Dissertations / Theses on the topic 'Single cell metabolic flux'

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2000.
Includes bibliographical references (p. 189-206).
Metabolic flux and population heterogeneity analysis were used to develop relations between mammalian cell physiology and specific culture environments and to formulate strategies for increasing cell culture performance. Mitochondrial characteristics associated with respiration, membrane potential, and apoptosis along with physiological state multiplicity involving both metabolism and apoptotic death played a key role in this research. Research involving the accurate calculation of metabolic flux and the analysis of cellular behavior occurring in continuous cultures set the stage for subsequent research on physiological state multiplicity. This phenomena was observed in continuous cultures when at the same dilution rate, two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations. Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the tri-carboxylic (TCA) cycle for energy generation. This metabolic adaptation was unlikely due to favorable genetic mutations and was implemented in subsequent research aimed at improving cell culture performance. A hypothesis stating that mitochondrial physiology and cellular physiology are correlated was tested and confirmed. A mammalian cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123. The MMP sorted subpopulations were subjected to apoptosis inducers, and the apoptotic death was characterized both morphologically through the determination of apoptosis related chromatin condensation and also biochemically through the measurement of caspase-3 enzymatic activity. The results showed dramatic differences in apoptotic death kinetics with the higher MMP subpopulations demonstrating a higher resistance to apoptotic death. These results were applied in the development of novel fed-batch feeding and operating strategies. The first strategy showed that overfeeding cells later in culture leads to an increase in culture viable cell concentration, viability, and productivity. The second strategy showed that cell populations with a higher mean MMP are able to resist apoptosis during fed-batch culture. These results indicate that mammalian cell populations have considerable flexibility in their ability to redistribute metabolic flux in central carbon metabolism. Furthermore, these cell populations contain subpopulations that vary in their resistance to apoptotic death. The analysis of mitochondrial physiology and metabolic flux led to these discoveries, and these areas will play a key role in future mammalian cell culture research.
by Brian D. Follstad.
Ph.D.

Dissertation presented to obtain the Ph.D degree in Biochemistry, Neuroscience
Brain energy metabolism results from a complex group of pathways and trafficking mechanisms between all cellular components in the brain, and importantly provides the energy for sustaining most brain functions. In recent decades, 13C nuclear magnetic resonance (NMR) spectroscopy and metabolic modelling tools allowed quantifying the main cerebral metabolic fluxes in vitro and in vivo. These investigations contributed significantly to elucidate neuro-glial metabolic interactions, cerebral metabolic compartmentation and the individual contribution of neurons and astrocytes to brain energetics. However, many issues in this field remain unclear and/or under debate.
To the financial support provided by Fundação para a Ciência a Tecnologia (SFRH/BD/29666/2006; PTDC/BIO/69407/2006) and to the Clinigene – NoE (LSHBCT2006- 010933). I further acknowledge the Norwegian Research Council for a fellowship that allowed me to perform part of my PhD work at NTNU, Norway.

The purpose of this research was to study the factors that affecting the growth and Taxol production by Taxus species. Taxol, a complex diterpene alkaloid, is approved by the FDA for the treatment of ovarian and breast cancer. It was originally isolated from the bark of Taxus brevifolia. However, the bark contains very low concentrations of Taxol. Currently, Taxol is manufactured via a semi-synthetic method, but the production is limited and Taxol is stilI an expensive drug. Plant cell culture method has been recognized as a promising alternative for Taxol production. Nevertheless, the low or unstable productivity has become an obstacle to attain high yield of Taxol. This research focused on the experimental and computational modelling aspects of Taxol production in Taxus cell cultures. Callus and cell suspension cultures were successfully initiated from the seedlings of T. baccata. The addition of 1.5 % (w/v) insoluble polyvinylpolypyrrolidone (PVPP) and use of half strength of picloram in callus maintenance medium reduced the problem of cell darkening considerably. In suspension cultures, the non-ionic XAD-4 adsorbent was added to overcome the same problem. Fructose was the best carbon source compared to sucrose and glucose. The addition of fructose (10 gIL) on day 8 and methyl jasmonate (lOO IlM) on day 10 increased Taxol production to 17 mgIL from the suspension cultures initiated from needle explants of the seedlings. The experimental data obtained from this study were used in the development of the computer models: the kinetic model, fundamental and integrated dynamic metabolic flux analysis models. The in silico Taxus metabolism was reconstructed and computational metabolic flux balancing method was used in order to obtain fluxes of all the metabolic reactions with linear programming and optimisation in GAMS environment (General Algebraic Modeling System). The objective function of optimisation was either the maximisation of the specific growth rate or the maximisation of the specific Taxol production rate. Experimental values of nutrient uptake rates such as glucose, fructose and oxygen during the course of the batch culture were used as constraints to obtain different sets of optimised flux distributions for different periods of the batch culture. The computational results indicated that the transhydrogenase reactions were important to balance the need of NADPH for biosynthetic reactions in Taxus metabolism. The variation in biomass composition, inclusion of starch biosynthesis and degradation reactions, and Taxol precursors did not affect the growth of cells significantly. When the secondary cell wall biosynthesis was incorporated in the model, both growth and Taxol production rates were reduced. An increase in in silico Taxol production was observed when methyl jasmonate elicitation was combined with phenylalanine addition. An integrated dynamic model was constructed in order to combine the abilities of Excel- VBA and GAMS to optimise the objective function, handle mathematical kinetic expressions and visualise the outputs. As an example of its application, Taxol concentrations were automatically computed after the optimisation during the time course of the batch culture. These models can be developed further and used in future in order to define some strategies such as media formulation, precursor addition and genetic engineering targets in silico in order to manipulate the metabolism and increase the Taxol yield.

In this work an intersection between optimization methods and animal cell culture modeling is considered. We present optimization based methods for analyzing and building models of cell culture; models that could be used when designing the environment cells are cultivated in, i.e., medium. Since both the medium and cell line considered are complex, designing a good medium is not straightforward. Developing a model of cell metabolism is a step in facilitating medium design. In order to develop a model of the metabolism the methods presented in this work make use of an underlying metabolic reaction network and extracellular measurements. External substrates and products are connected via the relevant elementary flux modes (EFMs). Modeling from EFMs is generally limited to small networks, because the number of EFMs explodes when the underlying network size increases. The aim of this work is to enable modeling with more complex networks by presenting methods that dynamically identify a subset of the EFMs. In papers A and B we consider a model consisting of the EFMs along with the flux over each mode. In paper A we present how such a model can be decided by an optimization technique named column generation. In paper B the robustness of such a model with respect to measurement errors is considered. We show that a robust version of the underlying optimization problem in paper A can be formed and column generation applied to identify EFMs dynamically. In papers C and D a kinetic macroscopic model is considered. In paper C we show how a kinetic macroscopic model can be constructed from the EFMs. This macroscopic model is created by assuming that the flux along each EFM behaves according to Michaelis-Menten type kinetics. This modeling method has the ability to capture cell behavior in varied types of media, however the size of the underlying network is a limitation. In paper D this limitation is countered by developing an approximation algorithm, that can dynamically identify EFMs for a kinetic model.
I denna avhandling betraktar vi korsningen mellan optimeringsmetoder och modellering av djurcellodling.Vi presenterar optimeringsbaserade metoder för att analysera och bygga modeller av cellkulturer. Dessa modeller kan användas vid konstruktionen av den miljö som cellerna ska odlas i, dvs, medium.Eftersom både mediet och cellinjen är komplexa är det inte okomplicerat att utforma ett bra medium. Att utveckla en modell av cellernas ämnesomsättning är ett steg för att underlätta designen av mediet. För att utveckla en modell av metabolismen kommer de metoder som används i detta arbete att utnyttja ett underliggande metaboliskt reaktions\-nätverk och extracellulära mätningar. Externa substrat och produkter är sammankopplade via de relevanta elementära metaboliska vägarna (EFM).Modellering med hjälp av EFM är i allmänhet begränsad till små nätverk eftersom antalet EFM exploderar när de underliggande nätverket ökar i storlek. Målet med detta arbete är att möjliggöra modellering med mer komplexa nätverk genom att presentera metoder som dynamiskt identifierar en delmängd av EFM. I artikel A och B betraktar vi en modell som består av EFM och ett flöde över varje EFM.I artikel A presenterar vi hur en sådan modell kan bestämmas med hjälp av en optimeringsteknik som kallas kolumngenerering.I artikel A undersöker vi hur robust en sådan modell är med avseende till mätfel. Vi visar att en robust version av det underliggande optimeringsproblemet i artikel A kan konstrueras samt att kolumngenerering kan appliceras för att identifiera EFM dynamiskt. Artikel C och D behandlar en kinetisk makroskopisk modell. Vi visar i artikel C hur en sådan modell kan konstrueras från EFM.Denna makroskopiska modell är skapad genom att anta att flödet genom varje EFM beter sig enligt Michaelis-Menten-typ av kinetik. Denna modelleringsmetod har förmågan att fånga cellernas beteende i olika typer av media, men storleken på nätverket är en begränsning.I artikel D hanterar vi denna begränsing genom att utveckla en approximationsalgoritm som identifierar EFM dynamiskt för en kinetisk modell.

