Practical implementation of robotic technologies for the inspection of ships

The ROBINS project aims at filling the technology and regulatory gaps that today still represent a barrier to the adoption of Robotics and Autonomous Systems (RAS) in activities related to inspection of ships, starting from understanding end user\u2019s actual needs and expectations and analyzing how existing or near-future technology can meet them. -Robotics technology: Improve the ability of RAS in sensing and probing; Improve capabilities in navigation and localization in confined spaces, access to and mobility within the environment; Improve safety and dependability of RAS in hazardous, harsh and dirty environments; Provide new tools for image and data processing (3D models, VR/AR environments); Provide the same level of information as obtained by direct human observation for the assessment of inspected structures. -Rules and Regulations: Provide a framework for the assessment of equivalence between the outcomes of RAS-assisted inspections and traditional inspection procedures; Define criteria, testing procedures and metrics for the evaluation of RAS performance; Design, implement and assess a testing environment where repeatable tests and measurements of RAS performance can be carried out; Improve confidence in technology capabilities by means of test campaigns to be performed both in the testing facilities and onboard. The expected impact can be summarized as follows: Wide scale adoption of RAS technology in marine industry Improved safety in ship surveys Economic advantages new supply chain and new potential markets particularly beneficial for SMEs; new services and products for data processing and knowledge management; reduction of costs related to inspection activities; improvement in the quality and variety of inspection services; new certification schemes for equipment, operators and procedures

Estimating causal networks in biosphere–atmosphere interaction with the PCMCI approach

Local meteorological conditions and biospheric activity are tightly coupled. Understanding these links is an essential prerequisite for predicting the Earth system under climate change conditions. However, many empirical studies on the interaction between the biosphere and the atmosphere are based on correlative approaches that are not able to deduce causal paths, and only very few studies apply causal discovery methods. Here, we use a recently proposed causal graph discovery algorithm, which aims to reconstruct the causal dependency structure underlying a set of time series. We explore the potential of this method to infer temporal dependencies in biosphere-atmosphere interactions. Specifically we address the following questions: How do periodicity and heteroscedasticity influence causal detection rates, i.e. the detection of existing and non-existing links? How consistent are results for noise-contaminated data? Do results exhibit an increased information content that justifies the use of this causal-inference method? We explore the first question using artificial time series with well known dependencies that mimic real-world biosphere-atmosphere interactions. The two remaining questions are addressed jointly in two case studies utilizing observational data. Firstly, we analyse three replicated eddy covariance datasets from a Mediterranean ecosystem at half hourly time resolution allowing us to understand the impact of measurement uncertainties. Secondly, we analyse global NDVI time series (GIMMS 3g) along with gridded climate data to study large-scale climatic drivers of vegetation greenness. Overall, the results confirm the capacity of the causal discovery method to extract time-lagged linear dependencies under realistic settings. The violation of the method's assumptions increases the likelihood to detect false links. Nevertheless, we consistently identify interaction patterns in observational data. Our findings suggest that estimating a directed biosphere-atmosphere network at the ecosystem level can offer novel possibilities to unravel complex multi-directional interactions. Other than classical correlative approaches, our findings are constrained to a few meaningful set of relations which can be powerful insights for the evaluation of terrestrial ecosystem models

SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES (SPIONS): DESIGN AND SYNTHESIS OF NEW NANOCONJUGATES

Superparamagnetic Iron Oxide Nanoparticles (SPIONs) have demonstrated great promise for diagnostic and therapeutic applications. Thanks to their magnetic properties and to their size, comparable to that of biologically objects, they are very useful for biomedical applications, such as, for example, automated DNA extraction, targeted gene delivery, magnetic resonance imaging (MRI), and magnetic field induced hyperthermia for cancer therapy.For these applications, SPIONs must be coupled with targeting agents, therapeutic drugs, and other functional probes. Hence, the need to develop efficient synthetic strategies for the conjugation of molecules to SPIONs is an important and appealing target. The strategies used can involve passive noncovalent adsorption on the outer particle surface or the formation of a more stable covalent bond by using appropriate heterobifunctional linkers, in which one functional group specifically binds the nanoparticle, while the other reacts with the biomolecule in order to form the new nanoconjugate. In this thesis, the use of an heterobifunctional linkers containing an isocyanate moiety as new functional group able to directly bind SPIONs will be shown. We were able to demonstrate that the NCO moiety is able to directly reacts with the surface hydroxyl groups exposed on the outer nanoparticles surface leading to a covalent carbamate-like bond. Comparison with classical non-covalent and covalent anchoring methodologies were also performed. To further confirm the possible application of this new anchoring methodology, new SPION-PNA (Peptide Nucleic Acid, mimics of natural oligonucleotides) nanoconjugates were synthesized and their binding affinity towards complementary DNAs were evalueted

