​Jan Van Erum​

• 18 December 2019
  
• Supervisors: Peter Paul De Deyn and Debby Van Dam

Abstract

Neurodegenerative diseases are often associated with comorbidities that worsen the disease phenotype. As our society might benefit from more effective therapeutic strategies for these comorbidities, we sought to provide a better characterization of the underlying mechanisms that play a role in their development. In this doctoral thesis, we focused on two comorbidities in particular, namely epilepsy and sleep disturbances.

Epilepsy is more prevalent in certain neurodegenerative diseases. Patients are more prone to the development of seizures and epilepsy than aged control individuals. As many of these neurodegenerative disorders are characterized by the presence of pathologic forms of tau and neurofibrillary tangles, studying the effects of tau pathology on the development of epilepsy might prove rewarding. To gain more insight into the relation between tau pathology and epileptogenesis, the Tau58/4 tauopathy mouse model was subjected to a seizure susceptibility analysis. We reported increased seizure susceptibility in young Tau58/4 mice, which are still in an early pathologic stage, but not in old Tau58/4 mice with full-blown tau pathology. Therefore, we hypothesized a crucial role for pre-fibrillar hyperphosphorylated tau in increasing the sensitivity towards PTZ-induced seizures.

Aside from epilepsy, many patients also suffer from other comorbidities, including sleep and circadian rhythm disturbances. In contrast to epilepsy, sleep abnormalities are among the first noticeable symptoms of the disease. To gain a better understanding of how certain dementia-specific pathologic proteins interact with sleep-regulating brain centers and influence cognition, two different transgenic mouse models were studied. We found that, at advanced stages of pathology, both models display severe sleep deficits. In APP23 mice, the sleep and activity profile hints towards a hyperactive condition during the dark phase. In Tau58/4 mice sleep time was reduced during both photoperiods. Most remarkably, disturbances in rapid eye movement sleep were already present from a young age. Based on the pathologic spread in the APP23 model and the spectral data of the EEG, we concluded that Aβ probably has toxic effects at the level of the cortex, resulting in disrupted patterns of cortical activation and sleep loss. By contrast, tau pathology might be more detrimental for the central regulation of sleep, regulated by sleep centers in the brainstem, because sleep disturbances are photoperiod-independent and arise around the time pathologic tau starts to accumulate in pontine regions. To conclude, Aβ and tau obviously have differential effects on sleep in these two distinct models.

​Elias Adriaenssens​

• 13 December 2019
  
• Supervisors: Vincent Timmerman and Stuart Maudsley

Abstract

Small heat shock proteins are molecular chaperones which prevent misfolded proteins from aggregating. Many aspects regarding the molecular mechanisms of small heat shock proteins, both in health and disease, remain to be unraveled.

In this thesis we found that mammalian small heat shock proteins are targeted to the mitochondrial intermembrane space. While small heat shock proteins of plants are targeted to virtually every membrane-enclosed compartment, this is the first example of mammalian small heat shock proteins to have an organelle-localization. This previously unknown mitochondrial function of HSPB1 is disturbed by CMT disease-causing mutations. One of the most severe HSPB1 mutations is located in a conserved IxI/V motif in the C-terminal domain. This mutation is known to cause the formation of extremely large oligomers, which we found to prevent HSPB1 from being imported into mitochondria.

The underlying structural changes, associated to the formation of these large mutant oligomers, were so far poorly characterized. In this thesis we identified an unexpected mechanism underlying the structural changes. The substitution of Pro182 with Leu allows the IxI/V peptide to sample a larger conformational space and the peptide therefore finds itself less frequently in the required conformation to bind the hydrophobic cleft. As a consequence, other IxI/V containing proteins can bind more to HSPB1, explaining the altered protein-protein interactions.

Finally, the co-chaperone BAG3 forms a protein scaffold which binds both small heat proteins and Hsp70. As such, it brings both arms of protein surveillance together. In this thesis we studied three different mutations in the IPV-motif of BAG3, which is where small heat shock proteins bind BAG3, and found that they induce aggresome formation. As a result of that, mutant BAG3 traps the small heat shock proteins and Hsp70 at the aggresome which leads to a general collapse in the chaperone-network.

