Improved diagnosis of drug-resistant tuberculosis: from implementation of simple phenotypic testing to whole genome sequencing - Elisa Ardizzoni (21/12/2023)
- 21 December 2023, 4:15pm - 6:15pm
- Aula Janssens, ITG
- Promoters: Leen Rigouts, Bouke de Jong
For many years, the diagnosis of tuberculosis (TB) and resistance to anti-TB drugs has relied on standard phenotypic methods, based on the culture of viable mycobacteria. These analyses are expensive and slow, and to contain biohazard, they must be conducted in high containment laboratories, not available in remote low-resource settings. During the last decade, diagnosis of TB has been revolutionized by the development of rapid molecular diagnostic tests, such as Xpert MTB/RIF, now rolled out worldwide. At the time of its endorsement, in 2011, Médecins sans Frontières has been one of the early implementers, installing the tests in over eighteen countries. The thesis reports the results of this experience, showing undoubtful increase of TB detection, but also challenges due to logistic constraints, mainly encountered in peripheral laboratories.
Despite the improvement of TB diagnosis, the need for phenotypic methods persists, particularly to monitor patients during treatment and to assess resistance to drugs not targeted in rapid molecular assays.
This thesis describes the improvement of thin layer agar (TLA) a rapid, simple, and inexpensive phenotypic non-commercial method for simultaneous detection of TB and drug resistance, applicable to low resources setting. In a first phase, the assay was assessed at the Institute of Tropical Medicine, demonstrating very good results. Next, TLA has been evaluated under field conditions in a Médecins sans Frontières project in Eswatini. The thesis presents the results, discusses the challenges and limitations, and proposes ways forward for further improvement of this technique.
Along with the great advancements in diagnostic tools, new anti-TB drugs have been approved. However, specifically for bedaquiline, results from both molecular and phenotypic susceptibility testing methods remain difficult to interpret. Our research generated data to contribute to closing this knowledge gap. Testing for susceptibility to new drugs before starting treatment, to avoid weak treatments and the risk to contribute to the development of resistance, should become a priority in routine practice. The development of a rapid molecular test however does not seem close, so that in peripheral settings, TLA could be further evaluated to provide rapid and accurate results for these new drugs.
DNA methylation dynamics during zebra finch vocal development - Jolien Diddens (15/12/2023)
- 15 December 2023, 4pm - 6pm
- Auditorium R2 (CDE)
- Promoters: Wim Vanden Berghe, Annemie Van der Linden, Manfred Gahr
Like humans, songbirds are among the few animal groups that learn their vocalizations. They learn their song from a tutor early in life, analogous to human speech learning. In zebra finches, only the males sing, and song learning is restricted to a specific developmental time window, called the “critical period”. This critical period consists of two overlapping phases: first, they memorize the song of an adult male tutor (sensory memorization phase), after which they actively start to vocalize themselves and gradually match their song to the memorized template (sensorimotor phase).
We investigated which molecular mechanisms could explain why zebra finches can learn singing during, but no longer after the end of the critical period. Since the DNA sequence itself does not change during brain development, we hypothesized that epigenetic changes may be involved, as they determine when and where genes are expressed by altering the accessibility of the DNA. We focused on DNA methylation, an epigenetic modification that is known to play a role in learning, memory, and adaptation to the environment.
Our results show that dynamic DNA methylation plays an important role in the brain regions controlling song learning and production, since these regions display very high levels of DNA methylation, as well as of the two antagonizing classes of enzymes that define the methylome: DNA methyltransferases (DNMT) catalyzing DNA methylation and ten-eleven translocation (TET) dioxygenases responsible for demethylation. DNA methylation levels were found to increase over the song learning period, specifically in these song control nuclei. In addition, we found many genes involved in brain plasticity to be regulated by DNA methylation. By the end of the critical period, the expression of DNMT and TET enzymes in the song control nuclei strongly decreased. A tutor song deprivation experiment showed that this decrease cannot explain the closing of the sensory phase of song learning. Whether it could explain the closing of the sensorimotor phase still remains to be investigated.
Given the many parallels between vocal learning in songbirds and humans, unraveling the molecular mechanisms of vocal learning in songbirds could provide insights for the treatment of speech disorders. Moreover, the principles of critical period regulation seem largely generalizable between systems, meaning that a deeper understanding of the role of DNA methylation would provide new insights about how to re-induce youth-like neuroplasticity in the adult brain to improve recovery after brain injury, treat developmental disorders, or to enhance lifelong learning.
