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Primär kutane Lymphome sind typische Krankheitsbilder in der Dermatologie. Obwohl diese Erkrankungen zu den seltenen Krankheiten zählen, sind sie jedoch von therapeutischem und wissenschaftlichen Interesse. Der erste Teil der Arbeit beschäftigt sich mit der Klassifikation und Therapie primär kutaner Lymphome. Die 1997 veröffentlichte EORTC-Klassifikation wird mit ihren wichtigsten Entitäten erläutert. Die EORTC-Klassifikation geht auf spezifische Besonderheiten der primär kutanen Lymphome ein und orientiert sich an der guten Prognose dieser Erkrankungen. Therapeutische Strategien wie der Einsatz von für kutane Lymphome typische Behandlungsmethoden (PUVA, Exzision, Radiatio) als auch gentechnisch hergestellte Medikamente wie therapeutische Antikörper und Vakzinen werden erklärt. Der zweiten Teil der Habilitationsschrift konzentriert sich auf experimentelle Arbeiten zur molekularbiologischen Untersuchung von primär kutanen Lymphomen. Im Mittelpunkt steht die Methode der Mikromanipulation und Einzelzell-PCR. Für die Mykosis fungoides konnte gezeigt werden, daß im initialen Ekzemstadium nur wenige klonale maligne T-Zellen in der Probe nachzuweisen sind. Mit Zunahme des Infiltrates (Plaque) sind die malignen Zellen in der Epidermis oder gruppiert in der Dermis lokalisiert. Im Tumorstadium dominieren die malignen Zellen das dermale Infiltrat (Gellrich S, J Invest Dermatol, 2000). Die Tumorzellen primär kutaner B-Zell-Lymphome weisen einen dem Keimzentrum ähnlichen Mutationsstatus, nämlich somatische Mutationen und intraklonale Diversifikation, auf (Gellrich S, J Invest Dermatol, 1997; Gellrich S, J Invest Dermatol, 2001). Die Daten sprechen für einen noch aktiven Mutationsmechanismus, sogenannte ongoing mutations (Golembowski S, Immunobiology, 2000). Eine Unterform der kutanen Lymphome stellen die primär kutanen CD30+ T-Zell Lymphome dar. In Untersuchungen mittels Mikromanipulation und Einzelzell-PCR wurden CD30+ Zellen aus primär kutanen CD30+ großzelligen Lymphomen hinsichtlich ihrer T-Zell-Klonalität untersucht. Dabei stellte sich heraus, daß nicht alle atypischen Zellen zur Tumorpopulation gehören. Es wird vermutet, daß ein unbekannter Stimulus zur Ausprägung der Zellmorphe und zur Expression des CD30-Moleküls führt (Gellrich S, J Invest Dermatol, 2003). Eine weitere Entität, bei welcher CD30+ Zellen eine Rolle spielen, ist die lymphomatoide Papulose. In den hier dargestellten Untersuchungen wurden CD30+ große atypische Zellen einzeln isoliert und anschließend mittels PCR für die Gene des T-Zell-Rezeptor-Gamma und des Immunglobulinrezeptors amplifiziert bei einem Patienten mit lymphomatoider Papulose und assoziierter Morbus Hodgkin-Erkankung. In zwei von drei Fällen waren diese CD30+ Zellen polyklonal. Die aus der Fragmentanalyse bekannte klonale T-Zell-Population konnte dagegen in CD3+CD30- kleinen Zellen gefunden werden. In einem dritten Fall enthielten die CD30+ Zellen klonale B-Zellen, welche die gleichen Immunglobulingene rearrangiert hatten wie Zellen aus einem zuvor bestehenden Hodgkin-Lymphom desselben Patienten. Diese Ergebnisse lassen vermuten, dass es für das Aufschießen und die Regredienz der Läsionen der lymphomatoiden Papulose einen Stimulus gibt und die klonalen T- und B-Zellen als Begleitinfiltrat ohne pathologische Bedeutung anzusehen sind. Insgesamt bilden die Daten dieser Arbeit eine Grundlage für eine Fortsetzung der Untersuchung zur Ätiopathogenese von primär kutanen Lymphomen und deren Therapie und bieten die Möglichkeiten vielfältiger wissenschaftlicher Kooperationen. / Primary cutaneous lymphomas present with typical clinical features in dermatology. Although these diseases are rare, they particularly are of scientific and therapeutic interest. The first part of this work deals with the classification and treatment of primary cutaneous lymphomas (PCBCL). The 1997 published EORTC classification will be explained according to the good prognosis of PCBCL. Therapeutic strategies as well as typical procedures (PUVA, excision, irradiation) and gene-technically produced drugs (therapeutic antibodies, vaccination) are illustrated in detail. The second part of this publication focuses on the experimental molecular biological work-out, done in primary cutaneous lymphomas by means of micromanipulation and single cell PCR. For the mycosis fungoides could be shown that in the patch stage only a few malignant T cells can be detected. Increasing infiltrates (plaque-stage) are characterized by epidermotrop or dermally grouped atypical cells. In tumor stage dermal atypical T cells are predominating (Gellrich S, J Invest Dermatol, 2003). The tumor cells of primary cutaneous B cell lymphomas are