High light stress in photosynthesis: the role of oxidative post-translational modifications in signaling and repair

Oxidative stress is a natural consequence of photosynthetic oxygen evolution and redox enzyme processes. Trp oxidation to N-formylkynurenine (NFK) is a specific, reactive oxygen species (ROS)-mediated reaction. This thesis work describes the identification and functional characterization of NFK in oxygen evolving Photosystem II (PSII). Although proteomics studies have confirmed NFK modifications in many types of proteins, limited knowledge on the biochemical significance exists. In vitro studies in thylakoids and PSII membranes were used to establish a correlation between oxidative stress, NFK formation, and photoinhibition. The in vivo effect of preventing Trp oxidation to NFK was assessed by site-directed mutation in the cyanobacteria Synechocystis sp. PCC 6803. This work provides insight into the role of NFK in photosynthetic oxygen evolution and photoinhibition. Based on the current knowledge of NFK, ROS, and repair, a new model is described. In this modified model for photoinhibition and repair, NFK plays a role in signaling for turnover of damaged proteins. NFK may play a similar role in replacement of damaged proteins in other systems.PhDCommittee Chair: Bridgette A. Barry; Committee Member: David Collard; Committee Member: Ingeborg Schmidt-Krey; Committee Member: Wendy Kelly; Committee Member: Yomi Oyeler

\u3cem\u3ePhytophthora cinnamomi\u3c/em\u3e Colonized Reclaimed Surface Mined Sites in Eastern Kentucky: Implications for the Restoration of Susceptible Species

Appalachian forests are threatened by a number of factors, especially introduced pests and pathogens. Among these is Phytophthora cinnamomi, a soil-borne oomycete pathogen known to cause root rot in American chestnut, shortleaf pine, and other native tree species. This study was initiated to characterize the incidence of P. cinnamomi on surface mined lands in eastern Kentucky, USA, representing a range of time since reclamation (10, 12, 15, and 20 years since reclamation). Incidence of P. cinnamomi was correlated to soil properties including overall soil development, as indicated by a variety of measured soil physical and chemical parameters, especially the accumulation of soil organic carbon. P. cinnamomi was detected in only two of the four sites studied, aged 15 and 20 years since reclamation. These sites were generally characterized by higher organic matter accumulation than the younger sites in which P. cinnamomi was not detected. These results demonstrate that P. cinnamomi is capable of colonizing reclaimed mine sites in Appalachia; additional research is necessary to determine the impact of P. cinnamomi on susceptible tree species at these sites

Engineering gold nanotubes with controlled length and near-infrared absorption for theranostic applications

Important aspects in engineering gold nanoparticles for theranostic applications include the control of size, optical properties, cytotoxicity, biodistribution, and clearance. In this study, gold nanotubes with controlled length and tunable absorption in the near-infrared (NIR) region have been exploited for applications as photothermal conversion agents and in vivo photoacoustic imaging contrast agents. A length-controlled synthesis has been developed to fabricate gold nanotubes (NTs) with well-defined shape (i.e., inner void and open ends), high crystallinity, and tunable NIR surface plasmon resonance. A coating of poly(sodium 4-styrenesulfonate) (PSS) endows the nanotubes with colloidal stability and low cytotoxicity. The PSS-coated Au NTs have the following characteristics: i) cellular uptake by colorectal cancer cells and macrophage cells, ii) photothermal ablation of cancer cells using single wavelength pulse laser irradiation, iii) excellent in vivo photoacoustic signal generation capability and accumulation at the tumor site, iv) hepatobiliary clearance within 72 h postintravenous injection. These results demonstrate that these PSS-coated Au NTs have the ideal attributes to develop their potential as effective and safe in vivo imaging nanoprobes, photothermal conversion agents, and drug delivery vehicles. To the best of knowledge, this is the first in vitro and in vivo study of gold nanotubes

A Convergent Synthetic Platform for Single-Nanoparticle Combination Cancer Therapy: Ratiometric Loading and Controlled Release of Cisplatin, Doxorubicin, and Camptothecin

The synthesis of polymer therapeutics capable of controlled loading and synchronized release of multiple therapeutic agents remains a formidable challenge in drug delivery and synthetic polymer chemistry. Herein, we report the synthesis of polymer nanoparticles (NPs) that carry precise molar ratios of doxorubicin, camptothecin, and cisplatin. To our knowledge, this work provides the first example of orthogonally triggered release of three drugs from single NPs. The highly convergent synthetic approach opens the door to new NP-based combination therapies for cancer.MIT Research Support CommitteeLincoln Laboratory. Advanced Concepts CommitteeUnited States. Dept. of Defense (Ovarian Cancer Research Program Teal Innovator Award)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32EB017614-01)Natural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship)Natural Sciences and Engineering Research Council of Canada (Graduate Fellowship)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

