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At the atomic-cluster scale, pure boron is markedly similar to carbon, forming simple planar molecules and cage-like fullerenes. Theoretical studies predict that two-dimensional (2D) boron sheets will adopt an atomic configuration similar to that of boron atomic clusters. We synthesized atomically thin, crystalline 2D boron sheets (i.e., borophene) on silver surfaces under ultrahigh-vacuum conditions. Atomic-scale characterization, supported by theoretical calculations, revealed structures reminiscent of fused boron clusters with multiple scales of anisotropic, out-of-plane buckling. Unlike bulk boron allotropes, borophene shows metallic characteristics that are consistent with predictions of a highly anisotropic, 2D metal.

Glioblastoma is a highly malignant and incurable brain tumor characterized by intrinsic and adaptive resistance to immunotherapies. However, how glioma cells induce tumor immunosuppression and escape immunosurveillance remains poorly understood. Here, we find upregulation of cancer-intrinsic chitinase-3-like 1 (CHI3L1) signaling modulating an immunosuppressive microenvironment by reprogramming tumor-associated macrophages (TAMs). Mechanistically, CHI3L1 binding with galectin 3 (Gal3) selectively promotes TAM migration and infiltration with a protumor M2-like, but not an antitumor M1-like, phenotype in vitro and in vivo, governed by a transcriptional program of NF-[kappa]B/CEBP[beta] in the CHI3L1/Gal3-PI3K/AKT/mTOR axis. Conversely, galectin 3-binding protein (Gal3BP) negatively regulates this process by competing with Gal3 to bind CHI3L1. Administration of a Gal3BP mimetic peptide in syngeneic glioblastoma mouse models reverses immune suppression and attenuates tumor progression. These results shed light on the role of CHI3L1 protein complexes in immune evasion by glioblastoma and as a potential immunotherapeutic target for this devastating disease.

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg 2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg 2+ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg 2+ and the primer 3′-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication. The intermediate states occurring during nucleotide addition by HIV-1 RT remain unclear. Here, authors use cryo-EM to capture five unique states that show how a mobile catalytic Mg 2+ drives phosphodiester bond formation.

DNA-directed RNA Polymerase II (Pol II) is one of the most important molecules in biology. Pol II is highly-conserved among eukaryotic organisms and plays a fundamental role in cellular life, the transcription of genes into messenger RNA. Through technology development and innovation in structural biology approaches we have uncovered the time evolution of the molecular events during Pol II transcription elongation using optochemical approaches. In order to perform real time studies of transcription, we demonstrate that 1) a photoactive caged ATP can bind to Pol II; and 2) that shinning a UV light source (365 nm) can break the nitrobenzyl group (NPE) in crystallo. Further experiments employing a UV light source coupled to data collection above the glass transition temperature (140 and 160 Kelvin) of Pol II crystals allowed NPE release, Mg coordination and nucleotide addition. We extrapolate this approach to the small GTPases NRAS and KRAS capturing conformational changes on the path to GTP hydrolysis. Taken together these experiments illustrate that it is possible to observe chemical transition states at temperatures above water’s glass transition temperature.

Reverse transcription of the HIV-1 retrovirus single-stranded RNA genome into double stranded DNA is an essential step in HIV-1 replication, and reverse transcriptase (RT), the enzyme that catalyzes this reaction, is a target for antiretroviral therapy. HIV-1 RT structures have been available for a number of years. However, these structures represent thermodynamically stable species and do not inform on kinetic intermediates. Moreover, structures for the RT-T/P-dNTP complexes utilized substrate analogs (i.e., a dideoxy-terminated primer or a non-hydrolyzable dNTP) which prevent phosphodiester (PDE) bond formation. Therefore, there are no available structures of a “live” dNTP in RT’s active site and existing structures may not reflect catalytically active complexes. To address this issue, we took advantage of the single particle cryo-EM framework devising “diffusion/blot experiments” where enzyme and substrates are applied on opposite sides of a grid allowing mixing of both reaction components by diffusion on the grid through the matrix of holes. By freezing grids after short diffusion times (4-12s), we have for the first time elucidated several intermediate kinetic states during HIV-1 Reverse Transcriptase (RT) mediated nucleotide (dATP) incorporation. First, dATP binding to RT-template/primer involves a single Mg2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg2+ can bind (site A). Then, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg2+ and the primer 3ʹ-OH for nucleophilic attack. Finally, we captured a product state comprising two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Collectively, this study provides insights into a fundamental chemical reaction that impacts polymerase fidelity, nucleoside inhibitor drug design, and mechanisms of drug resistance.

