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Ce travail de thèse porte sur l’étude de la réplication chez les archées, qui constituent le troisième domaine du vivant avec les bactéries et les eucaryotes. L’organisme modèle que nous avons utilisé est l'archée halophile Haloferax volcanii car les outils génétiques disponibles permettent d’exprimer des protéines fusionnées à la Protéine Fluorescente Verte (GFP) dans cet organisme mésophile et aérobe et ainsi de localiser les protéines d’intérêt dans des cellules vivantes. Nous nous sommes ainsi intéressés à la localisation cellulaire de quatre protéines de la réplication qui ont été fusionnées à la GFP et exprimées sous contrôle de leur propre promoteur : (i) la protéine ‘Flap Endonuclease 1’ (FEN1), qui intervient dans la maturation des fragments d’Okazaki, (ii) la protéine ‘Origin Recognition Complex’ (ORC1) impliquée dans la reconnaissance des origines de réplication, (iii) la protéine ‘Proliferating Cellular Nuclear Antigen’ (PCNA), anneau de processivité des ADN polymérases réplicatives, et (iv) la protéine de fixation à l’ADN simple-brin ‘Replication Protein A’ (RPA2) essentielle à la réplication chez H. volcanii. Seule la protéine PCNA n’a pu être exprimée en fusion avec la GFP, suggérant que la protéine fusion n’est pas fonctionnelle. GFP::Orc1 et GFP::Fen1 ont été exprimées dans la cellule mais ne présentent pas de localisation spécifique reflétant un rôle de ces protéines dans la réplication de l’ADN. En revanche des foyers de fluorescence de la protéine fusion GFP::Rpa2 ont été observés, dont le nombre augmente significativement dans des cellules exposées à l’aphidicoline, drogue inhibant la synthèse de l’ADN et induisant ainsi un stress réplicatif. Cependant une localisation différente de la protéine GFP::Rpa2 a été observée lorsque les cellules sont exposés à la phléomycine, qui induit notamment des cassures double-brin de l‘ADN. Dans ces cellules, GFP::Rpa2 forme un foyer de fluorescence massif qui colocalise avec l’ADN compacté dans la grande majorité des cellules observées. Nos résultats suggèrent donc que la localisation spécifique observée pour GFP::Rpa2 reflète son rôle dans la réparation de l’ADN et/ou le redémarrage des fourche de réplication arrêtées. / The aim of this thesis project was to improve our understanding of DNA replication in archaea, the third domain of life with bacteria and eukarya. The model organism chosen for these studies is the halophilic archaea Haloferax volcanii, a mesophilic aerobe for which genetics tools allow studying in living cells the localization of proteins fused to the Green Fluorescent protein (GFP). Four proteins involved in DNA replication were fused to the GFP and expressed under the control of their own promoter: (i) the ‘Flap Endonuclease 1’ (FEN1), involved in Okazaki fragments maturation, (ii) the ‘Origin Recognition Complex’ (ORC1), involved in DNA replication origin recognition, (iii) the ‘Proliferating Cellular Nuclear Antigen’ (PCNA), processivity factor of replicative DNA polymerases, and (iv) the ‘Replication Protein A’ (RPA2), single-stranded DNA binding protein essential for DNA replication in H. volcanii. Only the PCNA fusion to the GFP was not successful, suggesting that the GFP hinders essential roles of PCNA in DNA replication. Fen1 and Orc1 were successfully fused to the GFP and expressed in living cells, but specific localization in cells related to growth phase, reflecting different replication dynamics, were not observed. In contrast, we could observed fluorescent foci formed by the fully functional GFP::Rpa2 protein that actively responded to DNA damage in H. volcanii cells. The number of these fluorescent foci per cell was constant during cell growth but it significantly increased in cells exposed to aphidicoline, which inhibits DNA synthesis during replication. When cells were treated with phleomycine, a DNA damaging agent mainly causing double-strand breaks, formation of a massive fluorescent focus coinciding with DNA compaction was observed. Our results suggest that the specific cellular localization of GFP::Rpa2 observed reflects Rpa2 roles in DNA repair and/or DNA replication fork restart.

Indiana University-Purdue University Indianapolis (IUPUI) / The young field of gene therapy offers the promises of significant progress towards the treatment of many different types of human diseases. Gene therapy has been proposed as an innovative way to treat Acute Kidney Injury (AKI). Through proteomic analysis, the upregulation of two enzymes, IDH2 and SULT1C2, within the mitochondrial fraction has been identified following ischemic preconditioning, a treatment by which rat kidneys are protected from ischemia. Using the hydrodynamic fluid gene delivery technique, we were able to upregulate the expression of IDH2 and SULT1C2 in the kidney. We found that the delivery of IDH2 plasmid through hydrodynamic fluid delivery to the kidney resulted in increased mitochondrial oxygen respiration compared with injured kidneys without gene delivery. We also found that renal ischemic preconditioning altered the membrane fluidity of mitochondria. In conclusion, our study supports the idea that upregulated expression of IDH2 in mitochondria can protect the kidney against AKI, while the protective function of upregulated SULT1C2 needs to be further studied.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute Kidney Injury (AKI) is a prevalent and significant problem whose primary treatment is supportive care. Ischemic preconditioning is a strategy used to protect organs from ischemic injury via a prior injury. Ischemic preconditioning in the kidneys has been shown to confer protection onto kidneys from subsequent ischemic insults with attenuated serum creatinine values in treated rats. In the preconditioned kidneys, the enzyme IDH2 was discovered to be upregulated in the mitochondria. Hydrodynamic fluid delivery to the kidney was found to be a viable technique for delivering this gene to the kidney, resulting in artificially upregulated expression of IDH2. Via a two-pronged effort to discern the functional significance of ischemic preconditioning and hydrodynamic IDH2 fluid injections, we performed mitochondrial oxygen respiration assays on both preconditioned and injected kidneys. We found that renal ischemic preconditioning resulted in no significant difference between sham and preconditioned, subsequently injured kidneys, which is similar to the results from the serum creatinine studies. Hydrodynamically IDH2-injected, and subsequently injured kidneys respire significantly better than vehicle injected, and subsequently injured kidneys, which shows that hydrodynamic injections of IDH2 protects kidneys against injury, and partially mimics the effects of preconditioning.

ALBA proteins were discovered in Archaea more than 30 years ago. They were gradually identified to be well conserved in Eucaryotes as well. A functional dimeric form of these proteins with DNA and RNA-binding capability was claimed in both mentioned domains of organisms. However, their roles diversified during evolution and vary in between organisms. In Archaea, ALBAs are involved in the genome organization and RNA-protein interactions. In Eukaryotes, there are presented two different subfamilies of ALBA proteins - Rpp20 and Rpp25 subfamily. A sole protein from each subfamily was identified in some organisms though they were multiplied in plants, respectively. These proteins can interact with each other and participate in ontogenetic development and stress responses. According to several studies, ALBA proteins were found to be involved in DNA stability maintenance or pre-rRNA splicing in the nucleus of Arabidopsis thaliana. However, they have been shown to play a role in the cellular metabolism and stress responses in cytoplasm. Six ALBA proteins were identified in the genome of A. thaliana, three from each subfamily. In this study, all heterodimeric protein- protein interactions were investigated by the bimolecular fluorescence complementation (BiFC) assay which revealed positive results in...