​Paulo Arnaldo​

• 10 December 2019
  
• Supervisors: Luc Kestens and Anna Rosanas-Urgell

Abstract

Malaria in pregnancy (MiP) is an important public health problem in Sub-Saharan Africa. It is known to be the most common and preventable cause of harmful outcomes to both mothers and developing foetuses in malaria-endemic areas. In stable transmission areas, MiP typically does not cause clinical symptoms and is usually not detected.

This thesis aimed to evaluate the community coverage of IPTp-SP and factors potentially related to low IPTp-SP among women with non-institutional and institutional deliveries; investigate the factors associated with malaria infection and adverse pregnancy outcomes among pregnant women at delivery; explore the perceptions, views, experiences and behaviors of pregnant women and health workers on accessing IPTp-SP for malaria prevention in pregnancy and assess the frequency of dhfr/dhps mutations in P. falciparum isolates collected from pregnant women, analyze the association between mutant haplotypes with parasitological and pregnancy outcomes and, investigate the effect of IPTp-SP on the carriage of asexual and sexual stages.

We found that less than a half of the women reported taking the recommended ≥ 3 doses of SP during their last pregnancy. This coverage remained well below the national’s target of 80% of pregnant women receiving ≥ 3 doses of IPTp-SP. This low IPTp-SP coverage was associated with non-institutional births, late first ANC visit, lower awareness about IPTp and the level of education attained by pregnant women.

Pregnant women were not aware of the risks of MiP or the benefits of its prevention. Delays in accessing antenatal care, irregular attendance of visits, and insufficient time for proper antenatal care counselling by health workers may explain inadequate IPTp delivery.

We report the persistence of quintuple mutated parasites in the study area, while we provided the first evidence of the occurrence of super-resistant P. falciparum parasites (carrying the sextuple mutant parasites), although not associated with adverse pregnancy outcomes. The study further shows an important burden of submicroscopic gametocyte carriers infected with mutant parasites, indicating that pregnant women could constitute a non-negligible human reservoir of malaria transmission. Overall, these findings indicate that, in addition of a suboptimal IPTp-SP coverage in the study area, the emergence of highly-resistant parasites is a threat to IPTp-SP effectiveness for malaria prevention during pregnancy. Therefore, although efforts to improve the coverage of IPTp are important, alternatives to SP for IPTp, such as dihydroartemisinin-piperaquine should continue to be tested (specifically in the study area) and new interventions against MiP (e.g. community delivery of IPTp or screening and treatment of pregnant women) should be explored in Mozambique.

​Yalda Baradaran-Heravi​

• 22 November 2019
  
• Supervisors: Julie van der Zee and Christine Van Broeckhoven

Abstract

Frontotemporal dementia (FTD) is a leading cause of early-onset dementia (EOD). Like for other dementias, no effective treatments exist. The clinical and biological complexity of dementia has long been underestimated. To improve diagnosis and therapy development, we need a better understanding of the underlying disease causes, to which we aimed to contribute by studying the genetic etiology of FTD. Major insights into the pathological mechanisms underlying FTD have come from genetic studies, and have allowed identification of key genes and proteins in FTD.  Nevertheless, half of the patients with a positive family history remain unexplained, suggesting underlying causes in other not yet identified genes. Despite availability of massive sequencing technologies, gene hunting remains challenging, particularly because of the difficulties in rare-variant interpretation (the so-called variants of uncertain significance, VUS).

It is in this context that our research is situated, where we aimed to contribute to the further unravelling of the genetic etiology and molecular signatures of FTD, by family-based gene-hunting, validation of candidate genes in extended patient cohorts, and elucidation of the driving molecular mechanisms. One of these molecular mechanisms is the role of aberrant RNA-binding proteins (RBPs) in perturbing stress granule (SG) dynamics and seeding of toxic protein aggregation. Mutations in a growing number of RBP coding genes have been shown to interfere with SG formation, and cause or influence disease. We propose that aggregation-prone RBPs should be considered candidate genes, and offer opportunities for gene prioritization when mining sequencing data of unresolved FTD and related amyotrophic lateral sclerosis (ALS) patients. In line with this, we provide first genetic evidence for a causal implication of a novel RBP gene, RBM45, in the FTD-ALS spectrum. In a second family-based exome gene-hunting effort, we present two possible candidate genes that require further follow-up to demonstrate causality. Sharing these genetic findings, even though preliminary or affecting just a handful of patients, is of importance to move the field forward, as they may trigger replication studies and present converging evidence with other related research.

