Spectral imaging in preclinical research and clinical pathology.

Spectral imaging methods are attracting increased interest from researchers and practitioners in basic science, pre-clinical and clinical arenas. A combination of better labeling reagents and better optics creates opportunities to detect and measure multiple parameters at the molecular and cellular level. These tools can provide valuable insights into the basic mechanisms of life, and yield diagnostic and prognostic information for clinical applications. There are many multispectral technologies available, each with its own advantages and limitations. This chapter will present an overview of the rationale for spectral imaging, and discuss the hardware, software and sample labeling strategies that can optimize its usefulness in clinical settings

Development of a biological scaffold from adult human skin for cardiovascular repair and regeneration

Cardiovascular diseases (CVDs) are still the leading cause of death and disabilities globally. Among CVDs, ischemic heart disease (IHD) has remained the leading cause of death worldwide in the last 16 years. IHD is caused by a sudden blockage of blood flow through coronary arteries that prevents the supply of oxygen and nutrients to the region of myocardium fed by the affected vessels. This condition causes the necrosis of the myocardium that is followed by a reparative process that starts from the infarcted area, but then involves, at later stages, also the uninjured myocardium, causing progressive fibrosis that may lead eventually to heart failure. Unfortunately, there is no cure for IHD and therapy can at best control symptoms and prevent a second ischemic event. The induction of post-infarction cardiac regeneration by the means of three factors, cells, scaffold and signals, is currently the target of cardiac tissue engineering. However, the field is still at its infancy and all three factors are yet to be defined. Since the ECM is the naturally occurring scaffold loaded with uncountable biological and mechanical signals, we aimed at obtaining and characterizing a biological three-dimensional scaffold for cardiac repair and regeneration from the adult human skin. Our results provided evidence that the scaffold of decellularized human skin (d-HuSk) was acellular and had a preserved architecture, retained components of the ECM that are also typical of cardiac matrix and are critical for cardiac functions and mechanical properties of the ECM, like collagen, fibronectin, laminin, tenascin, elastin and GAGs. Additionally, growth factors stored in d-HuSk matrix were similar to those found in cardiac matrix and, as similar were the signals, similar were the effects of d-HuSk and cardiac matrix on human cardiac progenitor cells (hCPCs). Indeed, as emerged from cytocompatibility study, the environment offered by d-HuSk did not differ from the cardiac native one in supporting engraftment and survival of hCPCs. Furthermore, d-HuSk attracted hCPCs from the cardiac native matrix and sustained their differentiation and differentiation towards cardiac myocytes. Therefore, d-HuSk is a biological scaffold that is easily obtained and might be used as an autograft. It shares to a large extent the composition of the cardiac native matrix, exerts on hCPCs similar effects in vitro and is also capable of stimulating their mobilization and engraftment. Overall, d-HuSk fulfills the key requirements needed for a scaffold to warrant its use in tissue engineering and, then, holds great promise as substitute for cardiac environment. Additionally, consisting of ECM proteins and being a storage of growth factors, d-HuSk might alone provide two of the three pillars of tissue engineering, namely the scaffold and the signals, and might be exploited as stand-alone scaffold to boost cardiac regeneration by recruiting resident cardiac progenitor cells, or as a cellularized scaffold by preparing a cardiac engineered tissue in vitro with the cell population of choice

Improving treatment of glioblastoma: new insights in targeting cancer stem cells effectively

Glioblastoma is the most common primary malignant brain tumour in the adult population. Despite multimodality treatment with surgery, radiotherapy and chemotherapy, outcomes are very poor, with less than 15% of patients alive after two years. Increasing evidence suggests that glioblastoma stem cells (GSCs) are likely to play an important role in the biology of this disease and are involved in treatment resistance and tumour recurrence following standard therapy. My thesis aims to address two main aspects of this research area: 1) optimization of methods to evaluate treatment responses of GSCs and their differentiated counterparts (non-GSCs), with a particular focus on a tissue culture model that resembles more closely the tumoral niche; 2) characterization of cell division and centrosome cycle of GSCs, investigating possible differences between these cells and non-GSCs, that would allow the identification of targets for new therapeutic strategies against glioblastomas. In the first part of my project, I optimized a clonogenic survival assay, to compare sensitivity of GSCs and non-GSCs to various treatments, and I developed the use of a 3-dimentional tissue culture system, that allows analysis of features and radiation responses of these two subpopulations in the presence of specific microenvironmental factors from the tumoral niche. In the second part, I show that GSCs display mitotic spindle abnormalities more frequently than non-GSCs and that they have distinctive features with regards to the centrosome cycle. I also demonstrate that GSCs are more sensitive than non-GSCs to subtle changes in Aurora kinase A activity, which result in a rapid increase in polyploidy and subsequently in senescence, with a consistent reduction in clonogenic survival. Based on these findings, I propose that kinases involved in the centrosome cycle need to be explored as a novel strategy to target GSCs effectively and improve outcomes of glioblastoma patients

The nucleus under the microscope. A biophysical approach.

