Lung cancer is the leading cause of cancer death in the Netherlands. For years chemotherapy was the only (palliative) treatment, with a short survival of only months. Since the introduction of... Show moreLung cancer is the leading cause of cancer death in the Netherlands. For years chemotherapy was the only (palliative) treatment, with a short survival of only months. Since the introduction of immunotherapy in 2015, this survival has increased significantly, with the first results showing a survival of even a few years. However, the response rate is relatively low, the treatment is expensive and the (low percentage of) side effects are severe. Therefore a biomarker is needed to predict which patients would benefit of immunotherapy.This thesis is about the search for a new biomarker. With the use of the RNA of platelets, proteins, tumor markers in blood and a an electronic nose for exhaled breath, we tried to find a non-invasive biomarker for the prediction of response on immunotherapy and for the (future) use in clinical practice, some of which are promising. Show less
Protein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be... Show moreProtein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be associated with the development and progression of the disease paving the way for the development of new clinical biomarkers as well as providing specific targets for therapeutic intervention, patient stratification and personalized medicine. Protein glycosylation is also critical for the development of biopharmaceuticals, as even minor shifts in manufacturing procedures can substantially impact the bioactivity, safety, and efficacy of therapeutic proteins. Although a variety of mass spectrometric and chromatographic methods are available for the identification and characterization of glycans from complex sample mixtures, the lack of standardized protocols across platforms often results in inconsistent results, making data integration and comparison challenging. Furthermore, most of the current technology for the study of intact glycans would not be suitable for the rapid analysis of large sample sets, mainly due to limitations in sample throughput. The scope of this thesis is to establish standardized, high-throughput glycomics technologies for the quantitative analysis of protein N- and O-glycosylation and improve current methodologies in order to facilitate the characterization of intact oligosaccharides from in vitro established model systems. Show less
Wind, M.; Akker-van Marle, M.E. van den; Ballieux, B.E.P.B.; Cobbaert, C.M.; Rabelink, T.J.; Lith, J.M.M. van; ... ; Sueters, M. 2022
Background: This study investigated the clinical value of adding the sFlt-1/PlGF ratio to the spot urine protein/creatinine ratio (PCr) in women with suspected pre-eclampsia. Methods: This was a... Show moreBackground: This study investigated the clinical value of adding the sFlt-1/PlGF ratio to the spot urine protein/creatinine ratio (PCr) in women with suspected pre-eclampsia. Methods: This was a prospective cohort study performed in a tertiary referral centre. Based on the combination of PCr (< 30) and sFlt-1/PlGF (& LE;38) results, four groups were described: a double negative result, group A-/-; a negative PCr and positive sFlt-1/PlGF, group B-/+; a positive PCr and negative sFlt-1/PlGF, group C+/-; and a double positive result, group D+/+. The primary outcome was the proportion of false negatives of the combined tests in comparison with PCr alone in the first week after baseline. Secondary, a cost analysis comparing the costs and savings of adding the sFlt-1/PlGF ratio was performed for different follow-up scenarios. Results: A total of 199 women were included. Pre-eclampsia in the first week was observed in 2 women (2%) in group A-/-, 12 (26%) in group B-/+, 4 (27%) in group C+/-, and 12 (92%) in group D+/+. The proportion of false negatives of 8.2% [95% CI 4.9-13.3] with the PCr alone was significantly reduced to 1.6% [0.4-5.7] by adding a negative sFlt-1/PlGF ratio. Furthermore, the addition of the sFlt-1/PlGF ratio to the spot urine PCr, with telemonitoring of women at risk, could result in a reduction of 41% admissions and 36% outpatient visits, leading to a cost reduction of euro46,- per patient. Conclusions: Implementation of the sFlt-1/PlGF ratio in addition to the spot urine PCr, may lead to improved selection of women at low risk and a reduction of hospital care for women with suspected pre-eclampsia. Show less
Wind, M.; Akker-van Marle, M.E. van den; Ballieux, B.E.P.B.; Cobbaert, C.M.; Rabelink, T.J.; Lith, J.M.M. van; ... ; Sueters, M. 2022
