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Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 148-159). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references (leaves 181-233). Also available online through Digital Dissertations.

Conçues à partir d’une approche rationnelle, nous avons créé de nouvelles sondes membranaires permettant l’imagerie de l’organisation de la membrane plasmique cellulaire. Dans ce travail, nous avons d’abord développé un groupe d’outils, à partir du fluorophore solvatochrome Nile Red et de Black Hole Quencher-2, capable de marquer spécifiquement les domaines ordonnés et désordonnés (radeaux) en les identifiant par leur couleur d’émission. Les études cellulaires, à l’aide de ces sondes, suggèrent que la membrane plasmique est composée de deux phases distinctes. Puis dans le but de créer de nouvelles sondes basées sur Nile Red compatibles avec le sérum et fixables par formaldéhyde/glutaraldéhyde, nous avons modifié la sonde, préalablement développée, NR12S avec un groupement PEG ou amino, respectivement. Etonnamment, la sonde PEGylée est rapidement internalisée dans la cellule et le dérivé animo agrège avec l’agent fixant. D’un autre côté,basée sur Nile Red, nous avons conçu une sonde capable de détecter un récepteur donné et de visualiser son environnement lipidique. Initialement, nous avons obtenu des sondes capables d’allumer leur fluorescence en se liant sur le RCPG à l’ocytocine. Puis, nous avons conjugué NR12Spar l’intermédiaire d’un espaceur PEG(12) au ligand de l’intégrine, RGD. Les résultats préliminaires montrent que la molécule peut se lier à la membrane et détecter l’ordre lipidique, cependant les études cellulaires nécessitent un achèvement. Nous avons aussi travaillé sur des sondes membranaires fluorogéniques (turn-on) pour de l’imagerie multi-couleurs. Basées sur le fluorophore3-méthoxychromone, nous avons obtenu des sondes plus brillantes et plus photostables que la sonde développée originellement à partir de 3-hydroxychromone (F2N12S). Grâce à l’important déplacement de Stokes, elles permettent une imagerie de la membrane cellulaire avec une autofluorescence minimale dans la région spectrale bleue, compatible avec les marqueurs communs verts et rouges. Pour finir, basées sur le fluorophore squaraine, nous avons développé trois nouvelles sondes opérant dans la région rouge lointain, qui est particulièrement intéressante pour l’imagerie in vitro et in vivo. Ces sondes montrent une orientation parallèle avec la membrane lipidique, alors que les expériences cellulaires indiquent que seule la sonde avec deux ancres lipidiques est capable de marquer de façon stable la membrane plasmique. Ces sondes développées ici sont prévues pour être utilisées dans la recherche des radeaux lipidiques aussi bien que pour l’imagerie super-résolution et multi-couleurs de cellules vivantes. / Based on rational molecular design, we design new membrane probes that enable fluorescence imaging of cell plasma membrane organization. In this work, we first synthesized a toolkit, based on solvatochromic Nile Red dye and Black Hole Quencher-2, that can stain specifically ordered and disordered lipid domains (rafts) and identify them by the emission color. Cellular studies with these probes suggested that the plasma membrane is composed of two distinct phases. Then,with the idea to make Nile Red-based probes compatible with serum medium and fixable by formaldehyde/glutaraldehyde, we modified previously developed probe NR12S with PEG and aminogroups, respectively. Surprisingly, the PEGylated probe is quickly internalized inside the cell and the amino-derivative aggregates with the fixing agent. On the other hand, based on Nile Red we designed probes capable to detect a given receptor and visualize its lipid environment. Initially, we obtained probes that can turn-on fluorescence on binding to the oxytocin GPCR receptor. Then, we conjugated NR12S through a PEG(12) spacer to the ligand of intergrin, RGD. The first data show that the molecule can bind to the membrane and detect the lipid order, though cellular studies have to be completed. We also worked on fluorogenic (turn-on) membrane probes for multi-color imaging. Based on blue 3-methoxychromone dyes, we obtained probes that are brighter and more photostable than the originally developed probe based on 3-hydroxychromone (F2N12S). Due to large Stocks shift, they enabled cell membrane imaging with minimal auto-fluorescence in the blue spectral region, compatible with common green and red probes. At the end, based on squaraine fluorophore, we developed three new probes operating in the far red region, which is also very interesting for in vitro and in vivo imaging. These dyes show a parallel orientation with the lipid membrane, while the cellular experiments point out that only the probe with two anchor groups is able to stain stably the plasma membrane. The probes developed here are expected to be used for lipid rafts research as well as for super-resolution and multi-color imaging of living cells.

