffer from limitations such as low sensitivity, cost, long procedural time and requiring complex lab equipment. Thus, there is a critical need for rapid, sensitive and low-cost bacterial detection platform in various applications ranging from water and food safety to medical diagnosis. The quest to overcome these limitations have sparked significant interest in innovative biosensor development, with considerable emphasis on optical techniques. Among optical biosensors, label-free methods are highly desirable over label-based alternatives for eliminating the additional cost and sample processing required for labeling. Also, techniques for whole-cell bacteria detection are preferred to detection of pathogenic molecular components detection due to the requirement for extracting and isolating the desired bacterial components such as nucleic acids or proteins. Overall, label-free whole-cell detection of pathogenic bacteria has a significant advantage of simplicity in sample preparation that translates to time and cost reduction. An additional benefit of detecting whole-cell bacteria without labels, thus in their natural environment, is the ability of monitoring the growth and replication of individual pathogens with a potential application in antimicrobial susceptibility determination. Despite the significant advantages of antibiotics as one of our most powerful tools for fighting infections, their extensive misuse and overuse over the years, have resulted in the emergence of antibiotic resistant bacteria as the global health crisis of our time. The current gold-standard technique for antibiotic susceptibility testing (AST) used in clinics, is culture-based disk diffusion assays. The time-consuming diagnosis method of the common clinical susceptibility testing, which is an inherent limitation of culture-based techniques, have necessitated the need for an alternative AST analysis platform. A clinical diagnosis test that could perform rapid pathogenic bacteria identification and determine its susceptibility to a panel of selected antibiotics, would greatly reduce the hospital stay time for patients with bacterial infection, therefore decreasing mortality and morbidity rate. In addition, it will have a great economic impact on the global healthcare system by advising optimal antibiotic use and maintaining the value of existing drugs. In this dissertation, we describe the design and development of a rapid, sensitive, and multiplexed biosensor platform that can both identify pathogenic bacteria and perform image-based AST on a single reader instrument. The simple and low-cost design of our biosensing platform makes it a perfect candidate as a point-of-care (POC) diagnostic tool in clinical setting. The biosensor presented in this dissertation is based on interferometric enhancement of the visibility of individual biological particles, such as viruses and bacteria, afforded by Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS), previously developed in our group. The integration of SP-IRIS with microfluidic flow cells provides kinetic measurements capability, by enabling in-liquid imaging of the sensor surface in real-time, therefore making it a promising diagnostic platform. Here, we build upon the SP-IRIS platform and utilize it for pathogenic bacteria identification and image-based AST analysis. To validate our biosensor's functionality, we demonstrate E. coli detection and characterization in end-point and real-time measurement modality through particle detection and tracking analysis of the acquired images from sensor surface. In addition, we perform rapid image-based AST analysis for E. coli bacteria against two antibiotics, ampicillin and gentamicin, by monitoring single cell morphological variations and tracking their growth rate under various antibiotic challenges.

No description available.

Honey bees can form distinct spatiotemporal memories that allow them to return repeatedly to different food sources at different times of day. Although it is becoming increasingly clear that different behavioral states are associated with different profiles of brain gene expression, it is not known whether this relationship extends to states that are as dynamic and specific as those associated with foraging-related spatiotemporal memories. We tested this hypothesis by training different groups of foragers from the same colony to collect sucrose solution from one of two artificial feeders; each feeder was in a different location and had sucrose available at a different time, either in the morning or afternoon. Bees from both training groups were collected at both the morning and afternoon training times to result in one set of bees that was undergoing stereotypical food anticipatory behavior and another that was inactive for each time of day. Between the two groups with the different spatiotemporal memories, microarray analysis revealed that 1329 genes were differentially expressed in the brains of honey bees. Many of these genes also varied with time of day, time of training or state of food anticipation. Some of these genes are known to be involved in a variety of biological processes, including metabolism and behavior. These results indicate that distinct spatiotemporal foraging memories in honey bees are associated with distinct neurogenomic signatures, and the decomposition of these signatures into sets of genes that are also influenced by time or activity state hints at the modular composition of this complex neurogenomic phenotype.

A general procedure is described for the analysis of gene expression of Candida albicans cultured in a mouse infection model. This technique involves first infecting mice with Candida and subsequently harvesting blood and other tissue at specific time points during infection. The tissues are homogenized and the infecting Candida isolated. Finally, RNA is extracted from recovered Candida cells and subjected to microarray analysis.

Psychological and physical stress can alter the immune system in both humans and animals. We have reported that mice subjected to chronic 12-h daily physical restraint for 2 days showed dramatic apoptosis in splenocytes. To identify genes that contribute to the splenocyte apoptosis, we compare gene expression in the spleens of restrained and unstressed mice using oligo microarrays consisting of 226 genes. We report here that mice subjected to chronic 12-h daily physical restraint for 2 days exhibited significantly altered expression of 50 of 226 genes. These genes included pro-apoptotic genes. We selected 5 genes of interest and confirmed the microarray results by real-time PCR. In this study, we identify a potentially important component of pro-apoptotic activity in restraint stress and suggest a possible target for anti-apoptotic therapy to protect splenocytes against stress-induced apoptosis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23606号 / 医博第4793号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 古川 壽亮, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The lymphatic vasculature is composed of lymphatic endothelial cells (LECs) that coalesce into a branched hierarchy of small capillaries and larger collecting vessels that regulate interstitial fluids, lipid uptake and immunity. Few studies have focused on pulmonary lymphatic system. To fill these critical knowledge gaps, we interrogated the fetal maturation program of lymphatic endothelium, and we provide evidence that CSF1R-lineage progenitors contribute to LECs in the lung during a temporally defined period in early postnatal life. The pulmonary lymphatic system is required for fluid clearance and air breathing at birth, suggesting a prenatal maturation program. To interrogate this, we developed a cell sorting strategy to enrich pulmonary LECs by their unique cell surface immunophenotype (CD45-, EPCAM-, CD31+, VEGFR3+, PDPN+, LYVE1+) for transcriptional profiling. These experiments highlighted the coordinate down-regulation of genes involved in “cell cycle”, and “mRNA processing” along with coordinate upregulation of “complement/coagulation cascade”, “lipid metabolism”, and “angiogenesis” genes from embryonic day E16.5 to E18.5. The most significantly enriched gene set corresponded to the “interferon-alpha/beta signaling” pathway which was confirmed with qRT-PCR and in-situ hybridization. These data provide the first description of the transcriptional landscape of fetal pulmonary LEC maturation. During development, all LECs are thought to originate from embryonic veins, however multiple studies have suggested a myeloid origin for a subset of LECs. A relationship between myeloid cells and the pulmonary LECs has not been elucidated. Here, we used myeloid-specific inducible CSF1R-CreERtdTomato lineage tracing mice and identified rare, single cells that co-expressed CSF1R- CreERtdTomato and Prox1, the master lymphatic regulator, in the postnatal day 3 lung. This process was temporally restricted to the early postnatal period. Lineage tracing with additional myeloid-Cre mice (CSF1R-iCre and CX3CR1-Cre) also showed contribution to postnatal LECs. To determine the biological significance of CSF1R-derived LECs to postnatal lung biology, we performed conditional Prox1 loss of function experiments. CSF1R-CreER mediated deletion of Prox1 resulted in lymphatic hypoplasia, edematous foci and clotting. These findings suggest that early postnatal CSF1R+ progenitors contribute to the pulmonary lymphatic endothelium and that vascular clotting may result from lymphatic malformation/dysfunction. / 2021-06-14T00:00:00Z