HOST FACTORS ASSOCIATED WITH HEPATITIS B VIRUS LIFE-CYCLE: INSIDE AND OUT

An estimated 257 million people are chronically infected with hepatitis B virus (HBV), which is a major cause of mortality among viral hepatitis related deaths despite the availability of a safe and effective vaccine. Current antiviral therapies against HBV focus on the suppression of viral replication and although improving the quality of life and long-term survival of patients this therapy only rarely leads to functional cure. To comply with the goals of WHO (summarized in “Global Health Sector Strategy on viral hepatitis”), more potent HBV therapeutics are needed and thus, a better understanding of the basics of HBV biology is essential to explore novel antiviral strategies. Hepatocytes infected with HBV contain highly stable episomal viral genomes, called covalently closed circular DNA (cccDNA), acting as a template for the synthesis of all viral transcripts. In addition, genomic integration of replication-defective, partial HBV genomes is frequently observed early after infection, which is thought to contribute to the chronicity of hepatitis B by providing a stable reservoir for the production HBV antigens. The aim of my thesis was to delineate host cell factors contributing to the HBV replication cycle. To this end, I addressed two specific aspects: First, to identify host factors modulating the secretion of subviral particles (SVPs) that originate from sub-genomic HBV integrates; second, to determine host factors incorporated into HBV virions and subviral particles in the course of particle morphogenesis. With respect to the first aim, I conducted an RNAi-based mini-screen using a custom-made library. I found that the neddylation pathway components Nedd8 and Ube2m are required for hepatitis B surface antigen (HBsAg). Knock-down of these components or pharmacological inhibition with the small molecule neddylation inhibitor MLN4924 inhibited the secretion of HBsAg. Previously, MLN4924 was reported to re-instate an epigenetic cccDNA silencing complex called Smc5/6 (structural maintenance of chromosomes complex 5/6) by preventing its HBx dependent degradation. I found that in addition, neddylation also contributes to the secretion of HBsAg from integrated HBV genome sequences in an HBx-independent manner. To sum up, my siRNA screen identified a previously unknown role of the neddylation pathway on HBsAg secretion that is independent of cccDNA. In the second part of my thesis, I established a protocol combining affinity chromatography, size exclusion chromatography, isopycnic centrifugation, and HBsAg immune-precipitation to enrich HBV particles and SVPs derived from two independent cell lines. Obtained particles were analyzed by mass spectrometry to determine their proteome. In this way, we identified approximately 160 proteins associated with both particle fractions, 6 of them exclusively detected in HBV virions. In addition to the previously published HBV associated proteins, we could identify numerous novel host factors, laying the ground for mechanistic follow-up studies and the possible use of identified host factors as targets for novel antiviral therapy

Enhanced cytotoxicity of silver complexes bearing bidentate N-heterocyclic carbene ligands

A diverse library of cationic silver complexes bearing bis(N-heterocyclic carbene) ligands have been prepared which exhibit cytotoxicity comparable to cisplatin against the adenocarcinomas MCF7 and DLD1. Bidentate ligands show enhanced cytotoxicity over monodentate and macrocyclic ligands

The Effects of Tooth Extraction and Nonextraction on the Soft Tissue Profile in Patients With Class II Division 2 Malocclusion

Objective:To determine and compare change in the position and area of the upper and lower lips in patients with Class II division 2 malocclusion treated with and without tooth extraction.Materials and Method:This retrospective study included 40 female adolescent patients with Class II division 2 malocclusion who were divided into an extraction treatment group (group E, n=20) and a nonextraction treatment group (group NE, n=20). Pretreatment (T1) and posttreatment (T2) cephalometric variables, and upper-lower lip area (via digital planimeter) were measured on lateral cephalometric radiographs. For soft tissue profile area measurement the upper lip was divided into 2 parts (area 1 and 2), and the lower lip was divided into 3 parts (area 3, 4, and 5). The paired t test was used to determine intragroup differences, and the Student t test was used to determine intergroup differences.Results:Mean age in group E was 15.8 years vs 15.4 years in group NE. A wide range of individual responses to the treatment protocols were observed, and none of the skeletal or soft tissue changes differed significantly between the groups.Conclusion:Despite great variability in posttreatment soft tissue changes—including area measurements—the mean values for all measurements were similar in group E and group NE. Nonetheless, due to the observed variation in soft tissue response, we think that treatment planning should be carefully tailored to the needs of each patient

