Autophagy is a conserved biological stress response in mammalian cells that is responsible for clearing damaged proteins and organelles from the cytoplasm and recycling their contents via the lysosomal pathway. In cases where the stress is not too severe, autophagy acts as a survival mechanism. In cases of severe stress, it may lead to programmed cell death. Autophagy is abnormally regulated in a wide-range of diseases, including cancer. To integrate the existing knowledge about this decision process into a rigorous, analytical framework, we built a mathematical model of cell fate decision mediated by autophagy. The model treats autophagy as a gradual response to stress that delays the initiation of apoptosis to give the cell an opportunity to survive. We show that our dynamical model is consistent with existing quantitative measurements of time courses of autophagic responses to cisplatin treatment. To understand the function of this response in cancer cells we have provided a systems biology experimental framework to study dynamical aspects of autophagy in single cancer cells using live-cell imaging and quantitative uorescence microscopy. This framework can provide new insights on function of autophagic response in cancer cells. / Ph. D.

Enfermer le porteur de l’information génétique dans le noyau a obligée la cellule a créé un système de transport complexe, qui permet l’export d’un ARNm du noyau au cytoplasme. Le mécanisme général de l’export des ARNm est encore mal connu, même si les facteurs principaux ont été découverts il y a longtemps. De récents progrès en microscopie nous ont permis d’étudier directement le comportement des ARNm durant le processus d’export. Durant ma maitrise, nous avons été capables de localiser et suivre des ARNm en temps réel pour la première fois chez Saccharomyces cerevisiae. Nous avons créé un gène rapporteur en mettant le gène GLT1 sous le contrôle du promoteur GAL1. Nous avons aussi marqué l’ARNm de GLT1 avec plusieurs boucles PP7. L’ARNm sera visible après l’attachement de plusieurs protéines PP7-GFP aux boucles. En utilisant la technique d’imagerie en cellules vivantes, nous sommes capable de visualiser et suivre chaque ARNm, depuis son relâchement du site de transcription jusqu’à l’export. Une fois relâché du site de transcription, l’ARNm diffuse librement dans le nucléoplasme, mais une fois à la périphérie nucléaire, il commence à « scanner » l’enveloppe nucléaire avant d’être exporté. Nous avons trouvé que le « scanning » dépend de la présence des Myosin Like Proteins (Mlp1p et Mlp2p), protéines qui forment le panier nucléaire, car suite à la délétion de MLP1 et MLP2, les ARNm n’étaient plus capable de « scanner ». Nous avons également trouvé que la partie C-terminale de Mlp1p était nécessaire au « scanning ». De plus, suite à la délétion du gène TOM1, gène codant pour une ubiquitine ligase, les ARNm ont un comportement similaire aux ARNm d’une souche ∆mlp1/mlp2, suggérant que le « scanning » permet à Tom1p d’ubiquitiner Yra1p, ce qui causera son relâchement de l’ARNm. Également, nous avons montré que les ARNm endogènes MDN1 et CBL2 scannent aussi la périphérie nucléaire. Ensemble, nos résultats suggèrent que le scanning est un processus par lequel passent tout les ARNm nucléaire lorsqu’ils se retrouvent à la périphérie du noyau, pour initier plusieurs étapes de réarrangements nécessaires à leurs export. De plus, nous avons examiné le rôle de Yhr127p, une protéine nouvellement identifiée qui se lie à l’ARN. Après avoir marqué cette protéine avec la GFP, nous avons montré qu’elle forme des foci dans le noyau et que ces derniers vont disparaitre suite à l’arrêt de la transcription. La délétion de YHR127 à conduit à une augmentation de la transcription de quelques gènes spécifiques, mais n’affecte pas la capacité de la cellule à exporter les ARNm. Nos résultats suggèrent que cette protéine joue un rôle dans la régulation de la transcription et/ou dans la stabilité de l’ARNm. / In eukaryotic cells, the processes of RNA and protein synthesis are spatially separated into two distinct compartments. With this division, a complex pathway of nucleocytoplasmic RNA export has evolved, which to date remains poorly understood. Recent advances in single-molecule microscopy have enabled direct studies focused on investigating the dynamics and kinetics of RNA export. In this Master thesis, we present the first real time visualization of mRNA export in the yeast Saccharomyces cerevisiae. We first generated a GLT1 reporter under the control of the inducible GAL1 promoter, in which the GLT1 mRNA was tagged with an array of PP7 repeats and detected by exogenous PP7-GFP binding protein. Using a single-molecule live cell imaging approach, we were able to visualize and track the behavior of individual mRNAs from the site of transcription to the point of export. Interestingly, we found that once released from the transcription site, single mRNAs diffuse freely in the nucleoplasm, but once they reach the nuclear periphery, they scan the periphery before being exported to the cytoplasm. This scanning behavior was dependent on Myosin Like Proteins (Mlp1p and Mlp2p), which form the basket of the Nuclear Pore Complex (NPC), as mRNAs were not retained at the periphery and were rapidly released into the nucleoplasm in mlp1p/mlp2p double mutant cells. Specifically, we found that the C-terminal part of Mlp1p was important for scanning. Furthermore, mRNAs from cells depleted of the E3 ubiquitin ligase TOM1 had a similar phenotype to mRNAs in mlp1p/mlp2p double mutant cells, suggesting a role for scanning in the Tom1p-mediated release of Yra1p from the RNA. Lastly, we confirmed that endogenous MDN1 and CBL2 mRNAs also exhibit scanning behaviour. Taken together, our results suggest that mRNAs scanning the nuclear periphery is a general behaviour for all mRNAs to initiate the mRNA export process, allowing mRNP arrangement required for export to occur at the nuclear periphery. In addition, we investigated the role of YHR127, a newly identified RNA binding protein, in RNA biogenesis. Notably, we show that GFP-tagged YHR127p formed distinct foci in the nucleus, which were lost upon transcription arrest. Deletion of YHR127 led to an increase in transcript levels of specific genes, but not to a global accumulation of mRNAs in the nucleus, suggesting a role for this protein in regulating transcription and/or mRNA stability.