Terahertz thermometry: combining hyperspectral imaging and temperature mapping at terahertz frequencies

The accurate and non-invasive determination of multiple physical parameters, with well-defined spatial resolution, is crucial for applications in manufacturing, chemistry, medicine and biology. Specifically, the ability to simultaneously measure both temperature and spectral signatures is still experimentally unavailable. To this end, we propose a mapping technique for biological systems, which exploits a linear correlation between terahertz wave reflectivity and temperature, and allows to spatially and spectrally resolve thermal distributions. This method is applied to a model biological system in two relevant cases where in one example, nanoplasmonic-induced photothermal effects are imaged gaining new insights into collective heating phenomena. In the second example, we demonstrate a joint thermal-hyperspectral imaging approach to chemically map the presence of a model drug formulation and simultaneously investigate its thermal stability in our biological system. This concept can be easily extended and widely applied to all materials that demonstrate a measurable change in their dielectric properties

Hyperspectral darkfield microscopy of single hollow gold nanoparticles for biomedical applications

Hyperspectral microscopy is a versatile method for simultaneous spatial and spectroscopic characterization of nonfluorescent samples. Here we present a hyperspectral darkfield imaging system for spectral imaging of single nanoparticles over an area of 150 × 150 µm2 and at illumination intensities compatible with live cell imaging. The capabilities of the system are demonstrated using correlated transmission electron microscopy and single-particle optical studies of colloidal hollow gold nanoparticles. The potential of the system for characterizing the interactions between nanoparticles and cells has also been demonstrated. In this case, the spectral information proves a useful improvement to standard darkfield imaging as it enables differentiation between light scattered from nanoparticles and light scattered from other sources in the cellular environment. The combination of low illumination power and fast integration times makes the system highly suitable for nanoparticle tracking and spectroscopy in live-cell experiments

A Nanoparticle-Based Combination Chemotherapy Delivery System for Enhanced Tumor Killing by Dynamic Rewiring of Signaling Pathways

Exposure to the EGFR (epidermal growth factor receptor) inhibitor erlotinib promotes the dynamic rewiring of apoptotic pathways, which sensitizes cells within a specific period to subsequent exposure to the DNA-damaging agent doxorubicin. A critical challenge for translating this therapeutic network rewiring into clinical practice is the design of optimal drug delivery systems. We report the generation of a nanoparticle delivery vehicle that contained more than one therapeutic agent and produced a controlled sequence of drug release. Liposomes, representing the first clinically approved nanomedicine systems, are well-characterized, simple, and versatile platforms for the manufacture of functional and tunable drug carriers. Using the hydrophobic and hydrophilic compartments of liposomes, we effectively incorporated both hydrophobic (erlotinib) and hydrophilic (doxorubicin) small molecules, through which we achieved the desired time sequence of drug release. We also coated the liposomes with folate to facilitate targeting to cancer cells. When compared to the time-staggered application of individual drugs, staggered release from tumor-targeted single liposomal particles enhanced dynamic rewiring of apoptotic signaling pathways, resulting in improved tumor cell killing in culture and tumor shrinkage in animal models.National Institutes of Health (U.S.) (NIH and Center for Cancer Nanotechnology Excellence, grant no. P30-CA14051)National Institutes of Health (U.S.) (NIH and Center for Cancer Nanotechnology Excellence, grant no. U54-CA151884)National Institutes of Health (U.S.) (NIH and Center for Cancer Nanotechnology Excellence, grant no. U54-CA112967)National Institutes of Health (U.S.) (NIH and Center for Cancer Nanotechnology Excellence, grant no. R01-ES015339)National Institutes of Health (U.S.) (NIH and Center for Cancer Nanotechnology Excellence, grant no. R21-ES020466)Breast Cancer Alliance (Exceptional Project Grant)National Science Foundation (U.S.) (Graduate Research Fellowship)National Health and Medical Research Council (Australia) (CJ Martin Fellowship)National Institutes of Health (U.S.) (Kirschstein NRSA 1F32EB017614-01)Natural Sciences and Engineering Research Council of Canada (post-doctoral fellowship)Kathy and Curt Marble Cancer Research FundDavid H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute Frontier Research Program