QC 20150827

Humans are approximately 99% similar with inter-individual differences caused in part by single-nucleotide polymorphisms (SNPs), which poses a challenge for the effective treatment of disease. Bioinformatics resources can help to store and analyse gene and protein information to address this challenge, however these resources have limitations, so the collation and biocuration of gene and protein information is required. Using the large conductance calcium- and voltage-activated potassium channel, also known as the Big Potassium (BK) channel as an example, due to its ubiquitous expression and widespread varied role in human physiology, this study aimed to prioritise SNPs with the potential to affect the function of the channel. Using a BK channel resource created with bioinformatics tools and published literature, mSlo SNPs H55Q and G57A, located in the S0-S1 linker, were prioritised and selected for lab-based verification. These SNPs flank three cysteine residues proven to modulate channel cellular distribution via palmitoylation, a reversible process shown to increase protein association with the cell membrane. The SNPs alter the predicted palmitoylation status of C56, one of the cysteine residues located in the S0-S1 linker. The cellular distribution of BK channels incorporating the SNPs was assessed using confocal microscopy and revealed that the direction and magnitude of SNP mimetic cell membrane expression was closely related to the C56 predicted palmitoylation score; a 'C56 palmitoylation pattern' was observed. It was shown that exposure to metabolic substrates glucose, palmitate and oleate modulated SNP-mimetic cellular distribution and could invert the 'C56 palmitoylation pattern', indicating that there is interplay between the metabolic status of the cell and the amino-acid composition of the channel via palmitoylation. The creation of a novel BK channel resource in this thesis highlighted the limitations, and inter-dependency of bioinformatics and lab based experimentation, whilst SNP verification experiments solidified the link between S0-S1 cysteine residues and BK cellular distribution. BK channel function is linked with a number of physiological processes; thus, the potential clinical consequences of the SNPs prioritised in this thesis require further research.

Le système immunitaire adaptatif joue un rôle de premier plan dans la défense contre les infections. La réponse humorale, impliquant la production d'anticorps, est un élément important de la réponse immunitaire adaptative. Au cours d'une infection, des cellules B spécifiques du système immunitaire prolifèrent et libèrent de grandes quantités d'anticorps qui se lient sélectivement à la protéine cible (antigène) trouvée sur le pathogène invasif, induisant la destruction du pathogène.Cependant, le système immunitaire ne répond pas toujours suffisamment efficacement pour détruire les agents pathogènes, et les mécanismes de tolérance empêchent la génération d'anticorps contre les protéines humaines - comme les marqueurs de surface cellulaire sur les cellules cancéreuses ou les cytokines impliquées dans des maladies inflammatoires et auto-immunes - qui pourraient être des cibles thérapeutiques importantes. Par conséquent, il existe un grand intérêt pour la recherche et le développement d'anticorps spécifiques qui peuvent être utilisés pour le traitement des patients par immunothérapie. En raison de leur grande affinité et de leur liaison sélective aux antigènes, les anticorps monoclonaux (mAbs) sont apparus comme des agents thérapeutiques puissants. Les anticorps monoclonaux dérivés de cellules B individuelles ont une séquence unique et présentent une affinité de liaison pour un antigène spécifique. Cependant, jusqu'à maintenant, la découverte des mAbs a été limitée par l'absence de méthodes à haut débit pour le criblage direct et à grande échelle de cellules B primaires non immortalisées pour découvrir les rares cellules B qui produisent des anticorps spécifiques d'intérêt clinique. Ceci est maintenant possible avec l'émergence et l'amélioration des méthodes de compartimentation in vitro pour l'encapsulation et le criblage de cellules uniques dans des gouttelettes picolitriques. Dans mon projet de doctorat, je décris le développement d'immunodosages et de dispositifs microfluidiques pour le criblage phénotypique direct de cellules individuelles à partir de populations de cellules B enrichies. Ce développement a permis une analyse détaillée de la réponse immunitaire humorale, avec une résolution à l’échelle de la cellule unique. C’est aussi un élément essentiel d'un pipeline de détection d'anticorps couplant le criblage phénotypique de cellules individuelles au séquençage d'anticorps sur cellules uniques. Il est maintenant possible, pour la première fois, de cribler des millions de cellules B individuelles en fonction de l'activité de liaison des anticorps sécrétés et de récupérer les séquences d'anticorps
The adaptive immune system plays a leading role in defense against infection. The humoral response, involving the production of antibodies, is an important component of the adaptive immune response. During an infection, specific B cells of the immune system proliferate and release large amounts of antibodies which bind selectively to the target protein (antigen) found on the invading pathogen, inducing destruction of the pathogen. However, the immune system does not always respond efficiently enough to destroy pathogens, and tolerance mechanisms prevent the generation of antibodies against human protein - such as cell surface markers on cancer cells or cytokines involved in inflammatory and autoimmune disease - that could be important therapeutic targets. Hence, there is great interest in research and development of specific antibodies that can be used for immunotherapy of patients. Due to their high affinity and selective binding to antigens, monoclonal antibodies (mAbs) have emerged as powerful therapeutic agents. Monoclonal antibodies derived from single B cells have a unique sequence and display binding affinity for a specific antigen. However, until now, the discovery of mAbs has been limited by the lack of high-throughput methods for the direct and large-scale screening of non-immortalized primary B cells to uncover rare B cells which produce the specific antibodies of clinical interest. This is now becoming possible with the emergence and improvement of in vitro compartmentalization methods for single-cell encapsulation and screening in picoliter droplets. In my PhD project, I describe the development of binding immunoassays and microfluidic devices for the direct phenotypic screening of single-cells from enriched B cell populations. This development has enabled detailed analysis of the humoral immune response, with single-cell resolution and is an essential component of an antibody-discovery pipeline coupling single-cell phenotypic screening to single-cell antibody sequencing. It is now possible, for the first time, to screen millions of single B cells based on the binding activity of the secreted antibodies and to recover the antibody sequences