comparable with the stage of mutation of follicle centre cells: somatic mutations and intraclonal diversity (Gellrich S, J Invest Dermatol, 1997; Gellrich S, J Invest Dermatol, 2001). The data indicate, that there may be an active mutation mechanism, the so-called ongoing mutations (Golembowski S, Immunobiology, 2000). One subgroup of PCBCL, are presented by the CD30positve entities. By means of micromanipulation and PCR, single cells were investigated due to T cell clonality. Not all atypical cells belong to the malignant population. It is supposed that an unknown stimulus leads to morphological features and CD30 expression (Gellrich S, J Invest Dermatol, 2003). Another CD30positive entity is reflected by the lymphomatoid papulosis. In these experiments large atypical CD30positve cells were isolated and have been investigated via PCR for T cell receptor g or immunoglobuline heavy and light chain gene rearrangement. The majority of the large CD30postive cells (two cases) belong to a polyclonal T cell population. In the opposite, the small CD3positive cells are the cells persisting within the T cell clone. In another case B cells with the same immunoglobulin gene rearrangement like in a preceding Hodgkin disease of the same patient could be detected. The data seem to underline the fact that reactive polyclonal CD30positive cells are triggered by an unkown stimulus with clonal bystander cells without any pathological significance. In summary, this work could be the basis for further investigations about the etiopathogenesis of PCBCL.

Die zelluläre Antwort auf einen spezifischen Stimulus wird nicht nur durch den Stimulus selbst, sondern auch von dem Zustand der Zelle bestimmt. Um ein tieferes Verständnis für die Variabilität in einer Zellpopulation zu gewinnen, ist es notwendig, die verschiedenen zellulären Antworten mit definierten zellulären Zuständen zu verbinden. In dieser Arbeit wurde ein System etabliert, welches es ermöglicht, die zelluläre Antwort auf DNA-Schäden und den Einfluss unterschiedlicher zellulärer Zustände zu studieren sowie die zu Grunde liegenden molekularen Mechanismen zu identifizieren. Im Zuge dessen wurde eine auf CRISPR/Cas9 basierende Methode entwickelt, mit der Fluoreszenzreporter für endogene Signalproteine in nicht transformierten Brustepithelzellen (MCF10A) generiert wurden. Anhand dieses Reportersystems konnte durch time-lapse Mikroskopie die Dynamik des Tumorsuppressors p53 und eines seiner Zielgene, des Zellzyklusinhibitors p21, verfolgt werden. Dabei wurde deutlich, dass die p21 Antwort der einzelnen Zellen auf DNA-Schäden sehr heterogen ausfällt. Über eine Form-basierte Gruppierungsmethode wurden vier verschiedene Subpopulationen mit charakteristischen p21 Dynamiken identifiziert. Um den Einfluss der Zellzyklusphase zu untersuchen, wurde die Zellteilung vor Bestrahlung analysiert und so Rückschlüsse auf die initiale Zellzyklusphase gezogen. 24h nach Bestrahlung wurde ein EdU labeling durchgeführt und der Zellzyklus mittels semi-supervised Klassifizierung bestimmt. Durch Einführen einer Mutation in der Bindedomäne von p21 wurde gezeigt, dass proliferating cellular nuclear antigen (PCNA) für die Heterogenität der p21 Antwort verantwortlich ist. Alles in allem bietet mein Projekt eine Pipeline, um auf Einzelzellebene zu erforschen, wie zelluläre Antworten durch den Zellzyklus beeinflusst werden. Dieser Ansatz könnte zukünftig Anwendung in der Erforschung von Medikamentenresistenz finden, zumal zelluläre Heterogenität in der Tumortherapie zu fractional killing führt. / The cellular response to a given stimulus is not only governed by the stimulus itself, but also depends on the state of the cells. However, it remains obscure how cellular states influence cell fate decisions. In this thesis, I established a framework to study how the cellular response to DNA damage is affected by varying cell states and to identify the underlying molecular mechanisms. To this end, I generated fluorescent reporters using CRISPR/Cas9 in non-transformed breast epithelial cells (MCF10A) and measured the dynamics of the tumor suppressor p53 and one of its target genes, the cell cycle inhibitor p21 using time-lapse microscopy. I found DNA damage induced highly diverse p21 dynamics in individual cells. A shape-based clustering identified four subpopulations of characteristic p21 dynamics. To examine the source of variability, I analyzed initial cell cycle states by monitoring cell division prior to damage, and determined final cellular state by EdU labelling and a semi-supervised classification 24h post damage. The results suggested that p21 dynamics depend on cell cycle phases and determine cell cycle progression. Furthermore, proliferating cellular nuclear antigen (PCNA)--a cell cycle dependent factor-- was shown to determine p21 heterogeneity using a mutant p21 deficient in interaction with PCNA. Overall, my project provides a pipeline to study at the single cell level how cellular response is affected by cellular states. Considering that cellular heterogeneity leads to fractional killing in tumor therapies, this approach also suggests future application on studying drug-resistance in cancer therapy.