Risk factors for unsuccessful lumbar puncture in children

Background. This descriptive study provides the first information on an association between the use of sedation and a reduction in the prevalence of unsuccessful lumbar puncture (LP) in African children of all races.Objective. Our hypothesis was that children who do not receive any procedural sedation are more likely to have unsuccessful LPs.Methods. A cross-sectional observational study examined LPs performed from February to April 2013, including details of the procedure, sedation or analgesia used, and techniques. The setting was the Medical Emergency Unit at Red Cross War Memorial Children’s Hospital, Cape Town, South Africa, and the participants all children aged 0 - 13 years who had an LP in the unit during the time period.Results. Of 350 children, 62.9% were <12 months of age, the median age being 4.8 months (interquartile range 1.5 - 21.7). The prevalence of unsuccessful (traumatic or dry) LP was 32.3% (113/350). Sedation was used in 107 children (30.6%) and was associated with a reduction in the likelihood of unsuccessful LP (p=0.002; risk ratio (RR) 0.5 (95% confidence interval (CI) 0.34 - 0.78)) except in those <3 months of age, where sedation did not significantly reduce the likelihood (p=0.56; RR 1.20 (95% CI 0.66 - 2.18)).Conclusions. Unsuccessful LP was common. Sedation was not routinely used, but the results suggest that it may be associated with a reduction in the rate of unsuccessful LP. Unsuccessful LP may lead to diagnostic uncertainty, prolonged hospitalisation and unnecessary antibiotic use. Whether a procedural sedation protocol would reduce the rate of unsuccessful LP requires further study

A new complication registration system for errors in radiology:Initial 5-year experience in a tertiary care radiology department

Purpose: To describe and evaluate our initial 5-year experience with a new complication registration system for errors in radiology. Materials and methods: This study reviewed all cases that were submitted to a new complication registration system of a tertiary care radiology department between 2015–2019. Results: Sixty-seven cases were included. In the group of diagnostic complications/errors (n = 34), there were 21 perceptual errors and 13 cognitive errors. This 61.8 % (21/34) perceptual error rate was not significantly different (P = 0.297) from the 70 % perceptual error rate known from previous literature. In the group of interventional complications (n=19), most cases (47.4 % [9/19]) concerned symptomatic or major hemorrhage. In the group of organizational complications/errors (n=14), the leading incident type according to the International Classification System for Patient Safety was clinical process/procedure with wrong body part/side/site as subclassification (35.7 % [5/14]). Harm severities were none (n=35), mild (n=10), moderate (n=10), severe (n=6), death (n=5), and unknown (n=1). Harm severity of interventional complications was significantly higher (P < 0.05) than that of organizational complications, while there were no significant differences in harm severities between other groups of complications. Conclusion: It is feasible to implement the radiologic complication registration system that was described in this study. Perceptual mistakes, hemorrhage, and procedures on the wrong body part/side/site dominated in the categories of diagnostic, interventional, and organizational complications/errors, respectively, and these should be the topic of vigilance in clinical practice and further research. Future studies are also required to determine whether this complication registration system reduces radiologic errors and improves healthcare quality