Thesis (MScEng)--Stellenbosch University, 2011.
ENGLISH ABSTRACT: Rapid Single Flux Quantum (RSFQ) cells are key in the design of complex and applicable RSFQ electronic circuits. These cells are low-level circuit elements that are used repeatedly to build larger, applicable RSFQ circuitry. Making these cells simple to layout and manufacture, but reliable for extensive use demands a careful development process for RSFQ cells. Cell functionality is verified through simulations, thereafter the cell is laid out in special software packages. Inductance of on-chip superconductor structures is extracted through careful modelling with numerical field solver software. A cell library has been developed by incorporating existing or published cells after further analysis and optimization, as well as developing new cells. Cells that have been adapted into the library include the Josephson transmission line (JTL), Splitter, Merger, D-Flip Flop (DFF), T-Flip Flop (TFF), NOT, AND, OR and XOR, DC-SFQ and SFQ-DC and PTL Driver and Receivers. New cells include NOR, NAND and XNOR. The cells were designed for the IPHT’s RSFQ1D 1kA/cmª and Hypres’ 4.5kA/cmª processes. The cells in the library have good bias current operating margins obtained through simulations (> ±26%). All cells have all the parameters listed in the thesis including extracted inductance values. In order to have a complete and verified RSFQ cell library, cells have been sent for fabrication at IPHT and Hypres facilities. These cells can now be tested on-chip, in the laboratory, to establish functionality and practical bias current margins. All test signal patterns and bias currents required for testing are defined to allow co-workers or collaborators to test the cells.
AFRIKAANSE OPSOMMING: "Rapid Single Flux Quantum" (RSFQ) selle is van sleutelbelang in die ontwerp van komplekse en toepaslike RSFQ elektroniese stroombane. Hierdie selle is laevlak stroombaanelemente wat herhaaldelik gebruik word om groter RSFQ bane mee te bou. Versigtige ontwikkeling is nodig om hierdie selle eenvoudig vir uitleg en vervaardiging te hou terwyl dit ook betroubaar is vir wye gebruik. Selfunksionaliteit word geverifieer deur middel van simulasies, waarna selle vir vervaardiging uitgelê word in spesiale sagtewarepakette. Induktansie van supergeleierstrukture op vervaardigde skyfies word deur versigtige modellering met behulp van numeriese veldoplossingsagteware onttrek. In hierdie tesis is ’n selbiblioteek ontwerp deur bestaande (gepubliseerde) selle verder te analiseer en optimeer, en deur nuwe selle te ontwerp om die biblioteek volledig te maak. Selle wat aangepas is vir hierdie biblioteek sluit die Josephson-Transmissielyn (JTL), Verdeler, Samevoeger, DWipkring (DFF), T-Wipkring (TFF), NIE, EN, OF en XOF, asook die DC-SFQ en SFQ-DC selle en Passiewe Transmissielyn (PTL) drywers en ontvangers in. Nuwe selle sluit die NOF, NEN en XNOF hekke in. Die selle is ontwerp en uitgelˆe vir beide IPHT se RSFQ1D 1kA/cmª en Hypres se4.5kA/cmª prosesse. Die selle in die biblioteek toon goeie voorspanningstroom-werksmarges, soos verkry deur simulasie (> ±26%). Parameters en berekende induktansies vir alle selle word in die tesis gelys vir naslaandoeleindes. Vir die daarstel van ’n volledige en geverifieerde RSFQ selbiblioteek is selontwerpe vir vervaardiging na IPHT en Hypres gestuur. Aangesien vervaardiging slegs een maal per jaar by IPHT gedoen word, is die skyfies egter nog nie beskikbaar nie. Na vervaardiging kan die skyfies egter getoets word om selfunksionaliteit in die laboratorium te meet. Ten einde hierdie toetsing vir enige medewerker te vergemaklik, word alle toetsparameters soos voorspanningstroom en intreeseinpatrone in die tesis gedefinieer.

In the past five years, Single Molecule Real Time (SMRT) sequencing technology has been found to be a reliable indicator of certain epigenetic modifications in bacterial genomes. The genome of the model organism Saccharomyces cerevisiae has long been thought to be free of DNA level modification, but literature surrounding this subject is conflicting. Additionally, the mitochondria of S. cerevisiae control the transition between three distinct chronological life phases – exponential, postdiauxic, and stationary - as defined by their main metabolic processes. This study attempted to identify base modifications to mtDNA using PacBio sequencing while additionally establishing gene expression changes as a result of altered mitochondrial metabolic capabilities. PacBio results showed intriguing results but statistical analysis proved experimentation with improved protocols were necessary. Multiple genes with unknown or uncharacterized function were also shown to have significant differential expression between metabolic life phases.

pH is a key parameter in the optimization of animal cell processes, and has be linked to specific patterns of consumption and production of extracellular metabolites. However, the effect of extracellular pH on intracellular metabolism has not been fully elucidated. Metabolic flux analysis is a mathematical method that can be used to generate the intracellular flux distributions in cells, e.g. as a function of some environmental parameter. In this work, the overall objective was to develop a culture system and experimental protocol for cultivation of CHO cells, which can be used to generate data for analysis of the relationship between extracellular pH and intracellular fluxes in CHO cells by metabolic flux analysis. First, shake-flask culture of an IgG-producing cell line was performed to select an academic and chemically-defined medium with known composition. This was followed by subsequent adaptation of the cells. It was found that the originally selected medium had to be supplemented with a commercial medium to produce acceptable growth and viability. Shake-flask culture was also performed to evaluate the effect of the biological buffer HEPES on cell growth and viability, and the pH-stability during culture. HEPES-concentrations in the investigated range (7.5-45 mM) did not show an apparent effect on cell growth or viability. The higher concentrations gave slightly better buffering capacity at inoculation, however were not sufficient to keep pH stable during culture. As a result, the idea of using shake flask culture and similar techniques for cultivation of cells at various pH set-points was dismissed. Instead, a culture system and protocol based on a 100 mL Spinner flask with pH-regulation was custom-designed for the project. Features of the final design included continuous monitoring of pH and DO, stable temperature at 37 °C, adjustable agitation rate, as well as the option to incorporate inflow of air, O2 and CO2. In addition, the possibility to disconnect the flask unit to perform medium exchange and sample collection away from the reactor site (i.e. in a laminar flow workbench) was integrated into the design and protocol. The system was demonstrated for pseudo-perfusion culture with the adapted IgG-producing cell line at pH 7.0 during 24 days. Optimized regulation settings were identified. It was shown that the system could support viable cell densities of up to 11 MVC/mL and high viability (> 90 %). During the final phase of culture, stable growth, at specific growth rates of approximately 0.7 Day-1, was achieved. The specific rates of consumption and production of the key metabolites glucose, glutamine, lactate and NH4+, as well as 20 amino acids were analyzed. A majority of the rates were in accordance with CHO cell metabolism. The expected consumption of a majority of the essential amino acids and main carbon sources glucose and glutamine were confirmed, as well as the associated production of by-products lactate and NH4+. The system and protocol developed in this work can be used in future experiments to generate data describing metabolic profiles as a function of various pH-set points. This data may then be used in metabolic flux analysis to further elucidate the metabolism behind pH effects in CHO cells.
pH är en viktig parameter i optimeringen av animalcellsprocesser och har sammankopplats med specifika konsumtions- och produktionsmönster rörande extracellulära metaboliter. Det extracellulära pH-värdets effekt på den intracellulära metabolismen är dock inte fullt klarlagd. Metabolisk flux analys är en matematisk metod som kan användas för att generera intracellulära fluxfördelningar i celler, exempelvis som en funktion av någon yttre parameter. Det övergripande målet i detta arbete var att utveckla ett odlingssystem och experimentellt protokoll för odling av CHO-celler som kan användas för att generera data för metabolisk flux analys där målet är att studera effekten av pH på den intracellulära cellmetabolismen. En IgG-producerande CHO-cellslinje odlades först i skakkolv för att välja ut ett akademiskt kemiskt definierat medium med känd sammansättning. Därefter följde försök att anpassa cellerna till det valda mediet. Det visade sig att ett kommersiellt medium behövde tillsättas för att ge godtagbar tillväxt och viabilitet. Effekten av den biologiska bufferten HEPES på cellernas tillväxt och viabilitet, samt pH-stabiliteten under odling, undersöktes också genom odling i skakkolv. HEPES-koncentrationer i det undersökta intervallet (7.5 – 45 mM) hade ingen större effekt på tillväxt och viabilitet. För de högre koncentrationerna var buffertkapaciteten något bättre precis vid inokulering. Dessa koncentrationer var dock ej tillräckliga för att ge stabilt pH under odlingen. Baserat på dessa resultat övergavs tanken på att använda skakkolvsodling för att odla celler vid olika pH-värden. Ett odlingssystem och ett protokoll baserat på en 100 mL Spinnerflaska med pH-reglering specialdesignades istället för projektet. I det färdiga systemet fanns lösningar för kontinuerlig övervakning av pH och DO, stabil temperatur vid 37 °C, justerbar omrörningshastighet, samt valmöjligheten att flöda in luft, O2 och CO2. Dessutom infördes möjligheten att koppla loss flaskenheten från reglersystemet för byte av medium och provtagning. För att demonstrera systemet genomfördes en odling med den anpassade IgG-producerande cellinjen enligt principen för pseudo-perfusion vid pH 7.0. Odlingen pågick under 24 dagar och optimerade reglerinställningar identifierades. Det visades att systemet kunde understödja cellkoncentrationer upp till 11 miljoner celler per milliliter, samt hög viabilitet (> 90 %). Under den senare delen av odlingen uppnåddes stabil tillväxt, vid specifika tillväxthastigheter omkring 0.7 per dygn. Den specifika konsumtions- och produktionshastigheten för metaboliterna glukos, glutamin, laktat och NH4+, samt 20 aminosyror analyserades. Majoriteten av hastigheterna stämde överens med typisk CHO-cellsmetabolism. Den förväntade konsumtionen av majoriteten av de essentiella aminosyrorna och huvudsakliga kolkällorna glukos och glutamin konfirmerades, såväl som den associerade produktionen av bi-produkterna laktat och NH4+. Odlingssystemet och det experimentella protokollet som utvecklades i detta arbete kan användas i framtida experiment för att generera data som beskriver metaboliska profiler som funktion av extracellulärt pH. Dessa data kan sedan användas i metabolisk flux analys för att dra slutsatser om pH-effekter i CHO-celler.