Cette thèse concerne le développement d’outils microfluidique pour l’ingénierie d’enzymes d’intérêt thérapeutique. La microfluidique à base de gouttelettes présente un énorme potentiel dans le domaine de la biologie quantitative. Nous développons des outils microfluidiques pour l’évolution dirigée de l’enzyme L-asparaginase, enzyme utilisée comme traitement de laleucémie lymphoblastique aiguë. Ce traitement est basée sur une enzyme d’origine bactérienne,ce qui conduit à déclencher des réactions immunitaires qui se traduit par l’interruption du traitement, souvent fatale pour le patient. Cependant, une version humaine de l’enzyme L-asparaginase, qui est moins immunogénique, n’est à l’heure actuelle pas suffisamment active pour être utilisée. L’objectif principal de cette thèse est d’alors d’analyser et de cribler des banques de mutants d’enzymes en utilisant des méthodes classiques de mutagenèse et d’analyser chaque mutant individuellement par le biais de la microfluidique. Pour cela, plusieurs systèmes microfluidiques ont été développés et optimisés afin de répondre à différents critères de sélection pour l’analyse et la sélection de l’enzyme L-asparaginase. La version bactérienne a servi de contrôle positif pour l’optimisation des systèmes microfluidiques afin de pouvoir analyser et de cribler des banques de mutants de la version humaine de l’enzyme L-asparaginase. / This thesis deals with the development of microfluidic tools for the engineering ofenzymes of therapeutic interest. Droplet microfluidics has enormous potential in the field ofquantitative biology. We are developing microfluidic tools based on the directed evolutionof the enzyme L-asparaginase, an enzyme used to treat acute lymphoblastic leukemia. Thistreatment is based on an enzyme of bacterial origin, which leads to immune reactions thatresult in the interruption of treatment, often fatal for the patient. However, a human version ofthe enzyme L-asparaginase, which is less immunogenic, is currently not sufficiently active to beused. The main objective of this thesis is to analyze and screen enzyme mutant libraries usingstandard mutagenesis methods and to analyze each mutant individually through microfluidics.For this, several microfluidic systems have been developed and optimized for different selectioncriteria for the analysis and selection of the enzyme L-asparaginase. The bacterial versionserving as a positive control for the optimization of microfluidic workflows to analyze andscreen mutant libraries of the human version of the enzyme L-asparaginase.

There have been a number of computational modelling studies that aim to replicate the cerebellar Purkinje cell, though these typically use the morphology of rodent cells. While many species, including rodents, display intricate dendritic branching, it is not a universal feature among Purkinje cells. This study uses morphological reconstructions of 24 Purkinje cells from seven species to explore the changes that occur to the cell through evolution and examine whether this has an effect on the processing capacity of the cell. This is achieved by combining several modes of study in order to gain a comprehensive overview of the variations between the cells in both morphology and behaviour. Passive and active computational models of the cells were created, using the same electrophysiological parameters and ion channels for all models, to characterise the voltage attenuation and electrophysiological behaviour of the cells. These results and several measures of branching and size were then used to look for clusters in the data set using machine learning techniques. They were also used to visualise the differences within each species group. Information theory methods were also employed to compare the estimated information transfer from input to output across each cell. Along with a literature review into what is known about Purkinje cells and the cerebellum across the phylogenetic tree, these results show that while there are some obvious differences in morphology, the variation within species groups in electrophysiological behaviour is often as high as between them. This suggests that morphological changes may occur in order to conserve behaviour in the face of other changes to the cerebellum.