BackgroundThis study investigated the clinical value of adding the sFlt-1/PlGF ratio to the spot urine protein/creatinine ratio (PCr) in women with suspected pre-eclampsia.MethodsThis was a... Show moreBackgroundThis study investigated the clinical value of adding the sFlt-1/PlGF ratio to the spot urine protein/creatinine ratio (PCr) in women with suspected pre-eclampsia.MethodsThis was a prospective cohort study performed in a tertiary referral centre. Based on the combination of PCr (< 30) and sFlt-1/PlGF (≤38) results, four groups were described: a double negative result, group A−/−; a negative PCr and positive sFlt-1/PlGF, group B−/+; a positive PCr and negative sFlt-1/PlGF, group C+/−; and a double positive result, group D+/+. The primary outcome was the proportion of false negatives of the combined tests in comparison with PCr alone in the first week after baseline. Secondary, a cost analysis comparing the costs and savings of adding the sFlt-1/PlGF ratio was performed for different follow-up scenarios.ResultsA total of 199 women were included. Pre-eclampsia in the first week was observed in 2 women (2%) in group A−/−, 12 (26%) in group B−/+, 4 (27%) in group C+/−, and 12 (92%) in group D+/+. The proportion of false negatives of 8.2% [95% CI 4.9–13.3] with the PCr alone was significantly reduced to 1.6% [0.4–5.7] by adding a negative sFlt-1/PlGF ratio. Furthermore, the addition of the sFlt-1/PlGF ratio to the spot urine PCr, with telemonitoring of women at risk, could result in a reduction of 41% admissions and 36% outpatient visits, leading to a cost reduction of €46,- per patient.ConclusionsImplementation of the sFlt-1/PlGF ratio in addition to the spot urine PCr, may lead to improved selection of women at low risk and a reduction of hospital care for women with suspected pre-eclampsia. Show less
Levink, I.J.M.; Klatte, D.C.F.; Hanna Sawires, R.G.; Vreeker, G.C.M.; Ibrahim, I.S.; Burgt, Y.E.M. van der; ... ; Mesker, W.E. 2022
Background: Surveillance of individuals at risk of developing pancreatic ductal adenocarcinoma (PDAC) has the potential to improve survival, yet early detection based on solely imaging modalities... Show moreBackground: Surveillance of individuals at risk of developing pancreatic ductal adenocarcinoma (PDAC) has the potential to improve survival, yet early detection based on solely imaging modalities is challenging. We aimed to identify changes in serum glycosylation levels over time to earlier detect PDAC in high-risk individuals. Methods: Individuals with a hereditary predisposition to develop PDAC were followed in two surveillance programs. Those, of which at least two consecutive serum samples were available, were included. Mass spectrometry analysis was performed to determine the total N-glycome for each consecutive sample. Potentially discriminating N-glycans were selected based on our previous cross-sectional analysis and relative abundances were calculated for each glycosylation feature. Results: 165 individuals ("FPC-cohort" N = 119; Leiden cohort N = 46) were included. In total, 97 (59%) individuals had a genetic predisposition (77 CDKN2A, 15 BRCA1/2, 5 STK11) and 68 (41%) a family history of PDAC without a known genetic predisposition (>10-fold increased risk of developing PDAC). From each individual, a median number of 3 serum samples (IQR 3) was collected. Ten individuals (6%) developed PDAC during 35 months of follow-up; nine (90%) of these patients carried a CDKN2A germline mutation. In PDAC cases, compared to all controls, glycosylation characteristics were increased (fucosylation, tri-and tetra-antennary structures, specific sialic linkage types), others decreased (complex-type diantennary and bisected glycans).The largest change over time was observed for tri-antennary fucosylated glycans, which were able to differentiate cases from controls with a specificity of 92%, sensitivity of 49% and accuracy of 90%. Conclusion: Serum N-glycan monitoring may support early detection in a pancreas surveillance program.(c) 2022 The Authors. Published by Elsevier B.V. on behalf of IAP and EPC. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
The African savannas that many early hominins occupied likely experienced stark seasonality and contained mosaic habitats (i.e., combinations of woodlands, wetlands, grasslands, etc.). Most would... Show moreThe African savannas that many early hominins occupied likely experienced stark seasonality and contained mosaic habitats (i.e., combinations of woodlands, wetlands, grasslands, etc.). Most would agree that the bulk of dietary calories obtained by taxa such as Australopithecus and Paranthropus came from the consumption of vegetation growing across these landscapes. It is also likely that many early hominins were selective feeders that consumed particular plants/plant parts (e.g., leaves, fruit, storage organs) depending on the habitat and season within which they were foraging. Thus, improving our understanding of how the nutritional properties of potential hominin plant foods growing in modern African savanna ecosystems respond to season and vary by habitat will improve our ability to model early hominin dietary behavior. Here, we present nutritional analyses (crude protein and acid detergent fiber) of plants growing in eastern and southern African savanna habitats across both wet and dry seasons. We find that many assumptions about savanna vegetation are warranted. For instance, plants growing in our woodland habitats have higher average protein/fiber ratios than those growing in our wetland and grassland transects. However, we find that the effects of season and habitat are complex, an example being the unexpectedly higher protein levels we observe in the grasses and sedges growing in our Amboseli wetlands during the dry season. Also, we find significant differences between the vegetation growing in our eastern and southern African field sites, particularly among plants using the C4 photosynthetic pathway. This may have implications for the differences we see between the stable carbon isotope compositions and dental microwear patterns of eastern and southern African Paranthropus species, despite their shared, highly derived craniodental anatomy. Show less
