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Artificial Metalloenzymes
No full textIn de natuur wordt een groot deel van de chemische transformaties uitgevoerd door complexe moleculen, die bekend staan als enzymen. Deze enzymen zijn katalysatoren die vaak bouwstenen produceren van één spiegelbeeld. Het produceren van bouwstenen van slechts een spiegelbeeld is belangrijk voor het maken van bijvoorbeeld medicijnen. Echter natuurlijke enzymen kunnen slechts een gelimiteerde set van deze transformaties uitvoeren. Homogene katalyse, vaak gebruikt in organische chemie, kan een veel bredere set aan transformatie uitvoeren, maar niet altijd van één spiegelbeeld. Artificiële metaalenzymen maken gebruik van de voordelen van de twee eerder genoemde katalysatoren en combineert deze tot een. Zo’n katalysator bestaat uit een biomolecuul en een metaalcomplex. Toepassing van deze katalysatoren is al zeer succesvol gebleken, echter het aantal biomoleculen dat beschikbaar is voor de constructie van artificiële metaalenzymen is beperkt.
Dit proefschrift beschrijft een nieuwe benadering voor ontwikkeling en constructie van artificiële metaalenzymen.
Een actieve centrum, waar de chemische transformatie plaats vindt, werd gecreëerd op een interface van een eiwit dat bestaat uit twee gelijk delen. Hiervoor werd het eiwit LmrR gebruikt. Twee benaderingen om het metaal complex te verankeren aan LmrR werden gedemonstreerd, namelijk op een covalente en niet covalente manier. De artificiële metaalenzymen die hieruit ontstonden, konden verschillende katalytische transformaties uitvoeren waarbij zeer goede resultaten werden behaald. Dat wil zeggen dat een hoog percentage van een spiegelbeeld werden gevormd. Een verdere studie van deze katalysatoren gaf een beter inzicht hoe deze transformaties uitgevoerd werden.
In nature, a large scope of chemical transformations are performed by complex molecules, known as enzymes. Enzymes are catalysts that are able to produce building blocks of just one mirror image. The production of these building blocks possessing just one mirror image is important for the construction of medicine, for example. However, enzymes have a very limited scope of transformation they can achieve. On the other hand, homogenous catalysis, can perform a much wider scope of transformations. However, it does not always produce one mirror image of the building block. Artificial metalloenzymes utilizes the benefits of the catalysts mentioned previously. An artificial metalloenzymes consists of a metal complex and a biomolecule. Artificial metalloenzymes proved to be very successful, however the number of available biomolecules for the construction of artificial metalloenzymes is limited.
This thesis describes a new approach for the development and construction of artificial metalloenzymes.
An active site, in which the chemical transformation takes place, was constructed on the interface of a protein that consisted of two equal parts. The protein that was used was LmrR. Different approaches to anchor the metal complex covalent or non-covalent are described in this thesis. The resulting artificial metalloenzymes were able to catalyze a variety of chemical transformations with very good results, i.e. a high percentage of one of the two mirror images of the products were formed. A study of these artificial metalloenzyme gave more insight in how these transformations were performed.
What is diurnal mood variation
No full textDiurnal mood variation (DMV) is the change in mood over the course of the day, which is a characteristic in many depressed patients. There are two distinct forms of DMV, the classical form, with worse mood in the morning and an improvement towards the evening, and the reversed form, with a worsening of mood towards the evening. DMV is not just a response to environmental or behavioral conditions. Mood also follows an endogenous circadian rhythm. The lowest point in mood is observed during the night. DMV has been linked to a positive response to sleep deprivation. An explanation of why patients with DMV respond better to sleep deprivation has not yet emerged. Years of research have sought for an explanation what causes DMV. According to hypotheses that have been stated, DMV in depression can be understood from a weakened circadian function, or DMV is caused by increased susceptibility to stimuli. Patients who are more susceptible to stimuli tend to vary more in mood and have a higher chance of showing DMV. All these hypotheses treat the state of mood as a kind of black box. Specific regulatory relationships between brain areas and mood are not included in these studies. The direction of DMV seemed to be influenced by serotonin. Patients with a polymorphism in the promoter region of a serotonin transporter showed more reversed DMV than patients with classical DMV. Imaging studies showed that the balance between dorsal and ventral emotion neural systems is disrupted in DMV. In depressed patients, improvement of mood towards the evening is parallel with an increased metabolic activity in ventral limbic-paralimbic regions. These findings support the hypothesis, that limbic-paralimbic regions are involved in DMV. Further research is necessary to explain the exact mechanism underlying DMV. This knowledge is perhaps useful in designing personalized treatments for depression.