Multicellular life is formed by an orchestrated interplay of processes on different scales in space and time. Observing and quantitatively measuring these processes in an intact, living organism requires gentle and adaptive imaging. One example of such a process is the scaling of the mitotic spindle during early development. The spindle segregates the chromosomes during cell division and the spindle length determines the positioning of the chromosomes in the successive daughter cells. Thus, adaptation of spindle size to cell size is crucial for proper functioning. Early development is an excellent phase to study spindle scaling since cells rapidly divide in the absence of growth. In this phase, the spindle can be studied in cells of the same organism changing its volume orders of magnitude. During early zebrafish embryogenesis, the mitotic spindle only appears for three minutes out of the fifteen minutes cell cycle. Quantifying these short-lived events in a living embryo requires flexible and adaptive multi-resolution recordings, which are impossible with any state-of-the-art microscope. In this thesis, I present two new techniques to adaptively image biological samples based on light sheet fluorescence microscopy (LSFM). First, I present a remote, contact-free positioning technique based on magnetic forces to orient the sample in the microscope. When imaging biological samples, there is often only one sample orientation that offers the best view on the region of interest. This preferred orientation typically changes over time as the specimen grows and develops. The contact-free positioning technique allows to always image specimens from the optimal viewing angle. I demonstrate the functionality of this method by 3D orientation of zebrafish embryos and zebrafish larvae. Second, I present a new type of LSFM that autonomously adapts its detection scheme to the sample state. This microscope contains an adaptable magnification module to map the development of the millimeter-sized zebrafish embryo and measure single-molecule dynamics of individual spindles in a single experiment. To automatically adapt the detection scheme, I trained a Convolution Neural Network to detect the cell cycle state of individual cells from acquired fluorescence images. Using this new type of LSFM, I demonstrate autonomous measurements of the mitotic spindle scaling in freely developing zebrafish embryos. / Multizelluläres Leben wird durch ein orchestriertes Zusammenspiel von Prozessen auf verschiedenen Skalen in Raum und Zeit gebildet. Beobachtung und quantitative Messungen dieser Vorgänge in einem intakten, lebenden Organismus erfordern schonende und adaptive Bildgebung. Ein Beispiel für einen solchen Prozess ist die Größenanpassung der mitotischen Spindel während der frühen Entwicklung. Die Spindel trennt die Chromosomen während der Zellteilung und die Spindellänge bestimmt die Positionierung der Chromosomen in den Tochterzellen. Daher ist die Anpassung der Spindelgröße an die Zellgröße entscheidend für die ordnungsgemäße Funktion. Die Phase der frühen Entwicklung eignet sich hervorragend zur Untersuchung der Spindel-Skalierung, da die Zellen sich schnell teilen ohne zu wachsen. Während der frühen Zebrafischembryogenese erscheint die Spindel nur drei Minuten innerhalb des fünfzehnminütigen Zellzyklus. Die Quantifizierung dieser kurzlebigen Ereignisse in einem lebenden Embryo erfordert flexible und anpassungsfähige Aufnahmen mit variabler Auflösung, die mit keinem Mikroskop nach dem aktuellen Stand der Technik möglich sind. In dieser Arbeit präsentiere ich zwei neue Techniken zur adaptiven Abbildung biologischer Proben basierend auf der Lichtblatt-Fluoreszenzmikroskopie (LSFM). Zuerst stelle ich eine berührungslose Positionierungstechnik vor, die auf Magnetkräften basiert, um die Probe im Mikroskop zu orientieren. Bei der Abbildung biologischer Proben gibt es oft nur eine Probenorientierung, welche die beste Sicht auf die Region von Interesse bietet. Diese Vorzugsorientierung ändert sich typischerweise mit der Zeit, wenn die Probe wächst und sich entwickelt. Die Positionierungstechnik ermöglicht es, Proben immer aus dem optimalen Betrachtungswinkel abzubilden. Zweitens stelle ich einen neuen Typ von LSFM vor, der sein Detektionsschema autonom an den Probenzustand anpasst. Dieses Mikroskop enthält ein anpassbares Vergrößerungsmodul, um die Entwicklung des millimetergroßen Zebrafischembryos abzubilden und die Einzelmoleküldynamik einzelner Spindeln in einem einzigen Experiment zu messen. Um die Detektion automatisch anzupassen, trainierte ich ein Convolutional Neural Network, um den Zellzyklusstatus einzelner Zellen anhand der aufgenommenen Fluoreszenzbilder zu erkennen. Mit diesem neuen LSFM-Typ demonstriere ich autonome Messungen der Spindel-Skalierung in sich frei entwickelnden Zebrafischembryonen.

