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.

<p>Control of E2F transcription factor activity, regulated by the action of the retinoblastoma tumor suppressor, is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell cycle regulatory activities, has not been clearly defined. </p><p>Recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we made use of an integrated system to follow E2F transcriptional dynamics at the single cell level and in real time. We measured and characterized E2F temporal dynamics in the first cell cycle where cells enter the cell cycle after a period of quiescence. Quantitative analyses revealed that crossing a threshold of amplitude of E2F transcriptional activity serves as the critical determinant of cell-cycle commitment and division. </p><p>By using a developed ordinary differential equation model for Rb/E2F network, we performed simulations and predicted that Myc and cyclin D/E activities have distinct roles in modulating E2F transcriptional dynamics. Myc is critical in modulating the amplitude whereas cyclin D/E activities have little effect on the amplitude but do contribute to the modulation of duration of E2F transcriptional activation. These predictions were validated through the analysis of E2F dynamics in single cells under the conditions that cyclin D/E or Myc activities are perturbed by small molecule inhibitors or RNA interference. </p><p>In an ongoing study, we also measured E2F dynamics in cycling cells. We provide preliminary results showing robust oscillatory E2F expression at the single-cell level that aligns with the progression of continuous cell division. The temporal characteristics of the dynamics trajectories deserve further quantitative investigations.</p><p>Taken together, our results establish a strict relationship between E2F dynamics and cell fate decision at the single-cell level, providing a refined model for understanding the control logic of cell cycle entry.</p> / Dissertation

Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

Stem and progenitor cell fate (self-renewal, proliferation, survival, differentiation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell therapies. During erythropoiesis, erythroid progenitors at the colony forming unit-erythroid (CFU-E) stage are responsive to both stem cell factor (SCF) and erythropoietin (EPO); however, the joint action of SCF and EPO in these cells and the underlying mechanisms remain to be defined. In this study, quantitative data on the activation of signaling pathways and gene expression profiles provided definitive evidence for two parallel but complementary mechanisms that resulted in enhanced generation of red blood cells from mouse bone marrow-derived CFU-E culture in the presence of SCF and EPO. First, SCF and EPO signaling intersected within the extracellular signal-regulated kinase (ERK) pathway and the sustained ERK activation was required for the maximal changes in the expression levels of genes that are involved in the proliferation and survival of CFU-Es. Second, the apparent competition between SCF and EPO in regulating c-Kit expression was found to have a dramatic impact on the terminal differentiation of CFU-Es. The latter mechanism was, for the first time, reported in a primary cell system. In addition, a fetal liver-derived conditioned medium further enhanced the survival and proliferation of bone marrow CFU-Es in the presence of SCF and EPO by not only increasing the ERK signaling duration but also, the amplitude. The agents present in the conditioned media possess significant clinical potential to stimulate erythropoiesis both in vivo and in vitro. In conclusion, our study has provided novel insights into the mechanisms by which combinations of niche factors control the fate of erythroid progenitors at a unique transitional stage and highlighted the important role of the ERK signaling dynamics in adult erythropoiesis.

The local microenvironment of hematopoietic stem cells (HSCs) in the bone marrow -referred to as stem cell niche- is thought to regulate the balance of stem cell maintenance and differentiation by a complex interplay of extrinsic signals including spatial constraints, extracellular matrix (ECM) components and cell-cell interactions. To dissect the role of niche ECM components, a set of well-defined matrix biomolecular coatings including fibronectin, laminin, collagen IV, tropocollagen I, heparin, heparan sulphate, hyaluronic acid and co-fibrils of collagen I with heparin or hyaluronic acid were prepared and analyzed with respect to adhesive interactions of human CD133+ HSCs in vitro. ECM molecule dependent adhesion areas as well as fractions of adherent HSCs were assessed by reflection interference contrast microscopy and differential interference contrast microscopy. HSCs, so far mostly classified as suspension cells, exhibited intense adhesive interactions with fibronectin, laminin, collagen IV, heparin, heparan sulphate, and collagen I based co-fibrils. An integrin mediated adhesion on fibronectin and a L-selectin mediated adhesion on heparin pointed to specific interactions based on different adhesion mechanisms. As a consequence of HSC adhesion to molecules of the vascular and the endosteal regions, both regions were confirmed as possible stem cell niches and adhesive signals were suggested as potential regulators of stem cell fate. Furthermore, the impact of a spatially organized ECM on the HSC behavior was analyzed by single cell tracking. These studies required the development of engineered three-dimensional, ECM coated microcavities with the option for single cell tracking. A semi-automated cell-tracking tool was established to accelerate data access from time-lapse image sequences. From this analysis it was possible to reveal the genealogy, localization, morphology and migration of single HSCs over a time period of 4 days. A decreased cycling frequency was observed depending on the HSC localization in the spatially constraining microcavities. Besides the revealed impact of spatial constraints on HSC fate, the newly engineered ECM-coated microcavity setup and the semi-automated cell tracking tool provide new options to study the cell fate in engineered microenvironments at single cell level for other cell types ex vivo. / Die lokale Mikroumgebung von Blutstammzellen (BSZ) im Knochenmark, bezeichnet als Stammzellnische, reguliert das Gleichgewicht von Stammzellerhaltung und -differenzierung durch ein komplexes Zusammenspiel von extrinsischen Signalen wie räumliche Beschränkungen, Komponenten der extrazellulären Matrix (EZM) und Zell-Zell Wechselwirkungen. Um die Rolle der EZM-Komponenten zu analysieren, wurden definierte Beschichtungen von Fibronektin, Laminin, Kollagen IV, monomerem Kollagen I, Heparin, Heparan Sulphat, Hyaluronsäure und Co-Fibrillen aus Kollagen I und Heparin oder Hyaluronsäure hergestellt und in vitro bezüglich der adhäsiven Wechselwirkungen von humanen CD133+ BSZ untersucht. Die Adhäsionsflächen und der Anteil adhärenter Zellen wurden in Abhängigkeit von der EZM-Beschichtung mittels Reflexions- Interferenz-Kontrast-Mikroskopie und Differentieller Interferenz Kontrast Mikroskopie bestimmt. BSZ, bisher als Suspensionszellen definiert, zeigten intensive adhäsive Wechselwirkungen mit Fibronektin, Laminin, Kollagen IV, Heparin, Heparan Sulphat und den Co-Fibrillen. Eine Integrin abhängige Adhäsion auf Fibronektin und eine L-Selektin abhängige Adhäsion auf Heparin, wiesen auf spezifische Wechselwirkungen hin, die auf unterschiedlichen Mechanismen basieren. Aufgrund der Adhäsion von BSZ sowohl zu Molekülen der vaskulären als auch der endostealen Knochenmarkregion, wurden beide Bereiche als mögliche Stammzellnische bestätigt. Adhäsive Signale sind potentielle Regulatoren der Stammzellentwicklung. Im Weiteren wurde der Einfluss einer räumlich beschränkenden EZM auf das Verhalten der BSZ durch Einzelzellverfolgung untersucht. Diese Studien erforderten die Entwicklung von dreidimensionalen EZM-beschichteten Mikrokavitäten, die das Verfolgen einzelner Zellen ermöglichten. Es wurde ein halbautomatischer Algorithmus für die Zellverfolgung etabliert, um die Datengenerierung von den Zeitreihenaufnahmen zu beschleunigen. Die Analysen ermöglichten Aussagen über die Genealogie, Lokalisierung, Morphologie und Migration einzelner BSZ während einer Analysenzeit von 4 Tagen. Eine verringerte Zellteilungsaktivität wurde in Abhängigkeit von der BSZ Lokalisierung innerhalb der räumlich einschränkenden Mikrokavitäten festgestellt. Neben diesen Erkenntnissen bieten die entwickelten Mikrokavitäten und die etablierte Einzelzellverfolgung neue Möglichkeiten auch andere Zelltypen auf Einzelzellniveau ex vivo zu untersuchen.

Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications. This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective. / QC 20100728

