Microcoil MRI of plants and algae at ultra-high field : an exploration of metabolic imaging

This thesis investigates the relations between metabolism and anatomy through the use of Magnetic Resonance Imaging (MRI). Two model systems are studied: Botryococcus braunii, a green oleaginous algae and Medicago truncatula, a small leguminous plant in symbiosis with Sinorhizobium meliloti bacteria. In order to map the variation of metabolic profiles across tissues, MRI is used extensively, including Chemical Shift Imaging, Magnetic Resonance Spectroscopy and Diffusion-Weigthed Imaging. Home-built microcoils for ultra-high magnetic field strength are developed for studying the model organisms. Solid state NMR/Biophysical Organic Chemistr

Toetsontwikkeling op virussen in Zantedeschia

Zantedeschia (=Calla) heeft zich ontwikkeld tot een belangrijk siergewas. Voor de productie van snijbloemen en potplanten is een goede kwaliteit vereist. Virus kan een sterk negatieve invloed hebben op de kwaliteit door o.a. groeimisvorming en kleurbreking op blad en bloem. Een kleine tien jaar geleden is de BKD op verzoek van het vak gestart met een keuring op o.a. zichtbaar virus. Sinds het groeiseizoen van 2003 zijn de virusproblemen ondanks de keuring alleen maar groter geworden. Het beperken van virusverspreiding in Zantedeschia is daarom recent in detail bestudeerd (PT-project 12048). Daarnaast is een goed toetsenpakket belangrijk om virusvrij uitgangsmateriaal te kunnen realiseren. Zonder robuuste toetsen op virussen in Zantedeschia is dit haast onmogelijk te verwezenlijken. Er zijn veel verschillende virussen gevonden in Zanedeschia en dit aantal is de afgelopen jaren verder gestegen. Een aantal virussen in Zanedeschia kan prima via serologische methoden als ELISA worden aangetoond; andere virussen alleen door middel van PCR. Voor sommige virussen in Zantedeschia waren bij de Bloembollenkeuringsdienst (BKD) en Praktijkonderzoek Plant en Omgeving (PPO-BBF) geen goede detectiemethoden aanwezig, of was bekend dat ze slecht met de bestaande toetsmethoden te detecteren zijn. Dit was een onbevredigende situatie, met name voor bedrijven die schoon uitgangsmateriaal willen uitleveren. Daarom heeft dit project als belangrijkste doel de kennis over virussen in Zantedeschia te vergroten en het pakket aan toetsmogelijkheden compleet te maken. De ELISA- en PCR-toetsen zijn binnen dit project gevalideerd met praktijkmateriaal en het protocol voor het toetsen op uitgangsmateriaal is geëvalueerd

Benchmarking High-Fidelity Pedestrian Tracking Systems for Research, Real-Time Monitoring and Crowd Control

High-fidelity pedestrian tracking in real-life conditions has been an important tool in fundamental crowd dynamics research allowing to quantify statistics of relevant observables including walking velocities, mutual distances and body orientations. As this technology advances, it is becoming increasingly useful also in society. In fact, continued urbanization is overwhelming existing pedestrian infrastructures such as transportation hubs and stations, generating an urgent need for real-time highly-accurate usage data, aiming both at flow monitoring and dynamics understanding. To successfully employ pedestrian tracking techniques in research and technology, it is crucial to validate and benchmark them for accuracy. This is not only necessary to guarantee data quality, but also to identify systematic errors. Currently, there is no established policy in this context. In this contribution, we present and discuss a benchmark suite, towards an open standard in the community, for privacy-respectful pedestrian tracking techniques. The suite is technology-independent and it is applicable to academic and commercial pedestrian tracking systems, operating both in lab environments and real-life conditions. The benchmark suite consists of 5 tests addressing specific aspects of pedestrian tracking quality, including accurate line-based crowd flux estimation, local density estimation, individual position detection and trajectory accuracy. The output of the tests are quality factors expressed as single numbers. We provide the benchmark results for two tracking systems, both operating in real-life, one commercial, and the other based on overhead depth-maps developed at TU Eindhoven, within the Crowdflow topical group. We discuss the results on the basis of the quality factors and report on the typical sensor and algorithmic performance. This enables us to highlight the current state-of-the-art, its limitations and provide installation recommendations, with specific attention to multi-sensor setups and data stitching

Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host

Function of monocytes and monocyte-derived macrophages in α1-antitrypsin deficiency.

α1-antitrypsin deficiency is the most widely recognised genetic disorder causing chronic obstructive pulmonary disease (COPD). Mutant Z α1-antitrypsin expression has previously been linked to intracellular accumulation and polymerisation of this proteinase inhibitor. Subsequently, this has been described to underlie an exaggerated endoplasmic reticulum stress response and enhanced nuclear factor-κB signalling. However, whether monocyte-derived macrophages display the same features remains unknown. Monocytes from homozygous PiZZ α1-antitrypsin deficiency patients and PiMM controls were cultured for 6 days in the presence of granulocyte-macrophage or macrophage colony-stimulating factor to obtain pro- and anti-inflammatory macrophages (mφ-1 and mφ-2, respectively). We first showed that, in contrast to monocytes, pre-stressed mφ-1 and mφ-2 from healthy blood donors display an enhanced endoplasmic reticulum stress response upon a lipopolysaccharide trigger (XBP1 splicing, CHOP, GADD34 and GRP78 mRNA). However, this endoplasmic reticulum stress response did not differ between monocyte-derived macrophages and monocytes from ZZ patients compared to MM controls. Furthermore, these ZZ cells do not secrete higher cytokine levels, and α1-antitrypsin polymers were not detectable by ELISA. These data suggest that monocyte-derived macrophages are not the local source of Z α1-antitrypsin polymers found in the lung and that endoplasmic reticulum stress and pro-inflammatory cytokine release is not altered.This study was supported by a grant from the Netherlands Asthma Foundation (grant no. 3.2.08.0032). E.F.A. van't Wout is an European Alpha-1-Antitrypsin Laurell’s Training Awardee (sponsored by Grifols, Barcelona, Spain). D.A. Lomas is supported by the Medical Research Council (London, UK) and the NIHR UCLH Biomedical Research Centre (London). S.J. Marciniak is a Medical Research Council Senior Clinical Research Fellow (grant no. G1002610)

Organoid-based expansion of patient-derived primary alveolar type 2 cells for establishment of alveolus epithelial Lung-Chip cultures

Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.Microscopic imaging and technolog

A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress

Endoplasmic reticulum (ER) stress is increasingly recognized as an important mechanism in a wide range of diseases including cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's and Alzheimer's disease. Therefore, there is an increased need for reliable and quantitative markers for detection of ER stress in human tissues and cells. Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause ER stress, which leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of X-box binding protein-1 (XBP1) mRNA, which is frequently used as a marker for ER stress. In most studies, the splicing of the XBP1 mRNA is visualized by gel electrophoresis which is laborious and difficult to quantify. In the present study, we have developed and validated a quantitative real-time RT-PCR method to detect the spliced form of XBP1 mRNA

The positive prognostic effect of stromal CD8+tumor-infiltrating T cells is restrained by the expression of HLA-E in non-small cell lung carcinoma

Analysis and Stochastic

Acute cigarette smoke exposure leads to higher viral infection in human bronchial epithelial cultures by altering interferon, glycolysis and GDF15-related pathways

Background Acute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects. Methods Primary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air-liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and l-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, l-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge. Results CS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and l-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline. Conclusion Altogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15related pathways.Pathogenesis and treatment of chronic pulmonary disease