La prise de décision cellulaire se traduit par la capacité de toute cellule vivante à intégrer les différentes informations provenant de son environnement, et à les transformer en une réponse biologique cohérente. Il est aujourd'hui de plus en plus démontré que les populations cellulaires présentent une hétérogénéité quantitative et qualitative significative, qui pourrait jouer un rôle essentiel dans le fonctionnement des organismes vivants. La première partie de ma thèse a ainsi consisté à étudier la variabilité de l'expression génique au cours de la différenciation de progéniteurs érythrocytaires aviaires primaires, à l'échelle de la cellule unique. L'expression de 92 gènes a été analysée par RT-qPCR dans des cellules isolées à différents temps de différenciation. Les principaux résultats de cette étude ont montré que la variabilité de l'expression des gènes, mesurée par l'entropie de Shannon, atteint un niveau maximal à 8h-24h de différenciation, simultanément à une chute du nombre de gènes corrélés. Cette augmentation de la variabilité génique précède l'engagement irréversible des cellules dans le processus de différenciation érythrocytaire identifié entre 24 et 48h. Cette étude a également mis en lumière le gène LDHA (Lactate dehydrogenase A), codant pour une enzyme de la glycolyse anaérobie, dans les progéniteurs érythrocytaires en état d'auto-renouvellement et aux points critiques, 8h et 24h, de la différenciation. La deuxième partie de ma thèse a donc consisté à analyser le rôle précis de LDHA dans l'auto-renouvellement des progéniteurs érythrocytaires, ainsi que les variations du métabolisme du glucose au cours de la différenciation. Nos premiers résultats suggèrent que le processus de différenciation érythrocytaire s'accompagne d'un changement métabolique correspondant au passage de la glycolyse anaérobie dépendante de LDHA, vers une production d'énergie aérobie, reposant sur la phosphorylation oxydative / The meaning of cell decision making consists in the capacity of every living cell to integrate environmental information and to transform it in a coherent biological response. Nowadays it is increasingly demonstrated that cell populations present a significant quantitative and qualitative heterogeneity that could be involved in living organisms functions. Thus, the first part of my thesis consisted in studying gene expression variability at the single-cell level during the differentiation process of primary avian erythroid progenitor cells. The expression of 92 genes was analyzed using RT-qPCR in cells isolated at different differentiation time-points. The main results of this study showed that gene expression variability, as measured by Shannon entropy, reached a maximal level, simultaneously to a drop in the number of correlated genes, at 8-24h of differentiation. This increase of the gene expression variability preceded the irreversible commitment of cells into differentiation, identified between 24h and 48h. This analysis also highlighted the potential importance ofLDHA(Lactate dehydrogenase A) encoding a glycolytic enzyme, in erythroid progenitors self-renewal and at the critical differentiation time-point 8-24h. Therefore the second part of my thesis consisted in analyzing the role of LDHA in erythroid progenitors self-renewal and the variations of glucose metabolism during the differentiation process. Our first results suggested that erythroid differentiation might be accompanied with a metabolic change, corresponding to a switch from anaerobic glycolysisdepending upon LDHA, toward aerobic energy production, relying upon oxidative phosphorylation

Le néocortex possède un état activé dans lequel l'activité corticalemanifeste un comportement complexe. Au niveau cellulaire, l'activitéest caractérisée par de fortes fluctuations sous-liminaires dupotential membranaire et une décharge irrégulière à bassefréquence. Au niveau du réseau, l'activité est marquée par un faibleniveau de synchronie et une dynamique chaotique. Néanmoins, c'est dansce régime que l'information est traitée de manière fiable par lesréseaux neuronaux. Ce régime est donc crucial pour le traitement del'information par le cortex. Dans cette thèse, nous contribuons à sacompréhension en examinant comment les propriétés biophysiques auniveau cellulaire combinées avec les propriétés d'architecture desréseaux façonnent cette dynamique asynchrone.Cette thèse repose sur les modèles de dynamique de réseaux appelésmodèles de champ moyen, un formalisme théorique qui décrit ladynamique de population grâce à une approche auto-consistante. Aucoeur de ce formalisme se trouve la fonction de transfertneuronale : la fonction entrée-sortie d'un neurone. La première partiede cette thèse s'attache à dériver des fonctions de transfertbiologiquement réalistes en incorporant des caractérisationsexpérimentales.Dans un premier temps, nous avons examiné in vitro comment lesneurones néocorticaux pyramidaux de la couche V du cortex visuelrépondent à des fluctuations du potentiel membranaire. Nous avonsobservé que les neurones individuels ne diffèrent pas seulement entermes d'excitabilité, mais qu'ils diffèrent aussi par leurssensibilités aux paramètres des fluctuations. Dans un deuxième temps,nous avons étudié de manière théorique comment l'intégrationdendritique dans des structures arborescentes façonne les fluctuationsau soma. Nous avons observé que, en fonction des propriétés del'activité présynaptique, différentes comodulations des paramètres desfluctuations pouvaient être obtenues. En combinant cette observationavec nos mesures expérimentales, nous avons observé que cela induisaitdes couplages différents entre activité synaptique et déchargeneuronale pour chaque neurone. Nous proposons donc que, puisque cemécanisme offre un moyen d'activer spécifiquement certains neurones enfonction des propriétés de l'entrée, l'hétérogénéité biophysiquepourrait contribuer à l'encodage de propriétés des stimuli dans lestraitements de l'information sensorielle.La deuxième partie de cette thèse examine comment les propriétésd'architecture des réseaux neuronaux se combinent avec les propriétésbiophysiques et affectent les réponses sensorielles via des effets dedynamiques de populations.Nous avons tout d'abord examiné de manière théorique comment un hautniveau d'activité spontanée impactait les réponses post-synaptiquesdans le cortex. Nous avons observé que la compétition entre lerecrutement dans le réseau cortical activé et les effets deconductances associés prédisaient une relation non-triviale entrel'intensité des stimuli et l'amplitude des réponses. Cette prédictionfut observée dans des enregistrements de réponses post-synaptiquesdans le cortex auditif du rat in vivo en réponse à des stimulicorticaux, thalamiques et auditifs.Pour finir, en tirant avantage des approches de champ moyen, nousavons construit un modèle grande échelle du réseau des couches II-IIIincluant le réseau des fibres horizontales. Nous avons examiné lespropriétés intégratives spatio-temporelles du modèle et nous les avonscomparées avec des mesures par imagerie optique de l'activitécérébrale chez le singe éveillé. En particulier, nous avonsreconstruit une expérience typique du traitement sensoriel: lemouvement apparent. Le modèle prédit un fort signal suppressif dont leprofil spatio-temporel correspond quantitativement à celui observé invivo... / The neocortex of awake animals displays an activated state in whichcortical activity manifests highly complex, seemingly noisybehavior. At the level of single neurons the activity is characterizedby strong subthreshold fluctuations and irregular firing at lowrate. At the network level, the activity is weakly synchronized andexhibits a chaotic dynamics. Yet, it is within this regime thatinformation is processed reliably through neural networks. This regimeis thus crucial to neural computation. In this thesis, we contributeto its understanding by investigating how the biophysical propertiesat the cellular level combined with the properties of the networkarchitecture shapes this asynchronous dynamics.This thesis builds up on the so-called mean-field models of networkdynamics, a theoretical formalism that describes population dynamicsvia a self-consistency approach. At the core of this formalism lie theneuronal transfer function: the input-output description of individualneurons. The first part of this thesis focuses on derivingbiologically-realistic neuronal transfer functions. We firstformulate a two step procedure to incorporate biological details (suchas an extended dendritic structure and the effect of various ionicchannels) into this transfer function based on experimentalcharacterizations.First, we investigated in vitro how layer V pyramidal neocorticalneurons respond to membrane potential fluctuations on a cell-by-cellbasis. We found that, not only individual neurons strongly differ interms of their excitability, but also, and unexpectedly, in theirsensitivities to fluctuations. In addition, using theoreticalmodeling, we attempted to reproduce these results. The model predictsthat heterogeneous levels of biophysical properties such as sodiuminactivation, sharpness of sodium activation and spike frequencyadaptation account for the observed diversity of firing rateresponses.Then, we studied theoretically how dendritic integration in branchedstructures shape the membrane potential fluctuations at the soma. Wefound that, depending on the type of presynaptic activity, variouscomodulations of the membrane potential fluctuations could beachieved. We showed that, when combining this observation with theheterogeneous firing responses found experimentally, individual neuronsdifferentially responded to the different types of presynapticactivities. We thus propose that, because this mechanism offers a wayto produce specific activation as a function of the input properties,biophysical heterogeneity might contribute to the encoding of the stimulusproperties during sensory processing in neural