Using novel molecular approaches to understand transmission of Mycobacterium leprae in the Comoros - Sofie Braet (07/12/2023)
- 7 December 2023, 4pm - 6pm
- Aula Janssens, ITG
- Promoter: Leen Rigouts
This PhD thesis centers around the complex dynamics of leprosy, a debilitating disease caused by Mycobacterium leprae (M. leprae). Despite being among the first human pathogens identified, the means by which it spreads among individuals remain enigmatic, complicating leprosy control even with available treatments. With an annual diagnosis of over 200,000 new cases worldwide and a significant proportion children, indicating the ongoing spread of the disease. Innovative approaches to leprosy control are essential to definitively halt transmission.
This study revisited transmission questions in an innovative manner. Initially, it examines the specificity of RLEP qPCR for M. leprae. Subsequently, it evaluated non-invasive, field-applicable tests to quantify bacterial levels in patients. Findings show that αPGL-I IgM levels in fingerstick blood correlate with bacterial load. Combining αPGL-I R-values ≥ 0.81 with a lesion count ≥25 predicts high bacterial load in a patient.
Pioneering the field, the study introduced targeted Next Generation Sequencing of M. leprae through the innovative Deeplex Myc-Lep assay with a detection limit of 80 M. leprae genomes. This approach enabled the first drug resistance survey of M. leprae in the Comoros, revealing no drug resistance. The Deeplex Myc-Lep also characterized M. leprae diversity in the Comoros, classifying distinct genotypes linked to patient residences, aiding in understanding transmission patterns. SNP subtypes detected are 1D-Malagasy and 1A. Among 265 patients with a full VNTR pattern, 79.7% cluster with at least one other patient based on identical VNTR profiles.
Additionally, the study explored if wild ticks from the Comoros carry M. leprae DNA, building on earlier evidence of their potential to transmit the bacterium. No M. leprae DNA is detected in these ticks, suggesting a limited role in leprosy transmission. These insights contribute to refining strategies for effective leprosy control, within the Comoros and beyond.
On the hunt for a novel biomarker of Huntington’s disease: disentangling brain network activity from hemodynamic alterations across disease progression - Tamara Vasilkovska (25/09/2023)
- 25 September 2023, 4pm - 6pm
- Auditorium O1 (CDE)
- Promoters: Marleen Verhoye, Annemie Van der Linden
Huntington’s disease (HD) is a monogenic neurodegenerative disorder which is marked by an abnormal expansion of the CAG repeats in the huntingtin gene, resulting with the production of the mutated huntingtin protein (mHTT). The progressive accumulation and aggregation of mHTT is linked to neuronal dysfunction and loss in the striatum and the cortex – a hallmark of HD. Despite the known genetic background, HD has no successful treatment so far. The development of therapeutic strategies and testing of novel drug targets depends on the discovery of biomarkers. Although neuronal loss is the hallmark of HD, altered neuronal function precedes these changes. A biomarker that can detect functional abnormalities prior to atrophy can yield promising novel targets that can potentially reverse the inevitable course of neurodegeneration.
In this thesis, we aimed to elucidate functional alterations in whole-brain connectivity and brain hemodynamics along the course of HD progression and whether these changes are linked to underlying histopathology in the neurovascular unit components. For this purpose, we used a multimodal approach, where resting-state fMRI (rsfMRI), pseudocontinuous arterial spin labelling (pCASL) fMRI and immunofluorescence were applied in the zQ175 delta neo (DN) heterozygous (HET) knock-in mouse model of HD. This model has shown molecular, cellular, and behavioral properties that resemble several aspects of the human HD.
Using rsfMRI, we observed reduced functional connectivity (FC) in the default mode-like network (DMLN) at the onset of motor deficits, while reduced FC of other networks became more pronounced in advanced disease stages. Whole-brain network dynamics, as revealed by quasi-periodic patterns (QPPs) and co-activation patterns (CAPs), showed spatial hyperactivations occurring in HD relevant regions, at the earliest HD-like stage before motor disturbances, followed by gradually pronounced decrease in activations in the DMLN along the course of HD. These outcomes coincided with increased mHTT deposition in astrocytes and pericytes in these HD related regions. Resting-state brain perfusion, as measured with pCASL, revealed transiently increased perfusion in the cortex at the earliest HD-like stage. Moreover, cerebrovascular activity was impaired when the HD phenotype was already developed.