At the beginning of 1950, many researchers challenged the possibility to overcome the fundamental Abbe limit. An attempt was made by Giuliano Toraldo di Francia, who showed that the width of the point-spread function can be reduced applying a filtering technique (called apodozation) (1). In 1994, a revolutionary event took place in the field of optical microscopy: Hell described a method for circumventing the light diffraction barrier (2). In this way, details that were not visible in diffraction-limited techniques could be imaged using a fluorescence microscopy. Nowadays, these methods termed Far-field fluorescence microscopy or nanoscopy techniques, has become an indispensable tool for scientist to address important biological and biophysical questions at the single molecule level. To highlight the outstanding importance of such techniques, the Royal Swedish Academy of Sciences awarded Eric Betzig, Stefan W. Hell, and William E. Moerner the Nobel Prize in Chemistry 2014 \u201cfor the development of super-resolved fluorescence microscopy\u201d. In addition, several important technical improvements, including confocal laser scanning microscopy (CLSM) (3), multiphoton microscopy, 4Pi (4) and I5M (5) have had an important role in the field of optical microscopy. On the other side, in 2015 Boyden and colleagues developed a new method termed Expansion Microscopy (ExM), which allows expanding uniformly biological samples by increasing the relative distances among fluorescent molecules labelling specific cellular components (6). ExM permits to achieve a lateral resolution of about 65 nm, using a conventional - diffracted microscope. However, all super resolution methods demand a particular attention in the sample preparation. Achieving super resolved images require the optimization of every steps involved in the labelling process, from the expression of a fluorescent proteins to the fixation of the biological samples. In the last years, these labelling strategies have obtained a critical role in the field of fluorescence microscopy. In particular, the design and the localization precision of specific affinity probes are crucial features that can restrict the applicability of these techniques. In this work, several labelling approaches and optimization of different staining protocol for super resolution techniques were addressed. My effort was focused on STED nanoscopy and ExM, and how to optimize the labelling protocol, the fluorophores choice for a high labelling density. The optimization of the steps involved in the labelling processes allows me combining ExM with STED nanoscopy (ExSTED), to enhance the final resolution (7). In addition, these techniques were used to decipher molecular assemblies in the cellular nuclei. In particular, my attention was focused on an important layer termed nuclear envelope (NE) (8). This nuclear region encases the genetic material, maintains the regular shape of the nucleus and regulates the gene expression. NE is composed by two lipid bilayer and different class of proteins, which pass through or are strictly linked to the nuclear membranes. Nuclear pore complexes (NPCs) and nuclear lamins, two classes of proteins belonging to the NE, were investigated in this work. In particular, NPCs was used to evaluate the isotropy and calculate the expansion factor (EF) at the nanoscale level in ExM. In this work, we show that Nup153, a filamentous subunit localized in the nuclear pore basket (9), is a good reporter to verify the isotropy of the expansion process and its quantification. In addition, nuclear lamins, in particular lamin A (LA) and its mutation \u394LA50 (10), were used to investigate the physiological and pathological nuclear membrane invagination in normal and aging cells

Golgi apparatus and Golgi outposts in neurons studied by correlative microscopy

The Golgi apparatus is a highly dynamic organelle, which has many vital functions in cellular mechanisms like lipid metabolism, protein secretion, intracellular signaling, and regulation of cell division. First described in neurons, the ultrastructure and function of this complex network is still not well delineated. Recent studies have shown this secretory organelle also in dendrites of neurons, named Golgi outposts (GOs). To date, these GOs have been analyzed mainly by light microscopy while their ultrastructural appearance remained only poorly defined. To study the morphology of GOs in dendrites and to examine if GOs show a different ultrastructure to that of somatic Golgi apparatus, I established new applications of correlative light and electron microscopy in cultured neurons and brain slices, which combine fluorescence microscopy with the superior resolution of electron microscopy. Photo-oxidation-based approaches allow for the direct ultra-structural visualization of fluorescent markers in 3D, like genetically encoded green fluorescent proteins (GFPs). However, this challenging methodology had to be adapted to detect Golgi enzymes in neuronal cell cultures and brain tissue. Therefore, I first optimized the chemical fixation conditions for cultured hippocampal neurons to approximate the morphologic quality of high-pressure freeze fixation. Secondly, I enhanced sensitivity, reliability, and precision of photo-oxidation procedures on neurons, which allowed to analyse the 3D ultrastructure of GOs in dendrites of cultured neurons and mammalian brain slices. Correlative 3D microscopy showed that GOs are smaller in volume and have lower number of cisternae per stack compared to somatic Golgi apparatus of the same cell. Further, the number of peri-Golgi vesicles around GOs appears to be less. Additionally, while the stack polarity of somatic Golgi apparatus might switch along the ribbon, GOs have strictly unidirectional polarity. The technical achievements and enhanced sensitivity regarding correlative microscopy of GFPs in cultured neurons and brain slices allow for further ultrastructural investigations of GOs at different metabolic states to better understand their vital functions

Simplified Bioprinting-Based 3D Cell Culture Infection Models for Virus Detection

Studies of virus–host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus–host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.Peer Reviewe

Hydrogel-Tissue Chemistry: Principles and Applications

Over the past five years, a rapidly developing experimental approach has enabled high-resolution and high-content information retrieval from intact multicellular animal (metazoan) systems. New chemical and physical forms are created in the hydrogel-tissue chemistry process, and the retention and retrieval of crucial phenotypic information regarding constituent cells and molecules (and their joint interrelationships) are thereby enabled. For example, rich data sets defining both single-cell-resolution gene expression and single-cell-resolution activity during behavior can now be collected while still preserving information on three-dimensional positioning and/or brain-wide wiring of those very same neurons—even within vertebrate brains. This new approach and its variants, as applied to neuroscience, are beginning to illuminate the fundamental cellular and chemical representations of sensation, cognition, and action. More generally, reimagining metazoans as metareactants—or positionally defined three-dimensional graphs of constituent chemicals made available for ongoing functionalization, transformation, and readout—is stimulating innovation across biology and medicine

Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo