Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Probabilistic robust control : theory and applications

In this work, the development of a probabilistic approach to robust control is motivated by structural control applications in civil engineering. Often in civil structural applications, a system's performance is specified in terms of its reliability. In addition, the model and input uncertainty for the system may be described most appropriately using probabilistic or "soft" bounds on the model and input sets. The probabilistic robust control methodology contrasts with existing H∞/μ robust control methodologies that do not use probability information for the model and input uncertainty sets, yielding only the guaranteed (i.e., "worst-case") system performance, and no information about the system's probable performance which would be of interest to civil engineers.

The design objective for the probabilistic robust controller is to maximize the reliability of the uncertain structure/controller system for a probabilistically-described uncertain excitation. The robust performance is computed for a set of possible models by weighting the conditional performance probability for a particular model by the probability of that model, then integrating over the set of possible models. This integration is accomplished efficiently using an asymptotic approximation. The probable performance can be optimized numerically over the class of allowable controllers to find the optimal controller. Also, if structural response data becomes available from a controlled structure, its probable performance can easily be updated using Bayes's Theorem to update the probability distribution over the set of possible models. An updated optimal controller can then be produced, if desired, by following the original procedure. Thus, the probabilistic framework integrates system identification and robust control in a natural manner.

The probabilistic robust control methodology is applied to two systems in this thesis. The first is a high-fidelity computer model of a benchmark structural control laboratory experiment. For this application, uncertainty in the input model only is considered. The probabilistic control design minimizes the failure probability of the benchmark system while remaining robust with respect to the input model uncertainty. The performance of an optimal low-order controller compares favorably with higher-order controllers for the same benchmark system which are based on other approaches. The second application is to the Caltech Flexible Structure, which is a light-weight aluminum truss structure actuated by three voice coil actuators. A controller is designed to minimize the failure probability for a nominal model of this system. Furthermore, the method for updating the model-based performance calculation given new response data from the system is illustrated.

Applications of frequency modulation interference cancellers to multiaccess communications systems

Cancellation of interfering frequency-modulated (FM) signals is investigated with emphasis towards applications on the cellular telephone channel as an important example of a multiple access communications system. In order to fairly evaluate analog FM multiaccess systems with respect to more complex digital multiaccess systems, a serious attempt to mitigate interference in the FM systems must be made. Information-theoretic results in the field of interference channels are shown to motivate the estimation and subtraction of undesired interfering signals. This thesis briefly examines the relative optimality of the current FM techniques in known interference channels, before pursuing the estimation and subtracting of interfering FM signals.

The capture-effect phenomenon of FM reception is exploited to produce simple interference-cancelling receivers with a cross-coupled topology. The use of phase-locked loop receivers cross-coupled with amplitude-tracking loops to estimate the FM signals is explored. The theory and function of these cross-coupled phase-locked loop (CCPLL) interference cancellers are examined. New interference cancellers inspired by optimal estimation and the CCPLL topology are developed, resulting in simpler receivers than those in prior art. Signal acquisition and capture effects in these complex dynamical systems are explained using the relationship of the dynamical systems to adaptive noise cancellers.

FM interference-cancelling receivers are considered for increasing the frequency reuse in a cellular telephone system. Interference mitigation in the cellular environment is seen to require tracking of the desired signal during time intervals when it is not the strongest signal present. Use of interference cancelling in conjunction with dynamic frequency-allocation algorithms is viewed as a way of improving spectrum efficiency. Performance of interference cancellers indicates possibilities for greatly increased frequency reuse. The economics of receiver improvements in the cellular system is considered, including both the mobile subscriber equipment and the provider's tower (base station) equipment.

The thesis is divided into four major parts and a summary: the introduction, motivations for the use of interference cancellation, examination of the CCPLL interference canceller, and applications to the cellular channel. The parts are dependent on each other and are meant to be read as a whole.

Future of earthquake early warning : quantifying uncertainty and making fast automated decisions for applications

Earthquake early warning (EEW) systems have been rapidly developing over the past decade. Japan Meteorological Agency (JMA) has an EEW system that was operating during the 2011 M9 Tohoku earthquake in Japan, and this increased the awareness of EEW systems around the world. While longer-time earthquake prediction still faces many challenges to be practical, the availability of shorter-time EEW opens up a new door for earthquake loss mitigation. After an earthquake fault begins rupturing, an EEW system utilizes the first few seconds of recorded seismic waveform data to quickly predict the hypocenter location, magnitude, origin time and the expected shaking intensity level around the region. This early warning information is broadcast to different sites before the strong shaking arrives. The warning lead time of such a system is short, typically a few seconds to a minute or so, and the information is uncertain. These factors limit human intervention to activate mitigation actions and this must be addressed for engineering applications of EEW. This study applies a Bayesian probabilistic approach along with machine learning techniques and decision theories from economics to improve different aspects of EEW operation, including extending it to engineering applications.

Existing EEW systems are often based on a deterministic approach. Often, they assume that only a single event occurs within a short period of time, which led to many false alarms after the Tohoku earthquake in Japan. This study develops a probability-based EEW algorithm based on an existing deterministic model to extend the EEW system to the case of concurrent events, which are often observed during the aftershock sequence after a large earthquake.

To overcome the challenge of uncertain information and short lead time of EEW, this study also develops an earthquake probability-based automated decision-making (ePAD) framework to make robust decision for EEW mitigation applications. A cost-benefit model that can capture the uncertainties in EEW information and the decision process is used. This approach is called the Performance-Based Earthquake Early Warning, which is based on the PEER Performance-Based Earthquake Engineering method. Use of surrogate models is suggested to improve computational efficiency. Also, new models are proposed to add the influence of lead time into the cost-benefit analysis. For example, a value of information model is used to quantify the potential value of delaying the activation of a mitigation action for a possible reduction of the uncertainty of EEW information in the next update. Two practical examples, evacuation alert and elevator control, are studied to illustrate the ePAD framework. Potential advanced EEW applications, such as the case of multiple-action decisions and the synergy of EEW and structural health monitoring systems, are also discussed.

Multi-model inference ranking and applications to physics at the Large Hadron Collider

In the measurement of the Higgs Boson decaying into two photons the parametrization of an appropriate background model is essential for fitting the Higgs signal mass peak over a continuous background. This diphoton background modeling is crucial in the statistical process of calculating exclusion limits and the significance of observations in comparison to a background-only hypothesis. It is therefore ideal to obtain knowledge of the physical shape for the background mass distribution as the use of an improper function can lead to biases in the observed limits. Using an Information-Theoretic (I-T) approach for valid inference we apply Akaike Information Criterion (AIC) as a measure of the separation for a fitting model from the data. We then implement a multi-model inference ranking method to build a fit-model that closest represents the Standard Model background in 2013 diphoton data recorded by the Compact Muon Solenoid (CMS) experiment at the Large Hadron Collider (LHC). Potential applications and extensions of this model-selection technique are discussed with reference to CMS detector performance measurements as well as in potential physics analyses at future detectors.

Fast numerical methods for mixed, singular Helmholtz boundary value problems and Laplace eigenvalue problems - with applications to antenna design, sloshing, electromagnetic scattering and spectral geometry

This thesis presents a novel class of algorithms for the solution of scattering and eigenvalue problems on general two-dimensional domains under a variety of boundary conditions, including non-smooth domains and certain "Zaremba" boundary conditions - for which Dirichlet and Neumann conditions are specified on various portions of the domain boundary. The theoretical basis of the methods for the Zaremba problems on smooth domains concern detailed information, which is put forth for the first time in this thesis, about the singularity structure of solutions of the Laplace operator under boundary conditions of Zaremba type. The new methods, which are based on use of Green functions and integral equations, incorporate a number of algorithmic innovations, including a fast and robust eigenvalue-search algorithm, use of the Fourier Continuation method for regularization of all smooth-domain Zaremba singularities, and newly derived quadrature rules which give rise to high-order convergence even around singular points for the Zaremba problem. The resulting algorithms enjoy high-order convergence, and they can tackle a variety of elliptic problems under general boundary conditions, including, for example, eigenvalue problems, scattering problems, and, in particular, eigenfunction expansion for time-domain problems in non-separable physical domains with mixed boundary conditions.

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.