Proteins play a crucial role in life, taking part in all vital process in the body, and are therefore used as therapeutic agents in a diverse range of biomedical applications. When... Show more Proteins play a crucial role in life, taking part in all vital process in the body, and are therefore used as therapeutic agents in a diverse range of biomedical applications. When administrated into bodily fluids, most native proteins are prone to degradation or inactivation process. The challenges of protein delivery are overcoming poor stability, low permeability toward cell membrane. Among all existing materials for protein delivery, mesoporous silica nanoparticles (MSNs) are one of the most promising intracellular nanocarriers due to its key properties: biocompatible, straightforward synthesis, and surface modification. For various biomedical applications, monodisperse MSNs with a particle size in the 50-200 nm range,3 controllable surface chemistry,4 and a large pore size (> 5 nm) are desired. This thesis presents a new method to synthesize large disc-like pore (10 ± 1 nm) containing MSNs with an elongated cuboidal-like geometry (90 × 43 nm), which effectively encapsulate and release proteins. Show less
The work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the... Show moreThe work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the ubiquitin proteasome system (UPS) and activity‐based proteasome profiling. Chapter 2 is a literature review of some new achievements in the activity‐based protein profiling field in the recent years, focusing on application in biochemistry, molecular and cellular biology, medicinal chemistry, pathology, physiology and pharmacology research. Chapter 3 is a protocol for performing quantitative activity‐based proteasome profiling experiments. In the protocol, both high throughput fluorescent ABPP and biotinylated probe plus LC/MS approaches are described. Chapter 4 is a brief technical report about bioorthogonal chemistry in ABPP. The commonly used secondary azide group is compared with a primary azide group in proteasome ABPs performing Cu(I) catalyzed azide‐alkyne cycloaddition and Staudinger‐Bertozzi reaction under native/denatured protein conditions Chapter 5 is focusing on the application of quantitative activity‐based proteasome profiling in the prognosis of cancer therapeutics. A combination of ABPP and global proteomics is performed to elucidate the bortezomib sensitivity and resistance mechanisms in leukemia and solid tumor cells. Chapter 6 describes the characterization of the newly discovered proteasome subunit β5t by ABPP and LC/MS proteomics. The subunit is proven to be catalytically active. A hydrophilic Thr residue on the P2 position of the proteasome inhibitor improves the inhibitory efficiency of β5t, which indicates it might prefer to cleave hydrophilic peptides. Chapter 7 describes the identification of O‐GlcNAcylation modifications on the ubiquitin receptor protein hHR23B and characterization of how the sugar moiety influences the conformation and functions of the protein. Show less
Therapeutic proteins have become very successful in the treatment of various chronic and life-threatening diseases. However, besides their benefits, therapeutic proteins seem to have a common... Show moreTherapeutic proteins have become very successful in the treatment of various chronic and life-threatening diseases. However, besides their benefits, therapeutic proteins seem to have a common problem - the response of a patient’s immune system against the protein. This means that the immune system of the patient actively removes the drug from the body, thereby potentially decreasing or reversing the effect of the therapy. By now there is strong consensus that damaged and aggregated proteins are important risk factors. Protein aggregates are, due to their heterogeneity and often low quantity, challanging to characterize. Further, there is a large academic interest in understanding the mechanisms of aggregation and the role of non-proteinaceous particles in the process of protein aggregation and unwanted immunogenicity in order to design more effective and safe protein-based medicines. This PhD thesis supported that research effort by developing and improving analytical methodologies to detect the size, quantity and other properties of protein aggregates and particles, especially in the relevant nano- and micrometer size range. These techniques were then applied to study a so far unknown nanoparticulate impurity in pharmaceutical-grade sugars. Further, the results shown in this thesis revealed that these nanoparticulate impurities pose a threat to protein stability. Show less