Human cytomegalovirus (HCMV) is a highly prevalent opportunistic infection and a major pathogen in immune-compromised patients. The virus exhibits a wide cell tropism and is able to lytically infect virtually any cell type, with detectable gene expression and release of new virions, but not the neutrophil. This cell is the first immune cell to engage most pathogens, engulfing and killing them before undergoing apoptosis and clearance by macrophages. However certain viruses and bacteria are able to evade host defences and use the neutrophil as a “Trojan horse” for replication and dissemination. In this context, enhanced neutrophil survival may promote infection. This work describes a profound neutrophil survival phenotype elicited by contact with live or UV-inactivated HCMV, in the absence of lytic viral gene expression. The effect does not involve signalling through candidate Toll-like receptors, but is dependent on activation of the ERK MAPK and NFκB signalling pathways, and is viral strain-dependent, restricted to clinical strains of the virus. Furthermore, HCMV triggers the secretion of a bioactive secretome that induces a similar paracrine anti-apoptotic effect in fresh neutrophils, and stimulates monocyte chemotaxis and differentiation to a phenotype that is permissive for HCMV infection. This “transferrable” effect is not due to residual virus or the presence of well-known neutrophil survival factors such as IL-6 or IL-8, but is mediated by a heat-stable protein or lipid, secreted late in culture. These results are supported by data in neutrophils isolated from patients with CMV viraemia and pneumonitis which show increased survival ex vivo, and will be further investigated using plasma membrane profiling by amino-oxybiotinylation and tandem mass tag mass spectrometry. This technique, used for the first time here in a primary cell type, allows quantitative proteomics to be performed for the first time in the neutrophil. This work demonstrates that the technique provides a comprehensive readout of all neutrophil plasma membrane proteins in a sample, with high plasma membrane purity and minimal neutrophil activation and necrosis, validated by flow cytometry. Furthermore, this has been applied to generate plasma membrane profiles for the resting, inflammatory and apoptotic neutrophil, revealing a number of neutrophil cell surface molecules not found by previous membrane proteomic methods. This technique has the potential to analyse the effect of HCMV and other pathogens on the expression profile of the neutrophil surface membrane and to examine how neutrophil signalling and function is modulated. These data shed light on the role of neutrophil apoptosis as a potential promoter of HCMV infection, and have the potential to increase our understanding of both the neutrophil’s response to pathogen invasion and to generate future approaches to combating HCMV dissemination and pathogenesis.

Abstract The carbonic anhydrases (CAs) catalyse the hydration of CO2 to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. Carbonic anhydrases are involved in many physiological processes connected with respiration and transport of CO2/bicarbonate between metabolising tissues and the lungs, pH homeostasis and electrolyte secretion in a variety of tissues/organs. The present work was undertaken to study the distribution and expression of CA isoenzymes in the normal and neoplastic gastrointestinal tissues. The expression of CA I, II, IX and XII in the human intestine and colorectal tumours was investigated by immunohistochemistry and western blotting. In the present study, immunohistochemical methods were also used to examine the location of CA IX and XII in the human pancreas and pancreatic tumours. The expression of CA XIV in the murine liver and intestine was studied using immunostaining and northern blotting. The present results suggest that transmembrane CA XII is absent from the small intestine, but is expressed in all segments of the normal large intestine. The positive signal for CA XII was confined to the basolateral plasma membranes of the epithelial cells of the surface epithelial cuff. In tumours, the signal for CA XII became stronger in the deep part of the lesion. The intensity of the immunostaining for CA I and II was clearly found to decrease in benign lesions and became very weak in malignant colorectal tumours. The reciprocal pattern of expression observed for membrane-associated (CA IX and XII) and cytoplasmic (CA I and II) isoenzymes in intestinal samples suggests that CA IX and XII may be functionally involved in tumour progression to malignancy and/or in invasion. CA I and II, which are thought to play important physiological roles in the normal colorectal mucosa, may not be required for growth of colorectal cancers and their expression consistently diminishes with progression to malignancy. In the human pancreas CA IX and XII appeared to be sporadically expressed in the basolateral plasma membrane of the normal acinar and ductal epithelium. The increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis. CA XIV was expressed in the hepatocyte plasma membrane and its localization on both apical and basolateral membrane domains suggests an important role for this isoenzyme in the regulation of ion and pH homeostasis in the liver.