The findings in this thesis show the unprecedented detail of aberrations in the functional architecture of HD, improving our understanding of the disease, and also demonstrating its translational value and potential to be used as an early promising biomarker to test novel therapies. In the future, the state-of-the-art methods - QPPs and CAPs, can subserve as noninvasive diagnostic tools in clinical HD research.
Unraveling the genetic landscape of high bone mass disorders and Paget’s disease of bone: From variant identification and validation to zebrafish modeling - Yentl Huybrechts (5/9/2023)
- 5 September 2023, 4pm - 6pm
- Promotiezaal (CDE)
- Promoters: Wim Van Hul, Geert Mortier
The heterogeneous landscape of genetic skeletal disorders is a major burden on the healthcare system worldwide. The latest version of the Nosology of genetic skeletal disorders (2023) includes 771 disease entities associated with 552 different causal genes. These numbers reflect the progress made in molecular research over the past years. However, there remain still a number of unsolved cases. Therefore, we aimed to further elucidate the etiology of two types of genetic bone disorders, i.e. high bone mass disorders and Paget’s disease of bone. In Part A of this thesis, we focused on two cases with a high bone mass disorder. Here, we identified pathogenic variants in genes linked to canonical WNT signaling activity (i.e. LRP4 and CTNNB1), ultimately resulting in increased bone formation. In Part B of this thesis, we aimed to further unravel the genetic architecture of Paget’s disease of bone. Although much of the genetic basis of this metabolic bone disease is clear, additional research is essential to study possible genotype-phenotype correlations and pathophysiological mechanisms. Recently, two novel genes have been identified to be involved in Paget’s disease of bone, i.e. PFN1 and ZNF687. In this thesis, a mutation analysis was performed in order to investigate whether genetic variation in these genes also plays a role in the development of Paget’s disease of bone in a Belgian patient cohort. Eventually, our findings revealed a cluster of genetic variation in the nuclear localization signal of the encoded ZNF687 protein, most likely resulting in increased NF-κB signaling and the development of a Pagetic phenotype. Finally, we aimed to assess the use of zebrafish to generate a novel in vivo model for Paget’s disease of bone. Therefore, a sqsmt1(tmΔUBA) mutant zebrafish line, characterized by a loss-of-function of the ubiquitin-associated domain of the p62 protein, was generated and phenotyped. In carriers of a mutant allele, skeletal and cellular defects similar to those described in human patients were observed. Our findings therefore suggest that our sqstm1(tmΔUBA) line is a successful proof-of-concept zebrafish model for Paget’s disease of bone, paving the way for future testing of novel therapeutic targets/compounds, as well as further investigation of the pathophysiological mechanisms underlying Paget’s disease of bone. In conclusion, this thesis has further elucidated the molecular genetic landscape of bone disorders, which will undoubtedly have a valuable impact on the diagnostics and understanding of high bone mass disorders and Paget’s disease of bone in the future.
CGG short tandem repeats in unexplained intellectual disability and autism - Dale John Annear (6/7/2023)
Dale John Annear
- 6 July 2023, 4pm - 6pm
- Promotiezaal (CDE)
- Promoters: Frank Kooy, Geert Vandeweyer
STR-related disorders result from expanded specific STR loci. Fragile X Syndrome (FXS) is an example caused by an expanded CGG trinucleotide STR in the FMR1 gene. The link between CGG STRs and neurological disorders is growing as more CGG STR disorders are found. Primarily, neurodevelopmental, neurodegenerative, and neuropsychiatric disorders are associated with pathogenic CGG STR expansions. In CGG STR-related neurodevelopmental disorders (NDDs), CGG STR expansion causes hypermethylation of the repeat and surrounding DNA. This hyper-condenses the STR and adjacent chromatin, silencing genes. However, technological limitations challenge repeat expansion detection. Routine diagnostic techniques inadequately detect large STR expansions, and contemporary whole-genome sequencing (WGS) struggles due to reliance on sequence-matching algorithms. Nonetheless, recent statistical-based repeat genotyping advancements enable reliable STR interrogation within WGS.
This thesis leveraged WGS and bioinformatic methods to study the CGG STR in a genome-wide manner. A catalogue of CGG STRs was constructed, identifying over 6,000 CGG STR loci in the human genome. Around 90% of these loci displayed polymorphism within a control population, indicating variable repeat lengths across individuals. Two-thirds of CGG STRs were located near gene promoters, many of which were associated with neurodevelopmental disorders. Hundreds of CGG STRs were observed to resemble known disease-causing repeats.
