Functional interpretation of single cell similarity maps.

We present Vision, a tool for annotating the sources of variation in single cell RNA-seq data in an automated and scalable manner. Vision operates directly on the manifold of cell-cell similarity and employs a flexible annotation approach that can operate either with or without preconceived stratification of the cells into groups or along a continuum. We demonstrate the utility of Vision in several case studies and show that it can derive important sources of cellular variation and link them to experimental meta-data even with relatively homogeneous sets of cells. Vision produces an interactive, low latency and feature rich web-based report that can be easily shared among researchers, thus facilitating data dissemination and collaboration

Developing Zebrafish embryos as a model to study host-material Interactions and wound healing

Inappropriate wound healing represents a considerable medical challenge associated with high mortality. However, improving on current wound healing therapies has proven difficult due to the complex and dynamic wound environment. The complexity of the wound healing process also puts high demands on the animal models used in wound research, since ideally such models should encompass the full complexity of the wound healing process, and at the same time be accessible for advanced biomedical analysis methods. In this thesis, the aim was to further develop the use of zebrafish embryos in wound healing research. Key advantages of zebrafish embryo models are the ability to visualize complex biological processes in high detail in intact tissues, as well as highly tractable genetics. The first part of the work describes the development of a zebrafish embryo model for investigating the immunomodulatory properties of hydrogels derived from decellularized extracellular matrix (ECM). The results demonstrate that the hydrogels can be properly injected into the embryos and that the host-materials interactions can be explored in detail inside live zebrafish embryos during wound healing. This constitutes a new in vivo model for investigating immunomodulatory materials in a realistic wound healing context. The second part of the work describes the development of a confocal Raman spectrometry imaging (cRSI) method for biomolecular characterization and the study of biological processes in zebrafish. This represents a new imaging modality that enables simultaneous inspection of a multitude of biomolecules in a label-free manner. The use of cRSI was demonstrated for biomolecular discrimination of mycobacteria in a zebrafish infection model, and for live in vivo imaging of zebrafish during the early wound response. Taken together, the work in this thesis has provided a new methodologies and insight for the use in zebrafish embryo models in wound healing research.Open Acces

Using zebrafish larval models to study brain injury, locomotor and neuroinflammatory outcomes following intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a devastating condition with limited treatment options, and current understanding of pathophysiology is incomplete. Spontaneous cerebral bleeding is a characteristic of the human condition that has proven difficult to recapitulate in existing pre-clinical rodent models. Zebrafish larvae are frequently used as vertebrate disease models and are associated with several advantages, including high fecundity, optical translucency and non-protected status prior to 5 days post-fertilisation. Furthermore, other groups have shown that zebrafish larvae can exhibit spontaneous ICH. The aim of this study was to investigate whether such models can be utilised to study the pathological consequences of bleeding in the brain, in the context of pre-clinical ICH research. Here, we compared existing genetic (bubblehead) and chemically inducible (atorvastatin) zebrafish larval models of spontaneous ICH and studied the subsequent disease processes. Through live, non-invasive imaging of transgenic fluorescent reporter lines and behavioural assessment we quantified brain injury, locomotor function and neuroinflammation following ICH. We show that ICH in both zebrafish larval models is comparable in timing, frequency and location. ICH results in increased brain cell death and a persistent locomotor deficit. Additionally, in haemorrhaged larvae we observed a significant increase in macrophage recruitment to the site of injury. Live in vivo imaging allowed us to track active macrophage-based phagocytosis of dying brain cells 24 hours after haemorrhage. Morphological analyses and quantification indicated that an increase in overall macrophage activation occurs in the haemorrhaged brain. Our study shows that in zebrafish larvae, bleeding in the brain induces quantifiable phenotypic outcomes that mimic key features of human ICH. We hope that this methodology will enable the pre-clinical ICH community to adopt the zebrafish larval model as an alternative to rodents, supporting future high throughput drug screening and as a complementary approach to elucidating crucial mechanisms associated with ICH pathophysiology

The Immune System: the ultimate fractionated cyber-physical system

In this little vision paper we analyze the human immune system from a computer science point of view with the aim of understanding the architecture and features that allow robust, effective behavior to emerge from local sensing and actions. We then recall the notion of fractionated cyber-physical systems, and compare and contrast this to the immune system. We conclude with some challenges.Comment: In Proceedings Festschrift for Dave Schmidt, arXiv:1309.455

Macrophage transactivation for chemokine production identified as a negative regulator of granulomatous inflammation using agent-based modeling

Cellular activation in trans by interferons, cytokines and chemokines is a commonly recognized mechanism to amplify immune effector function and limit pathogen spread. However, an optimal host response also requires that collateral damage associated with inflammation is limited. This may be particularly so in the case of granulomatous inflammation, where an excessive number and / or excessively florid granulomas can have significant pathological consequences. Here, we have combined transcriptomics, agent-based modeling and in vivo experimental approaches to study constraints on hepatic granuloma formation in a murine model of experimental leishmaniasis. We demonstrate that chemokine production by non-infected Kupffer cells in the Leishmania donovani-infected liver promotes competition with infected KCs for available iNKT cells, ultimately inhibiting the extent of granulomatous inflammation. We propose trans-activation for chemokine production as a novel broadly applicable mechanism that may operate early in infection to limit excessive focal inflammation

The interplay between obesity and cancer: a fly view

Accumulating epidemiological evidence indicates a strong clinical association between obesity and an increased risk of cancer. The global pandemic of obesity indicates a public health trend towards a substantial increase in cancer incidence and mortality. However, the mechanisms that link obesity to cancer remain incompletely understood. The fruit fly Drosophila melanogaster has been increasingly used to model an expanding spectrum of human diseases. Fly models provide a genetically simpler system that is ideal for use as a first step towards dissecting disease interactions. Recently, the combining of fly models of diet-induced obesity with models of cancer has provided a novel model system in which to study the biological mechanisms that underlie the connections between obesity and cancer. In this Review, I summarize recent advances, made using Drosophila, in our understanding of the interplay between diet, obesity, insulin resistance and cancer. I also discuss how the biological mechanisms and therapeutic targets that have been identified in fly studies could be utilized to develop preventative interventions and treatment strategies for obesityassociated cancers

Longitudinal in vivo assessment of host-microbe interactions in a murine model of pulmonary aspergillosis

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (F-19 MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of F-19 MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression

The Biochemical and Biophysical Mechanisms of Macrophage Migration

The ability of macrophages to migrate is critical for a proper immune response. During an innate immune response, macrophages migrate to sites of infection or inflammation where they clear pathogens through phagocytosis and activate an adaptive immune response by releasing cytokines and acting as antigen-presenting cells. Unfortunately, improper regulation of macrophage migration is associated with a variety of dieases including cancer, atherosclerosis, wound-healing, and rheumatoid arthritis. In this thesis, engineered substrates were used to study the chemical and physical mechanisms of macrophage migration. We first used microcontact printing to generate surfaces specifically functionalized with fibronectin and functionally blocked against cell adhesion to study the migration of RAW/LR5 murine macrophages. Using these surfaces we found that macrophage migration is biphasic with respect to increasing surface ligand or soluble chemokine concentration, and that RAW/LR5 migration is dependent on PI3K and ROCK signaling. We then used traction force microscopy to measure the force generation capabilities of primary human macrophages and found that these cells generate strong forces at their leading edge in a stiffness-dependent manner. Through the use of chemical inhbitors we showed that force generation is dependent on myosin II contraction, PI3K signaling, and Rac signaling downstream of the GEF Vav1, but not the GEF Tiam1. Finally, we investigated the motility and force generation of M1 and M2 polarized primary human macrophages. We found that M1 macrophages are less motile and generate less force than M0 or M2 macrophages, and that M2 macrophages are more motile but do not have any change in force generation compared to M0 macrophages. We have been able to show that both chemical signals and mechanical mechanisms contribute to macrophage migration. This work contributes to the growing understanding of the mechanisms that govern macrophage migration and demonstrates the importance of mechanics when studying leukocyte migration