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

Most of microorganisms are ubiquitously able to live in both planktonic and biofilm states, which can be applied to dissolve the energy and environmental issues (e.g., producing biofuels and purifying waste water), but can also lead to serious public health problems. To better harness microorganisms, plenty of studies have been implemented to investigate the metabolism of planktonic and/or biofilm cells via multi-omics approaches (e.g., transcriptomics and proteomics analysis). However, these approaches are limited to provide the direct description of intracellular metabolism (e.g., metabolic fluxes) of microorganisms. Therefore, in this study, I have applied computational modeling approaches (i.e., 13C assisted pathway and flux analysis, flux balance analysis, and machine learning) to both planktonic and biofilm cells for better understanding intracellular metabolisms and providing valuable biological insights. First, I have summarized recent advances in synergizing 13C assisted pathway and flux analysis and metabolic engineering. Second, I have applied 13C assisted pathway and flux analysis to investigate the intracellular metabolisms of planktonic and biofilm cells. Various biological insights have been elucidated, including the metabolic responses under mixed stresses in the planktonic states, the metabolic rewiring in homogenous and heterologous chemical biosynthesis, key pathways of biofilm cells for electricity generation, and mechanisms behind the electricity generation. Third, I have developed a novel platform (i.e., omFBA) to integrate multi-omics data with flux balance analysis for accurate prediction of biological insights (e.g., key flux ratios) of both planktonic and biofilm cells. Fourth, I have designed a computational tool (i.e., CRISTINES) for the advanced genome editing tool (i.e., CRISPR-dCas9 system) to facilitate the sequence designs of guide RNA for programmable control of metabolic fluxes. Lastly, I have also accomplished several outreaches in metabolic engineering. In summary, during my Ph.D. training, I have systematically applied computational modeling approaches to investigate the microbial metabolisms in both planktonic and biofilm states. The biological findings and computational tools can be utilized to guide the scientists and engineers to derive more productive microorganisms via metabolic engineering and synthetic biology. In the future, I will apply 13C assisted pathway analysis to investigate the metabolism of pathogenic biofilm cells for reducing their antibiotic resistance.
Ph. D.

Les anthocyanes sont une famille de polyphénols très répandus chez les végétaux. Chez la vigne, elles sont responsables de la coloration des baies des cépages rouges, et sont impliquées dans les propriétés organoleptiques des vins. Une nutrition azotée faible induit la production des anthocyanes dans les cellules de la pellicule de raisin des cépages rouges via des mécanismes de régulation qui ne sont pas encore totalement élucidés. Dans ce contexte, nous avons étudié les mécanismes moléculaires impliqués dans la réponse de l’accumulation des anthocyanes pour différents niveaux d’apports azotés. Deux matériels biologiques complémentaires ont été utilisés : des suspensions cellulaires de vigne (lignée GT3) et des plants de Cabernet-Sauvignon, cultivés au vignoble.L’augmentation de la synthèse d’anthocyanes en réponse à la diminution de la nutrition azotée a été confirmée dans les baies et les cellules de vigne en culture. Les analyses transcriptomiques globales (génome complet) et ciblées (qPCR) ont mis en lumière des modifications de l’expression génique, notamment de gènes liés au métabolisme des flavonoïdes, en réponse à la nutrition azotée. L’expression de nombreux gènes structuraux impliqués dans la voie de biosynthèse des anthocyanes est induite par une faible nutrition azotée. La variation de l’apport azoté influence également de façon coordonnée l’expression des gènes régulateurs positifs (facteurs de transcription de type MYB) et négatifs (protéine de type Lateral organ Boundary Domain (LBD)) des gènes de la biosynthèse des flavonoïdes chez la Vigne. L’expression de gènes liés à la production d’énergie (NADH, NADPH), est également affectée.En parallèle, une approche intégrative a été développée sur les suspensions cellulaires, en combinant des mesures d’activités enzymatiques, des dosages de métabolites primaires et secondaires, avec un modèle de balance de flux (Flux Balance Analysis, FBA). Les cartes de flux obtenues prédisent que la diminution de l’apport azoté entraîne une augmentation des flux métaboliques dans la voie du shikimate et des phénylpropanoïdes ; ainsi qu’une répression de la majorité des flux dans les différentes voies du métabolisme primaire, à l’exception de la voie des pentoses phosphates, dont le flux est maintenu, et de la voie de synthèse de l’amidon qui est accrue. Les résultats obtenus plaident en faveur d’un lien fort entre synthèse des anthocyanes et statut énergétique (ATP, NADPH) des cellules vigne
Anthocyanins are polyphenol compounds very abundant in most of the plants. In grapevine, they give color to red berries and they improve red wine quality and increase the organoleptic properties of the wine. Low nitrogen supply stimulates anthocyanin production in berry skin cells of red grape varieties through regulation mechanisms that are far from being fully understood. In this context, we worked on the molecular mechanisms involved in anthocyanin biosynthesis response to nitrogen supply. Two complementary biological materials were used: grapevine cell suspensions (GT3 line) that originate from a teinturier cultivar and produce anthocyanins under normal conditions; and red grape berries of cv. Cabernet-Sauvignon cultivated in a commercial vineyard. Increases of anthocyanins synthesis in response to low nitrogen levels were confirmed in the field-grown berries and the cells suspensions. Both comparative global (microarrays) and targeted (qPCR) transcriptomic analysis showed different regulations on the expression of the genes involved in the secondary (especially the anthocyanin) and nitrogen metabolisms. The expression of most structural genes of the anthocyanin biosynthesis pathway was induced by a low nitrogen supply. Nitrogen controls also the expression of the positive (MYB transcription factors) and negative (Lateral organ Boundary Domain family protein LBD39) regulatory genes of the flavonoid pathway in grapevine. Furthermore, some genes improved in energy production (ATP, NADPH) were affected. In parallel, an integrative approach combining enzymatic activities and primary and secondary metabolites measurements with developing a Flux Balance Analysis (FBA) modeling approach was used on cells suspensions GT3. The flux maps deciphered that low nitrogen increases metabolic fluxes in shikimate and phenylpropanoid pathways and represses the majority metabolic fluxes in different pathways of primary metabolism. The two exceptions included the pentose phosphate pathway, which the flux metabolism was maintained, and the starch synthesis pathway, which was enhanced. The results obtained showed a strong link between anthocyanin synthesis and energy status (ATP, NADPH) in the berry cell suspensions

