A review of modelling and verification approaches for computational biology

This paper reviews most frequently used computational modelling approaches and formal verification techniques in computational biology. The paper also compares a number of model checking tools and software suits used in analysing biological systems and biochemical networks and verifiying a wide range of biological properties

Characterization of Early Biofilm Formation and Physiology in \u3ci\u3eNeisseria gonorrhoeae\u3c/i\u3e

Many bacteria rely on the dynamics of their extracellular appendages to perform important tasks, like motility and biofilm formation. Interestingly, these dynamics have been linked to physiological responses in some pathogenic bacteria; therefore, it is important to understand more about the role of physical forces in bacteria. I used the causative agent of the human disease gonorrhea, Neisseria gonorrhoeae, as a model system to study the role of physical force on early biofilm formation. The advantage of this system is that cell-cell interactions are controlled by extracellular filaments called type IV pili (tfp). Tfp is composed of monomers that give bacteria the ability to produce a dynamic filament undergoing cycles of elongations and retractions, and thus to exert forces on their surroundings. Through experiments and modeling, I demonstrated that pilus interactions produce motility gradients in microcolonies potentially establishing a force gradient across the microcolonies. I was interested in testing the biological implications of those motility and force gradients, so I utilized an established genetic mutant, ∆pilT, which lacks the pilus retraction motor pilT (Merz, So, and Sheetz 2000). A ∆pilT mutant allowed us to measure physiological response in cells that do not produce retractive force from its pilus. I measured the level of gene expression of seven pilus-related genes in two backgrounds: WT and a pilus retraction-deficient mutant, ∆pilT. I found that some WT microcolonies express pilus-related genes in a heterogeneous fashion, while others are homogeneous. Spatiotemporal patterns in the microcolony are modified in a ∆pilT background. The presence or absence of retraction forces between bacteria have a profound impact on bacterial physiology: the WT and ∆pilT background do not survive in a classical static biofilm assay at the same rate. Together these results point toward a fundamental role for intracellular forces in shaping bacteria physiology. The work of biologists has been dominated by a biochemical perspective. Although biochemical processes, like metabolism and information transfer, are certainly essential in all hierarchical levels of life, there is growing evidence that physical forces may provide an alternate physiological mechanism. The introduction in Chapter 1 provides context for understanding the role of force pattern formation in multicellular structures, in the hopes to extend this line of thinking to microbial communities. The development of microbial communities relies on self-assembly of single cells. The development of Neisseria gonorrhoeae cellular aggregates rely exclusively on type IV pili interactions (Taktikos et al. 2015a). In Chapter 2 is a transcription of the publication where I explore the dynamics of the microcolonies (W. Pönisch et al. 2018a). We found that cells have differential motility depending where in the microcolony cells are located. Differential motility is a result of fewer pili-pili interactions on the perimeter of the microcolony, and more pili-pili interactions closer to the center. Therefore, due to frequency of pili-pili interactions, a gradient of motility produces heterogeneous behavior in the microcolony. To investigate whether heterogenous behavior is extended beyond motility, I investigated whether there is a connection between retraction force and the physiology of microcolonies. In Chapter 3 I used a quantitative approach to analyze seven pilus-related genes using fluorescent reporters. Using fluorescence and confocal microscopy, I quantified fluorescence intensity within space and time in microcolonies. Here, I provide evidence that physical intracellular cues in a three-dimensional bacterial aggregate provide context for spatial organization, since spatiotemporal patterning and survival in ∆pilT background are compromised in comparison to WT microcolonies. This suggests the important role PilT retraction force plays in regulating spatiotemporal patterning during early biofilm development. Lastly, in Chapter 4 I characterized some physical features of microcolonies. I measured the formation size and survival rates of microcolonies when exposed to a range of osmotic pressures. These experiments were motivated by my interest in understanding the native context of developing microcolonies. Microcolonies inhabit the viscous mucosal membranes of epithelial cells; therefore, I measured one aspect of the environmental effects of microcolony when exposed to similar osmotic pressure created by mucus. I also measured the plasticity of WT and ∆pilT microcolonies through squeezing microplate experiments. The overall aim of this work is to understand the role of physical force on microbial development. I largely focused on role of tfp forces on Neisseria gonorrhoeae microcolony formation. Characterizing gene expression in microcolonies provided key evidence for spatiotemporal heterogeneity in developing WT microcolonies. Heterogeneity was minimized without pilus retraction forces, which suggests that retraction forces play a role in the early development of biofilm formation