Mechanical cues have been recognized to be critically important in the regulation of cells. A myriad of cellular processes including differentiation, proliferation, and gene expression are all affected by physical forces from the extra- and intra-cellular microenvironments. Despite recent advances in nano-technologies, many questions still surround how cells sense and respond to forces. Through a series of studies, we demonstrate how both the structure and inherent mechanical properties of the cell affect their response to mechanical cues. We first develop a methodology to mechanically manipulate cells while simultaneously characterizing their deformations. Using combined atomic force and confocal microscopy techniques and through systematic examination we demonstrate the role of the cytoskeleton and nucleus in the deformability and shape change of epithelial cells. Mechanical properties have been used in recent years to identify diseased states, including cancer. With this in mind, we used HeLa cells as a model and characterized significant deformability of their plasma membrane and underlying cortex. Importantly, we demonstrate and characterize their ability to recover from large shape changes, which we also observed in other epithelial cells. Shape recovery is shown to be rapid and reliant upon the actin cytoskeleton and intracellular fluid flow. Although the nucleus does not contribute significantly to the deformation and recovery of HeLa cells, the importance of nuclear mechanics cannot be forgone. In vitro studies have shown that mechanical forces transmitted through the cell’s cytoskeleton critically affect nuclear mechanics and gene transcription processes. Many others have used simple models and isolated nuclei in an attempt to characterize nuclear properties. Thus, in a subsequent study, we examine the nucleus within intact cells. Nuclear shape change, in response to force, is shown to be complex and cannot be well-characterized by isotropic mechanical properties. Characterization of the mechanics of the cell, as demonstrated through our findings, is crucial in the field of biological physics. The aforementioned studies, written as scientific articles, are presented in the body of this thesis (Chapters 2-5). A review article that focuses on mechanotransduction and relevant examples using AFM as a tool for its examination acts as an introductory chapter.

La conservation de microorganismes d’intérêt (ferments, probiotiques) sous forme sèche et revivifiable est très répandue dans l’industrie. Cependant, les procédés de déshydratation conduisent à des taux de survie variables en fonction du groupe, de l’espèce et de la souche de microorganismes considérée ainsi que du type de procédé utilisé (séchage, lyophilisation, congélation). La membrane plasmique (MP), de par sa position entre l’environnement intra et extracellulaire, est une cible privilégiée des perturbations hydriques. Les modifications de cette structure lors de stress hydriques sont décrites pour être directement impliquées dans la mort des microorganismes. La compréhension des réponses membranaires se produisant pendant un cycle de déshydratation/réhydratation est essentielle afin d’optimiser la survie des microorganismes lors des procédés de déshydratation. Les manipulations réalisées lors de cette étude ont visé à caractériser les modifications fonctionnelles (intégrité) et structurales (déformations, répartition latérale de microdomaines riches en stérols) de la MP de Saccharomyces cerevisiae lors de différents types de perturbations hydriques (déshydratations osmotiques de différentes amplitudes et cinétiques, séchage dans différentes ambiances gazeuses). L’impact de la composition de la MP sur la survie des levures a également été étudié par l’utilisation de mutants accumulant différents types de stérols au niveau membranaire. Ce travail a confirmé la forte implication de la MP dans la mort des cellules lors de perturbations hydriques. L’étude des modifications membranaires a permis d’élucider le lien entre la cinétique de déshydratation et la survie des levures. Il a également été montré que l’ergostérol est une molécule clé dans la survie des levures aux perturbations hydriques. / Preservation of microorganisms of interest (ferments, probiotics) in dry form is widespread in the industry. However, the dehydration processes lead to variable survival rates according to the group, species and strain of microorganism considered, but also according to the type of process used (drying, freeze drying, freezing). The plasma membrane (PM), by its position between the intra-and extracellular environment, is a target of hydric perturbations. Changes in this structure during hydric stresses are described to be directly involved in microorganism death. Understanding of membrane responses occurring during a dehydration/rehydration cycle is essential to maximize the survival of microorganisms in the process of dehydration.Manipulations performed in this study aimed to characterize functional (integrity) and structural (deformations, lateral distribution of sterol-rich microdomains) changes of the PM of Saccharomyces cerevisiae during different types of hydric perturbations (osmotic dehydration of different magnitudes and kinetics, drying in different gas atmospheres). The impact of the composition of PM on yeast survival was also studied by using mutants accumulating different types of sterols in membranes. This work confirmed the strong involvement of the PM in cell death during hydric perturbations. The study of membrane changes helped to elucidate the relationship between the kinetics of dehydration and survival of yeasts. It has also been shown that ergosterol is a key molecule for the survival of yeasts during hydric perturbations.