Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms

The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, in a multicellular organism, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. In this thesis, we describe methods that enable the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. We first engineered a eukaryotic phenylalanyl-tRNA synthetase (PheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). Using Caenorhabditis elegans, we expressed the engineered PheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells -- and only those cells -- to be labeled with azides. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affnity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins -- including proteins with previously unknown expression patterns -- expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; spatiotemporal selectivity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we next developed a two-component system to drive overlapping -- but not identical -- patterns of expression of engineered PheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-PheRS system for highly efficient PheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells at specific developmental stages.

Iso-specific Ziegler-Natta polymerization of α-olefins with a single-component organoyttrium catalyst

The synthesis of a sterically tailored ligand array (M)_2((C_5H_2-2-Si(CH_3)_3-4-C(CH_3)_3)S_2i(CH_3)_2]("M_2Bp") (M = Li, 16; K, 19) is described. Transmetallation of Li_2Bp with YCl_3(THF)_3 affords exclusively the C_2 symmetric product rac-[BpY(µ_2-Cl)_2Li(THF)_2], 20. A X-ray crystal structure of 20 has been determined; triclinic, P1, a= 13.110 (8), b = 17.163 (15), c = 20.623 (14) Å, α = 104.02 (7), β = 99.38 (5), γ = 100.24 (6)° , Z = 4, R = 0.056. Transmetallation of K_2Bp with YCl_3(THF)_3 affords the halide free complex rac-BpYCl, 23. The corresponding rac-BpLaCl, 28, is prepared in an anlogous manner. In all cases the achiral meso isomer is not obtained since only for the racemic isomers are the unfavorable steric interactions between the Si(CH3)_3 groups in the narrow portion of the [Cp-M'-Cp] wedge avoided. Alkylation of 20 or 23 with LiCH(Si(CH_3)_3)_2 affords rac-BpYCH(Si(CH_3)_3)_2, 26 in good yield. Alkylation of 28 with LiCH(Si(CH_3)_3)_2 affords rac-BpLaCH(Si(CH_3)_3)_2 29. Hydrogenation of 26 cleanly affords the bridging hydride species [BpY(µ_2-H)]_2, 27, as the homochiral (R,R) and (S,S) dimeric pairs. 26 is an efficient initiator for the polymerization of ethylene to high molecular weight linear polyethylene. 27 catalyzes the polymerization of propylene (25% v/v in methylcyclohexane) and neat samples of 1-butene, 1-pentene, 1-hexene to moderately high molecular weight polymers: polypropylene (M_n = 4,200, PDI 2.32, T_m 157 °C); poly-1-butene (M_n = 8,500, PDI 3.44, T_m 105 °C); poly-1-pentene (M_n = 20,000, PDI 1.99, T_m 73 °C); poly-1-hexene (M_n = 24,000, PDI 1.75, T_m < 25 °C). ^(13)C NMR spectra at the pentad analysis level indicates that the degree of isotacticity is 99% mmmm for all polymer samples. 27 is the first single component iso-specific α-olefin polymerization catalyst. The presumed origins of the high isospecificity are presented.

On-chip integrated label-free optical biosensing

This thesis investigates the design and implementation of a label-free optical biosensing system utilizing a robust on-chip integrated platform. The goal has been to transition optical micro-resonator based label-free biosensing from a laborious and delicate laboratory demonstration to a tool for the analytical life scientist. This has been pursued along four avenues: (1) the design and fabrication of high-$Q$ integrated planar microdisk optical resonators in silicon nitride on silica, (2) the demonstration of a high speed optoelectronic swept frequency laser source, (3) the development and integration of a microfluidic analyte delivery system, and (4) the introduction of a novel differential measurement technique for the reduction of environmental noise.

The optical part of this system combines the results of two major recent developments in the field of optical and laser physics: the high-$Q$ optical resonator and the phase-locked electronically controlled swept-frequency semiconductor laser. The laser operates at a wavelength relevant for aqueous sensing, and replaces expensive and fragile mechanically-tuned laser sources whose frequency sweeps have limited speed, accuracy and reliability. The high-$Q$ optical resonator is part of a monolithic unit with an integrated optical waveguide, and is fabricated using standard semiconductor lithography methods. Monolithic integration makes the system significantly more robust and flexible compared to current, fragile embodiments that rely on the precarious coupling of fragile optical fibers to resonators. The silicon nitride on silica material system allows for future manifestations at shorter wavelengths. The sensor also includes an integrated microfluidic flow cell for precise and low volume delivery of analytes to the resonator surface. We demonstrate the refractive index sensing action of the system as well as the specific and nonspecific adsorption of proteins onto the resonator surface with high sensitivity. Measurement challenges due to environmental noise that hamper system performance are discussed and a differential sensing measurement is proposed, implemented, and demonstrated resulting in the restoration of a high performance sensing measurement.

The instrument developed in this work represents an adaptable and cost-effective platform capable of various sensitive, label-free measurements relevant to the study of biophysics, biomolecular interactions, cell signaling, and a wide range of other life science fields. Further development is necessary for it to be capable of binding assays, or thermodynamic and kinetics measurements; however, this work has laid the foundation for the demonstration of these applications.