Tumor-Targeted Synergistic Blockade of MAPK and PI3K from a Layer-by-Layer Nanoparticle

Purpose: Cross-talk and feedback between the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR cell signaling pathways is critical for tumor initiation, maintenance, and adaptive resistance to targeted therapy in a variety of solid tumors. Combined blockade of these pathways—horizontal blockade—is a promising therapeutic strategy; however, compounded dose-limiting toxicity of free small molecule inhibitor combinations is a significant barrier to its clinical application. Experimental Design: AZD6244 (selumetinib), an allosteric inhibitor of Mek1/2, and PX-866, a covalent inhibitor of PI3K, were co-encapsulated in a tumor-targeting nanoscale drug formulation—layer-by-layer (LbL) nanoparticles. Structure, size, and surface charge of the nanoscale formulations were characterized, in addition to in vitro cell entry, synergistic cell killing, and combined signal blockade. In vivo tumor targeting and therapy was investigated in breast tumor xenograft-bearing NCR nude mice by live animal fluorescence/bioluminescence imaging, Western blotting, serum cytokine analysis, and immunohistochemistry. Results: Combined MAPK and PI3K axis blockade from the nanoscale formulations (160 ± 20 nm, −40 ± 1 mV) was synergistically toxic toward triple-negative breast (MDA-MB-231) and RAS-mutant lung tumor cells (KP7B) in vitro, effects that were further enhanced upon encapsulation. In vivo, systemically administered LbL nanoparticles preferentially targeted subcutaneous MDA-MB-231 tumor xenografts, simultaneously blocked tumor-specific phosphorylation of the terminal kinases Erk and Akt, and elicited significant disease stabilization in the absence of dose-limiting hepatotoxic effects observed from the free drug combination. Mice receiving untargeted, but dual drug-loaded nanoparticles exhibited progressive disease. Conclusions: Tumor-targeting nanoscale drug formulations could provide a more safe and effective means to synergistically block MAPK and PI3K in the clinic.United States. Department of Defense (OCRP Teal Innovator Award)National Institutes of Health (U.S.) (Grant NIBIB 1F32EB017614-02)Misrock FoundationNational Science Foundation (U.S.)Swiss National Science FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT (Support Grant P30-CA14051)National Cancer Institute (U.S.)National Science Foundation (U.S.) (Massachusetts Institute of Technology. Materials Research Science and Engineering Center. Shared Experimental Facilities Grant DMR-0819762)Breast Cancer Alliance (Exceptional Project Grant

Quantifying Lipid Contents in Enveloped Virus Particles with Plasmonic Nanoparticles

Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. Here, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different HIV-1 and Ebola virus-like particles (VLPs) using sample sizes of less than 3 × 106 particles is introduced. The assay evaluates both scattering intensity and resonance wavelength, and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. A correlation of the optical observables with absolute lipid contents is achieved by calibration of the plasmon coupling-based methodology with unilamellar liposomes of known PS or GM1 concentration. The performed studies reveal significant differences in the membrane of VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers.NIH grants RO1CA138509 (B.M.R.), RO1A1064099 (S. G., and 1R56Al104393 (B.M.R. and S. G.; Ethan Edmonds support (CHE 1156666

Redox-responsive branched-bottlebrush polymers for \u3ci\u3ein vivo\u3c/i\u3e MRI and fluorescence imaging

Stimuli-responsive multimodality imaging agents have broad potential in medical diagnostics. Herein, we report the development of a new class of branched-bottlebrush polymer dual-modality organic radical contrast agents—ORCAFluors—for combined magnetic resonance and near-infrared fluorescence imaging in vivo. These nitroxide radical-based nanostructures have longitudinal and transverse relaxation times that are on par with commonly used heavy-metal-based magnetic resonance imaging (MRI) contrast agents. Furthermore, these materials display a unique compensatory redox response: fluorescence is partially quenched by surrounding nitroxides in the native state; exposure to ascorbate or ascorbate/glutathione leads to nitroxide reduction and a concomitant 2- to 3.5-fold increase in fluorescence emission. This behaviour enables correlation of MRI contrast, fluorescence intensity and spin concentration with tissues known to possess high concentrations of ascorbate in mice. Our in vitro and in vivo results, along with our modular synthetic approach, make ORCAFluors a promising new platform for multimodality molecular imaging