networks.The second part of this thesis investigates how circuit properties,such as recurrent connectivity and lateral connectivity, combine withbiophysical properties to impact sensory responses through effectsmediated by population dynamics.We first investigated what was the effect of a high level of ongoingdynamics (the Up-state compared to the Down-state) on the scaling ofpost-synaptic responses. We found that the competition between therecruitment within the active recurrent network (in favor of highresponses in the Up-state) and the increased conductance level due tobackground activity (in favor of reduced responses in the Up-state)predicted a non trivial stimulus-response relationship as a functionof the intensity of the stimulation. This prediction was shown toaccurately capture measurements of post-synaptic membrane potentialresponses in response to cortical, thalamic or auditory stimulation inrat auditory cortex in vivo.Finally, by taking advantage of the mean-field approach, weconstructed a tractable large-scale model of the layer II-III networkincluding the horizontal fiber network. We investigate thespatio-temporal properties of this large-scale model and we compareits predictions with voltage sensitive dye imaging in awake fixatingmonkey...

This thesis reports the investigation of spontaneous Raman Spectroscopy (RS) for its applicability in early drug discovery. A key focus has been to develop an understanding of the applicability of RS for the quantification and localisation of compound concentration inside mammalian cells. Further investigation into the use of Surface Enhanced Raman Spectroscopy (SERS) for research on Visceral Leishmaniasis (VL) and Leishmania donovani as well as investigating applicability for cancer research are decisive parts of this work. The key work described in this thesis is the investigation of whole cell concentration of compounds inside THP-1 and Madin Darby Canine Kidney (MDCK) cells. For true quantification the Cell Silent Region (CSR) is used to measure without interference from cellular background signal. The model compound is erlotinib, an anti-cancer drug with an alkyne group expressing a peak in the CSR. The developed RS system is calibrated using the current gold standard technique Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS). However, because of the single cell nature of the RS information on inter cell variability can be extracted. The RS measurements suggest that there is a large variation of concentration within single cell populations. The RS measurements can therefore give insight in single cell behaviour within a large cell population. Findings shows that washing cycles, before fixation, alter the intra-cellular concentrations significantly. This is hypothesised to be caused by the sudden change in concentration on the outside of the cell that applies an osmotic pressure, leading to loss of substance from inside the cell wall. Localisation of erlotinib is shown within THP-1 cells and points towards an accumulation inside the cell nucleus. Later, internalised Au nano-particles in the range of 30 nm to 80 nm have been investigated for their enhancement effects and localisation inside THP-1 cells. Au nano-particles are found to be internalised easily by differentiated THP-1 cells and accumulate in lysosomes. This allows for a high local enhancement of the spontaneous Raman signal. However, no advantage for the detection of lysosomally trapped compounds (chloroquine, chlorpromazine) was achieved. The detection of substances without a signal in the CSR was achieved without enhancement. Nonetheless, compounds with intrinsic peaks in the CSR could benefit from this enhancement. Lastly the RS system is explored for alternative uses in early drug discovery. This includes the detection of toxicity as well as the discrimination of cell types. Toxicity has been detected using optically trapped THP-1 cells and doxorubicin. Utilising Principal Component Analysis (PCA) combined with Linear Discriminant Analysis (LDA) on these measured spectra, allowed for a clear discrimination of toxically influenced from healthy cells. Differences mainly show up in DNA content caused by the mode of action of doxorubicin and caused by the trapping, which generates most of the signal within the nucleus of the cell. Discriminating cancerogenic (DU145) from healthy prostate cells (PNT2) has been achieved by probing fixed cells and evaluating the acquired Raman spectra with a PCA/LDA combination. The accuracy of separation of these cells when tested with a 10-fold cross-validation technique, is above 98 %, allowing a good discrimination.

There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns. / Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos.

Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity. Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections. To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays. The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery.