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Microfluidic Analysis in Patient Biopsies: toward Precision Medicine for Glioblastoma Multiforme
Get PDFAlthough every individual has a unique biology, most medicine still relies on the one-size-fits-all approach, which often fails in the treatment of heterogeneous diseases like cancer. An emerging approach to disease treatment is precision medicine, in which a specific treatment is tailored for individual patients using their biological information, including their genome, phenome, and proteome. Two clinical actions are important for implementing precision medicine in cancer therapies: choosing the correct drugs via patient stratification and choosing a suitable drug dosage and duration via drug response monitoring.
After selecting the potential drug candidate, it is crucial to monitor tumor response to drug therapy because cancer is a dynamic disease that can develop drug resistance. Although non-invasive tumor imaging techniques such as magnetic resonance imaging, computed tomography, and positron emission tomography can assess physical size and metabolic activity of tumors, these techniques have poor time resolution and cannot capture the dynamic changes of bio-molecules implicated with drug resistance. Thus, to effectively monitor drug response, supplemental diagnostic or prognostic markers must be routinely measured from patient biopsies. Unfortunately, routine monitoring of multiple biomarkers from patient biopsies is impractical, as conventional analytical assays require large sample amounts (up to 100-1,000 mg of tissue or 10 mL of blood).
In response to this challenge, this thesis describes the development of various microfluidic technologies that can perform multiplexed measurements (up to 20-plex) using minute amounts of sample (10,000-100,000 cells or 30µL of blood) in a miniaturized analytical platform (maximum 75 × 26 × 1 mm footprint). We applied these technologies for drug screening and drug response monitoring in glioblastoma multiforme, a highly lethal brain tumor, assaying two different types of patient biopsies: cancer cells and blood.
First, we developed an integrated microfluidics-chip/beta particle imaging system that can screen for effective therapies using small amounts of patient-derived cell lines. Since glioblastoma cells have abnormally high glycolytic activity, this was used as a read-out for drug response. Single cells were isolated in micro-traps, and their glycolytic activity was quantitated using a radioactive probe. This platform can assess potential drug targets directly from patient biopsies without administering drugs to the patient.
Second, we developed an in vitro diagnostic test that can monitor tumor drug resistance by measuring up to 14 proteins in finger-prick volumes of blood. This test relies on microfluidics and microarray patterning of antibodies to carry out multiplexed sandwich-type immunofluorescence assays. Using this technology and conventional tumor imaging techniques, we linked proteomic signatures to tumor growth, establishing diagnostic and prognostic models in two clinical treatment cases of bevacizumab and buparlisib. Moreover, we adopted the multiplexed proteomic measurement platform to rapidly screen out small peptide binding agents that target an oncogenic protein in glioblastoma.
The microfluidic tools developed here are sample-efficient and highly informative, and we propose that these techniques could enable routine evaluation of drug response in a precision medicine workflow.</p
Improving the Performance and Sensitivity of Gravitational Wave Detectors
Get PDFThe field of observational gravitational wave astronomy has begun in earnest, starting with the detection of the strain signal from the binary black hole merger GW150914 by the Laser Interferometer Gravitational-wave Observatory (LIGO) in 2015. The current incarnation of the LIGO observatories, known as Advanced LIGO, has achieved strain sensitivities on the order of 10−23/√Hz in the hundreds of Hz region, which has enabled unambiguous detection of astrophysical gravitational wave signals. Nevertheless, the scientific output from the LIGO observatories is constrained by the instrumental performance and sensitivity, as there remain many more distant and exotic sources to be observed.
This thesis describes a few topics in experimental gravitational physics, broadly unified by the desire to improve the performance and sensitivity of gravitational wave interferometers. First, it describes an experimental effort to search for a novel form of nonlinear mechanical noise that may be relevant for the ultimate performance of the mirror sus- pension systems used throughout the instrument. Next, it summarizes work done at the CalTech 40m LIGO controls prototype to realize its fully operational state, and a novel automated controls algorithm developed and tested there that may be useful in simplifying the control of current and future interferometers. Finally, it describes work done on a system to identify and subtract unwanted noise couplings out of recorded aLIGO strain data in an automated fashion. The noise subtraction system applied to GW150914 is demonstrated to reduce the uncertainties of the black hole mass parameters by about 10%.</p
The Coherence Collapse Regime of High-Coherence Si/III-V Lasers and the Use of Swept Frequency Semiconductor Lasers for Full Field 3D Imaging
Get PDFThe semiconductor laser is the linchpin of optical communication and is now also penetrating a wide spectrum of new applications such as biomedical sensing, coherent communication, metrology, and time keeping. These require a higher degree of temporal coherence than is available from the present generation. Recently, it has been proposed and shown that heterogeneously integrated lasers on silicon and InGaAsP can be used to design high coherence single mode lasers with a much narrower linewidth than their all InGaAsP counterparts. Unfortunately, these lasers suffer from large thermal impedances and their optical feedback characteristics have not yet been explored. In the first part of this thesis, we will explore how flip chip bonding can help decrease the thermal impedance of these lasers to improve their overall performance and show that these lasers can provide up to 20 dB of optical isolation compared to their all III-V counterparts.
In the second part of this thesis, we will report on the use of commercially available semiconductor lasers, in conjunction with an optical modulator to obtain high-resolution tomographic images in one shot without any moving parts. The electronic control over the imaged depth of this novel tomographic imaging camera enables it to monitor arbitrary depth slices in rapid succession over a depth range limited only by the coherence length of the laser. Not only does this imaging modality acquire the transverse image intensity (x,y) distribution of the light reflected from a particular depth, but also the phase of the reflected light enabling imaging beyond the conventional depth of field of the lens. This has important implications in applications requiring high lateral resolution images where the shallow depth of field would often require mechanical scanning of the lens elements to change the imaged depth.</p
Photoinduced, Copper-Catalyzed C-N and C-C Bond Formation and Photocatalytic Co-Mediated Nitrite Reduction to N₂O: Reactivity and Mechanism
Get PDFPhotocatalytic reactions with first-row transition metals are presented as a method for sustainable chemistry with great potential for new forms of reactivity and mechanistic pathways. Chapters 2 and 3 of this thesis discuss mechanism and reactivity of photoinduced, copper-catalyzed bond constructions. The Peters and Fu groups have reported that a variety of couplings of nitrogen, sulfur, oxygen, and carbon nucleophiles with organic halides can be achieved under mild conditions (−40 to 30 °C) through the use of light and a copper catalyst. Insight into the various mechanisms by which these reactions proceed may enhance our understanding of chemical reactivity and facilitate the development of new methods. We apply an array of tools (EPR, NMR, transient absorption, and UV−vis spectroscopy; ESI−MS; X-ray crystallography; DFT calculations; reactivity, stereochemical, and product studies) to investigate the photoinduced, copper-catalyzed coupling of carbazole with alkyl bromides. Our observations are consistent with pathways wherein both an excited state of the copper(I) carbazolide complex ([CuI(carb)2]−) and an excited state of the nucleophile (Li(carb)) can serve as photoreductants of the alkyl bromide. The catalytically dominant pathway proceeds from the excited state of Li(carb), generating a carbazyl radical and an alkyl radical. The cross-coupling of these radicals is catalyzed by copper via an out-of-cage mechanism in which [CuI(carb)2]− and [CuII(carb)3]− (carb = carbazolide), both of which have been identified under coupling conditions, are key intermediates, and [CuII(carb)3]− serves as the persistent radical that is responsible for predominant cross-coupling. This study underscores the versatility of copper(II) complexes in engaging with radical intermediates that are generated by disparate pathways, en route to targeted bond constructions.
In Chapter 3, we establish that photoinduced, copper-catalyzed alkylation can also be applied to C−C bond formation, specifically, that the cyanation of unactivated secondary alkyl chlorides can be achieved at room temperature to afford nitriles, an important class of target molecules. In the presence of an inexpensive copper catalyst (CuI; no ligand coadditive) and a readily available light source (UVC compact fluorescent light bulb), a wide array of alkyl halides undergo cyanation in good yield. Our initial mechanistic studies are consistent with the hypothesis that an excited state of [Cu(CN)2]− may play a role, via single electron transfer, in this process. This investigation provides a rare example of a transition metal-catalyzed cyanation of an alkyl halide, as well as the first illustrations of photoinduced, copper-catalyzed alkylation with either a carbon nucleophile or a secondary alkyl chloride.
Chapter 4 presents a mechanistic study of the photocatalytic reduction of nitrite to nitrous oxide with the use of an Ir photocatalyst ([Ir(ppy)2(phen)][PF6]) and a bimetallic CoMg co-catalyst with a diimine-dioxime ligand platform. Insights into the mechanism of this reaction may enhance our current understanding of N–N coupling processes relative to other pathways of reactivity for nitrosyl ligands, such as nitroxyl (HNO) dimerization. We propose a mechanism in which a coordinated and an uncoordinated •NO are coupled at a single Co center. One electron reduction of [(Cl)(NO)Co(Medoen)Mg(Me3TACN)(H2O)][BPh4] ({CoNO}8), a species we show to be catalytically relevant, forms a {CoNO}9 species that is characterized by UV-Vis, EPR, and FT-IR spectroscopy and whose electronic structure is supported by density functional theory (DFT). We formulate the {CoNO}9 as a 5-coordinate, S = 3/2 Co(II) antiferromagnetically coupled with an anionic S = 1 3NO– ligand. Experimental data suggest a mechanism in which this {CoNO}9 intermediate can release •NO, thereby reducing the Co(II) center to Co(I). This free •NO can react with another {CoNO}9 complex to generate a Co(NONO) intermediate which was observed by step-scan time-resolved IR spectroscopy and whose assignment was supported with DFT calculations. This Co(NONO) species, which can generate N2O and H2O, is formulated as a neutral hyponitrite intermediate with significant neutral radical character on both nitrosyl nitrogen atoms and a weak N–N bond.</p
Using Heterogeneous 3D Earth Models to Constrain Interseismic and Postseismic Deformation in Southern California and Nepal
Get PDFWe characterize interseismic strain accumulation across the Los Angeles basin and postseismic deformation following the 2010 Mw=7.2 El Mayor-Cucapah and 2015 Mw=7.8 Nepal earthquakes using geodetic data. These settings are all characterized by strong 3D heterogeneities of elastic structure, ductile properties, fault geometries, and fault slip behavior, and we use constaints from seismology, long-term tectonic modeling, geology, and other sources to construct detailed models of these heterogeneities. Postseismic surface displacements following the 2010 El Mayor-Cucapah earthquake indicate viscoelastic relaxation in the shallow Salton Trough mantle and possibly the lower crust, a process that would have been enhanced by high heat flow induced by crustal extension at the tip of the Gulf of California. We find that a dense and prolonged aftershock sequence in the Yuha Desert may have been driven by aseismic afterslip coupled with fluid flow. Our study of interseismic strain accumulation across the Los Angeles basin shows that the soft sedimentary basin has a first-order effect on the elastostatic Green’s functions mapping fault creep and locking at depth to surface deformation, and therefore on the estimation of interseismic fault creep rates and strain accumulation at depth. We infer modest interseismic coupling on the three major thrust faults underlying the Los Angeles basin, corresponding to an annual seismic moment deficit buildup rate (to be presumably released in earthquakes) of 1.7 +1.2/-0.5 x 1017 Nm/yr. We estimate the long-term seismicity model needed to balance the rate of moment deficit accumulation assuming a truncated Gutenberg-Richter magnitude-frequency distribution of earthquakes. The long-term catalog is consistent with the instrumental rates of small and moderate earthquakes and tops out at a M~6.9 earthquake every ~430 years. Finally, we characterize the postseismic deformation following the 2015 Nepal earthquake using models of the thermal structure, state of stress, and rheology that are based on the long-term evolution and topography of the Himalaya. The rheological structure based on these models predicts negligible postseismic viscoelastic deformation. Afterslip on the downdip extension of the rupture cannot realistically explain the observed displacements either. We find that the postseismic deformation is well explained by a combination of afterslip on the downdip edge of the coseismic rupture (as well as a narrow zone in between the mainshock and a large aftershock) and, more prominently, transient viscoelastic relaxation in the hot Tibetan crust. These processes contribute to the stress loading of the Main Himalayan Thrust.</p
Circuits of Dynamically Interacting Sigma Factors in Single Cells
Get PDFHow do cells integrate multiple, dynamic genetic circuits? I study this question in the context of the alternative sigma factors of B. subtilis.
The first project proposes a novel mode of gene regulation called timesharing. The key idea is that a limited resource is shared dynamically in time. Here we show that the alternative sigma factors of B. subtilis use dynamic sharing to share a limited supply of core RNA Polymerase (RNAP). We show that 5 alternative sigma factors activate in pulses, and that these pulses operate in a competitive regime. Interestingly, we found that pairwise correlations between these sigma factors contained a mixture of positive and negative correlations, whereas one may naively expect all correlations to be negative. We show with a mathematical model that competitive pulsing can lead to non-intuitive sets of mixed correlations.
The second project take a closer, quantitative look at sigma factor competition. Although competition between the housekeeping sigma and a single alternative sigma has been well studied, competition between alternative sigmas themselves has been relatively unexplored. To address this issue, we systematically investigated the pairwise competitive relationships between 7 alternative sigma factors in B. subtilis. The main experimental tool was a 7x7 'deletion' matrix of strains, where every matrix strain was deleted for one sigma, and reported on another sigma via a fluorescent reporter. The deletion matrix revealed that competition is highly asymmetric. Deletion of any given sigma factor increased σW activity, but did not affect other sigma factors. These results are recreated by a minimal mathematical model of sigma factor competition, where importantly σW is relatively high in abundance but weak in affinity for core RNAP. We used the model to predict how overexpressing sigma factors affect each other, and these predictions were matched by experiments.