Generation of Multispecific Antibodies with Immune Cell Modulating Functions

Within the field of biologics, monoclonal antibodies have ruled the market with blockbuster and top selling drugs over the last decades. With the advancements in technological discoveries, lightspeed progress has been achieved to discover novel antibodies and mechanisms of action to address unmet needs and indications. Particularly, display technologies, in vitro systems, and high/ultra-high-throughput technologies have shown monumental progress to ensure the rapid discovery of novel biological entities. In the first study presented herein, the focus laid on the generation of a bidirectional plasmid for recombinant antibody production in mammalian cells to facilitate native antibody folding and post-translational modifications. Conventional approaches for transient antibody production utilise co-transfection of heavy and light chain genes encoded on separate plasmids. Here, a single plasmid under the control of two independent promoters, constructed in a bidirectional fashion, is used. This study assessed promoter combinations resulting in the best antibody yields of two U.S. Food and Drug Administration (FDA)-approved antibodies, durvalumab and avelumab. By comparing promoters with varying strengths (CMV, minCMV, EF-1α and enhanced CMV), gene expression of heavy and light chain genes and subsequent IgG1 yields gave rise to the 2xeCMV combination, consisting of two mirrored eCMV cassettes controlling the expression of the light and heavy chains individually in each direction. This combination effectuated the most promising mRNA synthesis for both chains in two regularly used mammalian cell lines, human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO) cells, and the highest yields after IgG quantification, comparable to the conventional co-transfection method. By substituting the co-transfection approach with this bidirectional plasmid, lower plasmid preparation efforts are required and further facilitates the handling of a higher number of mAb candidates simultaneously. In the second study, the described bidirectional plasmid was put into practise by generating a Fab-presenting yeast surface display (YSD) library from immunised OmniRats. After screening of antibody formats via fluorescence-activated cell sorting (FACS), reformation of single candidates into their final IgG format is required, rapidly converting itself into a cumbersome step, and often resulting in the bottleneck to proceed with further characterisation. Within this study, a novel workflow based on Golden Gate Cloning (GGC) was established, allowing the bulk reformatting of antibody candidates after YSD FACS screening. By using an OmniRat-derived Fab library against MerTK, two screening rounds of YSD were performed by FACS. Subsequently, the antibody-encoding genes were transferred into a Mammalian_Destination (MD) vector, which contained a partial hinge-CH2-CH3 sequence, resulting in a full-length heavy chain after GGC with Esp3I. In order to produce the full-length variants, the yeast-specific Gal1,10 promoter was exchanged for the described promoter cassette combinations from the first study, 2xeCMV, by a final GGC step with BbsI. After assembly, the resulting MD vector contained the variable domains from the second sorting round with the respective constant domains required for the production of full-length IgG molecules. Next generation sequencing (NGS) of the screening rounds confirmed that the entire VH family diversity was covered in the resulting clones after bulk reformatting. Ten candidates were subsequently transiently expressed in mammalian cells and characterised for target binding and biophysical properties. This workflow presented a two-pot, two-step, PCR-free method to transition from YSD to a mammalian expression vector, eliminating any unwanted polymerase-introduced mutations and allowing for bulk cloning of yeast display-enriched antibody fragments. By this procedure, heavy and light chain pairing is conserved, contrary to other reformatting approaches, and paves the way to accelerate antibody hit discovery campaigns with YSD. Furthermore, this platform is malleable to other antibody formats and immunisation hosts, such as single chain variable fragments (scFvs) and chickens, and has the potential to be developed for bispecific or multispecific antibodies. Next-generation antibodies, including bi- and multispecific antibodies, have been set under the spotlight for their ability to combine multiple modes of action simultaneously and result in higher efficacy, where monoclonal antibodies are lacking. A special class of such are immune cell engagers which target immune cells and tumour-associated antigens (TAAs) to create an immune synapse. Depending on the effector cell being targeted, specialised killing mechanisms are triggered to efficiently kill the targeted cells. Macrophage engagers are aimed at forcing targeted phagocytosis of the engaged cell type and have typically targeted the CD47/SIRPα axis up to date, known as the “do not eat me” signal. Nevertheless, targeting CD47 lacks specificity due to its ubiquitous expression pattern. On the other hand, T-cell engagers (TCEs) result in very specialised signals by targeting CD3 on T cells and additional TAAs. The hyperactivation of T cells results in a feedback loop through the activation of macrophages and the over-release of cytokines, resulting in cytokine storms or cytokine release syndrome (CRS). If left untreated, these can provoke life-threatening conditions. Thus, macrophage engagers and TCEs require novel cell-specific targets and widening of their therapeutic windows for restored patient alleviation. In the third study within this cumulative thesis, the first bispecific macrophage engager targeting the receptor tyrosine kinase MerTK and epidermal growth factor receptor (EGFR) is presented. From the 10 antibody candidates derived in the second study, one candidate displayed agonistic properties, detected by the dose-dependent activation of the downstream signalling molecule phospho AKT (pAKT). MerTK’s overexpression on macrophages and tumour-associated macrophages within the tumour microenvironment (TME) lays the foundation to generate macrophage-engaging bispecific antibodies for targeted phagocytosis of tumour cells. Therefore, tandem biparatopic EGFR-binding VHH molecules (termed 7D9G) were combined in different architectures to generate bispecific molecules. By using the Knob-into-Hole technology, a bispecific with a MerTK-binding Fab arm and an EGFR-binding tandem VHH arm were generated, abolishing the agonistic properties of the parental MerTK mAb. On the other hand, a tetravalent bispecific antibody was generated by fusing the tandem VHHs to the C-terminus of the CH3 domain, resulting in intact MerTK-binding Fabs. The bispecific antibodies were able to bind both targets simultaneously in their soluble form and engage macrophages with EGFR+ tumour cells. Furthermore, they were able to compete with the binding site of EGF and therefore inhibit EGF-mediated signalling transduction by inhibiting pAKT. EGFR domain mapping of 7D9G by YSD resulted in binding to domain III of the extracellular EGFR domain, confirming its ligand-inhibiting abilities. Moreover, the bispecific antibodies resulted in targeted phagocytosis of EGFR+ tumour cells by macrophage-like THP-1 cells. This work represents the first bispecific macrophage-engager targeting MerTK for immuno-oncology applications by harnessing its expression and role in the tumour microenvironment to selectively phagocytise tumour cells. In the last study presented here, a trispecific T-cell engager and cytokine release modulating antibody (TriTECM) was generated. In brief, a tetravalent, bispecific two-in-one antibody binding EGFR and PD-L1 simultaneously with a single Fab arm was combined with anti-CD3 and anti-IL-6R scFvs, derived from foralumab or sarilumab, respectively. By testing two TriTECM architectures varying mainly in the anti-CD3 scFv positioning and valency of IL-6R binding, tetraspecific molecules were generated with multiple mechanisms of action. Firstly, increased tumour specificity was ensured by targeting EGFR and PD-L1 with a low nanomolar on-cell affinity. Checkpoint inhibition by blockage of the PD-1/PD-L1 axis was mediated by binding to PD-L1. T-cell engagement and subsequent T-cell-mediated cytotoxicity was attenuated, resulting in reduced pro-inflammatory cytokine release. And lastly, inhibition of the IL-6/IL-6R pathway can modulate cytokine storms after T-cell activation. The attenuation of CD3 binding could allow existing CD3-binders to be used, that were previously shown to result in cytotoxicity. With cytokine release still putting obstacles in the way of novel immune cell engagers, TriTECM designs represent a novel class of therapeutics with the potential to inertly modulate over-activated immune responses and widen the therapeutic index of T-cell-engaging therapeutics