Analysing a cohort of nearly 2,000 families with an autism spectrum disorder (ASD)-affected individual, mutation rates and inheritance patterns of CGG STRs were explored. Nearly all CGG STRs exhibited some degree of polymorphism, with specific hypermutable CGG STRs clustering in particular genomic regions. Comparing ASD-affected probands with unaffected siblings, significantly higher rates and sizes of CGG STR mutations were observed in probands. Non-Mendelian inheritance patterns of CGG STRs were noted, with smaller repeat alleles being preferably inherited.
By analysing 16,000 WGS samples from NDD patients, we identified 419 large CGG repeat mutations in 118 genes. Individuals with ASD had a 2.9 times higher chance of having large CGG repeat mutations compared to their unaffected parents. We also found CGG repeat mutations in over 30 genes previously associated with neurodevelopmental conditions. Additionally, we discovered strong candidate genes, such as RGPD2 and SAMD1, potentially involved in previously unreported recessive neurodevelopmental disorders In conclusion, this research provided a comprehensive understanding of CGG STRs and their association with NDDs. We identified novel CGG STRs, uncovered new gene candidates for repeat disorders, established the broad involvement of CGG STRs in NDDs, and developed a methodology for future research and potential clinical applications.
Investigating aneuploidy and mosaicism as a mechanism for survival and adaptation of Leishmania - Gabriel Heringer Negreira (27/06/2023)
Gabriel Heringer Negreira
- 27 June 2023, 4pm - 6pm
- Aula Janssens, ITG
- Promoters: Jean-Claude Dujardin, Malgorzata Domagalskia
Leishmania is a protozoan parasite causing a neglected tropical disease known as leishmaniasis, which is fatal in visceral infections if untreated. Few drugs are available for treatment and the emergence of drug resistance poses a threat to current elimination efforts. Leishmania has a plastic genome characterized by dynamic modulation of chromosomes copy number (aneuploidy), which leads to different karyotypes co-existing in clonal populations (mosaic aneuploidy). It is hypothesized that Leishmania exploits mosaic aneuploidy as a strategy for early adaptation to environmental stresses, in particular to drug pressure. In this thesis, we aimed to investigate the extent and dynamics of mosaic aneuploidy during adaptation to standard in vitro conditions as well as to drugs.
We first applied a high throughput single-cell genome sequencing (SCGS) to reveal for the first time the complete karyotype of thousands of individual Leishmania parasites in two distinct clonal populations in standard in vitro culture. We observed that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid cell, with mosaic aneuploidy further increasing by moderate and gradual karyotypic alterations. We also found that all chromosomes are prone to somy changes, but only some polysomies can achieve high frequencies in the population, suggesting that natural selection determines which karyotypes emerge and propagate.
In a second set of experiments, we investigated the dynamics of mosaic aneuploidy during adaptation to high drug pressure (SbIII and miltefosine) in vitro. By combining SCGS with lineage tracing using cellular barcodes and longitudinal genome characterization, we revealed that early aneuploidy changes observed under SbIII pressure result from the polyclonal selection of pre-existing karyotypes, complemented by rapid cumulative karyotypic alterations which culminates in similar aneuploidy modifications, pointing to a process of convergent evolution. In the case of miltefosine, early parasite adaptation was associated with independent pre-existing point mutations in a miltefosine transporter gene and aneuploidy changes only emerged later, upon exposure to increased concentration of the drug. Different mutants of the miltefosine transporter gene were identified, also pointing out the polyclonal origin of miltefosine resistance.
In summary, the present thesis demonstrates that polyclonality and genome plasticity are hallmarks of Leishmania adaptation, with the scenario of aneuploidy dynamics being dependent on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.
Unravelling disease signatures in Charcot-Marie-Tooth disease using induced pluripotent stem cells - Jonas Van Lent (14/06/2023)
Jonas Van Lent
- 14 June 2023, 3pm - 5pm
- Auditorium O5 (CDE)
- Promoter: Vincent Timmerman
Charcot-Marie-Tooth (CMT) disease is an inherited peripheral neuropathy caused by mutations in various genes. It is a genetically diverse group of disorders with over 1000 mutations in more than 90 genes associated with the disease. CMT affects approximately 1 in 2500 individuals worldwide and is categorized into demyelinating (CMT1) and axonal (CMT2) forms based on the primary deficits occurring in Schwann cells or neuronal axons.