The third project reports a novel activator for alternative sigma factors. Alternative sigmas factors are activated by many forms of stress, such as nutrient limitation, temperature shifts, and molecular stresses like antibiotics. Here we show that surprisingly, cell lysis causes adjacent cells to specifically activate σX. This cell lysis-σX response is a general phenomenon, as it is observed under multiple experimental conditions. We show this relationship between cell death and σX is causal, since harvested cell extract activates σX. Finally, we hypothesize that cell death and σX play an important role in biofilm wrinkle formation.</p
Iron, Cobalt, and Nickel Metalloboranes: Reactivity, Catalysis, N2 Activation and Stabilization of Reactive N2Hx Ligands
Get PDFThe reactivity of Fe and Co compounds supported by a bisphosphinoborane (DPB) ligand ([(DPB)Fe]2(N2) and (DPB)Co(N2)) towards E-H bonds (E = C, N, S, O, Si) is reported along with the catalytic hydrosilylation of ketones and aldehydes. The Fe and Co compounds displayed a mix of 1-electron and 2-electron chemistry. In some cases [(DPB)Fe]2(N2) and (DPB)Co(N2) facilitated oxidative addition of the E-H bond across the M-B interaction, and in others evolution of H2 giving a 1-electron oxidized complex of the general form (DPB)M(E) was observed. The reaction of Ph2SiH2 with (DPB)Co(N2) was found to be reversible, similar to the previously reported related nickel complex (PhDPBMes)Ni. The reactivity of these Fe and Co compounds is compared to previously reported Ni compounds supported by a similar ligand which catalyze olefin hydrogenation and hydrosilylation of substituted benzaldehydes.
The synthesis and metalation with nickel of two new variants of the DPB ligand (DP*BPh and DP*BMes) is described. The primary modification introduced in DP*BPh and DP*BMes is the incorporation of a tertiary amine moiety into the secondary coordination sphere. This was done with the hypothesis that the amine moiety might act as a proton shuttle and facilitate proton reduction or hydrogen oxidation electrocatalysis. The process of screening these compounds for activity as proton reduction and hydrogen oxidation catalysts is also discussed. Additionally, the stoichiometric reactivity of [(DP*BPh)Ni]2(N2) and (DP*BMes)Ni(N2) with H2 was studied. We observed that [(DP*BPh)Ni]2(N2) slowly decomposed to an unidentified mixture of products while (DP*BMes)Ni(N2) dimerized to form a phosphine bridged Ni-borohydride dimer [(DP*BMesH)Ni]2. [(DP*BPh)Ni]2(N2) and (DP*BMes)Ni(N2) were also tested as precatalysts for olefin hydrogenation and found to be less active that their previously reported counterpart (PhDPBMes)Ni. [(DP*BPh)Ni]2(N2) and (DP*BMes)Ni(N2) correspondingly showed no activity for hydrogenation of polar substrates such as ketones, aldehydes, or CO2.
Lastly, the synthesis of a new trisphosphinoborane ligand (ArP3B) with bulky aryl substituents on the phosphines and its metalation with Fe is described. The anionic-N2 adduct [(ArP3B)Fe(N2)][Na(12-C-4)2] was observed to react with H+ sources to generate the first observed parent iron-diazenido (ArP3B)Fe(NNH) and an iron-hydrazido(2-) [(ArP3B)Fe(NNH2)]+. [(ArP3B)Fe(NNH2)]+ was found to have similar spectroscopic properties to the previously reported [(TPB)Fe(NNH2)]+. A thorough characterization of [(ArP3B)Fe(N2)][Na(12-C-4)2], (ArP3B)Fe(NNH), and [(ArP3B)Fe(NNH2)]+ by a variety of continuous wave and pulsed ERP techniques is presented along with 57Fe Mössbauer data. The new (ArP3B)Fe system was also canvassed for activity as a catalyst for conversion of N2 to NH3 and found to yield substoichiometric amounts of NH3 in the presence of KC8 and HBArF24•2Et2O while no NH3 was observed using CoCp*2 and [H2NPh2][OTf].</p
Exoplanetary Atmospheric Characterization Using Polarimetry and Other Radiative Transfer Modeling Problems
Get PDFThis thesis deals with a pair of current problems with the remote sensing of planetary atmospheres. First is the modeling of polarization of scattered light from the atmospheres of exoplanets. With the first such observations becoming possible in the last year, there is a need to understand what these measurements actually mean. To that end, we developed families of radiative transfer models that simulate polarized phase curves for different atmospheric scenarios on hot Jupiters. These models were then used in the interpretation of scattered light from HD 189733b and WASP 12b, two hot Jupiter exoplanets, to determine their albedos and gauge what type of scattering particles might be present in their atmospheres. The last part of this half deals with observing oceans on distant Earth-like exoplanets using polarization from glint off the water surface. Though this measurement is not possible with current telescopes, but it may become accessible in the next decade with a slew of high powered ground and space telescopes in the pipeline.
