Ribosome profiling reveals ribosome pausing in <i>Bacillus subtilis</i>

The optimization of protein production focusses on increasing the mRNA levels of the protein of interest, removal of proteases and the induction of protein folding chaperones. However, almost no research has been done on improving translation of the protein of interest, other than simple codon optimization. As far as we know, no ribosome profiling study has been performed focused at the production of industrial relevant enzymes. In Chapter 2, we describe the setting up and testing of the Ribo-seq procedure for B. subtilis, and the investigation of ribosome pausing during the secretion of α-amylase under repeated batch fermentation conditions. The stringent response, which reduces protein translation, is activated in the stationary phase of growth when B. subtilis is most active in the production of secreted enzymes. In Chapter 3, we have investigated how blocking the stringent response affects amylase production and various ribosome profiles. In Chapter 4, we tried to determine whether the ribosome pausing sites found in the amyM mRNA are caused by amino acid sequence motifs or by the nucleotide sequence of the mRNA. In the final experimental chapter, Chapter 5, we have benchmarked several different translation fixation conditions to further optimize Ribo-seq

Behind the webcam:Contested visibility in online sex work in the Netherlands, Romania and the United Kingdom

In webcamming online sex workers can make money through tips and per-minute fees for livestreamed digital sexual acts and interactions. Like other work that takes place on the internet, websites which host webcam performers have considerable influence over how performers make a living. At the same time webcamming also differs from other digital labour in that it is heavily stigmatized. In many places around the world sex work, including webcamming, is considered socially unacceptable and looked down upon. Drawing on interviews with 67 webcam performers and 13 other industry insiders in the Netherlands, Romania and the UK, this thesis makes sense of how workers deal with these competing forces, and the question how do webcam performers in The Netherlands, Romania and the United Kingdom conduct their labour within platform structures?To answer this question, it presents an analysis which explicitly recognises webcamming as work rather than as sex. This thesis draws on a Marxist-feminist perspective which is critical of the exploitative labour conditions webcam platforms and sex work stigma create, while also acknowledging that workers still have agency even in unfavourable circumstances. The problems it identifies with online sex work have much more to do with the context of contemporary labour, in which increasing amounts of workers need to ensure they are visible online to generate an income, which platforms then profit from. It is this visibility that becomes central to the above research question is answered. <br/

From dialogue to decision:Using technology to facilitate shared decision-making in a fall prevention context

Falls are a leading cause of traumatic injury among older patients, and medication intake is a big risk factor for falls. General practitioners and older patients should evaluate the patient’s medication usage together regularly while engaging in a shared decision-making conversation. However, this is currently often not done (sufficiently). This dissertation aims to describe the systematic development and evaluation of a technological intervention that can provide personalized decision support to general practitioners and older patients to facilitate shared decision-making regarding the medication-related fall risk. The intervention consists of a clinical decision support system with a prediction model that calculates individualized fall risks, and a patient portal that helps the patient prepare for the consultation with their general practitioner. The dissertation describes the systematic development of these systems, applying a user-centered design approach in which end users are involved in all phases of the development process. Prototypes were developed based on a systematic literature review, focus groups, and interviews. These prototypes were further improved by conducting usability testing with a think-aloud method. The dissertation also describes the evaluation of the intervention with a randomized controlled trial. The results are promising. The intervention enhanced shared decision-making, increased patients’ satisfaction with the communication during the consultation, and diminished the level of decisional conflict experienced by patients. The effects on the medication changes made during the consultations seem promising, but for now remain inconclusive and need further exploration. This dissertation has showcased the great potential of innovative technology-driven interventions to empower patients and guide healthcare providers to engage in effective shared decision-making

Scratching the surface:Preclinical studies on cognitive flexibility and deep-brain stimulation in obsessive-compulsive disorder