In the present work, electrical measurements using in situ transmission electron microscopy (TEM) on pentagonal silver nanowires were performed. Electrical biasing was applied to individual nanowires with and without simultaneous in situ TEM mechanical deformation. The response of the ohmic resistance was measured in the I-V curves. A reduction in the break voltage and the resistance was measured, when the nanowires were subjected to a bending deformation. In situ electric measurements on both, with and without deformation, show a typical semiconductor behavior. Surface scattering of electrons in the nanowires and movement of dislocations act as the main causes of the electrical properties reported herein. In this way, the determination of the surface morphology was carried out by using off-axis electron holography followed by a phase reconstruction and structural modeling. The high Miller-index facets were determined to be the (533) stepped surface plane on all five longitudinal sides of the nanowires. Additionally, due to electrical saturation, a breakdown of the nanowires was observed during the in situ electrical measurements without mechanical deformation.

Glioblastoma is a highly malignant and incurable brain tumor characterized by intrinsic and adaptive resistance to immunotherapies. However, how glioma cells induce tumor immunosuppression and escape immunosurveillance remains poorly understood. Here, we find upregulation of cancer-intrinsic chitinase-3-like 1 (CHI3L1) signaling modulating an immunosuppressive microenvironment by reprogramming tumor-associated macrophages (TAMs). Mechanistically, CHI3L1 binding with galectin 3 (Gal3) selectively promotes TAM migration and infiltration with a protumor M2-like, but not an antitumor M1-like, phenotype in vitro and in vivo, governed by a transcriptional program of NF-KB/CEBPß in the CHI3L1/Gal3-PI3K/AKT/mTOR axis. Conversely, galectin 3-binding protein (Gal3BP) negatively regulates this process by competing with Gal3 to bind CHI3L1. Administration of a Gal3BP mimetic peptide in syngeneic glioblastoma mouse models reverses immune suppression and attenuates tumor progression. These results shed light on the role of CHI3L1 protein complexes in immune evasion by glioblastoma and as a potential immunotherapeutic target for this devastating disease.

The research represented in this thesis has a relationship with my sustained interest in the subject of cultural difference that typified my earlier artistic practice. During my Ph.D. studies in Art and Visual Culture at Western University, I have advanced new perspectives on this problematic by elaborating on the potentialities of listening in dialogic and collaborative artistic practice. It comprises the discussion about the projects and activities developed within my doctoral studies, according to two main and related purposes. The first is the examination of hegemonic practices of production of meaning regarding cultural difference with a backdrop of the social, cultural and historical processes that underlie the constitution of the space of modernity in my home country, Ecuador, one of the five nations that integrate the Andean region. The second is the analysis of artistic and collaborative activities where the cultivation of modes of listening (sounding implication, acoustic presence) foregrounds subaltern agency and the production of community. These preoccupations inform the analytical core of the two essays composing my thesis. Mountains and Rivers without End––a collaborative project involving artists and scholars from Canada and Ecuador in research on the historical, social, and environmental effects of mining in the district of Portovelo and Zaruma in Ecuador. Soundscape Pasochoa––a collaborative project developed in collaboration with José Sangoquiza that expand critical perspectives on the practice of the soundscapes to focus on the conditions for subaltern cultural production in the Valle of the Chillos, Pichincha province (Ecuador). A second section involving the presentation of my practice dossier includes documentation of the art projects and activities carried out during my doctoral studies. It also provides a brief discussion of the conceptual lines of the activities I carried out while being part of the research team of Surviving Memory in Postwar El Salvador. A comprehensive documentation of these projects and activities is found at https://ulises-unda-phd.squarespace.com/.