​Ken Declerck​

• 13 November 2019
  
• Supervisor: Wim Vanden Berghe

Abstract

​Bassirou Diarra​

• 8 October 2019
  
• Supervisors: Leen Rigouts and Bouke de Jong

Abstract

Drug-resistance is threatening the global control of tuberculosis (TB), the leading infectious cause of death worldwide, with incomplete understanding of predictors of poor treatment outcome. First-line TB drugs are more powerful and less toxic compared to most second-line drugs, further stressing the importance of early identification of rifampicin-resistant (RR) TB and preventing the transmission of de novo resistance. If feasible, WHO recommends “universal drug-susceptibility testing”, i.e. the search and identification of RR among new TB patients.

As universal drug-susceptibility testing is not yet implemented in Mali, we conducted a cohort study in Bamako to detect RR-TB early on during first-line treatment, and monitor RR-TB treatment. As conventional microscopy by either Ziehl-Neelsen or auramine is less sensitive, and does not distinguish between live and dead bacilli, mycobacterial culture is required for treatment monitoring. As an alternative to culture, which is not widely available, we applied fluorescein di-acetate (FDA) vital stain for detection of live mycobacteria in monitoring treatment success. Moreover, we estimated the population structure of mycobacterial strains circulating in Bamako, and determined clinical characteristics and outcome by lineage.

Our data shows that TB patients in Bamako present with a high bacillary burden, thus with advanced disease. Increased sensitization of the population about TB symptoms and training of clinicians may contribute to earlier diagnosis and treatment initiation. Among new TB patients, the level of primary RR was moderately high at 2.6% (95% CI: 1.7-3.7). All RR were caused by mutations within the RR determining region of the rpoB gene, suggesting that the rapid Xpert MTB/RIF assay serves the diagnostic algorithm well.

A high proportion (64%) of auramine-based treatment failures were FDA negative, reflecting continued expectoration of death bacilli. However, FDA was insufficiently able to identify early RR-TB or predict culture-confirmed treatment failure.

Regarding drug-resistant TB, we identified multiple challenges, including evidence of nosocomial transmission, low coverage of monthly monitoring and attrition. Neither HIV nor diabetes mellitus were identified as risk factors for acquiring drug-resistance.

As for circulating mycobacterial strains, we found that all first-six lineages of human importance were represented in Bamako, in addition to 0.8% M. bovis. The most widely represented lineages were Lineage 4 (Euro-American) (57%), and Lineage 6 (M. africanum) (22.9%). Lineage 6-diseased patients - compared to Lineage 4 - showed slower disease progression, higher smear-microscopy grades, and slower smear conversion by auramine and FDA. Hence, more efficacious treatment regimens are needed for Lineage 6 patients.

​Chakirath N'Dira Sanoussi​

• 7 October 2019
  
• Supervisors: Leen Rigouts and Bouke de Jong

Abstract

Tuberculosis (TB) is caused by bacteria of the Mycobacterium tuberculosis complex (MTBC) which comprises 7 human-adapted phylogenetic lineages, including the two M. africanum lineages (West-African 1 (L5) and 2 (L6)) that are geographically restricted to West and Central-Africa, and cause up to 40% of TB in West-Africa. This thesis aimed to increase the understanding of L5 epidemiology, diagnosis and genomic characteristics, and included a nationwide prospective cohort study in Benin, the country with the highest prevalence of L5 worldwide.

We identified numerous novel epidemiological associations that shed new light on this distinct MTBC member and suggested technical advances for improved unbiased TB diagnosis and molecular epidemiological studies. We found that L5 is under-represented in positive cultures and identified a decreased performance of the rapid MPT64-antigen-based assay for the identification of L5 as a MTBC member among positive cultures, likely due to an L5-wide non-synonymous SNP (I43N) in the mpt64 gene. This risks misclassifying of positive L5 cultures as non-tuberculous mycobacteria. To overcome the culture bias observed, direct spoligotyping was used for lineage determination. The countrywide distribution of lineages differed significantly by patient’s treatment history, with M. africanum present in 39.2% of new and 26.3% of previously-treated patients (31.1% and 21% respectively for L5 alone). In Cotonou, over 10 years, the L5 prevalence among new patients significantly declined by 9.4 % (1.2-17.6) as did L1, while the L4 prevalence increased by 16% (7.4-24.6), and the L6 prevalence remained similar.

