Discovery of antibiotics and their targets in multidrug-resistant bacteria

Global healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel modes-of-action is therefore of high priority.This thesis contains two drug discovery projects, originating from a antibacterial screen of a compound library. In both projects chemical hits are first structurally optimized, after which their mode-of-action is determined.The first project entails optimizing a hit with potency against MRSA into a submicromolar active antibiotic. By using a chemical proteomics approach, the targets of this compound were elucidated, along with the targets that are most important in its antibacterial activity.The second project concerns Gram-negative bacteria, where a hit molecule is optimized into the conformationally restricted LEI-800. The target of LEI-800 is found to be DNA gyrase, a common antibiotic target. However, it is that LEI-800 inhibits DNA gyrase differently, and more potently, than the status quo. Bio-organic Synthesi

Activity-based protein profiling

Activity‐based protein profiling is a method to study a subset of the enzymatically active proteome. This method uses chemical probes that covalently react with active enzymes. These labelled proteins can subsequently be analysed by means of a detection tag on the probe. A diverse set of probes has been developed for many enzyme classes, such as serine hydrolases, proteases, glycosidases and kinases. Different analytical techniques are currently available to visualise, identify and quantify probe‐labelled proteins with high efficiency. Activity‐based protein profiling has well‐developed applications in discovering new drug targets and in profiling inhibitors for potency and selectivity. Activity‐based protein profiling will, therefore, continue to aid research both in fundamental biology and drug discovery.Bio-organic SynthesisMolecular Physiolog

Tumor slice culture system to assess drug response of primary breast cancer

Background The high incidence of breast cancer has sparked the development of novel targeted and personalized therapies. Personalization of cancer treatment requires reliable prediction of chemotherapy responses in individual patients. Effective selection can prevent unnecessary treatment that would mainly result in the unwanted side effects of the therapy. This selection can be facilitated by characterization of individual tumors using robust and specific functional assays, which requires development of powerful ex vivo culture systems and procedures to analyze the response to treatment. Methods We optimized culture methods for primary breast tumor samples that allowed propagation of tissue ex vivo. We combined several tissue culture strategies, including defined tissue slicing technology, growth medium optimization and use of a rotating platform to increase nutrient exchange. Results We could maintain tissue cultures for at least 7 days without losing tissue morphology, viability or cell proliferation. We also developed methods to determine the cytotoxic response of individual tumors to the chemotherapeutic treatment FAC (5-FU, Adriamycin [Doxorubicin] and Cyclophosphamide). Using this tool we designated tumors as sensitive or resistant and distinguished a clinically proven resistant tumor from other tumors. Conclusion This method defines conditions that allow ex vivo testing of individual tumor responses to anti-cancer drugs and therefore might improve personalization of breast cancer treatment

Draagvlak en betrokkenheid van burgers bij natuur en natuurbeleid

Deze WOt-paper beschrijft de resultaten van de publieksenquête ‘Draagvlak voor natuur en natuurbeleid 2013’ en vergelijkt de uitkomsten met eerdere publieksenquêtes over dit onderwerp. Het is namelijk de vierde keer sinds 1996 dat zo’n enquête is uitgevoerd. Daarbij wordt aan een representatieve groep Nederlanders gevraagd hoe zij de natuur waarderen en gebruiken, hoe ze denken over het natuurbeleid en beleidsmaatregelen en in hoeverre ze betrokken zijn bij de natuur in hun dagelijkse bestaan. Het Planbureau voor de Leefomgeving (PBL) heeft opdracht gegeven voor dit onderzoek. De kennis die deze longitudinale onderzoeken oplevert, helpt het ministerie van Economische Zaken, decentrale overheden en het maatschappelijk middenveld om het natuurbeleid beter aan te laten sluiten bij wat de burger belangrijk vindt

Maatschappelijk draagvlak voor natuur en natuurbeleid in 2013

In dit rapport staan de resultaten van de publieksenquête Draagvlak voor natuur en natuurbeleid 2013 die gehouden is onder een representatieve groep Nederlanders. Uit deze longitudinale studie blijkt dat een grote groep burgers natuurbescherming belangrijk vindt, regelmatig de natuur bezoekt en activiteiten verricht voor natuur en landschap. Slechts een beperkte groep is actief betrokken bij burgerinitiatieven voor natuur in de woonomgeving. In vergelijking met de voorgaande enquête uit 2006 is het draagvlak voor beleidsmaatregelen iets afgenomen. Verder zijn mensen positiever geworden over de hoeveelheid natuur in Nederland en bezoekt men vaker de natuu

Generalized glycogen storage and cardiomegaly in a knockout mouse model of Pompe disease

Glycogen storage disease type II (GSDII; Pompe disease), caused by inherited deficiency of acid alpha-glucosidase, is a lysosomal disorder affecting heart and skeletal muscles. A mouse model of this disease was obtained by targeted disruption of the murine acid alpha-glucosidase gene (Gaa) in embryonic stem cells. Homozygous knockout mice (Gaa -/-) lack Gaa mRNA and have a virtually complete acid alpha-glucosidase deficiency. Glycogen-containing lysosomes are detected soon after birth in liver, heart and skeletal muscle cells. By 13 weeks of age, large focal deposits of glycogen have formed. Vacuolar spaces stain positive for acid phosphatase as a sign of lysosomal pathology. Both male and female knockout mice are fertile and can be intercrossed to produce progeny. The first born knockout mice are at present 9 months old. Overt clinical symptoms are still absent, but the heart is typically enlarged and the electrocardiogram is abnormal. The mouse model will help greatly to understand the pathogenic mechanism of GSDII and is a valuable instrument to explore the efficacy of different therapeutic interventions

The natural course of infantile Pompe's disease: 20 original cases compared with 133 cases from the literature

OBJECTIVE: Infantile Pompe's disease is a lethal cardiac and muscular disorder. Current developments toward enzyme replacement therapy are promising. The aim of our study is to delineate the natural course of the disease to verify endpoints of clinical studies. METHODS: A total of 20 infantile patients diagnosed by the collaborative Dutch centers and 133 cases reported in literature were included in the study. Information on clinical history, physical examination, and diagnostic parameters was collected. RESULTS: The course of Pompe's disease is essentially the same in the Dutch and the general patient population. Symptoms start at a median age of 1.6 months in both groups. The median age of death is 7.7 and 6 months, respectively. Five percent of the Dutch patients and 8% of all reported patients survive beyond 1 year of age. Only 2 patients from literature became older than 18 months. A progressive cardiac hypertrophy is characteristic for infantile Pompe's disease. The diastolic thickness of the left ventricular posterior wall and cardiac weight at autopsy increase significantly with age. Motor development is severely delayed and major d

The transfer matrix in four-dimensional CDT

The Causal Dynamical Triangulation model of quantum gravity (CDT) has a transfer matrix, relating spatial geometries at adjacent (discrete lattice) times. The transfer matrix uniquely determines the theory. We show that the measurements of the scale factor of the (CDT) universe are well described by an effective transfer matrix where the matrix elements are labeled only by the scale factor. Using computer simulations we determine the effective transfer matrix elements and show how they relate to an effective minisuperspace action at all scales.Comment: 32 pages, 19 figure