While most of us can identify with obsessive-compulsive disorder (OCD) patients, our understanding of the disorder and potential treatments remains insufficient. We set out to explore the relationship between various OCD-like symptoms, the role of cognitive flexibility in OCD, and the therapeutic mechanism of deep-brain stimulation (DBS) using SAPAP3 knockout mice (SAPAP3-/-), a mouse model for OCD. At the core of this model is compulsive-like self-grooming, which continues despite negative consequences such as loss of fur or even skin lesions. Therefore, in chapter 2, we developed a versatile, open-source grooming classifier that reliably and under various conditions can detect grooming behavior. In chapter 3, we assessed cognitive deficits in SAPAP3-/- and found reduced behavioral flexibility, a core symptom of OCD, accompanied by an imbalance in sign- and goal-tracking behavior. Surprisingly, OCD-like behaviors (i.e., grooming, anxiety, behavioral inflexibility, and hypoactivity) did not correlate. To further explore the relationship between balanced sign and goal tracking and behavioral flexibility, we validated these findings in chapter 4 and found that sign and goal tracking before reversal learning could predict behavioral flexibility, suggesting a robust relationship. By altering reward-cue salience, we were able to induce balanced sign and goal tracking, which resulted in rescued behavioral flexibility. To better understand the neurobiology of compulsive-like grooming, we developed and validated a novel open-source single-photon miniaturized fluorescent microscope for calcium imaging in collaboration with Dr. Tycho Hoogland in chapter 5. The so-called NINscope allows real-time in vivo dual-site imaging, while manipulating distal regions. This technique records large datasets that provide many challenges to analyze due to the large size, many neurons, and substantial out-of-focus signals. In collaboration with the Levelt lab, we developed a novel preprocessing pipeline for calcium-imaging data in chapter 6. Direct comparison between our pipeline and a widely used pipeline revealed comparable results. In chapter 7, we compared ventral striatum and internal capsule (IC) DBS, and found IC-DBS to be more effective at reducing excessive grooming. Both DBS targets recruited similar prefrontal cortex regions. To better understand the therapeutic mechanism of IC-DBS, we employed calcium imaging in cortical and striatal regions in freely behaving animals in chapter 8, while varying clinically relevant parameters. We identified a number of general DBS principles, accompanied by specific involvement of the medial orbitofrontal cortex, which was validated by optogenetics. In conclusion, although our research only scratched the surface of this complex disorder, we gained a better understanding of OCD and its treatment, DBS

Innovations in cardiology:Towards patient centered care

The thesis consists of three parts: In Part 1, the effect of telemonitoring on patients with congenital heart defects or genetic cardiomyopathy was investigated. Telemonitoring does not lead to a reduction in the number of unplanned hospital visits. Part 2 researched the effect of patient education through virtual reality. Particularly, patients with no prior experience in the operating room or the hospital benefit from this comprehensive education. In patients with experience, we did not observe a decreased anxiety about the procedure (even if it was a new procedure to them). In part 3, it was explored whether artificial intelligence can contribute to precise data point localization of the R-wave on the electrocardiogram. Our technique was data-point precise and outperformed current techniques. Expanding this technique in the future could assist cardiologists in automatically detecting heart conditions

(Pre)malignant lesions of the biliary tree:Beyond conventional endoscopic management

Patients with (pre)malignant lesions of the biliary tree often present with biliary obstruction requiring endoscopic drainage. This thesis explores clinical research aimed at optimising conventional endoscopic management of these lesions and beyond.The first part focuses on the treatment of papillary adenomas, rare precursors to papillary carcinomas. Endoscopic papillectomy is the preferred method, but due to the rarity of these lesions, there is limited consensus on the optimal approach. This thesis proposes a treatment algorithm based on expert consensus and underscores the need for long-term follow-up, given that up to 20% of patients experience recurrence.The second part explores a promising new technique for biliary drainage of the distal bile duct: EUS-guided choledochoduodenostomy (EUS-CDS). Although EUS-CDS using a lumen-apposing metal stent (LAMS) achieves high technical and clinical success rates, stent dysfunction remains a common issue. The thesis evaluates EUS-CDS as an alternative to the current standard ERCP, considers options for improved stent design to reduce dysfunction, and shows that EUS-CDS is safe and effective even in a preoperative setting.The third part addresses the diagnosis and drainage of perihilar and intrahepatic cholangiocarcinoma, tumours that are challenging due to their location and growth patterns. By optimising cytology and the use of next-generation DNA sequencing (NGS), diagnostic sensitivity can be enhanced. While metal stents are preferred over plastic stents for biliary drainage, tumour ingrowth remains a problem. A novel technique, endobiliary radiofrequency ablation (eRFA), seems safe and shows promising results in preventing tumour ingrowth, justifying further clinical research

Better methods for diagnostic test evaluations in infectious diseases

The thesis focuses on the application of epidemiological and biostatistical methods for better test accuracy assessment in infectious diseases, particularly in the pediatric population and in population health surveillance. We explore and propose a modelling approach that includes clinical and/or population health relevant constructs in the assessment of test accuracy and apply this to the respiratory pathogen SARS-CoV-2. We examine the severity of the multi-inflammatory syndrome in children and the challenges in diagnosing past infections during the early pandemic. We provide seroprevalence estimates and risk factors for seropositivity from a prospective cohort study in Belgian schools. Additionally, we report on the development, initiation, and analysis of the data provided by the surveillance system of Flemish school COVID-19 cases.Throughout the thesis, we use Bayesian latent class analysis to estimate the sensitivity and specificity of tests to diagnose SARS-CoV-2 past-infection rates. We then improve the more classically used latent class model for the assessment of the prevalence and the accuracy estimates, via the decomposition of the test accuracy question into its elements i) the tests under evaluation, ii) their measurands and iii) the target condition(s). We use directed acyclic graphs to visualize the model and Bayesian inference to obtain the estimates. We find more realistic estimates for the accuracy of the tests with regards to the measurands and the tests with regards to the different target conditions, clarifying their clinical performance to diagnose cases depending on the moment of testing. This corroborates the importance of explicitness regarding the target condition