Aujourd’hui, la biologie des systèmes est confrontée aux défis de l’analyse de l’énorme quantité de données biologiques et à la taille des réseaux métaboliques pour des analyses à grande échelle. Bien que plusieurs méthodes aient été développées au cours des dernières années pour résoudre ce problème, ce sujet reste un domaine de recherche en plein essor. Cette thèse se concentre sur l’analyse des propriétés structurales, le calcul des modes élémentaires de flux et la détermination d’ensembles de coupe minimales du graphe formé par ces réseaux. Dans notre recherche, nous avons collaboré avec des biologistes pour reconstruire un réseau métabolique de taille moyenne du métabolisme cellulaire de la plante, environ 90 noeuds et 150 arêtes. En premier lieu, nous avons fait l’analyse des propriétés structurelles du réseau dans le but de trouver son organisation. Les réactions points centraux de ce réseau trouvés dans cette étape n’expliquent pas clairement la structure du réseau. Les mesures classiques de propriétés des graphes ne donnent pas plus d’informations utiles. En deuxième lieu, nous avons calculé les modes élémentaires de flux qui permettent de trouver les chemins uniques et minimaux dans un réseau métabolique, cette méthode donne un grand nombre de solutions, autour des centaines de milliers de voies métaboliques possibles qu’il est difficile de gérer manuellement. Enfin, les coupes minimales de graphe, ont été utilisés pour énumérer tous les ensembles minimaux et uniques des réactions qui stoppent les voies possibles trouvées à la précédente étape. Le nombre de coupes minimales a une tendance à ne pas croître exponentiellement avec la taille du réseau a contrario des modes élémentaires de flux. Nous avons combiné l’analyse de ces modes et les ensembles de coupe pour améliorer l’analyse du réseau. Les résultats montrent l’importance d’ensembles de coupe pour la recherche de la structure hiérarchique du réseau à travers modes de flux élémentaires. Nous avons étudié un cas particulier : qu’arrive-t-il si on stoppe l’entrée de glucose ? En utilisant les coupes minimales de taille deux, huit réactions ont toujours été trouvés dans les modes élémentaires qui permettent la production des différents sucres et métabolites d’intérêt au cas où le glucose est arrêté. Ces huit réactions jouent le rôle du squelette / coeur de notre réseau. En élargissant notre analyse aux coupes minimales de taille 3, nous avons identifié cinq réactions comme point de branchement entre différent modes. Ces 13 réactions créent une classification hiérarchique des modes de flux élémentaires fixés et nous ont permis de réduire considérablement le nombre de cas à étudier (approximativement divisé par 10) dans l’analyse des chemins réalisables dans le réseau métabolique. La combinaison de ces deux outils nous a permis d’approcher plus efficacement l’étude de la production des différents métabolites d’intérêt par la cellule de plante hétérotrophique
Nowadays, systems biology are facing the challenges of analysing the huge amount of biological data and large-scale metabolic networks. Although several methods have been developed in recent years to solve this problem, it is existing hardness in studying these data and interpreting the obtained results comprehensively. This thesis focuses on analysis of structural properties, computation of elementary flux modes and determination of minimal cut sets of the heterotrophic plant cellmetabolic network. In our research, we have collaborated with biologists to reconstructa mid-size metabolic network of this heterotrophic plant cell. This network contains about 90 nodes and 150 edges. First step, we have done the analysis of structural properties by using graph theory measures, with the aim of finding its owned organisation. The central points orhub reactions found in this step do not explain clearly the network structure. The small-world or scale-free attributes have been investigated, but they do not give more useful information. In the second step, one of the promising analysis methods, named elementary flux modes, givesa large number of solutions, around hundreds of thousands of feasible metabolic pathways that is difficult to handle them manually. In the third step, minimal cut sets computation, a dual approach of elementary flux modes, has been used to enumerate all minimal and unique sets of reactions stopping the feasible pathways found in the previous step. The number of minimal cut sets has a decreasing trend in large-scale networks in the case of growing the network size. We have also combined elementary flux modes analysis and minimal cut sets computation to find the relationship among the two sets of results. The findings reveal the importance of minimal cut sets in use of seeking the hierarchical structure of this network through elementary flux modes. We have set up the circumstance that what will be happened if glucose entry is absent. Bi analysis of small minimal cut sets we have been able to found set of reactions which has to be present to produce the different sugars or metabolites of interest in absence of glucose entry. Minimal cut sets of size 2 have been used to identify 8 reactions which play the role of the skeleton/core of our network. In addition to these first results, by using minimal cut sets of size 3, we have pointed out five reactions as the starting point of creating a new branch in creationof feasible pathways. These 13 reactions create a hierarchical classification of elementary flux modes set. It helps us understanding more clearly the production of metabolites of interest inside the plant cell metabolism

Bacterial proliferation has been studied for more than 100 years, but our knowledge of the mechanisms that control its dynamical aspects is still very limited. In this Thesis we have studied, at both singlecell and population levels, different aspects of the main regulators of bacterial proliferation, namely the cell cycle, biomass production and membrane stability. Our goal has been to shed light on how perturbation of these regulators affects their dynamical responses in a variety of situations. Specifically, we show that the periodic doubling of genes through the bacterial cell cycle partially entrains a genetic oscillator, but that significant coupling only arises when the oscillator is fed back to cell cycle. We have also studied perturbations in the ability of cells to produce biomass, for instance through antibiotics that affect ribosomal function. Our results suggest that survival in the presence of these antibiotics is determined by the ability of the cells to incorporate magnesium ions, which can be captured by membrane potential changes. Finally, we show that interplay between different bacterial species with diverse sensitivities to membrane-targeting antibiotics can have an unexpected outcome in co-culture, which can be explained in a simple manner by the sharing of the antibiotic between the two species.
La proliferació bacteriana ha estat estudiada durant més de 100 anys, però el nostre coneixement dels mecanismes que controlen els aspectes dinàmics d’aquesta encara són molt limitats. En aquesta Tesi hem estudiat, tant en cèl.lules individuals com a nivell poblacional, diferents aspectes dels principals reguladors de la proliferació cel.lular, concretament el cicle cel.lular, la producció de biomassa i la estabilitat de la membrana. El nostre objectiu ha estat ajudar a explicar com pertorbacions en aquests reguladors afecten la dinàmica de les seves respostes en una varietat de situacions. Específicament, mostrem que el doblament periòdic de gens a través del cicle cel.lular bacterià entrena parcialment un oscil.lador genètic, però aquest acoblament només és significatiu quan l’oscil.lador retorna la resposta al cicle cel.lular. Tamée hem estudiat pertorbacions en l’habilitat de les cèl.lules de produir biomassa, per exemple a través d’antibiòtics que afecten la funció ribosomal. Els nostres resultats suggereixen que la supervivència sota l’efecte d’aquests antibiòtics ve determinada per l’habilitat de les cèl.lules d’incorporar ions de magnesi, la qual pot ser capturada a través de canvis en el potencial de membrana. Finalment, mostrem que les interaccions entre diferents espècies bacterianes amb diverses sensitivitats a antibiòtics que afecten la membrana cel.lular poden donar lloc a resultats inesperats quan es troben en co-cultiu, els quals poden ser explicats de forma senzilla a partir del compartiment de l’antibiòtic entre les dues espècies.

Les systèmes microbiologiques naturels (colonne d’eau), semi-naturels (station d’épuration), mais surtout industriels ou de laboratoire (bioréacteurs) sont communément représentés par des modèles mathématiques destinés à l’étude, à la compréhension des phénomènes ou au contrôle des processus (de production, par exemple).

Dans l’énorme majorité des cas, lorsque les cellules (procaryotes ou eucaryotes) mises en jeu dans ces systèmes sont en suspension, le formalisme de ces modèles non structurés traite le système comme s’il était homogène. Or, en toute rigueur, il est clair que cette approche n’est qu’une approximation et que nous avons à faire à des phénomènes hétérogènes, formés de plusieurs phases (solide, liquide, gazeuse) intimement mélangées. Nous désignons ces systèmes comme « polyphasiques dispersés » (SPD). Ce sont des systèmes thermodynami-quement instables, (presque) toujours ouverts.

La démarche que nous avons entreprise consiste à examiner si le fait de considérer des systèmes dits « homogènes » comme des systèmes hétérogènes (ce qu’ils sont en réalité) apporte, malgré une complication du traitement mathématique, un complément d’information significatif et pertinent.

La démarche s’est faite en deux temps :

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Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

This dissertation aims to identify the functional characteristics and genetic factors present within breast cancers that contribute to intratumoural heterogeneity and therapy resistance. The study utilises breast cancer cell line model systems to address epithelial-mesenchymal plasticity (EMP) at the cellular and functional level and underpins its role in cancer biology and chemoresistance. This research also interrogates the EMP programme in single cell-generated clones and through shRNA mediated functional drug screening assay identifies inhibitors that provides significant synergistic drug combinations. A comprehensive review of the drugs that can clinically target EMP was also consolidated.