Despite its clinical diversity, there is currently no cure for CMT, highlighting the need for a better understanding of its mechanisms and the development of new treatments. Recent advances in cell biology and transgenic rodent models have identified potential drug targets for CMT. However, translating these findings into successful clinical treatments has proven challenging. To overcome this, it is important to validate discoveries using biologically and clinically relevant models that accurately represent the human disease.
This thesis focuses on utilizing the induced pluripotent stem cell (iPSC) technology to study multiple CMT subtypes. The first part is focused on CMT2, from which patient-derived iPSCs were differentiated into motor and sensory neurons. We identified common hallmarks of axonal degeneration and showed that targeting these shared pathomechanisms holds promise for developing a uniform treatment for CMT.
In the second part, we established iPSC-derived organoid cultures, containing various cell types of the peripheral nervous system (PNS), including myelinating human Schwann cells. We used this organoid-model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we supported the therapeutic strategy of reducing PMP22 expression.
While our organoids closely mimic the in vivo complexity, they lack directional cell growth. This differs from the in vivo situation, where the PNS stretches in a directional manner from the spinal cord towards the most distal nerve ends, making it challenging to study the neuromuscular junction (NMJ), as NMJs could be located throughout the organoid. To address this limitation, we developed human neuron sphere-muscle co-cultures and 3D neuromuscular assembloids, providing a directional growth from motor neuron to muscle and enabling the study of NMJs.
In summary, we generated human 2D and 3D model systems using patient-derived iPSCs to study common disease mechanisms and allowing to validate candidate therapeutic molecules, which could potentially be beneficial to a larger group of CMT patients, or for patients with other neurodegenerative and neuromuscular disease.
Multimodal approach to unravel network disturbances and compensatory mechanisms during pre- and early-plaque stages of Alzheimer’s Disease - Monica van den Berg (23/05/2023)
Monica van den Berg
- 23 May 2023, 4pm - 6pm
- Promotiezaal (CDE)
- Promoter: Marleen Verhoye
Due to the aging world population, the number of people that are suffering from Alzheimer´s Disease (AD) is expected to dramatically increase. In AD, an accumulation of both toxic amyloid-beta (Aβ) aggregates and neurofibrillary tau tangles disrupts neuronal function, leading to severe cognitive deficits and behavioral alterations. AD is characterized by a long presymptomatic phase, where the progressive accumulation of Aβ and tau interferes with neuronal function. Early network imbalance during the presymptomatic phase, caused by synaptic dysfunction induced by soluble Aβ species, drives the progression of AD. Therefore, researchers hypothesize that restoration of the network imbalance at early stages of the disease could slow down or stop the disease progression of AD.
In this thesis, we aimed to evaluate alterations in whole brain network activity and hippocampal oscillatory activity at pre- and early-plaque stages of AD and how this is linked to histopathological AD-related alterations and behavioral disturbances. For this purpose, we used a multimodal approach where we obtained results from resting state functional MRI, in vivo hippocampal measurements of neuronal activity in freely behaving animals, and ex vivo histological analysis. We investigated pre- and early-plaque stages of AD, using the TgF344-AD rat model displaying all phenotypical hallmarks of AD.
We observed alterations in network activity during recurrent patterns of brain activity, so-called quasi-periodic patterns (QPP), at the pre-plaque stage of AD. Large differences in spatial activation occurred mainly in the basal forebrain (BFB) of TgF344-AD rats, which coincided with soluble Aβ pathology and astrogliosis in the BFB. During the early-plaque phase, spatial and temporal properties of QPPs in TgF344-AD rats were similar to the QPPs of wild-type littermates, and the astrogliosis in the BFB was diminished. Similarly, electrophysiology studies demonstrated during the pre-plaque stage of AD, alterations in neuronal activity within the hippocampus, a region important for learning and memory, which were partially recovered at the early-plaque stage in TgF344-AD rats. This partial recovery coincided with an increased number of hippocampal cholinergic synapses, suggesting that the cholinergic sprouting originating from the BFB might be an important compensatory mechanism during the early-plaque stage of AD.
In summary, this thesis highlights the diagnostic potential of quasi-periodic pattern analysis as a noninvasive early biomarker for AD. In future studies, the assessment of quasi-periodic pattern analysis upon activation of cholinergic neurons in the BFB using chemogenetic tools could be a valuable strategy to gain insights into recovery of network activity in AD.