To facilitate such multicentric studies/surveys, cetylpyridinium chloride, OMNIgene.SPUTUM and ethanol were compared for up to 28-day storage of sputum in ambient temperature, with an algorithm proposed to help the user in the choice.

Using comparative genomics, we identified differences in gene content not only between L5 and the currently used M. tuberculosis reference genome H37Rv (L4), but also among L5 strains (complete genomes from Benin, Gambia, Nigeria). Some genes were absent in L5, and based on their function, can partly explain the reduced growth in culture and other suggested characteristics of M. africanum.

In conclusion, direct genotyping should be used for MTBC population structure studies, and the algorithm for TB diagnostics testing in M. africanum- endemic countries could be improved, favoring direct diagnostics for unbiased results. The high within-L5 gene content variability suggests the pangenome of MTBC may be larger than previously thought, implying a reference-free de novo genome assembly approach may be preferable over the currently used H37Rv-based genome analyses.

​Federica Perrone​

• 27 September 2019
  
• Supervisors: Christine Van Broeckhoven and Julie van der Zee

Abstract

One of the major attempts of medical research is to entirely comprehend the neurodegenerative brain diseases (NBDs) etiology. These widespread disorders have an enormous impact on patients and families. Available therapeutic strategies are only able to attenuate symptoms. Neurodegeneration in the brain therefore progresses towards an irremediable loss of neurons. The patients’ condition severely worsening to a state of incapability to perform everyday life activities. The most common disease NBDs subtypes are Alzheimer’s disease (AD) and Parkinson’s disease, followed by less frequent known as frontotemporal dementia and Amyotrophic Lateral Sclerosis.

The past three decades have witnessed major advances in understanding NBDs molecular etiology. Initial genetic and association studies on affected individuals have discovered causal and risk genes underlying the biological mechanisms of neurodegeneration. Unfortunately, these mutated genes could only explain a small proportion of the affected patients. After whole exome and genome sequencing tools became available, an unprecedented rate of novel NBDs genes has been discovered. A large number of rare variants has therefore been identified of which contribution to disease remains still unclear. To date, functional research aiming to demonstrate the effect of genetic mutations on the protein function is scarce and these variants of uncertain significance (VUS) remain uncharacterized. This leaves a large fraction of the genetic NBDs etiology unexplained.

The work performed during this PhD aimed to clarify whether rare variants in NBD genes, identified through next generation sequencing, contribute to the disease etiology. To achieve this, the main NBD disease genes were screened in Belgian early-onset dementia patients with unclear clinical symptoms. A more accurate diagnosis was given to patients with a known pathogenic mutation identified. VUS were also observed, two of which were further characterized using patient biomaterials. Results from this study highlighted the NBDs genetic heterogeneity and corroborated the hypothesis of an NBDs disease continuum, further supported by the screening of two recently identified ALS genes (TUBA4A, CHCHD10) in Belgian FTD-ALS patients. Finally, the established AD disease genes (APP, PSEN1, PSEN2) were screened in Belgian AD patients. Both known pathogenic mutations and VUS were discovered. Cerebral spinal fluid was available for a subset of mutation carriers to further investigate a possible pathogenic role for amyloid-β 43 peptide in AD pathogenesis, which has recently raised attention in this field.

The work performed during this PhD has provided evidence for the importance of deciphering the possible pathogenic effect of the VUS in the NBDs.

​Winke Van der Gucht​

• 27 September 2019
  
• Supervisors: Peter Delputte and Paul Cos 

Abstract

Respiratory syncytial virus (RSV) is the principal viral cause of serious lower respiratory tract infections in infants and is the primary cause of hospitalization during the first year of life. RSV disease severity in young children displays great variety, ranging from mild cases with common cold-like symptoms to severe cases in need of hospitalization, respiratory support, additional hydration and tube-feeding. The factors attributing to the observed differences in disease severity can be divided into host factors, such as genetic and environmental background, and virus-intrinsic factors. Multiple studies have indicated that genetic variations in the host inflammatory immune response to RSV are responsible for the wide range of disease severity. In contrast, the role of factors intrinsic to the virus itself in contributing to disease has only partially been researched. No vaccine or therapeutic has been market approved to prevent or treat RSV infections and associated disease, therefore, treatment is currently limited to supportive care. Research on vaccines has been severely hampered by a legacy of vaccine enhanced respiratory disease. Blocking RSV entry by small molecules is an attractive therapeutic pathway, but fully understanding the entry mechanisms of RSV into the host cell is important for their development. Two theories currently describe the RSV fusion event, fusion at the cell surface and fusion within endosomes. Questions remain about the event that triggers the fusion of the viral envelope with the cell membrane and two main propositions have been made: spontaneous conversion of the metastable prefusion F protein conformation to the stable postfusion F protein conformation, or post-translational modifications by furin-like proteases.

