Browsing by Author "Diehl, Michael"

Now showing 1 - 8 of 8
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    A 3D microfluidic tumor model to study immune cell infiltration into solid tumors
    (2017-08-07) Vu, Timothy Q; Diehl, Michael
    The tumor microenvironment consists of other cells besides cancer cells, including immune cells like T-cells, natural killer cells, and macrophages. The roles of these infiltrated cells, however, are diverse and can be both pro- or anti-tumorigenic. The effects of infiltrated natural killer cells are poorly characterized and highly misunderstood. To better study both the mechanics and effects of infiltrating NK cells, advances to 3D in vitro cancer models are necessary. To this end, I have developed a novel microfluidic device for culturing 3D tumor tissue, which can recapitulate the interstitial flow and gradient microenvironment. I utilize this device to create novel methods in quantifying NK cell infiltration and demonstrate the utility of this device for both infiltration and post-infiltration analysis.
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    A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function
    (Rockefeller University Press, 2019) Lam, Michael T.; Coppola, Simona; Krumbach, Oliver H.F.; Prencipe, Giusi; Insalaco, Antonella; Cifaldi, Cristina; Brigida, Immacolata; Zara, Erika; Scala, Serena; Di Cesare, Silvia; Martinelli, Simone; Di Rocco, Martina; Pascarella, Antonia; Niceta, Marcello; Pantaleoni, Francesca; Ciolfi, Andrea; Netter, Petra; Carisey, Alexandre F.; Diehl, Michael; Akbarzadeh, Mohammad; Conti, Francesca; Merli, Pietro; Pastore, Anna; Levi Mortera, Stefano; Camerini, Serena; Farina, Luciapia; Buchholzer, Marcel; Pannone, Luca; Cao, Tram N.; Coban-Akdemir, Zeynep H.; Jhangiani, Shalini N.; Muzny, Donna M.; Gibbs, Richard A.; Basso-Ricci, Luca; Chiriaco, Maria; Dvorsky, Radovan; Putignani, Lorenza; Carsetti, Rita; Janning, Petra; Stray-Pedersen, Asbjorg; Erichsen, Hans Christian; Horne, AnnaCarin; Bryceson, Yenan T.; Torralba-Raga, Lamberto; Ramme, Kim; Rosti, Vittorio; Bracaglia, Claudia; Messia, Virginia; Palma, Paolo; Finocchi, Andrea; Locatelli, Franco; Chinn, Ivan K.; Lupski, James R.; Mace, Emily M.; Cancrini, Caterina; Aiuti, Alessandro; Ahmadian, Mohammad R.; Orange, Jordan S.; De Benedetti, Fabrizio; Tartaglia, Marco
    Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
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    Long-lived Luminescent Metal Complexes for Molecules Sensing and Nanotube Dispersion
    (2014-09-19) Huang, Kewei; Marti, Angel A.; Wilson, Lon J; Diehl, Michael
    Phosphorescent heavy-metal complexes are one class of excellent photoluminescent materials. The heavy metal-induced spin–orbit coupling leads to singlet–triplet state mixing, thus decreases the “spin-forbidden” component of the radiative relaxation of the triplet state, and consequently improves the phosphorescence quantum efficiency and radiative emission lifetime. Moreover, the emission wavelength of metal complexes can be easily tuned through the ligand modification and the change of central metals. Ruthenium(II) and Iridium(III)-based complexes have d6 electronic structures. The advantageous photophysical properties including long lifetime, large Stokes shift and long wavelength excitation provide them to be good candidates for chemosensors. This thesis focuses on the development of novel iridium and ruthenium complexes for small molecules sensing. Their long-lived photoluminescence lifetime allows detecting analytes even in the presence of short-lived background fluorescence by using time-gating techniques. An overview of the developing trends in molecular beacon design and applications will be introduced in Chapter 1. In Chapter 2, the long-lived emission of Ir(III) and Ru(II) complexes are combined with time-resolved spectroscopic techniques for optimizing the sensitivity of molecular beacons. A novel iridium complex with long-lived photoluminescence will be discussed in Chapter 3, which can be used for the detection of thiol-containing amino acids in the presence of strong background fluorescence. In Chapter 4, pre-exponential factors derived from time-resolved experiments will be applied for quantifying free histidine in mixtures with histidine-containing proteins. The last Chapter is the development of the new application of Ruthenium complexes as a media for dispersion nanotubes in aqueous solution.
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    Mesenchymal stem cells and macrophages interact through IL-6 to promote inflammatory breast cancer in pre-clinical models
    (Impact Journals, LLC, 2016) Wolfe, Adam R.; Trenton, Nicholaus J.; Debeb, Bisrat G.; Larson, Richard; Ruffell, Brian; Chu, Khoi; Hittelman, Walter; Diehl, Michael; Reuben, Jim M.; Ueno, Naoto T.; Woodward, Wendy A.
    Inflammatory breast cancer (IBC) is a unique and deadly disease with unknown drivers. We hypothesized the inflammatory environment contributes to the IBC phenotype. We used an in vitro co-culture system to investigate interactions between normal and polarized macrophages (RAW 264.7 cell line), bone-marrow derived mesenchymal stem cells (MSCs), and IBC cells (SUM 149 and MDA-IBC3). We used an in vivo model that reproduces the IBC phenotype by co-injecting IBC cells with MSCs into the mammary fat pad. Mice were then treated with a macrophage recruitment inhibitor, anti-CSF1. MSC and macrophages grown in co-culture produced higher levels of pro-tumor properties such as enhanced migration and elevated IL-6 secretion. IBC cells co-cultured with educated MSCs also displayed enhanced invasion and mammosphere formation and blocked by anti-IL-6 and statin treatment. The treatment of mice co-injected with IBC cells and MSCs with anti-CSF1 inhibited tumor associated macrophages and inhibited pSTAT3 expression in tumor cells. Anti-CSF1 treated mice also exhibited reduced tumor growth, skin invasion, and local recurrence. Herein we demonstrate reciprocal tumor interactions through IL-6 with cells found in the IBC microenvironment. Our results suggest IL-6 is a mediator of these tumor promoting influences and is important for the IBC induced migration of MSCs.