Cette thèse fait référence au bruit de choc généré par des jets sousdétendus simple ou en configuration co-axial. Le bruit de choc est généré par l'interaction entre les structures turbulentes de la couche de cisaillement et le réseaux de cellules de choc développé dans le cône potentiel du jet. Afin d'étudier le bruit choc, simulations à grandes échelles adaptés pour l'aéro-acoustique sont effectué avec des schémas d'ordre élevé qui permet une approche nondissipative et non-dispersive. Les résultats sont analysés et comparés avec des résultats expérimentaux. Notamment, une filtrage hydrodynamique et acoustique est réalisé dans le champ proche pour analyser les modes azimutaux acoustiques et hydrodynamiques. En outre, un analyse basé sur la transformé en ondelettes est mis en oeuvre pour identifier les caractéristiques acoustiques et hydrodynamiques importants des jets supersoniques
This thesis deals with the shock-cell noise generated by under-expanded supersonic jets in single- and dualstream configurations. Shock-cell noise is generated by the interaction between the turbulent structures of the shear-layer and the shock-cell system developed in the potential core of the jet. In order to study shock-cell noise, large eddy simulations adapted to aeroacoustics are carried out using high-order compact schemes that allow for a non-dissipative nondispersive approach. The results are analyzed and compared to experimental results. In particular, an acoustic-hydrodynamic filtering is carried out in the near field in order to analyze the acoustic and hydrodynamic azimuthal modes. Moreover, a wavelet-based analysis is implemented in order to identify the relevant acoustic and hydrodynamic features of the supersonic jets

The proven ability to ferment Saccharomyces cerevisiae on a large scale presents an attractive target for producing chemicals and fuels from sustainable sources. Efficient and predominant carbon flux through to ethanol is a significant engineering issue in the development of this yeast as a multi-product cell chassis used in biorefineries. In order to evaluate diversion of carbon flux away from ethanol, combinatorial deletions were investigated in genes encoding the six isozymes of alcohol dehydrogenase (ADH), which catalyse the terminal step in ethanol production. The scarless, dominant and counter- selectable amdSYM gene deletion method was optimised for generation of a combinatorial ADH knockout library in an industrially relevant strain of S. cerevisiae. Current understanding of the individual ADH genes fails to fully evaluate genotype-by-genotype and genotype-by-environment interactions: rather, further research of such a complex biological process requires a multivariate mathematical modelling approach. Application of such an approach using the Design of Experiments (DoE) methodology is appraised here as essential for detailed empirical evaluation of complex systems. DoE provided empirical evidence that in S. cerevisiae: i) the ADH2 gene is not associated with producing ethanol under anaerobic culture conditions in combination with 25 g l-1 glucose substrate concentrations; ii) ADH4 is associated with increased ethanol production when the cell is confronted with a zinc-limited [1 μM] environment; and iii) ADH5 is linked with the production of ethanol, predominantly at pH 4.5. A successful metabolic engineering strategy is detailed which increases the product portfolio of S. cerevisiae, currently used for large-scale production of bioethanol. Heterologous expression of the cytochrome P450 fatty acid peroxygenase from Jeotgalicoccus sp., OleTJE, fused to the RhFRED reductase from Rhodococcus sp. NCIMB 978 converted free fatty acid precursors to C13, C15 and C17 alkenes (3.81 ng μl-1 total alkene concentration).

abstract: Within the last decade there has been remarkable interest in single-cell metabolic analysis as a key technology for understanding cellular heterogeneity, disease initiation, progression, and drug resistance. Technologies have been developed for oxygen consumption rate (OCR) measurements using various configurations of microfluidic devices. The technical challenges of current approaches include: (1) deposition of multiple sensors for multi-parameter metabolic measurements, e.g. oxygen, pH, etc.; (2) tedious and labor-intensive microwell array fabrication processes; (3) low yield of hermetic sealing between two rigid fused silica parts, even with a compliance layer of PDMS or Parylene-C. In this thesis, several improved microfabrication technologies are developed and demonstrated for analyzing multiple metabolic parameters from single cells, including (1) a modified "lid-on-top" configuration with a multiple sensor trapping (MST) lid which spatially confines multiple sensors to micro-pockets enclosed by lips for hermetic sealing of wells; (2) a multiple step photo-polymerization method for patterning three optical sensors (oxygen, pH and reference) on fused silica and on a polyethylene terephthalate (PET) surface; (3) a photo-polymerization method for patterning tri-color (oxygen, pH and reference) optical sensors on both fused silica and on the PET surface; (4) improved KMPR/SU-8 microfabrication protocols for fabricating microwell arrays that can withstand cell culture conditions. Implementation of these improved microfabrication methods should address the aforementioned challenges and provide a high throughput and multi-parameter single cell metabolic analysis platform.
Dissertation/Thesis
M.S. Electrical Engineering 2014

碩士
國立中正大學
化學工程研究所
107
Compared with normal cells, cancer cells have different metabolisms, and different cancers are clearly different from each other. Therefore, it is very important to establish a tissue-specific model to analyze the metabolism and subsequent research of cancer.In this study, we use the model optimization algorithm, Cost Optimization Reaction Dependency Assessment (CORDA) to construct the tissue-specific models. Base on model of human metabolic network ( Recon2.2 ) and the data of expression for protein from Human Protein Atlas (HPA). Use the above data, we can reconstruct genome-scale metabolic model of 19 tissues, and we use the matrix components of RPMI-1640 and DMEM as the standard for nutrient intake. Then, using Flux Variability Analysis (FVA) to calculate the maximum and minimum fluxes of the reactions in the cancer and normal cells as well as the flux of the compounds. We can discuss the similarities and differences in metabolic reprogramming in different cancers. This is quite helpful for searching for oncogenes, identifying drug targets and biomarkers.

Tese de mestrado. Biologia (Bioinformática e Biologia Computacional). Universidade de Lisboa, Faculdade de Ciências, 2012
The model organisms Caenorhabditis elegans and E. coli form one of the simplest gut microbe host interaction models. Interventions in the microbe that increase the host longevity including inhibition of folate synthesis have been reported previously. To find novel single gene knockouts with an effect on lifespan, a screen of the Keio collection of E. coli was undertaken, and some of the genes found are directly involved in metabolism. The next step in those specific cases is to understand how these mutations perturb metabolism systematically, so that hypotheses can be generated. For that, I employed dynamic Flux Balance Analysis (dFBA), a constraint-based modeling technique capable of simulating the dynamics of metabolism in a batch culture and making predictions about changes in intracellular flux distribution. Since the specificities of the C. elegans lifespan experiments demand us to culture microbes in conditions differing from most of the published literature on E. coli physiology, novel data must be acquired to characterize and make dFBA simulations as realistic as possible. To do this exchange fluxes were measured using quantitative H NMR Time-Resolved Metabolic Footprinting. Furthermore, I also investigate the combination of TReF and dFBA as a tool in microbial metabolism studies. These approaches were tested by comparing wild type E. coli with one of the knockout strains found, ΔmetL, a knockout of the metL gene which encodes a byfunctional enzyme involved in aspartate and threonine metabolism. I found that the strain exhibits a slower growth rate than the wild type. Model simulation results revealed that reduced homoserine and methionine synthesis, as well as impaired sulfur and folate metabolism are the main effects of this knockout and the reasons for the growth deficiency. These results indicate that there are common mechanisms of the lifespan extension between ΔmetL and inhibition of folate biosynthesis and that the flux balance analysis/metabolic footprinting approach can help us understand the nature of these mechanisms.
Os organismos modelo Caenorhabditis elegans e E. coli formam um dos modelos mais simples de interacções entre micróbio do tracto digestivo e hospedeiro. Intervenções no micróbio capazes de aumentar a longevidade do hospedeiro, incluindo inibição de síntese de folatos, foram reportadas previamente. Para encontrar novas delecções génicas do micróbio capazes de aumentar a longevidade do hospedeiro, a colecção Keio de deleções génicas de E. coli foi rastreada. Alguns dos genes encontrados participam em processos metabólicos, e nesses casos, o próximpo passo é perceber como as deleções perturbam o metabolismo sistémicamente, para gerar hipóteses. Para isso, utilizo dynamic Flux Balance Analysis (dFBA), uma técnica de modelação metabólica capaz de fazer previsões sobre alterações na distribuição intracelular de fluxos. As especificidades das experiências de tempo de vida em C.elegans obrigam-nos a trabalhar em condições diferentes das usadas na maioria da literatura publicada em fisiologia de E. coli, e para dar o máximo realismo às simulações de dFBA novos dados foram adquiridos, utilizando H NMR Time-Resolved Metabolic Footprinting para medir fluxos de troca de metabolitos entre microorganismo e meio de cultura. A combinação de TReF e dFBA como ferramenta de estudo do metabolism microbiano é também investigada. Estas abordagens foram testadas ao comparar E. coli wild-type com uma das estirpes encontradas no rastreio, ΔmetL, knockout do gene metL, que codifica um enzima bifunctional participante no metabolismo de aspartato e treonina, e que exibe uma taxa de crescimento reduzida comparativamente ao wild-type. Os resultados das simulações revelaram que os principais efeitos da deleção deste gene, e as razões para a menor taxa de crescimento observada, são a produção reduzida de homoserina e metionina e os efeitos que provoca no metabolismo de folatos e enxofre. Estes resultados indicam que há mecanismos comuns na extensão da longevidade causada por esta deleção e inibição de síntese de folatos, e que a combinação metabolic footprinting/flux balance analysis pode ajudar-nos a compreender a natureza desses mecanismos.