The second half of the thesis is devoted to the development of a fast radiative transfer model. The goal of this model is to be able to process the massive amounts of data coming in from Earth observing satellites such as GOSAT and OCO-2 in a timely and accurate manner. We refined the principal component analysis based fast radiative transfer model to be accurate enough to retrieve carbon dioxide concentrations to the part per million accuracy that is necessary to track spatial and temporal changes in this important greenhouse gas.</p
Definability and Classification of Equivalence Relations and Logical Theories
Get PDFThis thesis consists of four independent papers.
In the first paper, joint with Kechris, we study the global aspects of structurability in the theory of countable Borel equivalence relations. For a class K of countable relational structures, a countable Borel equivalence relation E is said to be K-structurable if there is a Borel way to put a structure in K on each E-equivalence class. We show that K-structurability interacts well with various preorders commonly used in the classification of countable Borel equivalence relations. We consider the poset of classes of K-structurable equivalence relations for various K, under inclusion, and show that it is a distributive lattice. Finally, we consider the effect on K-structurability of various model-theoretic properties of K; in particular, we characterize the K such that every K-structurable equivalence relation is smooth.
In the second paper, we consider the classes of Kn-structurable equivalence relations, where Kn is the class of n-dimensional contractible simplicial complexes. We show that every Kn-structurable equivalence relation Borel embeds into one structurable by complexes in Kn with the further property that each vertex belongs to at most Mn := 2n-1(n2+3n+2)-2 edges; this generalizes a result of Jackson-Kechris-Louveau in the case n=1.
In the third paper, we consider the amalgamation property from model theory in an abstract categorical context. A category is said to have the amalgamation property if every pushout diagram has a cocone. We characterize the finitely generated categories I such that in every category with the amalgamation property, every I-shaped diagram has a cocone.
In the fourth paper, we prove a strong conceptual completeness theorem (in the sense of Makkai) for the infinitary logic Lω1ω: every countable Lω1ω-theory can be canonically recovered from its standard Borel groupoid of countable models, up to a suitable syntactical notion of equivalence. This implies that given two theories (L,T) and (L',T'), every Borel functor Mod(L',T') → Mod(L,T) between the respective groupoids of countable models is Borel naturally isomorphic to the functor induced by some L'ω1ω-interpretation of T in T', which generalizes a recent result of Harrison-Trainor, Miller, and Montalban in the case where T, T' are ℵ0-categorical.</p
The Union of Quantum Field Theory and Non-Equilibrium Thermodynamics
Get PDFQuantum field theory is the language used to describe nature at its most fundamental scales; while thermodynamics is a framework to describe the collective behavior of macroscopic systems. Recent advances in non-equilibrium thermodynamics have enabled this framework to be applied to smaller systems operating out of thermal equilibrium. This thesis is concerned with both quantum field theory and non-equilibrium thermodynamics independently and with their intersection.
First, a purely phenomenological application of quantum field theory is explored in the context of the upcoming Mu2E experiment. This experiment will look for rare decays which would indicate the presence of physics beyond the Standard Model. Using the language of effective field theories, a next-to-leading order analysis of the conversion rate is performed.
The focus then shifts to an apparent paradox in the Bayesian interpretation of statistical mechanics. For a Bayesian observer making measurements of an open system, the Shannon entropy decreases, in apparent violation of the Second Law of Thermodynamics. It is shown that rather than utilizing the entropy, which can decrease under Bayesian updates, the Second Law for a Bayesian observer can be rephrased in terms
of a cross-entropy which is always non-negative.
Finally, the intersection of quantum field theory and non-equilibrium thermodynamics is examined. Using quantum work fluctuation theorems, an investigation of how these frameworks can be applied to a driven quantum field theory is performed. For a time-dependent variant of λφ4 , analytic expressions for the work distribution functions at one-loop order are derived. These expressions are shown to satisfy the quantum Jarzynski equality and Crooks fluctuation theorem.</p