This thesis explores biochemical and genetical changes associated with migraine and cluster headache. This research aims to increase knowledge of the pathophysiology and signaling pathways involved... Show moreThis thesis explores biochemical and genetical changes associated with migraine and cluster headache. This research aims to increase knowledge of the pathophysiology and signaling pathways involved in migraine and cluster headache, thereby identifying new targets for treatment. Uncovering the biological mechanisms on how patients differ from those without disease leads to a better understanding of the pathophysiology of primary headache disorders. The biological systems in our body are related to each other, and are based on the genomic blueprint and lead via epigenetics, transcription and translation to proteins and biomolecules. The first part of the dissertation focuses on the examination of biomolecules in body fluids (blood and cerebrospinal fluid) in migraineurs. Several studies showed that biomolecule concentrations differed between people with and without migraine for lipids, amino acids and metabolites of glucose metabolism. These results indicate a general disruption of the metabolic profile in migraineurs. For other substances, no differences were found. The second part of the thesis looked at the genetic blueprint of various headache disorders. There a genetic difference between people with migraine and people without migraine was found. In addition, a genetic difference between people with and without cluster headache was found. Show less
The venom of the Australian snake Pseudonaja textilis contains coagulation factors V (five) and X (ten) which have been adapted to derail the blood clotting system of its prey. Snake venom factor V... Show moreThe venom of the Australian snake Pseudonaja textilis contains coagulation factors V (five) and X (ten) which have been adapted to derail the blood clotting system of its prey. Snake venom factor V is unique in that is constitutively active, unlike its human counterpart. The snake liver transcriptome was found to contain alternatively spliced factor V mRNA that encoded for either the activated protein or its quiescent form. A potential pre-mRNA splicing mechanism was uncovered that may yield the active protein. Snake venom factor V is also particularly stable due to several modifications to its molecular structure. These modifications have been investigated in detail by engineering chimeras of human and snake venom factor V. The snake venom factor X molecule was also investigated in more detail. It was discovered that this enzyme is insensitive to the action of certain anticoagulant drugs (Factor Xa inhibitors) due to a unique molecular modification. A Human factor X can be modified in similar fashion, so that it is no longer sensitive to the action of Factor Xa inhibitors. This finding could make an important contribution to acute care for patients who experience life-threatening bleeding after the use of FXa inhibitors. Show less
Less than 1 in 10 drug candidates that enter phase 1 clinical trials actually gets approved for human use. The high failure rate is in part due to unforeseen side effects or toxicity. A better... Show moreLess than 1 in 10 drug candidates that enter phase 1 clinical trials actually gets approved for human use. The high failure rate is in part due to unforeseen side effects or toxicity. A better understanding of the role of selectivity and a better insight in the off-target activities of drug candidates could greatly aid in preventing candidates to fail for these reasons. This thesis has tried to address some aspects in this challenging part of drug discovery. The use of activity-based protein profiling as presented in Chapters 2 and 3 in drug discovery and hit-to-lead optimization, and in Chapter 5 and 6 for the interaction profiling of a drug candidate, highlights the versatility and importance of this chemical biology technique. Combined with knowledge derived from biochemical assays, such as that developed in Chapter 4, ABPP can greatly aid the medicinal chemist. The recent surge in popularity of machine learning algorithms, backed by exponential growth of the amount of biological data available, holds great promise for drug discovery. Chapters 7 and 8 showed the applicability of one such algorithm, which was able to quite reliably predict interaction profiles. The challenges in finding, determining and predicting selectivity are far from solved, but, by incrementally expanding our understanding of the binding of small molecules to their (off-)targets, truly selective inhibitors might at some point become a reality or their necessity might be mitigated. Show less
The aim of the research presented in this thesis was to develop new methods forchallenging systems in liquid-state NMR using paramagnetic effects generated by thetwo-armed probe CLaNP-5. Chapter 1... Show moreThe aim of the research presented in this thesis was to develop new methods forchallenging systems in liquid-state NMR using paramagnetic effects generated by thetwo-armed probe CLaNP-5. Chapter 1 is an introduction about NMR assignmentdevelopment and challenges and a brief opening to the theory of paramagnetic effectsand their applications. Chapter 2 describes the upgrade of PARAssign software and itsability to assign and assess methyl groups of a 25 kDa protein using experimentalpseudocontact shifts from 1 to 3 paramagnetic centers. Chapter 3 describes theimplementation of residual dipolar coupling into PARAssign software as extra datasetinput in addition to the pseudocontact shifts. Applications are given for residueselective isotope labeled protein. Chapter 4 depicts the use of pseudocontact shiftsused to probe small methyl group movements and re-orientation under ligand binding.Chapter 5 gives some clues about how paramagnetic effect such as RDC but also PCScan provide information for challenging highly dynamic system. Chapter 6 givesinsights for PCS application to very large multimeric protein, the challenges andlimitations are discussed. Chapter 7 provides a discussion and few perspectives aboutthe work carried out and presented in this thesis. Show less
Repair of damage in the DNA is essential for an organism. Therefore, several repair mechanisms have evolved. In this thesis, the mechanism of Transcription-Coupled Nucleotide Excision Repair (TC... Show moreRepair of damage in the DNA is essential for an organism. Therefore, several repair mechanisms have evolved. In this thesis, the mechanism of Transcription-Coupled Nucleotide Excision Repair (TC-NER) and the UV Damage Endonuclease repair pathway (UVDE) have been studied. Central to TC-NER is the protein Cockayne Syndrome protein A (CSA). Its biological importance can be seen in that mutations in CSA cause the human, serious disorder Cockayne Syndrome. This thesis describes structural and biochemical studies of this protein, which give insights into its substrate-binding and into how mutations in this protein cause the disease Cockayne Syndrome. Biochemical and structural studies of UVDE show the identity and role of its post-translational modification, a carboxylation. A cocrystal structure of UVDE with 6-4PP DNA shows how UVDE can recognize UV damaged DNA. Show less
Many cellular processes are studied by biochemical techniques. Usually, this involves experiments where large number of cells are lysed, protein content is subsequently isolated and studied using... Show moreMany cellular processes are studied by biochemical techniques. Usually, this involves experiments where large number of cells are lysed, protein content is subsequently isolated and studied using antibodies to detect changes in protein levels, post-translational modifications, pairing with partner molecules, etcetera. Although informative, these mass population analyses often lack the time resolution to study rapid alterations in protein state, and do not allow the characterisation of highly dynamic processes. Moreover, analysis of millions of cells at once evidently shows the average response in the population of cells, thereby obscuring cell-to-cell variation and the dynamic range of a process. With the availability of microscopic techniques in combination with genetically encoded fluorescent probes, many of these restraints have been overcome. Highly dynamic reactions can now be studied in detail in a relatively easy manner, and in the context of a living cell, hence "single cell biochemistry". In this way, we studied two different cellular processes, antigen presentation and drug resistance in unprecedented detail. Both parts seem at first unrelated, yet are interconnected through the use of similar techniques. Assessment of individual cells using sensitive microscopic measurements, has led to important and detailed understanding of the dynamic processes involved in both topics. Show less