abstract: In the past decades, single-cell metabolic analysis has been playing a key role in understanding cellular heterogeneity, disease initiation, progression, and drug resistance. Therefore, it is critical to develop technologies for individual cellular metabolic analysis using various configurations of microfluidic devices. Compared to bulk-cell analysis which is widely used by reporting an averaged measurement, single-cell analysis is able to present the individual cellular responses to the external stimuli. Particularly, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are two key parameters to monitor heterogeneous metabolic profiles of cancer cells. To achieve multi-parameter metabolic measurements on single cells, several technical challenges need to be overcome: (1) low adhesion of soft materials micro-fabricated on glass surface for multiple-sensor deposition and single-cell immobilization, e.g. SU-8, KMPR, etc.; (2) high risk of using external mechanical forces to create hermetic seals between two rigid fused silica parts, even with compliance layers; (3) how to accomplish high-throughput for single-cell trapping, metabolic profiling and drug screening; (4) high process cost of micromachining on glass substrate and incapability of mass production. In this dissertation, the development of microfabrication technologies is demonstrated to design reliable configurations for analyzing multiple metabolic parameters from single cells, including (1) improved KMPR/SU-8 microfabrication protocols for fabricating microwell arrays that can be integrated and sealed to 3 × 3 tri-color sensor arrays for OCR and ECAR measurements; (2) design and characterization of a microfluidic device enabling rapid single-cell trapping and hermetic sealing single cells and tri-color sensors within 10 × 10 hermetically sealed microchamber arrays; (3) exhibition of a low-cost microfluidic device based on plastics for single-cell metabolic multi-parameter profiling. Implementation of these improved microfabrication methods should address the aforementioned challenges and provide a high throughput and multi-parameter single cell metabolic analysis platform.
Dissertation/Thesis
Doctoral Dissertation Electrical Engineering 2017

The large-scale production of monoclonal antibodies (MAb) by mammalian cells in batch and fed-batch culture systems is limited by the unwanted decline in cell viability and reduced productivity that may result from changes in culture conditions. Therefore, it becomes imperative to gain an in-depth knowledge of the factors affecting cell growth and cell viability that in turn determine the antibody production. An attempt has been made to obtain an overall model that predicts the behaviour of both batch and fed-batch systems as a function of the extra-cellular nutrient/metabolite concentrations. Such model formulation will aid in identifying and eventually controlling the dominant factors in play to optimize monoclonal antibody (MAb) production in the future. Murine hybridoma 130-8F producing anti-F-glycoprotein monoclonal antibody was grown in D-MEM medium (Gibco 12100) with 2% FBS. A systematic approach based on Metabolic Flux Analysis (MFA) was applied for the calculation of intracellular fluxes for metabolites from available extracellular concentration values. Based on the set of identified significant fluxes (from MFA), the original metabolic network was reduced to a set of significant reactions. The reactions in the reduced metabolic network were then combined to yield a set of macro-reactions obeying Monod kinetics. Half saturation constants were fixed empirically to avoid computational difficulties that parameter estimation for an over-parameterized system of equations would cause. Using Quadratic Programming, the proposed Dynamic Model was calibrated and model prediction was carried out individually for batch and fed-batch runs. Flux distribution for batch and fedbatch modes were compared to determine whether the same model structure could be applied to both the feeding profiles. Correlation analysis was performed to formulate a Biomass Model for predicting cell concentration and viability as a function of the extracellular metabolite concentrations in batch and fed-batch experiments. Quadratic Programming was applied once again for estimation of growth and death coefficients in the equations for viable and dead cell predictions. The prediction accuracy of these model equations was tested by using experimental data from additional runs. Further, the Dynamic Model was integrated with the Biomass Model to get an Integrated Model capable of predicting concentration values for substrates, extracellular metabolites, and viable and dead cell concentration by utilizing only starting concentrations as input. It was found that even though the set of significant fluxes was the same for batch and fedbatch operations, the order of these fluxes was different between the two systems. There was a gradual metabolic shift in the fed-batch system with time indicating that under conditions of nutrient limitation, the available energy is channeled towards maintenance rather than growth. Also, available literature with regard to cell kinetics during fed-batch operation suggests that under nutrient limited conditions, the cells move from a viable, non-apoptotic state to a viable apoptotic state. This is believed to lead to variations in antibody production rates and might explain inaccurate predictions for MAb obtained from the model proposed in the current work. As a result more detailed analysis of the system and in particular, the switch from non-apoptotic to apoptotic state is required. As a continuation of efforts to study the system in-depth, fluorescence imaging is currently being applied as a tool to capture the changes in cell morphology along the course of experimental batch and fed-batch runs. These experiments maybe able to elucidate the transition from non-apoptotic to apoptotic cells and this information maybe used in the future to improve the accuracy of the existing mathematical model.

abstract: Cell-cell interactions in a microenvironment under stress conditions play a critical role in pathogenesis and pre-malignant progression. Hypoxia is a central factor in carcinogenesis, which induces selective pressure in this process. Understanding the role of intercellular communications and cellular adaptation to hypoxia can help discover new cancer biosignatures and more effective diagnostic and therapeutic strategies. This dissertation presents a study on transcriptomic and metabolic profiling of pre-malignant progression of Barrett's esophagus. It encompasses two methodology developments and experimental findings of two related studies. To integrate phenotype and genotype measurements, a minimally invasive method was developed for selectively retrieving single adherent cells from cell cultures. Selected single cells can be harvested by a combination of mechanical force and biochemical treatment after phenotype measurements and used for end-point assays. Furthermore, a method was developed for analyzing expression levels of ten genes in individual mammalian cells with high sensitivity and reproducibility without the need of pre-amplifying cDNA. It is inexpensive and compatible with most of commercially available RT-qPCR systems, which warrants a wide applicability of the method to gene expression analysis in single cells. In the first study, the effect of intercellular interactions was investigated between normal esophageal epithelial and dysplastic Barrett's esophagus cells on gene expression levels and cellular functions. As a result, gene expression levels in dysplastic cells were found to be affected to a significantly larger extent than in the normal esophageal epithelial cells. These differentially expressed genes are enriched in cellular movement, TGFβ and EGF signaling networks. Heterotypic interactions between normal and dysplastic cells can change cellular motility and inhibit proliferation in both normal and dysplastic cells. In the second study, alterations in gene transcription levels and metabolic phenotypes between hypoxia-adapted cells and age-matched normoxic controls representing four different stages of pre-malignant progression in Barrett's esophagus were investigated. Through differential gene expression analysis and mitochondrial membrane potential measurements, evidence of clonal evolution induced by hypoxia selection pressure in metaplastic and high-grade dysplastic cells was found. These discoveries on cell-cell interactions and hypoxia adaptations provide a deeper insight into the dynamic evolutionary process in pre-malignant progression of Barrett's esophagus.
Dissertation/Thesis
Ph.D. Biological Design 2014

Mammalian cell culture has gained importance in biotechnology for the development of therapeutic and diagnostic agents. Among them, Chinese hamster ovary (CHO) cells are regarded as the mammalian cell "workhorse". The use of CHO cell line for the production of recombinant proteins used in human therapy has reached a level of industrial production. However, a major problem encountered in in vitro cultures is cell death via apoptosis. Since apoptosis leads to the loss of viability of mammalian cells in vitro, especially in serum-free media. This is important and necessary to prevent the activation of apoptosis cascade and increase their cell viability and enhance their cellular robustness. The overall goal of this study is to improve our understanding of the cellular and physiological determinants of apoptosis and its relationship with other cellular functions. Apoptosis is a result of a very complex network of signaling pathways triggered from both inside and outside of the cell and a highly regulated pathway by both pro-apoptotic and anti-apoptotic proteins that promote cell survival or cell death. Although many causes of apoptotic process in mammalian cell cultures had been researched in the past and have been discussed in recent years, a lot need to be explored. In order to bring novel strategies to understand apoptosis in mammalian cell cultures, our study was not only focused on the apoptotic pathway but also expand to metabolic network to set up a link between cell growth and apoptosis. In our project, we applied systems biology methods in a mammalian cell line (CHO TF 70R), to understand the relationship between cellular metabolism and apoptosis in a typical serum free culture medium. After establishing the basic culture platform, the effects of culture conditions on initiating apoptosis will be evaluated. Healthy and apoptotic cell samples were identified and isolated using Fluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS), respectively. A comprehensive study of CHO cellular metabolism was made using a metabolic flux network to compare and analyze by metabolic flux analysis (MFA) to get more information on cell metabolism and apoptotic behavior. Furthermore, 2-NBDG combined with Annexin V-PE was also successfully applied to estimate the glucose uptake rate in real early apoptotic cells. In summary, we used the integration of the data generated by MFA to understand apoptotic behavior and establish a correlation between cell regulation and apoptosis. It will help us to identify the changes during the onset of apoptosis process will be studied by using proteomics tools to analyze the protein up-regulation or down-regulation in different cell status in the future.

