The research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of... Show moreThe research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of platinum and palladium compounds utilizing the [Pt(bapbpy)]2+ scaffold. The study identifies intercalation as the primary mechanism of action for these complexes. Furthermore, it provides a detailed structure-activity relationship analysis, highlighting the critical role of the complex's protonation state in influencing its biological activity and efficacy. Subsequently, the study delves into photoactivated chemotherapy (PACT) using ruthenium (II) complexes, where light activation of ruthenium complexes enables targeted drug delivery to tumor cells, thereby reducing adverse effects. This research emphasizes the development of ruthenium-based compounds that can photorelease a DNA repair inhibitor, specifically targeting the RAD51 protein, essential for Homologous Recombination (HR). By disrupting the DNA repair mechanisms in cancer cells, this approach seeks to enhance the cytotoxicity of the therapy and address drug resistance. Show less
Lee, M.E.; Albert, E.; Wessels, E.; Kim, S.K.; Chung, H.S.; Gimenez, E.; ... ; Navarro, D. 2023
Monitoring of cytomegalovirus (CMV) viral load is critical for informing treatment decisions in order to prevent the severe health consequences of CMV infection or reactivation of latent CMV in... Show moreMonitoring of cytomegalovirus (CMV) viral load is critical for informing treatment decisions in order to prevent the severe health consequences of CMV infection or reactivation of latent CMV in immunocompromised individuals. This first field evaluation examined the analytical and clinical performance of the Alinity m CMV assay. Analytical performance was assessed with a commercially available six-member panel, while the clinical performance evaluation compared the Alinity m CMV assay to the RealTime CMV assay and a laboratory-developed test (LDT) as the test of record at three large hospital-based clinical laboratories. Precision of the Alinity m CMV assay was demonstrated with total standard deviation (SD) between 0.08 and 0.28 Log IU/mL. A total of 457 plasma specimens were tested on the Alinity m CMV assay and compared to the test of record at each site (n = 304 with RealTime CMV and n = 153 with LDT CMV). The Alinity m CMV assay had excellent correlation (correlation coefficient r =0.942) in comparison to the RealTime CMV or LDT CMV assays. The mean observed bias ranged from -0.03 to 0.34 Log IU/mL. Median onboard turnaround time of Alinity m CMV was less than 3 h. When the CMV assay is run on the Alinity m system, it has the capacity to shorten time to result and, therefore, to therapy. Show less
Detection and monitoring of acute infection or reactivation of Epstein-Barr virus (EBV) are critical for treatment decision-making and to reduce the risk of EBV-related malignancies and other... Show moreDetection and monitoring of acute infection or reactivation of Epstein-Barr virus (EBV) are critical for treatment decision-making and to reduce the risk of EBV-related malignancies and other associated diseases in immunocompromised individuals. The analytical and clinical performance of the Alinity m EBV assay was evaluated at two independent study sites; analytical performance was assessed by evaluating precision with a commercially available 5-member EBV verification panel, while the clinical performance of the Alinity m EBV assay was compared to the RealTime EBV assay and a laboratory-developed test (LDT) as the routine test of record (TOR). Analytical analysis demonstrated standard deviation (SD) between 0.08 and 0.13 Log IU/mL. A total of 300 remnant plasma specimens were retested with the Alinity m EBV assay, and results were compared to those of the TOR at the respective study sites (n = 148 with the RealTime EBV assay and n = 152 with the LDT EBV assay). Agreement between Alinity m EBV and RealTime EBV or LDT EBV assays had kappa values of 0.88 and 0.84, respectively, with correlation coefficients r of 0.956 and 0.912, while the corresponding observed mean bias was -0.02 and -0.19 Log IU/mL. The Alinity m EBV assay had a short median onboard turnaround time of 2:40 h. Thus, the Alinity m system can shorten the time to results and, therefore, to therapy. Show less
Taxonomy as a science has accumulated data and knowledge for more than 250 years. The quality and usefulness of the facts recorded in taxonomic literature has greatly improved from the early... Show moreTaxonomy as a science has accumulated data and knowledge for more than 250 years. The quality and usefulness of the facts recorded in taxonomic literature has greatly improved from the early descriptive texts to the modern data-rich, hypothesis-driven works. Our work illustrates the application of some of the “e-taxonomic” tools and the “New Taxonomy” thinking explored in the introduction. Here, we analyzed specimen data contained in legacy taxonomic literature in Chapters 2 and 3—to observe species distribution of one spider group and genital evolution, respectively—and also explored an integrative perspective that involves describing new taxa and testing phylogenetic hypotheses using molecular and morphological data, as done in Chapter 4 and 5. Show less