Sequence specific complexation of b DNA at sites containing g,c base pairs

A series of eight related analogs of distamycin A has been synthesized. Footprinting and affinity cleaving reveal that only two of the analogs, pyridine-2- car box amide-netropsin (2-Py N) and 1-methylimidazole-2-carboxamide-netrops in (2-ImN), bind to DNA with a specificity different from that of the parent compound. A new class of sites, represented by a TGACT sequence, is a strong site for 2-PyN binding, and the major recognition site for 2-ImN on DNA. Both compounds recognize the G•C bp specifically, although A's and T's in the site may be interchanged without penalty. Additional A•T bp outside the binding site increase the binding affinity. The compounds bind in the minor groove of the DNA sequence, but protect both grooves from dimethylsulfate. The binding evidence suggests that 2-PyN or 2-ImN binding induces a DNA conformational change.

In order to understand this sequence specific complexation better, the Ackers quantitative footprinting method for measuring individual site affinity constants has been extended to small molecules. MPE•Fe(II) cleavage reactions over a 10^5 range of free ligand concentrations are analyzed by gel electrophoresis. The decrease in cleavage is calculated by densitometry of a gel autoradiogram. The apparent fraction of DNA bound is then calculated from the amount of cleavage protection. The data is fitted to a theoretical curve using non-linear least squares techniques. Affinity constants at four individual sites are determined simultaneously. The distamycin A analog binds solely at A•T rich sites. Affinities range from 10^(6)- 10^(7)M^(-1) The data for parent compound D fit closely to a monomeric binding curve. 2-PyN binds both A•T sites and the TGTCA site with an apparent affinity constant of 10^(5) M^(-1). 2-ImN binds A•T sites with affinities less than 5 x 10^(4) M^(-1). The affinity of 2-ImN for the TGTCA site does not change significantly from the 2-PyN value. At the TGTCA site, the experimental data fit a dimeric binding curve better than a monomeric curve. Both 2-PyN and 2-ImN have substantially lower DNA affinities than closely related compounds.

In order to probe the requirements of this new binding site, fourteen other derivatives have been synthesized and tested. All compounds that recognize the TGTCA site have a heterocyclic aromatic nitrogen ortho to the N or C-terminal amide of the netropsin subunit. Specificity is strongly affected by the overall length of the small molecule. Only compounds that consist of at least three aromatic rings linked by amides exhibit TGTCA site binding. Specificity is only weakly altered by substitution on the pyridine ring, which correlates best with steric factors. A model is proposed for TGTCA site binding that has as its key feature hydrogen bonding to both G's by the small molecule. The specificity is determined by the sequence dependence of the distance between G's.

One derivative of 2-PyN exhibits pH dependent sequence specificity. At low pH, 4-dimethylaminopyridine-2-carboxamide-netropsin binds tightly to A•T sites. At high pH, 4-Me_(2)NPyN binds most tightly to the TGTCA site. In aqueous solution, this compound protonates at the pyridine nitrogen at pH 6. Thus presence of the protonated form correlates with A•T specificity.

The binding site of a class of eukaryotic transcriptional activators typified by yeast protein GCN4 and the mammalian oncogene Jun contains a strong 2-ImN binding site. Specificity requirements for the protein and small molecule are similar. GCN4 and 2-lmN bind simultaneously to the same binding site. GCN4 alters the cleavage pattern of 2-ImN-EDTA derivative at only one of its binding sites. The details of the interaction suggest that GCN4 alters the conformation of an AAAAAAA sequence adjacent to its binding site. The presence of a yeast counterpart to Jun partially blocks 2-lmN binding. The differences do not appear to be caused by direct interactions between 2-lmN and the proteins, but by induced conformational changes in the DNA protein complex. It is likely that the observed differences in complexation are involved in the varying sequence specificity of these proteins.

Gut microbiota promote hematopoiesis to control bacterial infection

The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential of specific myeloid cell progenitors of both yolk sac and bone marrow origin. Homeostatic innate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral antibiotic-treated mice display increased pathogen burden and acute death. Recolonization of germ-free mice with a complex microbiota restores defects in myelopoiesis and resistance to Listeria. These findings reveal that gut bacteria direct innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota.

Part I: Latent heat of vaporization of n-decane. Part II: Heat and mass transfer from a cylinder placed in a turbulent air stream - Sherwood and Frössling numbers as a function of free stream turbulence level and Reynolds number

Part I

The latent heat of vaporization of n-decane is measured calorimetrically at temperatures between 160° and 340°F. The internal energy change upon vaporization, and the specific volume of the vapor at its dew point are calculated from these data and are included in this work. The measurements are in excellent agreement with available data at 77° and also at 345°F, and are presented in graphical and tabular form.

Part II

Simultaneous material and energy transport from a one-inch adiabatic porous cylinder is studied as a function of free stream Reynolds Number and turbulence level. Experimental data is presented for Reynolds Numbers between 1600 and 15,000 based on the cylinder diameter, and for apparent turbulence levels between 1.3 and 25.0 per cent. n-heptane and n-octane are the evaporating fluids used in this investigation.

Gross Sherwood Numbers are calculated from the data and are in substantial agreement with existing correlations of the results of other workers. The Sherwood Numbers, characterizing mass transfer rates, increase approximately as the 0.55 power of the Reynolds Number. At a free stream Reynolds Number of 3700 the Sherwood Number showed a 40% increase as the apparent turbulence level of the free stream was raised from 1.3 to 25 per cent.

Within the uncertainties involved in the diffusion coefficients used for n-heptane and n-octane, the Sherwood Numbers are comparable for both materials. A dimensionless Frössling Number is computed which characterizes either heat or mass transfer rates for cylinders on a comparable basis. The calculated Frössling Numbers based on mass transfer measurements are in substantial agreement with Frössling Numbers calculated from the data of other workers in heat transfer.