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.

No description available.

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.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.

L'analyse de l'organisation à grande échelle des chromosomes, par des approches d'imagerie et de biologie moléculaire, constitue un enjeu important de la biologie. Il est maintenant établi que l'organisation structurelle du génome est un facteur déterminant dans tous les aspects des " transactions " génomiques: transcription, recombinaison, réplication et réparation de l'ADN. Bien que plusieurs modèles aient été proposés pour décrire l'arrangement spatial des chromosomes, les principes physiques qui sous-tendent l'organisation et la dynamique de la chromatine dans le noyau sont encore largement débattus. Le noyau est le compartiment de la cellule dans lequel l'ADN chromosomique est confiné. Cependant, la mesure quantitative de l'influence de la structure nucléaire sur l'organisation du génome est délicate, principalement du fait d'un manque d'outils pour déterminer précisément la taille et la forme du noyau. Cette thèse est organisée en deux parties: le premier axe de mon projet était d'étudier la dynamique et les propriétés physiques de la chromatine dans le noyau de la levure S. cerevisiae. Le deuxième axe visait à développer des techniques pour détecter et quantifier la forme et la taille du noyau avec une grande précision. Dans les cellules de levure en croissance exponentielle, j'ai étudié la dynamique et les propriétés physiques de la chromatine de deux régions génomiques distinctes: les régions codant les ARN ribosomiques regroupés au sein d'un domaine nucléaire, le nucléole, et la chromatine du nucléoplasme. Le mouvement de la chromatine nucléoplasmique peut être modélisé par une dynamique dite de " Rouse ". La dynamique de la chromatine nucléolaire est très différente et son déplacement caractérisé par une loi de puissance d'exposant ~ 0,7. En outre, nous avons comparé le changement de la dynamique de la chromatine nucléoplasmique dans une souche sauvage et une souche porteuse d'un allèle sensible à la température (ts) permettant une inactivation conditionnelle de la transcription par l'ARN polymérase II. Les mouvements chromatiniens sont beaucoup plus importants après inactivation transcriptionnelle que dans la souche témoin. Cependant, les mouvements de la chromatine restent caractérisés par une dynamique dite de " Rouse ". Nous proposons donc un modèle biophysique prenant en compte ces résultats : le modèle de polymère dit "branched-Rouse". Dans la deuxième partie, j'ai développé "NucQuant", une méthode d'analyse d'image permettant la localisation automatique de la position de l'enveloppe nucléaire du noyau de levures. Cet algorithme comprend une correction post-acquisition de l'erreur de mesure due à l'aberration sphérique le long de l'axe Z. "NucQuant" peut être utilisée pour déterminer la géométrie nucléaire dans de grandes populations cellulaires. En combinant " NucQuant " à la technologie microfluidique, nous avons pu estimer avec précision la forme et la taille des noyaux en trois dimensions (3D) au cours du cycle cellulaire. "NucQuant" a également été utilisé pour détecter la distribution des regroupements locaux de complexes de pore nucléaire (NPCs) dans des conditions différentes, et a révélé leur répartition non homogène le long de l'enveloppe nucléaire. En particulier, nous avons pu montrer une distribution particulière sur la région de l'enveloppe en contact avec le nucléole. En conclusion, nous avons étudié les propriétés biophysiques de la chromatine, et proposé un modèle dit "branched Rouse-polymer" pour rendre compte de ces propriétés. De plus, nous avons développé "NucQuant", un algorithme d'analyse d'image permettant de faciliter l'étude de la forme et la taille nucléaire. Ces deux travaux combinés vont permettre l'étude des liens entre la géométrie du noyau et la dynamique de la chromatine. / Chromosome high-order architecture has been increasingly studied over the last decade thanks to technological breakthroughs in imaging and in molecular biology. It is now established that structural organization of the genome is a key determinant in all aspects of genomic transactions. Although several models have been proposed to describe the folding of chromosomes, the physical principles governing their organization are still largely debated. Nucleus is the cell’s compartment in which chromosomal DNA is confined. Geometrical constrains imposed by nuclear confinement are expected to affect high-order chromatin structure. However, the quantitative measurement of the influence of the nuclear structure on the genome organization is unknown, mostly because accurate nuclear shape and size determination is technically challenging. This thesis was organized along two axes: the first aim of my project was to study the dynamics and physical properties of chromatin in the S. cerevisiae yeast nucleus. The second objective I had was to develop techniques to detect and analyze the nuclear 3D geomtry with high accuracy. Ribosomal DNA (rDNA) is the repetitive sequences which clustered in the nucleolus in budding yeast cells. First, I studied the dynamics of non-rDNA and rDNA in exponentially growing yeast cells. The motion of the non-rDNA could be modeled as a two-regime Rouse model. The dynamics of rDNA was very different and could be fitted well with a power law of scaling exponent ~0.7. Furthermore, we compared the dynamics change of non-rDNA in WT strains and temperature sensitive (TS) strains before and after global transcription was actived. The fluctuations of non-rDNA genes after transcriptional inactivation were much higher than in the control strain. The motion of the chromatin was still consistent with the Rouse model. We propose that the chromatin in living cells is best modeled using an alternative Rouse model: the “branched Rouse polymer”. Second, we developed “NucQuant”, an automated fluorescent localization method which accurately interpolates the nuclear envelope (NE) position in a large cell population. This algorithm includes a post-acquisition correction of the measurement bias due to spherical aberration along Z-axis. “NucQuant” can be used to determine the nuclear geometry under different conditions. Combined with microfluidic technology, I could accurately estimate the shape and size of the nuclei in 3D along entire cell cycle. “NucQuant” was also utilized to detect the distribution of nuclear pore complexes (NPCs) clusters under different conditions, and revealed their non-homogeneous distribution. Upon reduction of the nucleolar volume, NPCs are concentrated in the NE flanking the nucleolus, suggesting a physical link between NPCs and the nucleolar content. In conclusion, we have further explored the biophysical properties of the chromatin, and proposed that chromatin in the nucleoplasm can be modeled as "branched Rouse polymers". Moreover, we have developed “NucQuant”, a set of computational tools to facilitate the study of the nuclear shape and size. Further analysis will be required to reveal the links between the nucleus geometry and the chromatin dynamics.

This master’s thesis focuses on the image reconstruction from holographic microscope. The first part of the thesis summarizes problems of holography, describes its principle and application particularly in live cell biology. In the second part the description of several methods used for off-axis hologram reconstruction is provided. The last part describes the implementation of the basic step in holographic reconstruction – the elimination of autocorrelation and twin image – and then also the reconstruction of 3D information of the sample.