Firstly, this thesis focuses on the isolation and phenotypical and genetic characterization of recent clinical isolates. RSV was isolated from nasal samples derived from sick children and characterized in vitro. These experiments showed that the clinical isolates differ from laboratory strains isolated in 1961, and from each other on several characteristics. Additionally, these clinical isolates were then continuously cultured to assess their adaptation, which was found limited after 30 passages in most cases.

Secondly, this thesis aimed to gain more insight in the role of host proteases during the RSV entry process. RSV infected cells were first treated with several protease inhibitors to examine their effects on viral entry and the replication cycle of RSV. Serine protease AEBSF was found to inhibit the RSV infection during early entry, and the process was further elucidated using electron microscopy showing that fusion was inhibited.

​Pieter Pannus​

• 19 September 2019
  
• Supervisor: Guido Vanham

​Bruno Cesar Bremer Hinckel

• 28 June 2019
  
• Supervisors: Jean-Claude Dujardin and Xaveer Van Ostade
  

Abstract

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the Leishmania donovani complex. Without treatment, VL is fatal. Although diagnostic techniques, mainly based on the detection of anti-Leishmania antibodies are available, invasive procedures such as microscopy from spleen or bone marrow aspirates are still required for the diagnosis of seronegative VL suspects, for the detection of recurrent cases and to confirm cure after successful treatment. The rK39, the most widely used antigen for VL diagnosis has been an essential point-of-care tool for the detection of active VL cases, although it cannot be used as a test of cure due to antibody persistance (IgG) even after succeessful chemotherapy. Furthermore, this test has a decreased sensitivtiy in Eastern Africa, where the direct agglutination test (DAT) is still an intergral part of the algorithm to diagnose active VL cases. Previous investigations showed the potential of IgG1 as a biomarker of post-chemotherapeutic relapse for VL in rapid diagnostic tests (RDTs) sensitised with crude lysate antigen (CLA).

We employed in silico tools to search for desired protein features in a large number of L. donovani antigens detected by human IgG1 in western blots. We then employed prediction algorithms to profile epitopes from the shortlisted proteins. We screened a panel of high-scoring peptide epitopes in a high-throughput manner using arrays, with low reagent consumption. The most reactive peptide was adapted to RDTs, showing promising results of both sensitivity and specificity. This peptide has the potential of replacing the CLAs in IgG1 RDTs.

Using cure paired serum samples from India we evaluated the potential of IgG1 against the rK39 to monitor treatment outcome. ELISAs with rK39 and IgG1-specific conjugate gave a far more discriminative decrease in post-treatment antibody response when compared to IgG. Novel IgG1 rK39 RDTs provided strong evidence for decreased IgG1 response in patients who had successful treatment. Furthermore, both IgG1 rK39 RDTs and ELISAs showed a highly significant difference in test outcome between cured patients and those who relapsed. RDTs were more sensitive than corresponding ELISAs.

Considering VL diagnosis in eastern Africa, we showed that the pool of Leishmania antigens located at the surface of the parasite (membrane proteins - MPs) represent an interesting target for additional investigations. This protein extract might contain the DAT antigen while the identification of specific antigens would enable the development of an RDT with improved sensitivity for active cases detection in eastern Africa.

​Arne De Roeck​

• 20 Juni 2019
  
• Supervisor: Kristel Sleegers

Abstract

Dementia is the fifth leading cause of death in the world. To find a therapy, we need a better understanding of the underlying disease causes, to which we aimed to contribute by studying the genetics of Alzheimer’s disease (AD), the predominant form of dementia. In particular, we focused our work on the identification and characterization of complex genetic variants in the ATP Binding Cassette Subfamily A Member 7 (ABCA7) gene.