The genetic information of all living organisms is contained in their DNA. Cells modify the degree of DNA compaction by epigenetics, which largely determines what genes are read out and which genes... Show moreThe genetic information of all living organisms is contained in their DNA. Cells modify the degree of DNA compaction by epigenetics, which largely determines what genes are read out and which genes are transcriptionally silent. Despite decades of research into this mechanism, there is no consensus on how cells realize the various degrees of DNA compaction in vivo. Eukaryotes, such as humans, compact their DNA into higher-order structures called compact chromatin fibers. We characterize these fibers through a combination of single-molecule force spectroscopy techniques like magnetic tweezers, and rigid base pair Monte Carlo simulations. We show that, for instance, the length and sequence of the linker DNA, the DNA between adjacent nucleosomes, control the mechanical properties of chromatin fibers. Our measurements suggest the formation of more than one higher-order fiber structure. A deeper understanding of the chromatin fiber and its compaction mechanism is important because the dysfunction of such regulation results in various medical conditions such as the epigenetic disorder type 1 diabetes, fragile X syndrome, or various cancers. Show less
In human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional... Show moreIn human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional proteins. Such plasticity of the genome is maintained by dynamic folding and unfolding of DNA-protein spools called nucleosomes. It is unclear, however, how this process is controlled when multiple nucleosomes stack on top of each other and form compact chromatin fibers. This is particularly important since nucleosomes are rarely present in isolation inside a densely packed cell nucleus. Therefore, the aim of this thesis was to increase the understanding of the chromatin fiber structure and its dynamics. Knowing these details would provide many new insights into the mechanisms of gene expression (epigenetic regulation) which, upon malfunction, may cause severe diseases. The presented work consists of an experimental approach involving the application of single-molecule force spectroscopy, and makes use of theoretical modelling based on statistical mechanics. By using magnetic tweezers, we stretched and twisted individual chromatin fibers reconstituted in vitro in order to unfold its nucleosomes. These studies show that folding of nucleosomes into chromatin fibers opens up a plethora of regulatory pathways for controlling the level of DNA organization in cells. Show less
Large carnivores and humans, along with their livestock, have co-existed for thousands of years. However, human population growth and an increase in economic activities are modifying the landscape... Show moreLarge carnivores and humans, along with their livestock, have co-existed for thousands of years. However, human population growth and an increase in economic activities are modifying the landscape for large carnivores and their prey. I studied the interaction between tiger and leopard in a human dominated landscape around Bardia National Park, Nepal. Due to the increase in number of tigers inside the park leopards may be pushed out of the park, where they become involved in conflicts while there may also be a spill-over of young tigers moving to the buffer zone. Tigers were not directly involved in conflict with people which were found from the presence of prey remains in their scats. Elephants were killing most of the humans whereas leopards were found to kill most of the livestock. For future conservation programs we need to focus on the conservation of tiger and leopard in human dominated landscape. Due to interaction with tigers, the leopards are living in close proximity with people and thereby get more involved in conflicts with local communities. Tigers enjoy a religious and cultural tolerance among the people living in the buffer zone whereas leopards lack such religious and cultural tolerance. Show less
ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM... Show moreORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure. Show less
Unambiguous sequence variant descriptions are important in reporting the outcome of clinical diagnostic DNA tests. The standard nomenclature of the Human Genome Variation Society (HGVS)... Show moreUnambiguous sequence variant descriptions are important in reporting the outcome of clinical diagnostic DNA tests. The standard nomenclature of the Human Genome Variation Society (HGVS) describes the observed variant sequence relative to a given reference sequence. We propose an efficient algorithm for the extraction of HGVS descriptions from two DNA sequences. Our algorithm is able to compute the HGVS~descriptions of complete chromosomes or other large DNA strings in a reasonable amount of computation time and its resulting descriptions are relatively small. Additional applications include updating of gene variant database contents and reference sequence liftovers. Next, we adapted our method for the extraction of descriptions for protein sequences in particular for describing frame shifted variants. We propose an addition to the HGVS nomenclature for accommodating the (complex) frame shifted variants that can be described with our method. Finally, we applied our method to generate descriptions for Short Tandem Repeats (STRs), a form of self-similarity. We propose an alternative repeat variant that can be added to the existing HGVS nomenclature. The final chapter takes an explorative approach to classification in large cohort studies. We provide a ``cross-sectional'' investigation on this data to see the relative power of the different groups. Show less