Patterns in Nuclear and Mitochondrial DNA Reveal Historical and Recent Isolation in the Black-Tailed Godwit (Limosa limosa)
No full textOn the basis of morphological differences, three subspecies of Black-tailed Godwit (Limosa limosa) have been recognized (L. l. limosa, L. l. islandica and L. l. melanuroides). In previous studies mitochondrial DNA (mtDNA) sequence data showed minimal genetic divergence between the three subspecies and an absence of sub-structuring within L. l. limosa. Here, population genetic structure and phylogeographic patterns have been analyzed using COI, HVR1 and HVR2 mtDNA sequence data as well as 12 microsatellite loci (nuDNA). The nuDNA data suggest genetic differentiation between L. l. limosa from Sweden and The Netherlands, between L. l. limosa and L. l. islandica, but not between L. l. limosa and L. l. melanuroides. However, the mtDNA data were not consistent with the nuDNA pattern. mtDNA did support a split between L. l. melanuroides and L. l. limosa/L. l. islandica and also demonstrated two L. l. limosa haplotype clusters that were not geographically isolated. This genetic structure can be explained by a scenario of isolation of L. l. melanuroides from L. l. limosa in Beringia during the Last Glacial Maximum. During the Pleistocene separation of L. l. islandica from L. l. limosa occurred, followed by colonization of Iceland by the L. l. islandica during the Holocene. Within L. l. limosa founder events, followed by population expansion, took place during the Holocene also. According to the patterns observed in both markers together and their geographic separation, we propose that the three traditional subspecies indeed represent three separate genetic units.
DyP‑type peroxidases: a promising and versatile class of enzymes
No full textDyP peroxidases comprise a novel superfamily of heme-containing peroxidases, which is unrelated to the superfamilies of plant and animal peroxidases. These enzymes have so far been identified in the genomes of fungi, bacteria, as well as archaea, although their physiological function is still unclear. DyPs are bifunctional enzymes displaying not only oxidative activity but also hydrolytic activity. Moreover, these enzymes are able to oxidize a variety of organic compounds of which some are poorly converted by established peroxidases, including dyes, β-carotene, and aromatic sulfides. Interestingly, accumulating evidence shows that microbial DyP peroxidases play a key role in the degradation of lignin. Owing to their unique properties, these enzymes are potentially interesting for a variety of biocatalytic applications. In this review, we deal with the biochemical and structural features of DyP-type peroxidases as well as their promising biotechnological potential.
Research Report 1: Incubation rhythm in common redshanks Tringa totanus
No full textDe begeleider en/of auteur heeft geen toestemming gegeven tot het openbaar maken van de scriptie.
The supervisor and/or the author did not authorize public publication of the thesis.
Testing adaptive maternal effects in Drosophila melanogaster
No full textDe begeleider en/of auteur heeft geen toestemming gegeven tot het openbaar maken van de scriptie.
The supervisor and/or the author did not authorize public publication of the thesis.
The therapeutic potential of indoleamine 2.3-dioxygenase in kidney transplantation
No full textIn de loop der jaren, is niertransplantatie aanzienlijk verbeterd en is zowel de levenskwaliteit als de levensduur van miljoenen patiënten met eindstadium nierfalen verbeterd. Echter, overleving van de getransplanteerde nier is afhankelijk van het chronisch innemen van immunosuppressieve geneesmiddelen met mogelijk levensbedreigende bijwerkingen voor de patiënt. Zelfs met het gebruik van meerdere gecombineerde immunosuppressieve therapieën falen de meeste transplantaties door de acute afstoting en chronische transplantaat dysfunctie (CTD). Ondanks verschillende klinische studies bestaat er tot dusver geen effectieve therapie ter voorkoming of behandeling van CTD. In dit proefschrift hebben we het therapeutische effect van Indoleamine 2.3-dioxygenase (IDO), een immuun modulerend molecuul, op het voorkomen van afstoting en CTD onderzocht. IDO is een snelheid beperkend enzym in het tryptofaan (aminozuur) metabolisme. Tevens speelt IDO een cruciale rol in de foetus-maternale tolerantie. Eerst hebben we in een ratmodel van acute nier afstoting de effecten van lokale gentherapie met IDO bepaald. Op basis van de veelbelovende resultaten van dit onderzoek hebben we vervolgens ook het effect van IDO op CTD onderzocht in een rat model voor CTD. Omdat dendritische cellen (DC) een belangrijke rol spelen bij de initiatie van adaptieve immuunreacties betrokken bij transplantaat afstoting en CTD, hebben we de haalbaarheid en het therapeutisch potentieel bepaald van genetisch gemodificeerde DCs die IDO tot expressie brengen. Tot slot, aangezien IDO als enzym betrokken is bij de omzetting van tryptofaan naar kynurenines, hebben we tevens onderzocht of deze stoffen geschikte biomarkers zijn om nier afstoting en CTD na niertransplantatie in patiënten te voorspellen.
Over the years, kidney transplantation has considerably improved both quality of life and lifespan of millions of patients with end-stage renal failure. However, graft survival relies on patients chronically taking strong immunosuppressive drugs with possible life-threatening side effects. Moreover, even with the advanced use of multiple, complementary immunosuppressive therapies, most of the grafts fail due to the acute rejection and chronic transplant dysfunction (CTD). So far, no effective therapy exists to prevent or treat CTD despite several clinical attempts.
Here we have chosen as target of intervention the immunomodulatory molecule Indoleamine 2.3-dioxygenase (IDO). IDO is the rate-limiting enzyme in the metabolism of the aminoacid tryptophan and it is crucial in the foeto-maternal tolerance. We first investigated the effects of local gene therapy with IDO in a rat model of acute kidney transplant rejection. Based on the promising results of this study we have further tested the same approach in animal rat model of CTD. Moreover, we explored the feasibility of producing and the therapeutic potential of genetically modified IDO-expressing dendritic cells in vitro. Finally, we studied the tryptophan metabolism and its possible usefulness as a biomarker tool in patients after renal transplantation. Our results suggest IDO holds great therapeutical potential in renal transplantation, as it has beneficial effect on both immune and non-immune mechanisms of graft rejection.