Our research group was one of the first to demonstrate a strong AD risk increasing effect of rare ABCA7 premature termination codon (PTC) variants. We first expanded on this striking finding by next-generation sequencing (NGS) analysis of coding ABCA7 variants in early-onset AD patients. We identified novel PTC variants and observed an overall fivefold PTC enrichment in patients. Because of incomplete disease penetrance and a wide onset age range, we characterized these PTC mutations on the transcript level by long-read sequencing on a MinION sequencer. We detected frequent transcript alterations which can rescue a deleterious PTC effect and are a possible target for therapeutic interventions.

NGS, however, has limitations. By analysis of DNA sequences that are disregarded by conventional NGS processing, we were able to identify a highly polymorphic intronic ABCA7 variable number tandem repeat (VNTR). Subsequent Southern blotting revealed that VNTR expansions had a striking fourfold AD risk increasing effect. In downstream analyses we demonstrated that increasing VNTR lengths correlated with reduced ABCA7 expression and increased alternative splicing of a defunct ABCA7 isoform. Furthermore, we observed reduced amyloid β levels in cerebrospinal fluid of AD patients carrying long VNTR lengths.

Subsequently we aimed to develop a novel genotyping method based on long-read sequencing to enable improved characterization of this ABCA7 VNTR and other tandem repeats. We made use of the recently released high-throughput PromethION sequencing platform, and were the first to establish human whole genome long-read sequencing using only a single flow cell per individual. Finally, we developed “NanoSatellite”, a dynamic time warping-based tandem repeat calling algorithm, which demonstrated accurate length estimation for all ABCA7 VNTR lengths, including clinically relevant expansions, and detection of tandem repeat motif interruptions.

In conclusion, we contributed to the establishment of ABCA7 as a strong risk factor of AD, which opens opportunities for novel drug targets and smarter AD clinical trials. In the process, we established novel methods, which will hopefully enable other researchers to study complex alterations in our genome and transcriptome.

​Jan Detrez​

• 6 June 2019
  
• Supervisors: Winnok De Vos and Jean-Pierre Timmermans

Abstract

The neuropathology of Alzheimer’s disease is among other hallmarks characterised by stereotypical, progressive spreading pattern of tau pathology in the brain. It is however not known how exactly this spreading process occurs or how it correlates with cellular and functional deficits to date. The classical approach for studying cell biology in the brain, makes use of sectioned tissue, which leads to a significant loss of biological material and complicates downstream image analysis procedures.

With the advent of tissue clearing and light sheet microscopy, in toto imaging of whole organs at cellular resolution has become feasible. We have benchmarked and used this approach in a variety of transgenic mouse models and showed that targeted injection of tau fibrils in a Tau.P301L mouse model triggers a connectome-dependent spreading pattern of tau pathology that resembles the pattern observed in Alzheimer patients. We further found that fibril injection-induced tau pathology was associated with a specific microglial phenotype, typified by rod-like and swollen cell bodies. We showed that co-injection of an antibody targeting the aggregation domain of the tau could reduce the all-over pathological tau load.

One disadvantage of whole-brain microscopy is the fact that it can only be performed post-mortem and therefore does not allow assessing functional defects. To resolve this, we have performed resting state functional (rsf)MRI, enabling functional layering on volumetric histological data. However, despite overt tau pathology, no major change was observed in the resting state networks, suggesting that the accumulation of tau pathology does not affect functional connectivity per se.

In conclusion, we have established a whole-brain microscopy approach and gained insight in tau pathology development in mouse models. The combination of whole-brain microscopy with rsfMRI allowed for correlating morphological with functional information, representing a innovative imaging platform for a variety of preclinical studies, also beyond the AD domain.

​Sven Bervoets​

• 3 June 2019
  
• Supervisors: Albena Jordanova and Marie-Luise Petrovic-Erfurth

Abstract

Aminoacyl-tRNA synthetases (aaRS) catalyse the ligation of tRNAs with their cognate amino acid, the first step in protein translation, making them indispensable in every organism. Despite being ubiquitously expressed, dominant mutations in these enzymes cause a peripheral neuropathy. This incurable disease affects the longest motor and sensory neurons in the human body leading to a progressive impairment of motor abilities and sensation, ultimately causing loss of ambulation. Patients affected by dominant mutations in six different aaRSs have a similar clinical presentation, suggesting a shared disease mechanism. Yet, this pathomechanism still remains to be elucidated.