Στην παρούσα διπλωματική εργασία εξετάζεται το πρόβλημα του διαμοιρασμού των διαθέσιμων πόρων ενός ασύρματου συστήματος επικοινωνίας στους χρήστες μέσα στην περιοχή μιας κυψέλης. Γίνεται η υπόθεση ότι το ασύρματο σύστημα κάνει χρήση της τεχνικής πολλαπλής πρόσβασης (multiple–access technique) OFDMA, δηλαδή ότι χρησιμοποιεί έναν αριθμό ορθογώνιων υποφορέων τον οποίο αναθέτει στους χρήστες για τη μετάδοση της πληροφορίας. Η μελέτη εστιάζεται στην κάτω ζεύξη (downlink) του συστήματος OFDMA, δηλαδή στη μετάδοση δεδομένων από το σταθμός βάσης της κυψέλης στους χρήστες – δέκτες. Οι διαθέσιμοι πόροι του συστήματος είναι οι ορθογώνιοι υποφορείς και η συνολική διαθέσιμη ισχύς στο σταθμό βάσης. Θεωρούμε ότι ο σταθμός βάσης έχει στη διάθεσή του τέλεια γνώση του καναλιού που τον συνδέει με κάθε χρήστη (channel state information – CSI) μέσω καναλιών ανάδρασης. Η πληροφορία κατάστασης του καναλιού χρησιμοποιείται από τον σταθμό βάσης για την κατανομή των πόρων προς τους χρήστες με δυναμικό τρόπο με χρήση κατάλληλων αλγορίθμων. Εναλλακτικά, εάν ο πομπός δε διαθέτει αξιόπιστη πληροφορία σχετικά με την κατάσταση του καναλιού ή για απλοποίηση του σχεδιασμού η ανάθεση των πόρων μπορεί να πραγματοποιηθεί στατικά, δηλαδή γίνεται μόνιμη ανάθεση ενός συνόλου υποφορέων ανά χρήστη δίχως να λαμβάνεται υπόψη η CSI. Η ισχύς μπορεί να κατανεμηθεί στους υποφορείς είτε ισόποσα ή, βάσει της CSI, με χρήση του αλγορίθμου water–filling. Στην παρούσα διπλωματική εργασία εξετάζονται διαφορετικά σενάρια κατανομής των πόρων που περιλαμβάνουν αλγορίθμους για την ανάθεση των υποφορέων και το διαμοιρασμό των κατάλληλων ποσοτήτων ισχύος στους υποφορείς. Ανάλογα με το στόχο και τις ανάγκες των χρηστών του συστήματος, καθώς και με το ποσό της διαθέσιμης πληροφορίας καναλιού στο σταθμό βάσης, χρησιμοποιούνται διαφορετικοί αλγόριθμοι. Συγκεκριμένα, ο αλγόριθμος Maximum Sum Rate (MSR) επιδιώκει τη μεγιστοποίηση του συνολικού ρυθμού μετάδοσης του συστήματος χωρίς, όμως, να ενδιαφέρεται για την πιθανότητα κάποιοι από τους χρήστες να υποεξηπηρετούνται. Αντιθέτως, ο αλγόριθμος μέγιστης δικαιοσύνης ή αλγόριθμος max–min (ΜΜ) έχει ως κύριο μέλημά του την όσο το δυνατόν καλύτερη εξυπηρέτηση όλων των πελατών στο σύστημα. Τέλος, οι στατικές στρατηγικές ανάθεσης, αν και υποδεέστερες όσον αφορά την επίδοση, είναι πιο απλές στην υλοποίησή τους και μπορούν να εφαρμοστούν σε περιπτώσεις όπου η CSI δεν είναι διαθέσιμη ή δεν είναι επαρκώς αξιόπιστη. / In this diploma thesis, the downlink of a single-cell system is considered in the downlink transmission. The multiple–access technique that is used in this system is OFDMA and the objective is the allocation of the resources among users that access the system. The available resources to be distributed among the users of the OFDMA system comprise the subcarriers over which the signals of the users are transmitted and the available power that is allocated among subcarriers. It is assumed that users estimate and feedback perfectly the channel state information (CSI) to the base station of the cell, where subcarrier and power allocation are determined according to the CSI of the users and the resource–allocation algorithm. Subcarrier assignment to each user can be implemented in a static or a dynamic fashion. When static subcarrier allocation is employed, the CSI is not taken into account, and a fixed set of subcarriers is assigned to each user. On the other hand, dynamic allocation uses CSI and results in improved achievable rates compared to static allocation. Regarding transmit power adaptation, it can be allocated to subcarriers either equally or, provided that the CSI is perfectly known to the transmitter (the base station), using the water–filling procedure. In this diploma thesis, different resource allocation strategies for the downlink of an OFDMA system are compared. Each algorithm has a different objective. For instance, the objective of the Maximum Sum Ratio (MSR) algorithm is to maximize the sum rate of all users on the system with no regard to underserved users. On the contrary, the Max–Min (MM) algorithm, also known as Maximum Fairness Algorithm, has a different philosophy. Specifically, the objective of MM is the better service of the underserved users allocating an amount of resources to them to provide the maximum possible fairness to all users. Despite the total sum rate penalty it experiences, the MM algorithm leads to improved fairness compared to MSR. Finally, although inferior to their dynamic counterparts, static resource allocation strategies are easier to implement and can be employed even in the absence of CSI.