Bis{1,4-bis­[(3-butyl­imidazolium-1-yl)meth­yl]benzene}­silver(I) bis­(hexa­fluoridophosphate)

The asymmetric unit of the title complex, [Ag2(C22H30N4)2](PF6)2, consists of one AgI ion, one 1,4-bis­[(3-butyl­imidazolium-1-yl)meth­yl]benzene ligand and one discrete hexa­fluoridophosphate anion. The formula unit is generated by an inversion center. The unique AgI ion is coordinated by two C atoms of two heterocyclic carbene ligands in an essentially linear geometry. In the crystal structure, cations and anions are linked through weak C—H⋯F hydrogen bonds, forming a three-dimensional network

Terahertz Bandpass Frequency Selective Surfaces on Glass Substrates Using a Wet Micromachining Process

This paper presents terahertz (THz) frequency selective surfaces (FSS) implemented on glass substrate using standard microfabrication techniques. These FSS structures are designed for frequencies around 0.8 THz. A fabrication process is proposed where a 100-μm-thick glass substrate is formed through the HF etching of a standard 500-μm-thick low cost glass wafer. Using this fabrication process, three separate robust designs consisting of single-layer FSS are investigated using high-frequency structural simulator (HFSS). Based on the simulation results, the first design consists of a circular ring slot in a square metallic structure on top of a 100-μm-thick Pyrex glass substrate with 70% transmission bandwidth of approximately 0.07 THz, which remains nearly constant till 30° angle of incidence. The second design consists of a tripole structure on top of a 100-μm-thick Pyrex glass substrate with 65% transmission bandwidth of 0.035 THz, which remains nearly constant till 30° angle of incidence. The third structure consists of a triangular ring slot in a square metal on top of a 100-μm-thick Pyrex glass substrate with 70% transmission bandwidth of 0.051 THz, which remains nearly constant up to 20° angle of incidence. These designs show that the reflections from samples can be reduced compared to the conventional sample holders used in THz spectroscopy applications, by using single layer FSS structures manufactured through a relatively simple fabrication process. Practically, these structures are achieved on a fabricated 285-μm-thick glass substrate. Taking into account the losses and discrepancies in the substrate thickness, the measured results are in good agreement with the electromagnetic simulations. © 2017, Springer Science+Business Media New York

Rotational coherence of encapsulated ortho and para water in fullerene-C₆₀ revealed by time-domain terahertz spectroscopy

We resolve the real-time coherent rotational motion of isolated water molecules encapsulated in fullerene-C60 cages by time-domain terahertz (THz) spectroscopy. We employ single-cycle THz pulses to excite the low-frequency rotational motion of water and measure the subsequent coherent emission of electromagnetic waves by water molecules. At temperatures below ~ 100 K, C60 lattice vibrational damping is mitigated and the quantum dynamics of confined water are resolved with a markedly long rotational coherence, extended beyond 10 ps. The observed rotational transitions agree well with low-frequency rotational dynamics of single water molecules in the gas phase. However, some additional spectral features with their major contribution at ~2.26 THz are also observed which may indicate interaction between water rotation and the C60 lattice phonons. We also resolve the real-time change of the emission pattern of water after a sudden cooling to 4 K, signifying the conversion of ortho-water to para-water over the course of 10s hours. The observed long coherent rotational dynamics of isolated water molecules confined in C60 makes this system an attractive candidate for future quantum technology

Unprecedented use of silver(I) N-heterocyclic carbene complexes for the catalytic preparation of 1,2-bis(boronate) esters

Catalytic diboration of internal and terminal alkenes with Ag(I) N-heterocyclic carbene complexes leads to 1,2-bis(boronate) esters as single intermediates, that can be oxidised towards the corresponding diols.Sanau Torrecilla, Mercedes, [email protected]