The study of biological analytes in their native environment is a major challenge for biochemistry and molecular biology.  Luminesce spectroscopy is well suited for this task due to its non-invasiveness, high spatial and temporal resolution, and high signal to noise ratio. This thesis describes the development and applications of Ln-based luminescent probes for detecting small molecules and enzymes.  Specifically the probes presented are based on coumarin sensitizers coupled to a DO3A chelated LnIII center. The 1st generation of these probes employ 7-OH coumarins, caged at the 7-O position (Chapter 2). By use of p-pinacolatoboron benzyl or p-methoxybenzyl cages, this design allowed the construction of ratiometric EuIII-based probes capable of detecting the reactive oxygen species H2O2, NO and ONOO−. The second and third part of the thesis describes a further improvement of the design (Chapters 3 and 4). By employing caged coumarin precursors EuIII and TbIII-based probes were developed for a variety of different analytes (F−, Pd0, H2O2, β-galactosidase, β-glucosidase, α-mannosidase and phosphatase). Most of these probes displayed excellent turn-on responses when treated with their respective analytes. Furthermore they could be used for detecting multiple analytes simultaneously (Chapter 4). By use of one Eu-based and another Tb-based probe, the simultaneous detection of two analytes was possible. This could further be extended to simultaneous three analyte detection by the additional employment of an organic coumarin-based probe. The last part of the thesis (Chapter 5) describes protocols for the rapid and efficient access to triazole-linked lanthanide-antenna complexes by use of the copper-catalyzed azide-alkyne cycloaddition reaction. For robust substrates, microwave heating at 100 °C enabled rapid (15-60 min) access to various lanthanide complexes, which could be isolated via simple precipitation. Using these conditions pure bi- and tri-homometallic lanthanide complexes could be prepared. A second protocol, for substrates carrying sensitive functionalities was also developed. The application of catalytic amounts of CuOAc, BimPy2 ligand, and a large excess of NaAsc afforded a variety of lanthanide complexes, among them caged responsive probes, in moderate to good yields.

Fluorescens In Situ Hybridisering (FISH) är en cytogenetisk teknik som kan detektera genetiska sjukdomar och avvikelser i Deoxyribonukleinsyra (DNA) och Ribonukleinsyra (RNA). FISH börjar med kromosomutvinning av önskat analyspreparat, därefter får en direkt- eller indirekt fluorescensinmärkt probe (15-30 baspar lång) binda in till sin genetiska målsekvens via hybridisering. Preparatet kan sedan analyseras i fluorescensmikroskop för att bedöma om proben bundit in till sin målsekvens. I kursen ”Fördjupad laboratoriemetodik” för Biomedicinska analytikerprogrammet, Linnéuniversitetet utförs en FISH-laboration där resultaten varit otydliga och ej reproducerbara. Syftet med examensarbetet var att förbättra kurslaboration FISH som ges under kursen ”Fördjupad laboratoriemetodik”. Adherenta celler odlades till ≥ 3 x 106 i antal och till viss konfluens; primära endotelcell-linjen HCMEC till konfluenserna 60 % och 80 % och cancercell-linjerna VMM1 och H1915 till 80 % konfluens. Därefter skördades cellerna och dess respektive kromosomer utvanns. Kromosomerna undersöktes sedan med metoderna G-bandsfärgning, DNA-FISH och Multicolor-FISH (24X-probe) för tydliga och reproducerbara resultat. G-bandsfärgningen av HCMEC visade många hela interfasceller och få fria kromosomer för celler i 60 % konfluens. Både G-bandsfärgningen och DNA-FISH visade att HCMEC odlade till 80 % konfluens visade fria kromosomer från celler i metafas (celldelningsfas) där det fanns en svag signal för X-kromosomen. Multicolor-FISH-analys av VMM1 och H1915 gav tydliga resultat i fluorescensmikroskop där fria kromosomer var Multicolor-probeinmärkta; blå/aqua, röd och grön. Konklusionen är att vid kromosomutvinning från odlade adherenta celler bör dessa vara 80 % konfluenta. Detta för att ge tydliga och reproducerbara probeinfärgningar av kromosomer i metafas vid analys med Multicolor-FISH. Analys av 80 % konfluenta celler och användning av Multicolor-FISH-analys är en klar förbättring av kurslaborationen. / Fluorescens in situ hybridization (FISH) is used to detect cytogenetic aberrations and abnormalities of Deoxyribonucleic acid (DNA) and Ribonucleic Acid (RNA). The FISH begins with chromosome extraction of the desired cell preparation then a direct or indirect fluorescently labeled probe (15-30 base pair long) is hybridized to its genetic target sequence. The preparation can thereafter be analyzed in fluorescence microscope to see bound probe at chromosome level. In the course “Advanced laboratory methodology” for the Biomedical Scientist program, Linnaeus University, a FISH laboratory experiment is conducted where results have not been clear nor reproducible. The aim of this study was to improve the laboratory experiment FISH. Human Cardiac Microvascular Endothelial Cells (HCMEC) was grown to 60 % and 80 % confluence, to an estimated number of ≥ 3 x106, and analyzed by G-band staining and DNA-FISH. G-band staining showed many cells in interphase and few free chromosomes of cells with 60 % confluence. G-band staining and DNA-FISH showed that cells grown to 80 % confluence showed more free chromosomes from metaphase. The cancer cell lines VMM1 and H1915 were therefore grown to 80 % confluence and ≥ 3 x106. Multicolor-FISH on VMM1 and H1915 showed results from all painting probes blue/aqua, red and green. The conclusion is that in chromosomal extraction from cultured adherent cells should be 80 % confluent to give clear and reproducible probe staining of chromosomes in metaphase when assayed with Multicolor FISH. Analysis of 80 % confluent cells and the use of Multicolor FISH technology is a clear improvement to the “Advanced laboratory methodology” course.