Tese no âmbito do doutoramento em Biociências, área de Bioquímica e apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia.
Há cada vez mais evidências sobre alterações metabólicas significativas ao longo do ciclo de divisão celular (CDC) em células eucarióticas. No entanto, devido a limitações técnicas, não há informação quantitativa sobre a distribuição dos fluxos metabólicos (DFM) nas fases distintas do CDC. Nomeadamente, os métodos de sincronização do CDC perturbam o metabolismo sendo propensos a artefactos e os métodos de separação de células têm baixa eficiência e uma capacidade limitada para separar adequadamente as células de acordo com o CDC. Procurando ultrapassar estas limitações, idealizámos uma metodologia para marcar e seguir o perfil de distribuição de fluxos metabólico (DFM) de células eucarióticas numa fase específica do CDC, contornando a necessidade de sincronização ou de separação de células. A metodologia idealizada assenta no uso de marcadores isotópicos para seguir o metabolismo intermediário partindo da abundância dos isótopos de monómeros constituintes dos biopolímeros que são polimerizados apenas durante as fases-alvo do CDC. Como estudo preliminar para validar o conceito, analisámos a abundância de isótopos de desoxinucleósidos e nucleósidos a partir de DNA nuclear e RNA citoplasmático, respectivamente, de Saccharomyces cerevisiae cultivada com glicose marcada com 13C afim de determinar o metabolismo na fase S e fora da fase S, respectivamente, do CDC. Neste sentido, primeiro implementámos uma abordagem baseada em GC-MS para elucidar isotopómeros posicionais dos desoxinucleósidos e nucleósidos de DNA e RNA, inspeccionando os espectros de fragmentação de seus derivados de trimetilsilil. Identificámos a porção do ião molecular que constitui os respectivos fragmentos, focando particularmente nos átomos de carbono do esqueleto molecular. Os nucleósidos fragmentados ao nível da ligação N-glicosídica geram nucleobases e / ou iões de fragmentos de ribose ou desoxirribose e seus fragmentos. Também se observaram fragmentos de nucleósidos compostos pela nucleobase e alguns carbonos do anel de ribose. No total, atribuímos inequivocamente 31 fragmentos. A fim de avaliar a viabilidade da determinação da DFM em estudo a partir de informações obtidas a partir do método GC-MS anteriormente descrito e optimizar as condições experimentais, desenvolvemos um modelo computacional do metabolismo intermediário de S. cerevisiae; é um modelo desenvolvido a partir de levantamento genómico e adequado à investigação da DFM ao longo do CDC. Conceptualizámos duas subpopulações de células – células em fase S e fora da fase S do CDC. O modelo é viável e está terminado, pronto para ser usado para uma análise de sensibilidade meticulosa tendo como fim o desenho de experiências e a respectiva análise dos fluxos metabólicos por marcação com 13C (13C-MFA). S. cerevisae foi cultivada em cultura contínua em meio enriquecido com [1,2-13C] glicose. Os desoxinucleósidos e nucleósidos de DNA e RNA, respectivamente, foram isolados separadamente e a distribuição dos isótopos de massa (DIM) dos seus fragmentos foi medida por GC-MS. Uma análise qualitativa dessa DIM mostrou que: i) a porção de ribose dos nucleosídeos pirimidínicos foi biossintetizada via ramo oxidativo da via das pentoses juntamente com vai-e-vem no ramo não oxidativo; ii) desoxirribose de desoxinucleósidos pirimidínicos foram biossintetizados via glicólise e ramo não oxidativo da via das pentoses; iii) as nucleobases das desoxipirinas foram biossintetizadas via através de um fluxo concertado de vai-e-vem entre a glicólise e o ramo não oxidativo da via das pentoses seguindo-se fluxo pelo ramo oxidativo da via da pentoses; iv) o DIM da nucleobases da citidina, desoxicitidina e timidina revelam atividade do ramo oxidativo da via das pentoses, carboxilase do piruvate e ciclo dos ácidos tricarboxílicos. Esta evidência de multiplicidade de vias metabólicas contribuintes para a biosíntese de nucleobases da citidina, desoxicitidina e timidina pode ser resultante de um tempo de meia vida mais longo dos reservatórios de aspartato. A atividade combinada da carboxilase do piruvato e do ciclo dos ácidos tricarboxílicos pode dever-se à necessidade de satisfazer o recrutamento simultâneo de biossíntese de aminoácidos e ácidos gordos a partir dos intermediários do ciclo dos ácidos tricarboxílicos, exigindo, assim, um ciclo dos ácidos tricarboxílicos activo e o reabastecimento dos seus respectivo reservatórios. Os isotopómeros 13C dos monómeros do DNA diferem dos do RNA, indicando que uma DFM heterogénea ao longo do CDC.
There is increasing evidence of extensive metabolic changes over the division cycle of eukaryotic cells. However, quantitative information about how flux redistributes in distinct phases of this cycle is lacking, due to technical difficulties. Namely, cell division cycle (CDC) synchronization methods disrupt metabolism, and are thus artifact-prone, and cell sorting methods have low throughput and a limited ability to adequately separate cells by CDC. Seeking to bypass these shortcomings, we devised a methodology to profile the metabolic flux distribution (MFD) of eukaryotic cells in a specific phase of CDC without requiring CDC synchronization or cell sorting. The general principle consists in using isotopic tracers to back trace intermediary metabolism from the isotopomer abundances of building-blocks of biopolymers that are polymerized only during the target phases of the CDC. As a proof of principle, we analyzed the isotopomer abundances of building-blocks from nuclear DNA and cytoplasmic RNA of Saccharomyces cerevisiae grown on 13Clabeled glucose to profile the metabolism in S phase and non-S-phase (respectively) of the CDC. Towards this goal, we first implemented a Gas Chromatography – Mass Spectrometry (GC-MS) based approach to elucidate positional isotopomers of nucleosides from RNA and DNA by screening the fragmentation spectra of their trimethylsilyl derivatives. We identified the molecular ion moieties retained in the respective fragment ions, focusing particularly on the carbon backbone. Nucleosides fragmented at the N-glycosidic bond provide nucleobase and/or ribose or deoxyribose fragment ions and fragments thereof. Nucleoside fragments composed of the nucleobase plus some carbons of the ribose ring were also observed. In total, we unequivocally assigned 31 fragments. In order to assess the viability of determining the sought MFD from information obtainable from the previous GC-MS method and to optimize the experimental conditions, we developed a customized genome-wide computational model of intermediary metabolism of S. cerevisiae. Its design accounts for two sub-populations of cells – in and out of S-phase of the CDC. The model is feasible, ready to be used for a meticulous sensitivity analysis, to design further experiments and to perform 13C-metabolic flux analysis (13C-MFA). A continuous culture of S. cerevisae was fed with [1,2-13C]glucose. deoxynucleosides and nucleosides from DNA and RNA, respectively, were isolated separately and the mass isotopomer distribution (MID) of their fragments was measured in GC-MS. A qualitative analysis of these MID showed that: i) the ribose moiety of pyrimidinic nucleosides was biosynthesized via the oxidative branch of pentose phosphate pathway (PPP) followed by shunting back and forward in the non-oxidative branch of PPP; ii) deoxyribose of pyrimidinic deoxynucleosides was biosynthesized via glycolysis followed by the non-oxidative branch of PPP; iii) nucleobases of deoxypurines were biosynthesized via a concerted flux of shunting back and forward between glycolysis and non-oxidative PPP followed by the oxidative branch of PPP; iv) the MID of nucleobases of cytidine, deoxycytidine and thymidine reveal activity of the oxidative branch of PPP, PC and TCA cycle. This would come from the longer turnover of TCA cycle related pools. The concerted activity of PC and TCA cycle may satisfy the joint demand for amino acids and fatty acids biosynthesis from the intermediaries of TCA cycle, thus requiring an active TCA cycle and the respective replenishing of its pools. The 13C-isotopomers of DNA building blocks differ from those of RNA, indicating that the MFD is heterogeneous over CDC.