Abundância e diversidade da macrofauna do solo em diferentes fases de um sistema de integração lavoura-pecuária no arenito paranaense.

A macrofauna do solo exerce importante papel na ciclagem de nutrientes e agregação do solo, pois é responsável pela fragmentação dos resíduos orgânicos, mistura das partículas minerais e orgânicas, redistribuição da matéria orgânica e abertura de canais (bioporos). A abundância e diversidade da macrofauna do solo foram avaliadas em áreas submetidas a diferentes fases de um sistema de integração lavoura-pecuária, implantado em 2006, em Jardim Olinda/PR, sobre um Latossolo Vermelho arenoso (110 g kg-1 de argila). A área foi dividida em quatro módulos de 6 ha: P01 ? pasto/pasto/pasto/pasto/soja; P02 ? pousio/braquiária safrinha/ soja/milho + braquiária/soja; P03 ? pousio/milho + braquiária/soja/milho + braquiária/pasto; e P04 ? pousio/milho + braquiária/pasto/pasto/pasto. A espécie de braquiária utilizada na pastagem e no consórcio com o milho foi a Brachiaria brizantha cv. ?Xaraes?. Os módulos caracterizados por uma maior proporção de culturas anuais de grãos (P02 e P03) apresentaram menor abundância e diversidade da macrofauna em relação ao P01 e P04, explorados com pastagens perenes por mais tempo. O módulo P04 caracterizou-se por uma menor abundância total de organismos da macrofauna do solo em comparação ao P01, sendo essa diferença associada à densidade populacional de oligoquetas. Esses dois módulos apresentaram similaridade em relação à diversidade da macrofauna. Assim, concluiu-se que o uso de forrageiras tropicais em sistemas de integração lavoura-pecuária melhora a qualidade biológica do solo em termos da abundância e diversidade da macrofauna