This thesis focuses on super resolution STED optical imaging. STED provides a wealth of new informational content to the acquired images by using stimulated emission to surpass the diffraction limit in optical fluorescence microscopy. To further increase the informational content, a new method to perform multicolor STED imaging by exploiting differences in the photostability and excitation spectra of dyes is presented. In order to extract information from the images, computational algorithms which handle the new type of high resolution informational content are developed. We propose that multicolor super resolution imaging in combination with image analysis can reduce the amount of clinical samples required to perform accurate cancer diagnosis. To date, such diagnosis is based mainly on significant amounts of tissue samples extracted from the suspected tumor site. The sample extraction often requires anesthetics and can lead to complications such as hematoma, infections and even cancer cell ceding along the needle track. We show that by applying multicolor STED and image analysis, the information gained from single cells is greatly increased. We therefore propose that accurate diagnosis can be based on significantly less extracted tissue material, allowing for a more patient friendly sampling. This approach can also be applied when studying blood platelets, where we show how the high informational content can be used to identify platelet specific activational states. Since platelets are involved in many different types of diseases, such analysis could provide means of performing truly minimally invasive diagnostics based on a simple blood test. In addition, our data makes it possible to understand in finer detail the underlying mechanisms rendering cells metastasis competent. We combine the high resolution spatial information provided by STED with information regarding the adhesive forces of cells measured by TFM (Traction Force Microscopy) and the cell stiffness measured by AFM (Atomic Force Microscopy). Such comparisons provide a link between the specific highly resolved protein distributions and different cellular mechanics and functions. This thesis also includes STED imaging and analysis on the spatial organization of neuronal synaptic regulating proteins, implicating the speed with which neuronal signaling can be regulated. / <p>QC 20140207</p>

