Pharmaceutical, Biomedical and Veterinary Sciences

Defences 2021

Attented a phd of find the archive of concluded doctoral research

Towards a proteome based description of the Withaferin A antitumor properties - Martin Dom (12/03/2021)

Martin Dom

  • 12 March 2021, 4pm - 6pm
  • Online defence
  • Promoters: Xaveer Van Ostade, Wim Vanden Berghe

Abstract

Despite the promising multifunctional antitumor properties of WA, a general overview of its underlying molecular antitumor mechanisms is lacking and hampers its potential clinical use. Since multifunctional chemotherapy is a promising strategy in combating multidrug-resistant tumors, we wanted to fill up the gap between WA treatment and the observed cancer cell death.

To understand what happens in between, we optimized a quantitative chemoproteomic approach to characterize the direct protein targets of WA. Together with differential protein expression data, we hypothesize WA is an acute stress inducer and simultaneously target stress homeostasis responses like the oxidative stress response, the ubiquitin-proteasome system response, autophagy and ERAD responses. Even if cancer cells restore basal cellular stress levels, WA also affects more structural, cellular effects. These include cytoskeleton impairment, cell cycle arrest, and Akt/NFkB pathways. We furthermore identified a novel mode of WA proteasome inhibition via binding the deubiquitinating enzymes USP14 and UCHL5.

Taken together, we believe our results demonstrate that technological advances allow us to combine target-based and observation-based ideas together and with this, a whole new field of antitumor therapies can be exploited, securing our future strategy against multidrug-resistant tumors.

Strategies for efficient acquisition and reconstruction of structural and quantitative MRI - Maarten Naeyaert (11/02/2021)

Maarten Naeyaert

  • 11 February 2021, 5pm - 7 pm
  • Online defence
  • Promoters: Marleen Verhoye, Jan Sijbers

Abstract

Magnetic resonance imaging (MRI) is a valuable tool for investigating the brain, both in the clinic and in research. Its main drawback is its long acquisition time, which can be shortened by minimising the amount of data which is acquired and by using it efficiently. Three objectives were set for this thesis, each of which makes efficient use of the acquired data.

Two objectives relate to compressed sensing (CS). CS algorithms allow the reconstruction of signals or images from incomplete data, by acquiring this data in a pseudo-random way and finding the most compressible image still consistent with the acquired data. The final image quality depends on which data is sampled, and several acquisition strategies were tested on both simulated signals, test objects, and using actual acquisitions. Firstly, CS was implemented for a fast spin echo (FSE) sequence, and it was found that optimal results occur when the majority of the data is sampled in the centre of k-space, with progressively less data being sampled closer to the edge of k-space.

Secondly, a method to accelerate the acquisition of isotropic in-vivo high-angular radial diffusion imaging (HARDI) data using an FSE sequence was developed. While slow, the FSE sequence results in few artefacts, and CS can be used to acquire the large number (>50) of volumes required for HARDI faster. In this case, CS can also be used to subsample the diffusion signal, in the q-space, to acquire fewer volumes. Using knowledge from the first objective, strategies for subsampling the data, either only in q-space or both in k-space and q-space, were tested. We found that subsampling the q-space only is most efficient, and 15 to 20 volumes proved sufficient to yield high quality reconstructions without major differences in quantitative diffusion measures or tractography results.

Finally, a method was developed to quantify micron-sized iron-oxide particles (MPIO) in MRI-images. These particles are used to label cells and cause a negative contrast, i.e. hypo-intensities. However, positive contrast images can be constructed to improve the visibility and localisation of MPIO, which was done efficiently from a single acquisition. To quantify MPIO robustly and reliably the normalized average range (nAR) is introduced. The nAR compares the average value of regions of interest (ROI’s) to that of a control ROI in range filtered images, and showed a higher sensitivity in optimized positive contrast images and is not sensitive to the bias field of the receiver coil.