Dynamische zelluläre Prozesse wie Zellzyklus, Signaltransduktion oder Transkription zu analysieren wird Live-cell-imaging mittels Zeitraffermikroskopie verwendet. Um nun aber Zellabstammungsbäume aus einem Zeitraffervideo zu extrahieren, müssen die Zellen segmentiert und verfolgt werden können. Besonders hier, wo lebende Zellen über einen langen Zeitraum betrachtet werden, sind Fehler in der Analyse fatal: Selbst eine extrem niedrige Fehlerrate kann sich amplifizieren, wenn viele Zeitpunkte aufgenommen werden, und damit den gesamten Datensatz unbrauchbar machen. In dieser Arbeit verwenden wir einen einfachen aber praktischen Ansatz, der die Vorzüge der manuellen und automatischen Ansätze kombiniert. Das von uns entwickelte Live-cell-Imaging Datenanalysetool ‘eDetect’ ergänzt die automatische Zellsegmentierung und -verfolgung durch Nachbearbeitung. Das Besondere an dieser Arbeit ist, dass sie mehrere interaktive Datenvisualisierungsmodule verwendet, um den Benutzer zu führen und zu unterstützen. Dies erlaubt den gesamten manuellen Eingriffsprozess zu rational und effizient zu gestalten. Insbesondere werden zwei Streudiagramme und eine Heatmap verwendet, um die Merkmale einzelner Zellen interaktiv zu visualisieren. Die Streudiagramme positionieren ähnliche Objekte in unmittelbarer Nähe. So kann eine große Gruppe ähnlicher Fehler mit wenigen Mausklicks erkannt und korrigiert werden, und damit die manuellen Eingriffe auf ein Minimum reduziert werden. Die Heatmap ist darauf ausgerichtet, alle übersehenen Fehler aufzudecken und den Benutzern dabei zu helfen, bei der Zellabstammungsrekonstruktion schrittweise die perfekte Genauigkeit zu erreichen. Die quantitative Auswertung zeigt, dass eDetect die Genauigkeit der Nachverfolgung innerhalb eines akzeptablen Zeitfensters erheblich verbessern kann. Beurteilt nach biologisch relevanten Metriken, übertrifft die Leistung von eDetect die derer Tools, die den Wettbewerb ‘Cell Tracking Challenge’ gewonnen haben. / Live cell imaging based on time-lapse microscopy has been used to study dynamic cellular behaviors, such as cell cycle, cell signaling and transcription. Extracting cell lineage trees out of a time-lapse video requires cell segmentation and cell tracking. For long term live cell imaging, data analysis errors are particularly fatal. Even an extremely low error rate could potentially be amplified by the large number of sampled time points and render the entire video useless. In this work, we adopt a straightforward but practical design that combines the merits of manual and automatic approaches. We present a live cell imaging data analysis tool `eDetect', which uses post-editing to complement automatic segmentation and tracking. What makes this work special is that eDetect employs multiple interactive data visualization modules to guide and assist users, making the error detection and correction procedure rational and efficient. Specifically, two scatter plots and a heat map are used to interactively visualize single cells' visual features. The scatter plots position similar results in close vicinity, making it easy to spot and correct a large group of similar errors with a few mouse clicks, minimizing repetitive human interventions. The heat map is aimed at exposing all overlooked errors and helping users progressively approach perfect accuracy in cell lineage reconstruction. Quantitative evaluation proves that eDetect is able to largely improve accuracy within an acceptable time frame, and its performance surpasses the winners of most tasks in the `Cell Tracking Challenge', as measured by biologically relevant metrics.