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Indiana University-Purdue University Indianapolis (IUPUI) / A crucial task in computer vision and biomedical image applications is to represent images in a numerically compact form for understanding, evaluating and/or mining their content. The fundamental step of this task is the segmentation of images into regions, given some homogeneity criteria, prior appearance and/or shape information criteria. Specifically, segmentation of cells in microscopic images is the first step in analyzing many biomedical applications. This thesis is a part of the project entitled "Construction and profiling of biodegradable cardiac patches for the co-delivery of bFGF and G-CSF growth factors" funded by National Institutes of Health (NIH). We present a method that simultaneously segments the population of cells while partitioning the cell regions into cytoplasm and nucleus in order to evaluate the spatial coordination on the image plane, density and orientation of cells. Having static microscopic images, with no edge information of a cytoplasm boundary and no time sequence constraints, traditional cell segmentation methods would not perform well. The proposed method combines deformable models with a probabilistic framework in a simple graphical model such that it would capture the shape, structure and appearance of a cell. The process aims at the simultaneous cell partitioning into nucleus and cytoplasm. We considered the relative topology of the two distinct cell compartments to derive a better segmentation and compensate for the lack of edge information. The framework is applied to static fluorescent microscopy, where the cultured cells are stained with calcein AM.

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?-Conjugated systems have been the focus of study in recent years in order to understand their charge transport and optical properties for use in organic electronic devices, fluorescence bioimaging, sensors, and 3D optical data storage (ODS), among others. As a result, several molecular building blocks have been designed, allowing new frontiers to be realized. While various successful building blocks have been fine-tuned at both the electronic and molecular structure level to provide advanced photophysical and optoelectronic characteristics, the azulene framework has been under-appreciated despite its unique electronic and optical properties. Among several attributes, azulenes are vibrant blue naturally occurring hydrocarbons that exhibit large dipolar character, coupled with stimuli-responsive behavior in acidic environments. Additionally, the non-toxic nature and the accompanying eco-friendly feature of some azulenes, namely guaiazulene, may set the stage to further explore a more "green" route towards photonic and conductive materials. The first part of this dissertation focuses on exploiting guaiazulene as a natural building block for the synthesis of chromophores with varying stimuli-responsiveness. Results described in Chapter 1 show that extending the conjugation of guaiazulene through its seven-membered ring methyl group with aromatic substituents dramatically impacts the optical properties of the guaiazulenium carbocation. Study of these ?–stabilized tropilium ions enabled establishing photophysical structure-property trends for guaiazulene-terminated ?-conjugated analogs under acidic conditions, including absorption, emission, quantum yield, and optical band gap patterns. These results were exploited in the design of a photosensitive polymeric system with potential application in the field of three dimensional (3D) optical data storage (ODS). Chapter 2 describes the use of guaiazulene reactive sites (C-3 and C-4 methyl group) to generate a series of cyclopenta[ef]heptalenes that exhibit strong stimuli-responsive behavior. The approach presents a versatile route that allows for various substrates to be incorporated into the resulting cyclopenta[ef]heptalenes, especially after optimization that led to devising a one-pot reaction toward such tricyclic systems. Examining the UV-vis absorption profiles in neutral and acidic media showed that the extension of conjugation at C(4) of the cyclopenta[ef]heptalene skeleton results in longer absorption maxima and smaller optical energy gaps. Additionally, it was concluded that these systems act as sensitizers of a UV-activated (< 300 nm) photoacid generator (PAG), via intermolecular photoinduced electron transfer (PeT), upon which the PAG undergoes photodecomposition resulting in the generation of acid. In a related study, the guaiazulene methyl group at C-4 was employed to study the linear and nonlinear optical properties of 4-styrylguaiazulenes, having the same ?–donor with varying ?-spacer. It was realized that the conjugation length correlates with the extent of bathochromic shift of the protonated species. On the other hand, a trend of decreasing quantum yield was established for this set of 4-styrylguaiazulenes, which can be explained by the increasingly higher degree of flexibility. The second part of this dissertation presents a comprehensive investigation of the linear photophysical, photochemical, and nonlinear optical properties of diketopyrrolopyrrole (DPP)-based derivatives, including two-photon absorption (2PA), femtosecond transient absorption, stimulated emission spectroscopy, and superfluorescence phenomena. The synthetic feasibility, ease of modification, outstanding robustness, and attractive spectroscopic properties of DPPs have motivated their study for fluorescence microscopy applications, concluding that the prepared DPP's are potentially suitable chromophores for high resolution stimulated emission depletion (STED) microscopy.