Further insights into the pathogenetic mechanisms of melorheostosis and Paget’s disease of bone - Raphaël De Ridder (26/01/2021)

Raphaël De Ridder

  • 26 January 2021, 4:30pm - 6:30pm
  • Online defence
  • Promoters: Wim Van Hul, Geert Mortier

Abstract

Dysregulation of bone remodeling can lead to a large number of skeletal disorders. Elucidating the molecular background of such pathologies has the potential of improving diagnosis and can be of great value in the identification of novel targets for the development of novel therapies. The general aim of this thesis was therefore to investigate the contribution of genetic factors in the pathogenesis of two bone disorders, melorheostosis and Paget’s disease of bone (PDB).

In a first part, we performed a mutation analysis in a small cohort of melorheostosis cases. This is a very rare condition characterized by asymmetric increased density of the cortical bone and alteration in surrounding soft tissues. This analysis confirmed the importance of somatic pathogenic variation in the negative regulatory region of MAP2K1 and showed the presence of a novel melorheostosis-associated somatic p.Cys121Ser variant in the catalytic core. Gene-set enrichment of transcriptomic data indicates that this variant leads to hyperactivation of proliferative pERK signaling. 

The second part of this thesis was focused on PDB. Follow-up of families with a history of PDB further supports the importance of genetic variation in SQSTM1. Based on the molecular background of a number of PDB-related phenotypes, targeted sequencing was performed for a panel of 52 candidate genes. This indicated a significant association for variation in the RIN3 gene with the phenotype, with the rs117068593 variant conferring a protective effect that modifies the age of onset in patients. A panel-wide gene burden analysis highlighted association for a number of genes (TNFRSF11A, NUP205, VCP and NFKBIA) with PDB pathogenesis and suggested an enrichment of rare genetic variation in several other genes (NR4A1, NUP205 and PRKCI). Finally, the use of next-generation sequencing technologies is rapidly expanding the number of candidate genes that requires further functional evaluation. Therefore, we generated a loss-of-function model of the ubiquitin-associated domain in the sqstm1 gene of zebrafish. Preliminary results support the presence of a skeletal phenotype in our model. Further studies will be needed to elucidate whether this reflects an osteoclast-driven Pagetic phenotype.

Together, our results provide further insights into the regulation of bone remodeling and explore a novel platform to study the pathogenesis of skeletal disease in vivo.

Drug-Resistance in Visceral Leishmaniasis: Impact on parasite infectivity in the vertebrate host and sand fly vector - Dimitri Bulté (26/01/2021)

Dimitri Bulté

  • 26 January 2021, 4pm - 6pm
  • Online defence
  • Promoters: Guy Caljon, Louis Maes

Abstract

Visceral leishmaniasis (VL) is causing 50,000 to 90,000 new cases annually and more than 20,000 deaths which makes it the deadliest parasitic disease after malaria. In the absence of a successful vaccine, the main VL control strategy is dependent on chemotherapy, however, only a few antileishmanial treatment options are available. Miltefosine (MIL) is currently the only oral drug for VL but is failing to fully clear parasites in an increasing number of patients. These treatment failures could initially not be linked to the emergence of resistance, although more recently a few MIL-resistant (MIL-R) clinical isolates have been described. In order to safeguard the use of MIL for future VL therapies, it is essential to evaluate the impact of MIL-resistance on parasite fitness in the vertebrate host and sand fly vector as this could indicate the potential spread of resistant parasites into the population.

In accordance with previous studies using a laboratory MIL-R L.infantum, a decreased infectivity of the vertebrate host was observed for a natural MIL-R L. infantum. However, no impact on the development of the parasite in the sand fly vector was observed. These results indicate that the impact of resistance on parasite fitness in the vertebrate host and insect vector may not necessarily be the same and indicate the potential transmission of MIL-R parasites. To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo infectivity and drug  efficacy, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of laboratory MIL-R and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a relatively lower burden of MIL-R parasites in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites which contributed to the attenuated MIL-R phenotype. In addition, it was demonstrated that the presence of MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production. These results highlight the potential risk of MIL treatment in sustaining infections with resistant parasites. Close monitoring of parasite drug susceptibility and adjusted treatment protocols would therefore be beneficial.