Mathematical modeling has made great strides since the Lotka-Volterra predator-prey models. Newer models attempt to describe sub-cellular signal transduction pathways, such as the JAK-STAT and NF-κB pathways. However, the tools to accurately determine reaction and translocation rates in these pathways still have a number of drawbacks, including the effects of concentration scale on determining reaction rates and the effects of bulky additions to translocation rates. One method of overcoming these problems in signal transduction rate determination is to sample and stain cells from a full population at specific time points. However, fixed cell methods can only generate an average population rate. This could become an issue if the rate depends on the genotype of one of the proteins in the pathway. Another method of overcoming these problems in signal transduction rates is to use unmarked nuclei in live-cell imaging techniques. However, live cell imaging methods poses different problems, primarily how to find and track nuclei and cytoplasm when cells are actively moving and the nuclear and cytoplasmic intensities are by necessity fluctuating. To date, there is only one software package designed for tracking cells under these conditions - Cell Tracker (Shen et al., 2006). Cell Tracker is designed to handle the tracking of live cell images for protein translocation studies. They recommend using a separate color channel to mark the nucleus, although results can be obtained using unmarked nuclei. The results from Cell Tracker with unmarked nuclei are often less than optimal. We have developed a novel segmentation scheme and variation of the particle filter algorithm to allow more accurate tracking in time series with unmarked nuclei. The proposed segmentation scheme uses a non-parametric level set algorithm to refine a fast initial thresholding step. The tracking scheme uses a dense optical flow calculation to assist the particle filter algorithm in continuing to follow the true positions of the nuclei. To test the proposed algorithm, a novel mimicry of cell movement has been developed using random perturbations of a triangular mesh structure through the use of the finite element method.

Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.

In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate. / Chemistry and Chemical Biology

The goal of my research was to obtain a quantitative understanding of the mechanisms of DNA double-strand break (DSB) repair, and the activation of the tumor suppressor p53 in response to DSBs in human cells. In Chapter 2, we investigated how the kinetics of repair, and the balance between the alternate DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), change with cell cycle progression. We developed fluorescent reporters to quantify DSBs, HR and cell cycle phase in individual, living cells. We show that the rates of DSB repair depend on the cell cycle stage at the time of damage. We find that NHEJ is the dominant repair mechanism in G1 and in G2 cells even in the presence of a functional HR pathway. S and G2 cells use both NHEJ and HR, and higher use of HR strongly correlates with slower repair. Further, we demonstrate that the balance between NHEJ and HR changes gradually with cell cycle progression, with a maximal use of HR at the peak of active replication in mid-S. Our results establish that the presence of a sister chromatid does not affect the use of HR in human cells. Chapter 3 examines the sensitivity of the p53 pathway to DNA DSBs. We combined our fluorescent reporter for DSBs with a fluorescent reporter for p53, to quantify the level of damage and p53 activation in single cells. We find that the probability of inducing a p53 pulse increases linearly with the amount of damage. However, cancer cells do not have a distinct threshold of DSBs above which they uniformly induce p53 accumulation. We demonstrate that the decision to activate p53 is potentially controlled by cell-specific factors. Finally, we establish that the rates of DSB repair do not affect the decision to activate p53 or the dynamical properties of the p53 pulse. Collectively, this work emphasizes the importance of collecting quantitative dynamic information in single cells in order to gain a comprehensive understanding of how different DNA damage response pathways function in a coordinated manner to maintain genomic integrity.

Throughout their life cycle, plants adjust growth in response to their developmental and environmental situation within the limits of their energetic capacities. This capacity is defined by the local sugar availability, which is constantly modulated through synthesis, transport and consumption of sugar. The monitoring of sugar presence is carried out by a complex signalling network in which simple sugars (e.g. glucose, fructose and sucrose) act as metabolic signals for the modulation of physiological processes. However, often it remains unclear whether the regulation is induced by the simple sugars themselves or by their derivatives generated during sugar metabolism. This thesis focuses on the dissection of distinct sugar signals, their generation, perception and impact on the modulation of gene expression and cell division both in cell culture and young seedlings. Based on a stem-cell-like A. thaliana cell culture, which could be sustained in a hormone-free media, a new biological system, supplied with Xyl as the only carbon source was developed. The performance of a variety of sugar and sugar analogue treatments in this novel system allowed for the identification of sugar-responsive candidate genes, which were specifically regulated by glucose, fructose and sucrose. For several genes (e.g. bZIP63, AT5g22920, TPS9, MGD2 and BT2), this regulation required both sugar transport into the cytosol and metabolisation for the generation of the signal. Furthermore, gene expression analyses in young A. thaliana seedlings indicated the requirement for the catalytic activity of hexokinase 1 in the regulation of bZIP63, Atg22920 and BT2 under conditions of a perturbed carbohydrate balance. These findings have been combined in a proposed model for the transcriptional regulation of bZIP63, AT5g22920, TPS9, MGD2 and BT2, which further proposes a function of those genes in the regulation of cell division. The optimisation of a protocol for long-term real-time live-cell imaging provided a valuable tool to show that, similar to gene expression, the progression of cell division depended on a sugar-type-specific regulation at the single-cell level; this regulation was most likely caused by prolongation of the interphase. Together with the observation of cell death and growth arrest of the primary root in intact seedlings in response to the glucose analogue 2dog, this led to the conclusion that sugar signals themselves were sufficient to induce cell division. However, the continuation of cell cycle progression and consequently organ growth over long-time required the availability of the energy contained in the sugar.

No description available.

Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.