From Molecules to the Masses : Visual Exploration, Analysis, and Communication of Human Physiology

Det overordnede målet med denne avhandlingen er tverrfaglig anvendelse av medisinske illustrasjons- og visualiseringsteknikker for å utforske, analysere og formidle aspekter ved fysiologi til publikum med ulik faglig nivå og bakgrunn. Fysiologi beskriver de biologiske prosessene som skjer i levende vesener over tid. Vitenskapen om fysiologi er kompleks, men samtidig kritisk for vår forståelse av hvordan levende organismer fungerer. Fysiologi dekker en stor bredde romlig-temporale skalaer og fordrer behovet for å kombinere og bygge bro mellom basalfagene (biologi, fysikk og kjemi) og medisin. De senere årene har det vært en eksplosjon av nye, avanserte eksperimentelle metoder for å detektere og karakterisere fysiologiske data. Volumet og kompleksiteten til fysiologiske data krever effektive strategier for visualisering for å komplementere dagens standard analyser. Hvilke tilnærminger som benyttes i visualiseringen må nøye balanseres og tilpasses formålet med bruken av dataene, enten dette er for å utforske dataene, analysere disse eller kommunisere og presentere dem. Arbeidet i denne avhandlingen bidrar med ny kunnskap innen teori, empiri, anvendelse og reproduserbarhet av visualiseringsmetoder innen fysiologi. Først i avhandlingen er en rapport som oppsummerer og utforsker dagens kunnskap om muligheter og utfordringer for visualisering innen fysiologi. Motivasjonen for arbeidet er behovet forskere innen visualiseringsfeltet, og forskere i ulike anvendelsesområder, har for en sammensatt oversikt over flerskala visualiseringsoppgaver og teknikker. Ved å bruke søk over et stort spekter av metodiske tilnærminger, er dette den første rapporten i sitt slag som kartlegger visualiseringsmulighetene innen fysiologi. I rapporten er faglitteraturen oppsummert slik at det skal være enkelt å gjøre oppslag innen ulike tema i rom-og-tid-skalaen, samtidig som litteraturen er delt inn i de tre høynivå visualiseringsoppgavene data utforsking, analyse og kommunikasjon. Dette danner et enkelt grunnlag for å navigere i litteraturen i feltet og slik danner rapporten et godt grunnlag for diskusjon og forskningsmuligheter innen feltet visualisering og fysiologi. Basert på arbeidet med rapporten var det særlig to områder som det er ønskelig for oss å fortsette å utforske: (1) utforskende analyse av mangefasetterte fysiologidata for ekspertbrukere, og (2) kommunikasjon av data til både eksperter og ikke-eksperter. Arbeidet vårt av mangefasetterte fysiologidata er oppsummert i to studier i avhandlingen. Hver studie omhandler prosesser som foregår på forskjellige romlig-temporale skalaer og inneholder konkrete eksempler på anvendelse av metodene vurdert av eksperter i feltet. I den første av de to studiene undersøkes konsentrasjonen av molekylære substanser (metabolitter) ut fra data innsamlet med magnetisk resonansspektroskopi (MRS), en avansert biokjemisk teknikk som brukes til å identifisere metabolske forbindelser i levende vev. Selv om MRS kan ha svært høy sensitivitet og spesifisitet i medisinske anvendelser, er analyseresultatene fra denne modaliteten abstrakte og vanskelige å forstå også for medisinskfaglige eksperter i feltet. Vår designstudie som undersøkte oppgavene og kravene til ekspertutforskende analyse av disse dataene førte til utviklingen av SpectraMosaic. Dette er en ny applikasjon som gjør det mulig for domeneeksperter å analysere konsentrasjonen av metabolitter normalisert for en hel kohort, eller etter prøveregion, individ, opptaksdato, eller status på hjernens aktivitetsnivå ved undersøkelsestidspunktet. I den andre studien foreslås en metode for å utføre utforskende analyser av flerdimensjonale fysiologiske data i motsatt ende av den romlig-temporale skalaen, nemlig på populasjonsnivå. En effektiv arbeidsflyt for utforskende dataanalyse må kritisk identifisere interessante mønstre og relasjoner, noe som blir stadig vanskeligere når dimensjonaliteten til dataene øker. Selv om dette delvis kan løses med eksisterende reduksjonsteknikker er det alltid en fare for at subtile mønstre kan gå tapt i reduksjonsprosessen. Isteden presenterer vi i studien DimLift, en iterativ dimensjonsreduksjonsteknikk som muliggjør brukeridentifikasjon av interessante mønstre og relasjoner som kan ligge subtilt i et datasett gjennom dimensjonale bunter. Nøkkelen til denne metoden er brukerens evne til å styre dimensjonalitetsreduksjonen slik at den følger brukerens egne undersøkelseslinjer. For videre å undersøke kommunikasjon til eksperter og ikke-eksperter, studeres i neste arbeid utformingen av visualiseringer for kommunikasjon til publikum med ulike nivåer av ekspertnivå. Det er naturlig å forvente at eksperter innen et emne kan ha ulike preferanser og kriterier for å vurdere en visuell kommunikasjon i forhold til et ikke-ekspertpublikum. Dette påvirker hvor effektivt et bilde kan benyttes til å formidle en gitt scenario. Med utgangspunkt i ulike teknikker innen biomedisinsk illustrasjon og visualisering, gjennomførte vi derfor en utforskende studie av kriteriene som publikum bruker når de evaluerer en biomedisinsk prosessvisualisering målrettet for kommunikasjon. Fra denne studien identifiserte vi muligheter for ytterligere konvergens av biomedisinsk illustrasjon og visualiseringsteknikker for mer målrettet visuell kommunikasjonsdesign. Særlig beskrives i større dybde utviklingen av semantisk konsistente retningslinjer for farging av molekylære scener. Hensikten med slike retningslinjer er å heve den vitenskapelige kompetansen til ikke-ekspertpublikum innen molekyler visualisering, som vil være spesielt relevant for kommunikasjon til befolkningen i forbindelse med folkehelseopplysning. All kode og empiriske funn utviklet i arbeidet med denne avhandlingen er åpen kildekode og tilgjengelig for gjenbruk av det vitenskapelige miljøet og offentligheten. Metodene og funnene presentert i denne avhandlingen danner et grunnlag for tverrfaglig biomedisinsk illustrasjon og visualiseringsforskning, og åpner flere muligheter for fortsatt arbeid med visualisering av fysiologiske prosesser.The overarching theme of this thesis is the cross-disciplinary application of medical illustration and visualization techniques to address challenges in exploring, analyzing, and communicating aspects of physiology to audiences with differing expertise. Describing the myriad biological processes occurring in living beings over time, the science of physiology is complex and critical to our understanding of how life works. It spans many spatio-temporal scales to combine and bridge the basic sciences (biology, physics, and chemistry) to medicine. Recent years have seen an explosion of new and finer-grained experimental and acquisition methods to characterize these data. The volume and complexity of these data necessitate effective visualizations to complement standard analysis practice. Visualization approaches must carefully consider and be adaptable to the user's main task, be it exploratory, analytical, or communication-oriented. This thesis contributes to the areas of theory, empirical findings, methods, applications, and research replicability in visualizing physiology. Our contributions open with a state-of-the-art report exploring the challenges and opportunities in visualization for physiology. This report is motivated by the need for visualization researchers, as well as researchers in various application domains, to have a centralized, multiscale overview of visualization tasks and techniques. Using a mixed-methods search approach, this is the first report of its kind to broadly survey the space of visualization for physiology. Our approach to organizing the literature in this report enables the