Characterizing the bone marrow as a parasitological niche responsible for antileishmanial treatment failure - Laura Dirkx (12/05/2023)
- 12 May 2023, 4pm - 6pm
- Promotiezaal (CDE)
- Promoters: Guy Caljon, Louis Maes
Visceral leishmaniasis (VL) is a lethal parasitic disease caused by the intracellular protozoan Leishmania and is transmitted through the bites of sand flies. In the vertebrate host, parasites infect cells of the liver, spleen and bone marrow (BM). Treatment options are scarce, as are novel leads in the R&D pipeline. Moreover, toxicity, resistance and post-treatment relapse are common and currently no effective test-of-cure exists. There is no human vaccine available and knowledge on protective immunity is limited. All of these obstruct disease elimination and constitute a major clinical concern.
In this thesis, a specific cell type was uncovered as source of relapse, namely the long-term hematopoietic stem cell (LT-HSC) in the BM. An enormous number of parasites reside within this hospitable niche that is characterized by low oxidative burst levels and a higher tolerance to antileishmanial drugs. Infected LT-HSC express a unique transcriptional signature, termed StemLeish. Cross-species analyses revealed significant overlap with human VL and HIV co-infected blood transcriptomes. Using siRNA silencing, knockdown of major upregulated StemLeish genes Rgs1, Cxcr4, Ell2, Vav1 and Twistnb confirmed their prominence in regulating LT-HSC infection. Cxcr4 proved important for the observed decrease in oxidative burst, suggesting a central position of this gene in shaping the LT-HSC niche, corroborated by in vivo therapeutic exploration. In LT-HSC, amastigotes rapidly enter quiescence, a phenotype with a higher capacity to survive antileishmanial treatment, providing an informal link to relapse. Parasites that transitioned through quiescence displayed an increased cellular infectivity and high sand fly transmission capacity. Transcriptional profiling revealed a novel set of markers and potential drivers of quiescence.
Lastly, the impact of VL on B cell lymphopoiesis and humoral immunity was investigated. Corroborating the documented VL clinical manifestation of polyclonal hypergammaglobulinemia, infection was found to increase B cell progenitors in the BM, and all analyzed B cell subtypes of the spleen. Using immunization against a fluorescent heterologous antigen, it was shown that VL infection does not impair humoral immune memory. Surprisingly, the B cell interacts with Leishmania, carrying amastigotes, extracellularly attached while remaining in a resting, non-replicative state.
Collectively, the results in this thesis deliver unprecedented insights regarding post-treatment relapse and humoral immunity during VL infection, providing novel drug targets and biomarkers for both host-directed and parasite-directed therapeutics using differential genes from quiescent amastigotes residing in a relapse-prone niche. This data will have a direct impact on drug screening efforts tweaked to overcome relapse.
First-line anti-tuberculosis drugs revisited - Pauline Lempens (12/04/2023)
- 12 April 2023, 4pm - 6pm
- Aula Janssens, ITG
- Promoters: Leen Rigouts, Bouke de Jong
Drug-resistant tuberculosis (DR-TB) continues to be a major challenge in the control of the worldwide TB epidemic. One of the strategies to oppose DR-TB has been to optimize the use of existing drugs, including first-line drugs. In this thesis, we investigated the association between genotypic and phenotypic Mycobacterium tuberculosis resistance levels, and the impact of resistance on treatment outcome and transmission of rifampicin-resistant or multidrug-resistant TB (RR/MDR-TB).
We found that high-confidence mutations in katG, inhA, and the inhA promoter largely predict the level of phenotypic isoniazid resistance, with the combination of inhA promoter region and katG mutations being associated with the highest-level resistance (≥19.2 mg/L), exceeding peak level concentrations of the drug even at the highest doses in clinical use.
Secondly, we found that in patients with initially fluoroquinolone-/second-line-injectable-susceptible RR/MDR-TB who were treated with the shorter MDR-TB treatment regimen, resistance to other drugs in the regimen (either ethambutol, ethionamide, or pyrazinamide) was not associated with having a clinically adverse outcome. High-level isoniazid resistance (vs any other level) predicted treatment failure in patients with high-level fluoroquinolone resistance.
Thirdly, in a dataset of 394 whole genome sequenced MDR-TB isolates from Bangladesh, gathered over 6 years, we found a relatively low percentage of transmission clustering (34.8%) but no difference in clustering between isolates with borderline rpoB mutations and those with common rpoB mutations. Compensatory mutations in rpoA, rpoB, and rpoC were associated with higher levels of transmission clustering as were lineages 2, 3, and 4 relative to lineage 1. Young people as well as patients with high sputum smear positive TB were more likely to be in a transmission cluster.
Lastly, we characterised a set of 50 phenotypically RR-TB isolates with wild type rpoB, of which 33 belonged to the same lineage (lineage 4.7). Several mutations in genes previously associated with rifampicin resistance potentially explained resistance in these isolates. Retesting the isolates on Löwenstein-Jensen medium yielded minimum inhibitory concentrations around the critical concentration, as did retesting of rifampicin-susceptible isolates of lineage 4.7.
Our studies contributed to and stressed the importance of accurate detection of drug resistance, including to first-line drugs, in order to optimise treatment of RR/MDR-TB and minimise its transmission.
Implementation and evaluation of new tools for Ebola virus disease response during outbreaks in eastern Democratic Republic of the Congo: from rapid diagnostic tests to genomic sequencing - Daniel Mukadi (27/03/2023)
- 27 March 2023, 4pm - 6pm
- Aula P.G. Janssens, ITG
- Promoters: Kevin Ariën, Johan van Griensven
The Democratic Republic of the Congo has faced fifteen Ebola virus disease (EVD) outbreaks, among which the 2018-2020 was the most widespread and deadliest so far. During that outbreak associated with chronic insecurity, community mistrust and resistance, thirteen field laboratories were deployed to support the EVD diagnosis with the GeneXpert®. As this latter cannot be used everywhere in remote areas due to its additional requirements, rapid diagnostic tests (RDTs) were proposed as an alternative tool to support quick decision-making at the point-of-care. However, questions regarding RDTs diagnostic performance and usability were raised due to their disparate performances.
In the first part of this PhD thesis, we showed how decentralized and strategically positioned diagnostic laboratories quickly helped to mitigate the risk of Ebola virus spread through rapid, efficient, accurate and well-structured response. Quick hand over of competences and capacities to local teams led to successful management of further health emergencies (EVD flare-ups and Covid-19 pandemic), as those laboratories had dedicated equipment and well-trained local personnel. Sequencing data guided public health decision-making, helped understanding the outbreak dynamics, at risk populations and exposed health zones.
In the second part of this PhD, QuickNaviTM RDT had high specificity and quite good sensitivity than OraQuick® and Coris® tests in outbreaks conditions. QuickNavi-EbolaTM was less impressive compared to previous studies; OraQuick® test was almost in line with previous findings, although it performed better for the middle and lower Ct-values in laboratory conditions. None of the four Ebola RDTs evaluated throughout our studies, achieved the desired (sensitivity >98%, specificity >99%) or acceptable (sensitivity >95%, specificity >99%) levels of performance as stated by the WHO Target Product Profile for EBOV tests. However, respective specificities of the QuickNaviTM (>99%) and OraQuick® Ebola (98%) in most our studies were close to the acceptable level of performance (>99%).
Based on overall performances, QuickNaviTM and Oraquick® Ebola RDTs were proposed as a screening panel at the point-of-care to triage and isolate suspect-cases waiting for the RT-qPCR results. RDTs results expectancy will be done separately i.e. individuals with at least one positive RDT isolated in the high-risk area and those with negative RDTs results in low-risk area.
For postmortem surveillance, OraQuick® RDT effectively complemented the response efforts, improved the community engagement, and decreased the number of systematic safe and dignified burials (SDBs) in corpses with non-reactive test. Trust towards postmortem testing led families to voluntarily request for SDBs despite OraQuick® non-reactive results.
Metformin: an old drug offering new opportunities in the battle against chronic kidney disease - Raphaëlle Corremans (23/02/2023)
- 23 February 2023, 4:30pm - 6:30pm
- Promotiezaal (CDE)
- Promoters: Anja Verhulst, Benjamin Vervaet
Chronic kidney disease (CKD) is a major renal health issue, which has a high socio-economic impact by its far-reaching health consequences. This often results in the need for renal replacement therapy (dialysis or transplantation). Currently, disease management remains mainly supportive in nature due to the lack of efficient treatments that directly act on the kidney and improve its outcome upon injury. However, recent research on the antidiabetic drug metformin revealed a consistent beneficial effect of this drug on the challenged kidney. This PhD thesis further explores the renoprotective effects of metformin and compares these effects to that of canagliflozin, an antidiabetic drug that was recently approved by the US Food and Drug Administration (FDA, 2021) to treat CKD.
The first part of this PhD project compared the therapeutic efficacy of metformin and canagliflozin, on the progression of CKD in the non-diabetic rat model with adenine-induced CKD. Metformin, but not canagliflozin, therapeutically halted functional renal decline of established CKD, as well as progressive expansion of the interstitium and interstitial inflammation. Proteomic analyses revealed that metformin’s renoprotective effect was associated with the activation of the Hippo signaling pathway. In the future, this might open up new perspectives for the development of novel, uncontested, more effective and safe targeted treatment strategies which can serve as interventions in CKD.
Since diabetes is one of the most important risk factors to develop CKD, the second part of the PhD project focused on diabetic kidney disease (DKD). Therefore, a suitable rat model of DKD, that develops the clinical and pathological features of human DKD within an acceptable experimental time frame, was optimized. We discovered that L-NAME-induced NO-deficiency in diabetic rats importantly contributed to the development of all desired characteristic features of human DKD. Additionally, also in this preclinical model the ability of metformin and canagliflozin to prevent or slow down the progression of DKD was compared. Metformin exerted renoprotective effects beyond its capacity to reduce blood glucose. Metformin was able to protect renal proximal tubular cells, reduce tubular damage and prevent collagen accumulation. Canagliflozin exerted similar protective actions, which however, could clearly be linked to its blood glucose lowering activity. In this preclinical model, both metformin and canagliflozin were able to protect the kidney, in a non-synergistic way, against DKD development by, at least in part, inhibiting the NF-κB signaling pathway.
The versatile nature of cytoglobin, the Swiss army knife among globins, with a preference for oxidative stress - Joey De Backer (6/2/2023)
Joey De Backer
- 6 February 2023, 4pm - 6pm
- Promotiezaal (CDE)
- Promoters: Wim Vanden Berghe, Annemie Bogaerts, David Hoogewijs
Since its discovery 20 years ago, many studies have been performed to gain insight into the functional role of cytoglobin (Cygb). However, Cygb has been proven to be a promiscuous protein. Yet, there is a consensus that Cygb is a cytoprotective protein involved in redox homeostasis. CYGB is a ubiquitously expressed hexacoordinated globin that is highly expressed in melanocytes and is often found to be downregulated during melanocyte-to-melanoma transition.
In Chapter III, we investigated the molecular mechanism through which CYGB could be involved in redox regulation. Here, we showed that CYGB contains two redox-sensitive cysteine residues and that the formation of an intramolecular disulfide bridge resulted in the heme group becoming more accessible to external ligands. This supports the hypothesis that Cys38 and Cys83 serve as sensitive redox sensors. In Chapter IV we showed that CYGB mRNA and protein levels were elevated upon exposure to hypoxia. Interestingly, this upregulation was most likely HIF-2α-dependent. We propose that in melanoma, HIF-2α, rather than HIF-1α, positively regulates CYGB under hypoxic conditions in a cell type specific way. In Chapter V, the cytotoxic effect of indirect NTP treatment in two melanoma cell lines with divergent endogenous CYGB expression levels was investigated. We confirmed that NTP endows cytotoxicity that induces cell death through apoptosis and that this was mediated through the production of ROS. Moreover, we showed that CYGB protects melanoma cells from ROS-induced apoptosis by the scavenging of ROS. Interestingly, CYGB expression influenced the expression of NRF2 and HO-1. We identified the lncRNA MEG3 as a possible mechanism through which NRF2 expression and its downstream target HO-1 can be regulated by CYGB. In chapter VI, increased basal ROS levels and higher degree of lipid peroxidation upon RSL3 treatment contributed to the increased sensitivity of CYGB knockdown G361 cells to ferroptosis. Furthermore, transcriptome analysis demonstrates the enrichment of multiple cancer malignancy pathways upon CYGB knockdown, supporting a tumor-suppressive role for CYGB. Remarkably, CYGB expression regulation was identified as a critical determinant of the ferroptosis–pyroptosis therapy response. This suggests that CYGB is involved in the regulation of multiple modes of programmed cell death. FInally, we sought to delineate the RONS that are responsible for plasma-induced ICD. Our results highlight the importance of the short-lived species. Furthermore, we are first to demonstrate that NTP-created vaccine is safely prepared and offers complete protection. Moreover, we provide conclusive evidence that direct application of NTP induces ICD in melanoma.