Esta tesis constituye un estudio detallado y exhaustivo de las propiedades de una variedad específica de ondas ópticas solitarias. Observadas experimentalmente por primera vez en 1995, estas ondas estan formadas por un haz óptico a frecuencia fundamental y su segundo armónico que están ligados entre sí y viajan juntos en el material cuadrático; y son debidas al equilibrio entre la difracción lineal que sufre el haz al propagarse y un término no lineal de segundo orden en la susceptibilidad del medio. Las llamamos por eso solitones ópticos en medios cuadráticos o simplemente 'solitones cuadráticos'. También se les conoce como 'Solitones Multicolor' aludiendo al hecho de que requieren de haces a diferentes frecuencias para formarse.Un medio no-lineal cuadrático tiene por fuerza que ser no-centrosimétrico, lo cual es una variedad de anisotropía. Una gran parte de los materiales no-lineales cuadráticos (los que tienen mayor interés para la industria) son uniaxiales lo que significa que presentan un eje de simetría que suele llamarse eje óptico. De la dirección de un haz relativa a ese eje óptico dependen las características de la propagación del haz en el medio cuadrático no-lineal. Una consecuencia de eso en configuraciones de interés es un desvío ('walk-off') sufrido por el haz respecto a su dirección de propagación inicial al entrar en el material no-lineal.Las propiedades de los solitones cuadráticos 'caminantes' son también estudiadas en la tesis, estableciendo que existe una relación entre la potencia inyectada en el medio y el ángulo de desvío (walking angle).Una parte importante de la tesis está dedicada al estudio a través de exhaustivos experimentos numéricos del potencial de estas ondas solitarias para constituir la base de dispositivos de conmutación y encaminamiento totalmente ópticos que puedan hacer realidad la promesa de la red transparente totalmente óptica. Los experimentos han permitido identificar varias configuraciones de interés con niveles de potencia y dimensiones que permiten plantearse el diseño y construcción de dispositivos comerciales de conmutación y encaminamiento totalmente ópticos basados en solitones ópticos cuadráticos. / This thesis is a comprehensive study of the fundamental properties of a specific kind of optical spatial solitary waves. First observed experimentally in 1995, these solitary waves are formed by an optical beam at a fundamental frequency and its second harmonic which propagate together and are mutually entangled; and are due to a balanced interplay between the beams' linear diffraction and a second-order nonlinear susceptibility of the medium. They are thereby referred as 'Optical Solitons in Quadratic Nonlinear Media' or simply 'Quadratic Solitons', They are also known as 'Multicolor Solitons' recalling that they are formed by beams at different frequencies.A quadratic nonlinear media needs to be non centrosymmetric which is a special kind of anisotropy. A great deal of quadratic nonlinear materials (the most used by industry such as lithim niobate, KTP, etc.) are uniaxial meaning that they feature a symmetry axis known as 'optical axis'. The direction of propagation of an optical beam relative to that axis determines the characteristics of the beam's propagation through the quadratic nonlinear material. A main result of that in some configurations of interest is a walk-off suffered by the beam as it enters the quadratic material.The properties of the families of quadratic solitons in the presence of a linear walk-off (quadratic walking solitons) are studied as well in the thesis stating that there is a relationship between the power injected into the medium and the walking angle, suitable to applications of all-optical switching and routing.An important last part of the thesis is devoted to the study from a practical viewpoint and through extensive numerical experiments of the potential of these solitary waves as the basis of practical all-optical switches and routers which could take the all-optical transparent network to a reality. The experiments have allowed to identify several configurations of interest with power level and dimensions suited to practical applications which could allow the production of commercial all-optical switching and routing devices based on quadratic solitons.

Les Cellules Folliculaires Dendritiques (FDC) régulent l'homéostasie des lymphocytes B et sont indispensables à la mise en place des réponses immunes humorales. Les FDC s'organisent, au sein des organes lymphoïdes secondaires, en réseaux tridimensionnels denses, nécessaires à leur fonctionnement. Les études s'intéressant aux FDCs, empruntent classiquement des approches in vitro ou ex vivo, peu adaptées à la nature de ce type cellulaire. Au cours de mon travail de thèse, nous avons utilisé plusieurs systèmes de 'multicolor fate mapping' dans le but de déchiffrer in situ les mécanismes à l'origine du développement initial, et du remodelage des réseaux de FDCs en contexte inflammatoire. Nous avons démontré que les FDCs provenaient de la prolifération clonale et de la différentiation des Cellules Marginales Réticulaires (MRC), un autre sous-type cellulaire stromal résidant près des sinus sous-capsulaires ganglionnaires, et dont les fonctions étaient à ce jour, inconnues. Lors des réponses immunes, nous avons prouvé que les FDCs nouvellement formées, ne dérivaient ni du recrutement de progéniteurs circulants ni de la prolifération de FDCs matures, mais plutôt de la prolifération clonale des MRCs, suivie de leur différentiation en FDCs. Au-delà de l'étude de la biologie des FDCs, notre travail a révélé une fonction importante des MRCs dans le soutien de la plasticité des réseaux de FDCs. / Follicular Dendritic Cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods may thus be of little interest to understand the genuine immunobiology of FDCs in their native habitat. Herein, we utilised various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of Marginal Reticular Cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. In contrast, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population.

Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR). In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment. The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection. In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary. In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.