lookup of topics of interest according to spatio-temporal scale. It further subdivides works according to any combination of three high-level visualization tasks: exploration, analysis, and communication. This provides an easily-navigable foundation for discussion and future research opportunities for audience- and task-appropriate visualization for physiology. From this report, we identify two key areas for continued research that begin narrowly and subsequently broaden in scope: (1) exploratory analysis of multifaceted physiology data for expert users, and (2) communication for experts and non-experts alike. Our investigation of multifaceted physiology data takes place over two studies. Each targets processes occurring at different spatio-temporal scales and includes a case study with experts to assess the applicability of our proposed method. At the molecular scale, we examine data from magnetic resonance spectroscopy (MRS), an advanced biochemical technique used to identify small molecules (metabolites) in living tissue that are indicative of metabolic pathway activity. Although highly sensitive and specific, the output of this modality is abstract and difficult to interpret. Our design study investigating the tasks and requirements for expert exploratory analysis of these data led to SpectraMosaic, a novel application enabling domain researchers to analyze any permutation of metabolites in ratio form for an entire cohort, or by sample region, individual, acquisition date, or brain activity status at the time of acquisition. A second approach considers the exploratory analysis of multidimensional physiological data at the opposite end of the spatio-temporal scale: population. An effective exploratory data analysis workflow critically must identify interesting patterns and relationships, which becomes increasingly difficult as data dimensionality increases. Although this can be partially addressed with existing dimensionality reduction techniques, the nature of these techniques means that subtle patterns may be lost in the process. In this approach, we describe DimLift, an iterative dimensionality reduction technique enabling user identification of interesting patterns and relationships that may lie subtly within a dataset through dimensional bundles. Key to this method is the user's ability to steer the dimensionality reduction technique to follow their own lines of inquiry. Our third question considers the crafting of visualizations for communication to audiences with different levels of expertise. It is natural to expect that experts in a topic may have different preferences and criteria to evaluate a visual communication relative to a non-expert audience. This impacts the success of an image in communicating a given scenario. Drawing from diverse techniques in biomedical illustration and visualization, we conducted an exploratory study of the criteria that audiences use when evaluating a biomedical process visualization targeted for communication. From this study, we identify opportunities for further convergence of biomedical illustration and visualization techniques for more targeted visual communication design. One opportunity that we discuss in greater depth is the development of semantically-consistent guidelines for the coloring of molecular scenes. The intent of such guidelines is to elevate the scientific literacy of non-expert audiences in the context of molecular visualization, which is particularly relevant to public health communication. All application code and empirical findings are open-sourced and available for reuse by the scientific community and public. The methods and findings presented in this thesis contribute to a foundation of cross-disciplinary biomedical illustration and visualization research, opening several opportunities for continued work in visualization for physiology.Doktorgradsavhandlin

Sounding the reef: comparative acoustemologies of underwater noise pollution / Pejling af revet: komparativ akustemologi af undersøisk støjforurening

Matthew Buttacavoli studied the development and detection of the category of underwater noise pollution in the Great Barrier Reef. He examined the phenomenon using a multi-species ethnographic approach. He found that bodily affordances and species’ boundaries that make sensing underwater noise difficult, can be overcome through technology and skilled practice. [Extract from Danish abstract] Denne afhandling vælger en etnografisk strategi til at undersøge, hvordan interesserede lyttere forsøger at opfatte og rekonstruere det akustiske havmiljø. Observation og interviews af deltagere sættes sammen med optagelse og kreative metoder for at kortlægge de (ufuldkomne) måder, hvorpå lyttere (herunder forfatteren) forsøger at forstå havskabningers lydverdener. I fokus er de lyttemetoder, der blev udviklet af dykkere, havforskere, akustikere og interesseorganisationer

The global organization and topological properties of Drosophila melanogaster

The fundamental principles governing the natural phenomena of life is one of the critical issues receiving due importance in recent years. Most complex real-world systems are found to have a similar networking model that manages their behavioral pattern. Recent scientific discoveries have furnished evidence that most real world networks follow a scale-free architecture. A number of research efforts are in progress to facilitate the learning of valuable information by recognizing the underlying reality in the vast amount of genomic data that is becoming available. A key feature of scale-free architecture is the vitality of the highly connected nodes (hubs). This project focuses on the multi-cellular organism Drosophila melanogaster, an established model system for human biology. The major objective is to analyze the protein-protein interaction and the metabolic network of the organism to consider the architectural patterns and the consequence of removal of hubs on the topological parameters of the two interaction networks. Analysis shows that both interaction networks pursue a scale-free model establishing the fact that real networks from varied situations conform to the small world pattern. Similarly, the topology of the two networks suffers drastic variations on the removal of the hubs. It is found that the topological parameters of average path length and diameter show a two-fold and three-fold increase on the deletion of hubs for the protein-protein interaction and metabolic interaction network, respectively. The arbitrary exclusion of the nodes does not show any remarkable disparity in the topological parameters of the two networks. This aberrant behavior for the two cases underscores the significance of the most linked nodes to the natural topology of the networks

The role of cellular morphogenesis in the pathogenicity of the rice blast fungus Magnaporthe oryzae

Appressorium-mediated plant infection is a common strategy used by many plant pathogenic fungi. Understanding the underlying genetic network that controls cellular differentiation of appressorium is therefore pivotal to design durable resistance strategies for these devastating pathogens. This thesis describes four published studies, which investigate the role of septin GTPases in infection and the role of secretion during plant tissue invasion by the rice blast pathogen Magnaporthe oryzae. Appressorium development involves a series of morphogenetic changes that are tightly regulated by cell cycle checkpoints. Entry into mitosis allows differentiation of an appressorium, while penetration peg emergence appears to require progression through subsequent cell cycle checkpoints and cytokinesis. The studies presented here show that symmetry-breaking events that occur during appressorium differentiation are mediated by scaffold proteins, named septins. Septin GTPases recruit actomyosin ring components during septation and define the site of cytokinesis. They also recruit a toroidal cortical F-actin network to the appressorium pore that provides cortical rigidity to facilitate plant infection. Septins act as diffusion barriers for proteins that mediate membrane curvature necessary for penetration peg formation. Repolarization of the F-actin cytoskeleton at the appressorium pore is essential for plant penetration and is controlled by cell polarity regulators, such as Cdc42 and Chm1. Septin-mediated plant infection is regulated by NADPH oxidase (Nox) dependent generation of reactive oxygen species (ROS). The Nox2/NoxR complex is essential for septin organization at the appressorium pore. Septins are therefore key determinants of appressorium repolarization. I also report an investigation of fungal secretory processes during tissue invasion and present evidence that distinct pathways are involved in effector secretion by Magnaporthe oryzae. A BrefeldinA-sensitive pathway is necessary for secretion of apoplastic effectors, such as Bas4 and Slp1, while a BrefeldinA-insensitive pathway is necessary for secretion of effectors destined for delivery to rice cells.The Halpin Scholarship for Rice Blast Researc

Recent advances in petri nets and concurrency

CEUR Workshop Proceeding

Using the RISCI Genetic Screening Platform for Elucidating Apoptosis Signalling Network

Considerable development in the field of nanotechnology is increasingly yielding novel applications of nanoparticles. The unique properties of nanoparticles in particular their high aspect ratio (length : width ratio), however could pose potential risks to the user. A high throughput genetic screening platform, RISCI (robotic single cDNA investigation), was previously established for the systematic evaluation of single gene activities. Here, RISCI was utilised to identify pro-apoptotic genes as well as genes involved in the positive and negative regulation of silica nanoparticle-induced cell death. This project describes the further development of the screening platform by harnessing its capability to screen a cDNA library comprising approximately 30,000 full length, completely annotated, and sequenced human genes for novel regulators of apoptosis. It integrates an extensive skill sets and is broadly organised into three major phases: Setup, Screen and Analysis. The integration of a pro-apoptosis treatment to screen for inhibitors and sensitizers is a novel aspect of the current experimental setup, along with the low redundancy library. The extensive setup phase focused on technical aspects. The cDNA library, acquired as plasmid DNA, was transformed into a bacterial host for replication and subsequent DNA isolation. A new high-throughput process was developed encompassing the production of competent bacteria and a heat shock transformation protocol, which was subsequently transferred onto the robotic platform. In parallel, the software controlling the robots was redeveloped to allow for execution of user-defined protocols while novel transfection protocols were adapted for automation. The screen identified 699 apoptosis inducers, 1,141 inhibitors and 626 sensitizers. Bioinformatics analysis revealed that the inducers were highly enriched for cell death associated terms, while the inhibitors were strongly associated with cancer profiles. Both inducers and sensitizers were predominantly achieving the functional effect on the protein level, but inhibitors were mainly transcription based. Enriched metal response genes also suggest that the silica nanoparticles were causing their toxicity through reactive oxygen species generation. Intriguingly, the screen identified many noncoding sequences as being functionally capable of regulating apoptosis. These noncoding candidates are capable of regulating the protein coding counterparts identified from the screen. The truly interesting part of the project outcome remains those unknown candidates that were implicated in apoptosis regulation for the first time. Dissemination of the consolidated candidate list would help accelerate the experimental validation of these candidates and aid other researchers in deriving novel hypotheses when the candidates are placed in their research context. [For supplementary files please contact author]

MODEL DRIVEN SOFTWARE PRODUCT LINE ENGINEERING: SYSTEM VARIABILITY VIEW AND PROCESS IMPLICATIONS

La Ingeniería de Líneas de Productos Software -Software Product Line Engineerings (SPLEs) en inglés- es una técnica de desarrollo de software que busca aplicar los principios de la fabricación industrial para la obtención de aplicaciones informáticas: esto es, una Línea de productos Software -Software Product Line (SPL)- se emplea para producir una familia de productos con características comunes, cuyos miembros, sin embargo, pueden tener características diferenciales. Identificar a priori estas características comunes y diferenciales permite maximizar la reutilización, reduciendo el tiempo y el coste del desarrollo. Describir estas relaciones con la suficiente expresividad se vuelve un aspecto fundamental para conseguir el éxito. La Ingeniería Dirigida por Modelos -Model Driven Engineering (MDE) en inglés- se ha revelado en los últimos años como un paradigma que permite tratar con artefactos software con un alto nivel de abstracción de forma efectiva. Gracias a ello, las SPLs puede aprovecharse en granmedida de los estándares y herramientas que han surgido dentro de la comunidad de MDE. No obstante, aún no se ha conseguido una buena integración entre SPLE y MDE, y como consecuencia, los mecanismos para la gestión de la variabilidad no son suficientemente expresivos. De esta manera, no es posible integrar la variabilidad de forma eficiente en procesos complejos de desarrollo de software donde las diferentes vistas de un sistema, las transformaciones de modelos y la generación de código juegan un papel fundamental. Esta tesis presenta MULTIPLE, un marco de trabajo y una herramienta que persiguen integrar de forma precisa y eficiente los mecanismos de gestión de variabilidad propios de las SPLs dentro de los procesos de MDE. MULTIPLE proporciona lenguajes específicos de dominio para especificar diferentes vistas de los sistemas software. Entre ellas se hace especial hincapié en la vista de variabilidad ya que es determinante para la especificación de SPLs.Gómez Llana, A. (2012). MODEL DRIVEN SOFTWARE PRODUCT LINE ENGINEERING: SYSTEM VARIABILITY VIEW AND PROCESS IMPLICATIONS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/15075Palanci

Microfluidics-Based Single-Cell Functional Proteomics Microchip for Portraying Protein Signal Transduction Networks within the Framework of Physicochemical Principles, with Applications in Fundamental and Translational Cancer Research

Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing unique opportunities. This thesis describes an emerging microfluidics-based toolkit for single cell functional proteomics, focusing on the development of the single cell barcode chips (SCBCs) with applications in fundamental and translational cancer research. The microchip designed to simultaneously quantify a panel of secreted, cytoplasmic and membrane proteins from single cells will be discussed at the beginning, which is the prototype for subsequent proteomic microchips with more sophisticated design in preclinical cancer research or clinical applications. The SCBCs are a highly versatile and information rich tool for single-cell functional proteomics. They are based upon isolating individual cells, or defined number of cells, within microchambers, each of which is equipped with a large antibody microarray (the barcode), with between a few hundred to ten thousand microchambers included within a single microchip. Functional proteomics assays at single-cell resolution yield unique pieces of information that significantly shape the way of thinking on cancer research. An in-depth discussion about analysis and interpretation of the unique information such as functional protein fluctuations and protein-protein correlative interactions will follow. The SCBC is a powerful tool to resolve the functional heterogeneity of cancer cells. It has the capacity to extract a comprehensive picture of the signal transduction network from single tumor cells and thus provides insight into the effect of targeted therapies on protein signaling networks. We will demonstrate this point through applying the SCBCs to investigate three isogenic cell lines of glioblastoma multiforme (GBM). The cancer cell population is highly heterogeneous with high-amplitude fluctuation at the single cell level, which in turn grants the robustness of the entire population. The concept that a stable population existing in the presence of random fluctuations is reminiscent of many physical systems that are successfully understood using statistical physics. Thus, tools derived from that field can probably be applied to using fluctuations to determine the nature of signaling networks. In the second part of the thesis, we will focus on such a case to use thermodynamics-motivated principles to understand cancer cell hypoxia, where single cell proteomics assays coupled with a quantitative version of Le Chatelier's principle derived from statistical mechanics yield detailed and surprising predictions, which were found to be correct in both cell line and primary tumor model. The third part of the thesis demonstrates the application of this technology in the preclinical cancer research to study the GBM cancer cell resistance to molecular targeted therapy. Physical approaches to anticipate therapy resistance and to identify effective therapy combinations will be discussed in detail. Our approach is based upon elucidating the signaling coordination within the phosphoprotein signaling pathways that are hyperactivated in human GBMs, and interrogating how that coordination responds to the perturbation of targeted inhibitor. Strongly coupled protein-protein interactions constitute most signaling cascades. A physical analogy of such a system is the strongly coupled atom-atom interactions in a crystal lattice. Similar to decomposing the atomic interactions into a series of independent normal vibrational modes, a simplified picture of signaling network coordination can also be achieved by diagonalizing protein-protein correlation or covariance matrices to decompose the pairwise correlative interactions into a set of distinct linear combinations of signaling proteins (i.e. independent signaling modes). By doing so, two independent signaling modes – one associated with mTOR signaling and a second associated with ERK/Src signaling have been resolved, which in turn allow us to anticipate resistance, and to design combination therapies that are effective, as well as identify those therapies and therapy combinations that will be ineffective. We validated our predictions in mouse tumor models and all predictions were borne out. In the last part, some preliminary results about the clinical translation of single-cell proteomics chips will be presented. The successful demonstration of our work on human-derived xenografts provides the rationale to extend our current work into the clinic. It will enable us to interrogate GBM tumor samples in a way that could potentially yield a straightforward, rapid interpretation so that we can give therapeutic guidance to the attending physicians within a clinical relevant time scale. The technical challenges of the clinical translation will be presented and our solutions to address the challenges will be discussed as well. A clinical case study will then follow, where some preliminary data collected from a pediatric GBM patient bearing an EGFR amplified tumor will be presented to demonstrate the general protocol and the workflow of the proposed clinical studies.</p