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    MSANTD3, a noval transcription factor, recruits PRC2 complex to regulate neuron differentiation in mouse P19 cells
    (2014-12-05) Gou, Yufeng; Ma, Jianpeng; Diehl, Michael; Tao, Yizhi
    Polycomb repressive complex (PRC) 2 functions to repress thousands of target genes, and they are responsible for stem cell differentiation and carcinogenesis. However, how PRC2 are recruited to specific regions of their target genes remains elusive. In Drosophila, nine sequence-specific transcription factors including Zeste have been shown to act as PRC2 recruiters, but little is known about their homologues in mammalian cells, as a straightforward homology search failed to work in most cases. Aided by three-dimensional structure prediction and the use of the genomes of intermediate bridging species, we have identified a new protein, Myb/SANT-Like DNA-Binding Domain-Containing Protein 3 (MSANTD3), as the human homologue of Drosophila Zeste. Gel shift assays indicated that MSANTD3 binds to Zeste recognition sequence via its N-terminal DNA binding domain. MSANTD3 physically interacted with the components of PRC2 in GST-pull down assays and can be co-purified from native mouse P19 cells with active tri-methylation activity. Chip-seq and Chip-qPCR experiments showed the co-localization of MSANTD3 with PRC2 complex on neuron differentiation genes. Together, these findings revealed the critical function of MSANTD3 in recruiting PRC2 complexes to regulate neuron differentiation.
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    Multiplexed in situ molecular analyses and programmable molecular probes for regulated single amplification
    (2018-12-11) Diehl, Michael; Zimak, Jan; Schweller, Ryan; Samson, Edward B.; Duose, Dzifa Y.; Rice University; United States Patent and Trademark Office
    The present invention generally relates to methods for detecting a target in a sample; methods for modulating the reporting intensity of a labeled target in a sample of fixed cells or tissues; methods for detecting the location of at least two targets in a sample; and related compositions.
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    The role of actin-binding proteins in shaping the dynamics and structures of actomyosin networks
    (2022-12-01) Liman, James; Cheung, Margaret S.; Diehl, Michael; Wolynes, Peter G.
    Actomyosin networks give cells the ability to move and divide. These networks contract and expand while being driven by active energy-consuming processes such as motor protein walking and actin polymerization. Actin dynamics is also regulated by actin-binding proteins, such as the actin-related protein 2/3 (Arp2/3) complex and calcium/calmodulin-dependent protein kinase II (CaMKII) complex. Arp2/3 generates branched filaments while CaMKII binds and bundles actin filaments, thereby changing the overall organization and dynamics of the network. While the structures of Arp2/3 and CaMKII have been explored extensively, how these complexes change the architectural dynamics of the actomyosin network raises many questions. In this work, the spatiotemporal patterns of dynamical actin assembly accompanying reorganization caused by Arp2/3 and CaMKII were studied using both a mass action kinetic model and a computational model (mechanochemical dynamics of active networks or MEDYAN). This computational model simulates actomyosin network dynamics as a result of chemical reactions whose rates are modulated by rapid mechanical equilibration. We show that branched actomyosin networks relax significantly more slowly than do unbranched networks. Also, branched networks undergo rare convulsive movements, termed “avalanches”, that release strain in the network. These avalanches are associated with the more heterogeneous distribution of mechanically-linked filaments displayed by branched networks. These far-from equilibrium events arising from the marginal stability of growing actomyosin networks provide a possible mechanism of the “cytoquakes” recently seen in experiments. We show that the multivalent interactions between CaMKII and actin enable rich structural arrangements of actin filaments. Unlike branched networks, networks with CaMKII resemble crystalline networks due to abundant connections between CaMKII and actin filaments.
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    Understanding Receptor-Mediated NK Cell Adhesion and Motility Throughout development
    (2019-12-05) Lee, Barclay; Diehl, Michael; Mace, Emily
    Human natural killer cells originate from hematopoietic stem cells and can be differentiated in vitro through coculture with EL08.1D2 cells, a developmentally supportive stromal cell line. It is thought that these stroma support NK cell maturation by providing signaling cues and structural support that the stem cell would normally experience within tissue in vivo. These cues from the local microenvironment induce changes in cell motility and adhesion to guide cell fate. Although these signals are well studied in T and B lymphocytes, the full extent of these interactions is currently not well understood for NK cell development. In this dissertation, I describe changes in NK cell motility and adhesion that occur throughout development. Specifically, I show that developing NK cells undergo continuous changes in cell migration velocity and displacement that define their differentiation from precursor to functionally mature cell. In addition, I present extracellular matrix (ECM) derived from EL08.1D2 stroma as an alternate substrate for supporting NK cell development. This cell-derived matrix is composed of naturally secreted ECM components from the stroma and contains expected ECM components such as collagen and fibronectin. I show that this cell-derived matrix can support NK cell differentiation from hematopoietic precursor cells, and I further define the migratory and adhesive behaviors of NK developmental intermediates on EL08.1D2 and EL08.1D2-derived cell free matrix. Particularly, NK cell motility is acquired through development and NK cells undergo similar motility on both EL08.1D2 stroma and cell-derived matrix. The information presented in this work defines the extent to which developmentally supportive stroma are required for NK cell development in vitro and provides an avenue into research for alternative substrates and conditions for NK cell development.