At first it was postulated that loss of the aaRS’ enzymatic activity is pathogenic. However, recent work reveals that a toxic gain of function is likely contributing to the neuropathy. Outside their central role in protein translation, aaRSs have acquired multiple non-canonical functions, which often relate to cellular compartments where protein translation is generally not taking place. Interestingly, all aaRSs associated with a peripheral neuropathy localise to the nucleus, but the relationship between their nuclear presence and the peripheral neuropathy has not been explored thus far.

This study focuses on the nuclear role of tyrosyl-tRNA synthetase (TyrRS). This aaRS causes a subtype of a peripheral neuropathy, named Dominant Intermediate Charcot-Marie-Tooth type C (DI-CMTC). Conformational changes in the mutant TyrRS protein result in aberrant binding to components of the nuclear transcription machinery, supporting a toxic gain-of-function. As a result, a specific set of genes is uniquely misexpressed. The transcriptional changes particularly involve genes related to mitochondria. Furthermore, cellular and Drosophila models of DI-CMTC show fragmentation of mitochondria. Excluding the mutant protein from the nucleus, both genetically and pharmacologically, prevents the disease phenotypes in the models for TyrRS-induced peripheral neuropathy. Taken together, our data reveal the nucleus as the site of lesion in TyrRS-induced peripheral neuropathy, opening opportunities to study this putatively shared pathomechanism in aaRS-induced neurodegeneration. Our work paves the way for further development of new therapeutic strategies to treat peripheral neuropathies.

​Raquel José Matavele Chissumba​

• 8 May 2019
  
• Supervisors: Luc Kestens and Jani Ilesh

Abstract

The human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS). Globally, more than 36 million individuals are infected with HIV-1. During HIV-1 infection, high levels of systemic immune activation occurs which stimulates massive viral replication. Unfortunately, there is not an effective vaccine yet against HIV-1.

Evidence from previous efficacy trials of HIV-1 vaccine candidates, shows that a good HIV-1 should induce pronounced HIV-specific memory T and B cell responses, but at the same time, needs to limit responses that favor infection. A higher risk of HIV-1 acquisition was observed in participants of a previous HIV-1 efficacy trial which was associated to higher levels of immune activation and augmented expression of HIV binding molecules in vector specific CD4 T cells. Regulatory T cells (Tregs) are a heterogeneous population of CD4 T cells with the potential to suppress exacerbated immune activation. Furthermore, Tregs also participate in mechanisms of immune memory development. However, Tregs can also suppress the development of protective antigen-specific immune responses. Little is known regarding Tregs during early infection by HIV and particularly during vaccination against HIV-1. Tregs share some differentiation pathways with Th17 cells and reciprocal development between these two CD4 T cells populations has been demonstrated.

We aimed to assess the frequencies and phenotypic alterations of Tregs, in terms of expression of activation markers, suppression markers and HIV-1 binding molecules, during HIV-infection and HIV vaccination. We also assessed how these alterations correlated with indicators of HIV disease progression and vaccine-induced immune responses.

Our findings suggest that during HIV-1 infection, an increase of Tregs in relation to total CD4 T cells correlates with systemic immune activation.  Certain subsets of Tregs like those expressing the transcription factor Helios may have a beneficial role in mechanisms controlling the levels of viral replication. However higher levels of Helios expressing Tregs can be associated with deficiency in production of antibodies against HIV-1. During DNA-HIVIS/MVA-CMDR-HIV/±CN54rgp140 vaccination, pre- and post-vaccination Tregs proportions, their activation status, the Th17/Tregs ratio and other host factors affecting Tregs relative abundance, may have an impact on the magnitude of HIV vaccine-induced immune responses. Furthermore, the DNA-HIVIS/MVA-CMDR-HIV/±CN54rgp140 does not induce increased susceptibility to HIV-1 infection of Tregs and total CD4 T cells.

Therapeutic and prophylactic HIV-1 vaccine trials should consider the evaluation of Tregs for a better understanding of the mechanistic impact of these experimental interventions.

​Sylvie Slaets​

• 8 May 2019
  
• Supervisors: Sebastiaan Engelborghs and Peter Paul De Deyn

​Aya Hefnawy​

• 11 April 2019
  
• Supervisors: Jean Claude Dujardin and Dirk Valkenborg

Abstract

Visceral leishmaniasis (VL) is a neglected tropical disease that is lethal if left untreated. Drug resistance (DR) is a major problem for VL in the Indian sub continent (ISC). New chemotherapeutic agents are needed and there is no implementation of DR studies into the industrial R&D pipeline. In this study we reviewed the utility of DR studies for two compounds, antimonials (SSG) and miltefosine (MIL), both of which are no longer used as first line treatment options for VL in the ISC. We investigated how implementation of DR studies could have extended their use. Based on that we recommended the implementation of DR studies in an early stage of the R&D process through two approaches.

(A) Testing the efficacy of the newly discovered compounds on clinically isolated resistant strains.

(B) Utilizing DR studies to understand mechanisms of resistance to new compounds and possibly identify their mode of action. These two recommendations were tested in this PhD thesis. In collaboration with GSK, newly identified compounds from the Leishbox were tested on recently isolated ISC strains including an SSG-Resistant one. Only 45 percent of the compounds showed panactivity. This finding shows the importance of validation of the results of High-Throughput Screens (HTS) with a panel of recent clinical isolates of different geographical origin and isolates with demonstrated resistance to currently used compounds. For approach B one of the panactive compounds of the Leishbox with a promising profile was chosen (further-called compound X).

Resistance to X was selected in a stepwise manner in promastigotes. The time to develop resistance to compound X was found to be longer than developing resistance to MIL or SSG. X-Resistance was stable in the absence of drug pressure and amastigotes were also found to be resistant to X. Genomic and metabolomic characterization was performed to understand molecular adaptations of X-resistant parasites. In contrast to vast metabolic variations between X-resistant and X-sensitive parasites, only few genomic changes have been identified including a SNP in the gene encoding dynamin-1 like protein. Both these genomic and metabolic adaptations could give cues to the mechanism of resistance to X.

This work highlights the importance of academic and industrial collaboration. DR in Leishmania is an inevitable threat to any new drug candidate so implementing DR studies is the next logical step in the industrial drug discovery pipeline.

​Marlene Jara Portocarrero​

• 27 March 2019
  
• Supervisors: Jean Claude Dujardin and Paul Cos

Abstract

Leishmaniasis encompasses mild to fatal diseases caused by the infection with protists of the genera Leishmania. Parasitological evidence suggests that Leishmania amastigotes can remain in tissues of infected individuals as subclinical infections. These latent infections represent a threat as the onset or recrudescence of the disease is unpredictable. Latent infection in other microorganisms are associated with a downregulated physiological stage called quiescence. Although quiescence is a known process for bacteria it has been scarcely studied among protists.

The goal of this thesis was to unravel if Leishmania amastigotes can be quiescent. Our first hypothesis was that Leishmania infection can produce long term persistent but subclinical infections after the chemotherapy and clinical cure of mucosal leishmaniasis. Accordingly, we found that 41.2 % of the patients had subclinical persistence of a low number of parasites on their healed mucosa 6 to 18 months after treatment. The results highlight that Leishmania overcome the host immune system and chemotherapy to remain in their host. These subclinical infections resemble the well-known latent infections of Mycobacterium tuberculosis characterized by quiescent stages.

Secondly, we hypothesized that Leishmania amastigotes should share common parasitological and molecular features of others quiescent microorganism. We found that the steady infection index of amastigotes in vitro was associated with downregulation in the content of proteins, kDNA minicircle and with a dramatic drop in rDNA expression. Metabolomics showed amastigotes are characterized by a downregulation in the levels of biosynthetic molecules as amino acids and polyamines. Altogether, these results showed that amastigotes are endowed with molecular features of quiescence.

Thirdly, we hypothesized that the expression of GFP within the rDNA locus (rGFP expression) could be a biosensor to monitor the potential diversity of quiescent stages among amastigotes at single cell level.  We found a positive and stage specific relation between rGFP expression and the proliferative condition of Leishmania. Moreover, rGFP expression showed that among amastigotes different quiescent stages may coexist; the population could include shallow and deep quiescent stages that may be sorted by their levels rGFP expression.

 In summary this work supports the occurrence of quiescence among Leishmania amastigotes and offers the use of rGFP expression as a tool for further molecular and metabolic characterization of quiescent subpopulations. This may contribute to the understanding of the mechanisms that drive the maintenance of this clinically and epidemiologically relevant stage.