DNA-hosted silver clusters (Ag:DNAs) have attracted a lot of attention due to their small size (~20 atoms), wide range of applications in chemistry and biology, and sequence-dependent optical... Show moreDNA-hosted silver clusters (Ag:DNAs) have attracted a lot of attention due to their small size (~20 atoms), wide range of applications in chemistry and biology, and sequence-dependent optical tunability. Most of the previous studies are focused on the ensemble of emitters in solution. However, little is known about the optical properties of individual emitters, which is a crucial step towards understanding of their real nature, otherwise lost in ensemble averaging. We show that the excitation and emission spectra of individual emitters are broad even at 1.7 K (FWHM ~25 nm). Also, polarization measurements indicate that the excitation is not strongly dependent on the polarization of excitation light, whereas the emission is highly linearly polarized. Furthermore, from time-resolved measurements, we can conclude that the emission of single emitters can be fitted with single exponential decay curve, whereas the emitters organized with nanometer precision on the DNA scaffolds show double–exponential decay. This indicates the interaction between densely packed Ag:DNAs. Finally, we show that the DNA tubes can be used as a nano-contact glue between the colloidal particles functionalized with short DNA strands. Show less
The aim of this thesis was to investigate long term population dynamic processes in Black-tailed Godwit populations resulting from habitat fragmentation on three different spatial scales by using... Show moreThe aim of this thesis was to investigate long term population dynamic processes in Black-tailed Godwit populations resulting from habitat fragmentation on three different spatial scales by using genetics. We demonstrated that egg shell membranes provide a good alternative DNA source for population genetic studies compared to blood samples. Through the usage of egg shell membranes we could conduct the genetic studies in this thesis a large spatial scales. Genetic population structuring was studied here because Black-tailed Godwit breeding areas seemed geographically fragmented and it was unclear if this had led to genetic separation and inbreeding as well. On a local scale we did not detect any genetic populations structuring as an effect of habitat fragmentation. We showed that habitat fragmentation, although geographically visible, has not yet lead to genetic differences between Dutch breeding populations. As such it seems that Dutch breeding areas of the Black-tailed Godwit consist of a single panmictic population. On a global scale this thesis confirmed the presence of three genetically distinct groups now recognized as subspecies. However, no clear genetic differences were found between L. l. limosa across most of its current breeding range. Possibly, there is some genetic differentiation between L. l. limosa breeding areas in the Netherlands and Sweden. Show less
Pathology laboratories throughout the world have compiled large archives of unique collections of tissue specimens. These tissue samples are used for patient diagnostics and research. Novel... Show morePathology laboratories throughout the world have compiled large archives of unique collections of tissue specimens. These tissue samples are used for patient diagnostics and research. Novel molecular insights into alterations in normal cellular function have led to the identification of targets for innovative therapies. Testing for biomarkers combined with molecular pathology has created the potential for __personalized medicine__ and improved diagnosis, treatment and prognosis. New technologies for molecular analysis in molecular tumor diagnostics and research must be developed and implemented to keep pace with the latest insights, resulting in a constant cycle of change. Such translational research can only progress if patient material can be accessed from the archives for further study. The resulting new insights and strategies will eventually be implemented in patient care. This thesis describes three important issues in this cycle of change with a focus on molecular pathology. Tissue preparation, method development and data analysis. Show less
The exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective... Show moreThe exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective group in the solution-phase synthesis of (phosphorothioate) oligonucleotides. Pioneered by de Koning et al. a Solution-Phase Extraction Method (SPEM) amendable to scale-up was developed for the preparation of DNA and phosphorthioate oligonucleotide fragments. This approach utilizes extractive work-up procedures as the only tool in the isolation of oligonucleotides. Next, the design and synthesis of diamoniod decorated iminosugars as potential inhibitors of glycoside proccesing enzymes are described. Several libraries of natural iminosugars such as deoxynojirimycin and castanospermine decorated with an N-pentyloxy spacer were prepared. These iminosugar derivatives were evaluated as inhibitors of the enzymes involved in glucosylcramide metabolism, namely glucosylceramide synthase, beta-glucocerebrosidase and beta-glucosidase. Show less