abstract: The technology expansion seen in the last decade for genomics research has permitted the generation of large-scale data sources pertaining to molecular biological assays, genomics, proteomics, transcriptomics and other modern omics catalogs. New methods to analyze, integrate and visualize these data types are essential to unveil relevant disease mechanisms. Towards these objectives, this research focuses on data integration within two scenarios: (1) transcriptomic, proteomic and functional information and (2) real-time sensor-based measurements motivated by single-cell technology. To assess relationships between protein abundance, transcriptomic and functional data, a nonlinear model was explored at static and temporal levels. The successful integration of these heterogeneous data sources through the stochastic gradient boosted tree approach and its improved predictability are some highlights of this work. Through the development of an innovative validation subroutine based on a permutation approach and the use of external information (i.e., operons), lack of a priori knowledge for undetected proteins was overcome. The integrative methodologies allowed for the identification of undetected proteins for Desulfovibrio vulgaris and Shewanella oneidensis for further biological exploration in laboratories towards finding functional relationships. In an effort to better understand diseases such as cancer at different developmental stages, the Microscale Life Science Center headquartered at the Arizona State University is pursuing single-cell studies by developing novel technologies. This research arranged and applied a statistical framework that tackled the following challenges: random noise, heterogeneous dynamic systems with multiple states, and understanding cell behavior within and across different Barrett's esophageal epithelial cell lines using oxygen consumption curves. These curves were characterized with good empirical fit using nonlinear models with simple structures which allowed extraction of a large number of features. Application of a supervised classification model to these features and the integration of experimental factors allowed for identification of subtle patterns among different cell types visualized through multidimensional scaling. Motivated by the challenges of analyzing real-time measurements, we further explored a unique two-dimensional representation of multiple time series using a wavelet approach which showcased promising results towards less complex approximations. Also, the benefits of external information were explored to improve the image representation. / Dissertation/Thesis / Ph.D. Industrial Engineering 2011

Over recent years microbial oil has attracted much attention due to its potential to replace traditional oil sources in the production of biofuels and nutraceuticals. Its advantages arise from its independence of the food supply chain and its ease of production compared to conventional plant oils. Also, as concerns for the environment grow, microbially-synthesized oil emerges as potential competitor for the sustainable production of biodiesel. However, the high cost of its production currently hinders its large scale application. The bottlenecks to industrial microbial oil production are the cost of substrate and cultivation. Current research is focusing on process improvements to make microbial oil more competitive and worthwhile to produce. Several types of microorganisms have been explored so far and waste substrates have been utilised as cheap feedstocks. The overall cost is affected by the fermentation stage, therefore it is imperative to design cultivations with little operating requirements and high yields. Consequently, the present thesis aims to contribute to the field by developing and investigating a simple process for oleaginous yeast cultivation, focusing mainly on enhancing the yields during the bioreactor stage. Oleaginous yeasts were screened for their ability to grow on glycerol and the most promising strain was selected for further research. Then, the necessary conditions for its growth and oil accumulation were defined. Shake-flask cultivations showed that the specific growth rate and glycerol consumption of Rh. glutinis were higher at lower glycerol concentrations (smaller or equal to40 g/L), while higher C/N elemental ratios enhanced oil content. Experimental data were used to construct an unstructured kinetic model to describe and predict the system's behaviour. The Monod-based model took into account double substrate growth dependence and substrate inhibition. Following that, bioreactor cultivations extended the range of parameters studied, to include the influence of aeration rate and oxygen supply on cellular growth and microbial oil production. Cultivations at different air flow rates were performed in a 2 L bioreactor and showed that a low aeration rate of 0.5 L/min gave the best glycerol and nitrogen uptake rates, resulting in a concentration of biomass of 5.3 g/L with oil content of 33% under simple batch operation. This was improved by 68% to 16.8 g/L (cellular biomass) with similar oil content (34%) by applying a fed-batch strategy. Finally, different glycerol feeding schemes were evaluated in terms of their effect on oil accumulation. The concept of targeting first a cell proliferation stage, limited by the availability of nitrogen, followed by a lipid accumulation stage, fuelled by glycerol was tested. Continual feeding and pulsed feedings, delivering the same total amount of nitrogen (and glycerol), resulted in similar elevated values of both cellular biomass (~25 g/L) and oil content (~40%). Addition of glycerol at higher rates but giving the same total amount of nitrogen led to a further increase in oil content to 53%, resulting in an overall oil yield of more than 16 g/L (the highest achieved throughout the project). With comparable yields to those reported in the literature but achieved with a much poorer medium, there is every reason to be optimistic that microbial oil production from glycerol could be commercially viable in the future.

No description available.