In the past decade, advances in structure determination with electron microscopy of organic, beam sensitive, materials have been significant. The newly developed techniques, triggered by new... Show moreIn the past decade, advances in structure determination with electron microscopy of organic, beam sensitive, materials have been significant. The newly developed techniques, triggered by new microscope systems and new cameras, made it possible to acquire 3D structural information from these samples to a resolution which was impossible to achieve before. Knowledge is required to improve structure solution and every aspect of the process involved, from treatment of radiation sensitive materials, sample preparation, TEM imaging and diffraction systems all the way to how data must be interpreted. In this thesis I explained multiple new techniques and methods developed by us, using both new microscopes as well as a new type of detector: Timepix. I describe how these tools can help to overcome (what were) the most important problems and bottlenecks in detection of very low dose electron diffraction. Show less
The ultimate goal of translational colon and rectal cancer research is to turn these types of cancer into curable or manageable chronic diseases. The approach to achieve this is to enable... Show moreThe ultimate goal of translational colon and rectal cancer research is to turn these types of cancer into curable or manageable chronic diseases. The approach to achieve this is to enable clinicians to make (adjuvant) treatment decisions, based on the individual patient characteristics and individual characteristics of a patient__s tumor. Identification of new prognostic and predictive biomarkers, based on the biology of individual tumor characteristics, is therefore warranted to further refine the current TNM classification. This thesis describes the use of molecular techniques for the identification of prognostic biomarkers for clinical outcome in (sporadic) colon and rectal cancer. We here present compelling candidate biomarker combinations for validation in further studies. Retrospective and prospective validation of these prognostic biomarker combinations in international and independent patient series is therefore the crucial next step. Additionally, the presented studies stress the importance of -1- combining biomarkers based on tumor biology, -2- integrative analysis of cancer hallmark related processes at all different cellular regulatory levels (genetics, epigenetics and protein level), -3- assessment of tissue specificity between colon and rectal tumors, and -4- studying age-related effects in future colorectal cancer research. Show less
By utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and... Show moreBy utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and P450cam, while the results of PRE demonstrated the presence of a transient encounter complex. Furthermore, the significant insights of the interaction in the interface were uncovered by X-ray crystallography. Currently, the nature of Pdx effector activity is under debate. Since paramagnetic NMR experiments are applicable to solution studies at ambient temperature, PCS, RDC and PRE methods can further resolve the molecular mechanism of P450cam in the future. Show less
Navarrete, M.A.; Bertinetti-Lapatki, C.; Michelfelder, I.; Veelken, H. 2013
In this thesis the prospects of electron diffraction and imaging techniques are discussed to solve the structure of three dimensional nano-crystals. These crystals are beyond the scope of current X... Show moreIn this thesis the prospects of electron diffraction and imaging techniques are discussed to solve the structure of three dimensional nano-crystals. These crystals are beyond the scope of current X-ray techniques. Show less
Huisman, E.J.; Hoek, B. van; Soest, H. van; Nieuwkerk, K.M. van; Arends, J.E.; Siersema, P.D.; Erpecum, K.J. van 2012
The aim of this study is to prepare in situ forming hydrogels based on biocompatible polymers for the controlled release of hydrophobic drug and proteins. In order to load hydrophobic drug to the... Show moreThe aim of this study is to prepare in situ forming hydrogels based on biocompatible polymers for the controlled release of hydrophobic drug and proteins. In order to load hydrophobic drug to the hydrophilic hydrogel matrix, beta-cyclodextrin and human serum albumin was introduced to the hydrogel network respectively and acted as the primary accommodation for those hydrophobic molecules within the hydrogel network. Furthermore, supramolecular crosslinked and covalently crosslinked light sensitive hydrogels were prepared whose potential application for light controlled protein release system has been shown. Show less
The growing field of bio-electronics holds many promises with regard to the integration of various organic molecules onto printed circuit-boards. These include a decrease in the cost of production,... Show moreThe growing field of bio-electronics holds many promises with regard to the integration of various organic molecules onto printed circuit-boards. These include a decrease in the cost of production, an increased sensitivity and specificity to molecular detection from various solutions (i.e. blood) and ultra-miniaturization. However, numerous challenges still face such prospects, chief among which is the retention of biological activity of the adsorbed molecules. To circumvent the possible harmful effects of the bare surfaces, we have made use of self-assembled molecular films that not only shield the proteins (i.e. azurin) off surfaces, but also help establish a spatially-defined conductive path to electrodes. At the same time, the protein itself was engineered such that the active cavity is directly connected via such molecular __wires__. Our results may help in the adsorption of more complex enzymes into future molecular devices, that will retain their activity on the surfaces and are able to integrate into biosensors. Show less
UVDE is a repair enzyme discovered for the first time in the fission yeast Schizaromyces pombe. The initial biochemical characterization of this enzyme showed that its substrate specificity... Show moreUVDE is a repair enzyme discovered for the first time in the fission yeast Schizaromyces pombe. The initial biochemical characterization of this enzyme showed that its substrate specificity includes not only UV lesions, but also abasic sites and some nucleotide mismatches. The mechanism, however, of UVDE damage recognition and DNA cleavage was not clarified. S. pombe UVDE was seen to require Mn2+ and Mg2+ for its function, but the utilization of divalent metals in its cleavage reaction was empirical and without a clear vision of the nature, the number and the exact role of the metal cofactors. UVDE homologues were found in many fungal species and in a number of bacteria such as Bacillus subtilis and Thermus thermophilus. Although the S. pombe UVDE was partially characterized, no information was available for the substrate specificity or the cofactor requirements its bacterial homologues Show less
Despite major improvements in immunosuppressive agents and a reduction in acute rejection episodes, there has been no significant improvement in overall kidney transplant survival beyond the first... Show moreDespite major improvements in immunosuppressive agents and a reduction in acute rejection episodes, there has been no significant improvement in overall kidney transplant survival beyond the first 3 months after transplantation1. Up to sixty percent of all renal allografts are lost within 10 years after transplantation. Apart from death, the main cause of graft loss is chronic allograft nephropathy (CAN)2;3. CAN is characterized by the deterioration of graft function and structure as a consequence of immunological processes (i.e., chronic rejection) and/or a variety of often co-existing non-immunological factors that include: advanced donor age, ischemic injury to the graft during implantation, chronic calcineurin inhibitor induced nephrotoxicity, hypertension, reflux, infection, increased ureteral pressure, and glomerular hyperfiltration. The initial histological characteristics of CAN are the presen ce of tubulointerstitial fibrosis and tubular atrophy (IF/TA)4. Over time, additional features evolve including vasculopathy, glomerulopathy, and glomerulosclerosis. Unfortunately, functional studies significantly underestimate the incidence of graft injury. One of the largest studies with repeated protocol biopsies clearly showed that CAN is a process that develops early after transplantation. Up to 94% of protocol biopsies obtained one year after renal transplantation exhibited IF/TA in patients with stable graft function5. In addition, Solez et al. have shown that 2 years after transplantation two thirds of all kidney allografts exhibit CAN without deterioration of renal function 6. The most significant predictors for the development of CAN were the occurrence of acute rejection episodes, acute calcineurin toxicity, and the initial quality of the transplanted kidney. Often, the first clinical sign of CAN is the progressive decline in renal function as measured by increasing serum creatinine or the development of overt proteinuria2. Although renal function correlates with glomerulosclerosis, unfortunately the clinical tests currently available for renal function are not sensitive enough to detect early lesions associated with CAN. Inulin and iothalamate clearances are more reliable and precise techniques for measuring renal function, but they are expensive, difficult to perform, and time consuming; thus they are unlikely to become routine in daily practice. This situation has given rise to intensified interest in studies using protocol biopsies that may identify surrogate markers and provide insight into the development of CAN. Ideally, a surrogate marker is minimally invasive and amenable to frequent assessment. However, the least invasive markers in blood or urine samples can only identify qualities and quantities of structural lesions i n the renal allograft that typically occur in the advanced stages of disease. Currently, protocol biopsies are considered __the gold standard__ for discovering novel surrogate markers that better predict long-term outcomes for patients. In this thesis, we focused on identifying molecular markers in kidney transplant biopsies that could predict long-term allograft survival. The identification of molecular markers provides a means for superior monitoring of the transplant condition in order to maintain drug efficacy and limit drug-related nephrotoxicity. Moreover, potential new therapeutic targets might be discovered for developing therapies that improve long-term graft survival. Show less