La cryoconservation de tissu ovarien peut être proposée, avant traitements hautement gonadotoxiques, à des patientes afin de préserver leur fertilité. L'autogreffe de tissu ovarien est actuellement la seule méthode de réutilisation du tissu ovarien disponible, mais en cas de pathologie néoplasique, elle présente un risque de réintroduction d'éventuelles cellules malignes via le greffon. L'objectif de ce travail a été de développer une méthode pour détecter la maladie résiduelle (MRD) dans le tissu ovarien par cytométrie en flux multicouleurs (CMF) en cas de leucémie aiguë. Une technique de dissociation de cortex ovarien a été mise au point à partir de tissu ovarien de référence provenant de résections percoelioscopiques. Un modèle expérimental de détection de la MRD a été validé et consistait à ajouter des cellules de leucémie aiguë lymphoblastique (LAL) ou myéloïde (LAM) à une suspension de cellules ovariennes isolées de référence. La méthode a ensuite été utilisée pour rechercher la MRD dans le tissu ovarien cryoconservé de 11 patientes atteintes de leucémie aiguë (7 LAL et 4 LAM).Le modèle expérimental a permis de valider une sensibilité de 10"4 et une spécificité élevée tant pour les LAL que pour les LAM. Lorsqu'un marqueur moléculaire était disponible pour la recherche de la MRD, nous avons observé une bonne corrélation entre la CMF et la PCR quantitative. La détection par CMF de la MRD dans le tissu ovarien des patientes leucémiques était positive chez 3 des 11 patientes étudiées.Cette technique est essentielle pour évaluer le risque carcinologique avant de proposer la réutilisation du tissu ovarien cryoconservé par technique d'autogreffe. / Ovarian cryopreservation together with autograft of frozen/thawed ovarian tissue is a real option to preserve and restorefertility in cancer patients. However in cases of leukemia, there is a real concern regarding the presence of metastaticcells in the ovarian tissue, which could lead to the recurrence of the primary disease. The aim was to validate multicolorflow cytometry (MFC) as an original technique for minimal residual disease (MRD) detection in ovarian cortex fromacute leukemia patients.We developed an experimental model which consisted in adding serial dilutions of leukemic cells into isolated ovariancell suspensions obtained from healthy cortex. The modelization was validated for acute lymphoblastic leukemia (ALL)and acute myeloid leukemia (AML). Then the method was applied to MRD detection of leukemic cells in cryopreservedovarian cortex from 11 leukemia patients (7 ALL and 4 AML).This experimental model made it possible to obtain a high specificity and a robust sensitivity of 10~4 for MRD detectionby MFC for both types of acute leukemic cells. When a molecular marker was available, we observed a good correlationbetween CMF and quantitative PCR. Ovarian MRD was positive by MFC in one T-ALL and 2 AML patients.MRD detection in the ovarian cortex is essential to evaluate the risk of cancer reseeding before ovarian tissue autograft.

Le glioblastome (GBM) est la tumeur cérébrale primaire la plus agressive comportant une sous-population de cellules souches tumorales (CSG). Elles sont capables d’auto-renouvellement, de prolifération, de différenciation en cellules exprimant les marqueurs neuraux et de trans-différenciation en cellules de types vasculaires. Dans ce contexte, j’ai dérivé et caractérisé plusieurs lignées de CSG à partir de biopsies de patients. Puis j’ai évalué l’impact des peptides thérapeutiques transmembranaires développés au laboratoire, visant les plateformes de récepteurs de neuropiline-1 et de plexine-A1 surexprimées dans les CSG. Les deux peptides diminuent la croissance des CSG in vitro et in vivo. Finalement, j’ai développé un outil génétique fluorescent permettant de suivre le destin des CSG en direct. Basé sur l’expression de 4 rapporteurs fluorescents contrôlés par des promoteurs spécifiques des types cellulaires, il permet d’identifier l’hétérogénéité de ces cellules en différenciation. / The glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and includes a subpopulation of tumoral stem cells (CSG). Those cells can self-renew, proliferate and differentiate by expressing specific neural markers and/or transdifferentiate into vascular-like cells. In this context, my work consisted first to produce and characterize several CSG lines from patient biopsies to constitute a bank of cell lines with different properties. We also evaluated the impact of in house therapeutic transmembrane peptides targeting the neuropilin-1 / plexin-A1 receptor platforms overexpressed in GBM. We thus showed that both targeting peptides decrease the growth of GSC in in vitro and in vivo models. Finally, I developed an inducible mosaic expression system to track the live differentiation of CSG. This system is based on the expression of four different fluorescent reporters controlled by the activity of cell type specific promoters.

Background: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. Methods: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). Results: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10−17), the VC (p = 4.404 × 10−178), and both combined (p = 7.74 × 10−199). Conclusions: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination.