Protein Design Based on a PHD Scaffold

The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society

Organisational design for an integrated oncological department

OBJECTIVE: The outcomes of a Strength, Weakness, Opportunities and Threat (SWOT) analysis of three Integrated Oncological Departments were compared with their present situation three years later to define factors that can influence a successful implementation and development of an Integrated Oncological Department in- and outside (i.e. home care) the hospital. RESEARCH DESIGN: Comparative Qualitative Case Study. METHODS: Auditing based on care-as-usual norms by an external, experienced auditing committee. RESEARCH SETTING: Integrated Oncological Departments of three hospitals. RESULTS: Successful multidisciplinary care in an integrated, oncological department needs broad support inside the hospital and a well-defined organisational plan

Treatment of bacteriological infections in neutropenic patients with special emphasis on ceftazidime monotherapy

Contains fulltext : mmubn000001_038049627.pdf (publisher's version ) (Open Access)Promotores : C. Haanen en D. van der Waay131 p

Trombo-embolische complicaties van centraal-veneuze katheters

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Mantelzorgers van oncologiepatiënten in de palliatieve fase ervaren belasting en coping

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When cancer symptoms cannot be controlled: the role of palliative sedation.

Contains fulltext : 79832.pdf (publisher's version ) (Closed access)PURPOSE OF REVIEW: Palliative sedation, the intentional lowering of consciousness for refractory and unbearable distress, has been much discussed during the last decade. In recent years, much research has been published about this subject that will be discussed in this review. The review concentrates on: a brief overview of the main developments during the last decade, an exploration of current debate regarding ethical dilemmas, the development of clinical guidelines, and the application of palliative sedation. RECENT FINDINGS: Main findings are that palliative sedation is mostly described in retrospective studies and that the terminology palliative sedation in now common in the majority of the studies. In addition, life-shortening effects for palliative sedation are scarcely reported, although not absent. A number of guidelines have been developed and published, although systematic implementation needs more attention. Consequently, palliative sedation has become more clearly positioned as a medical treatment, to be distinguished from active life shortening. SUMMARY: Caregivers should apply palliative sedation proportionally, guided by the symptoms of the patient without striving for deep coma and without motives for life shortening. Clinical and multidisciplinary assessment of refractory symptoms is recommended as is patient monitoring during sedation. Future research should concentrate on proportional sedation rather than continuous deep sedation exclusively, preferably in a prospective design

Sedation in palliative care.

Contains fulltext : 52133.pdf (publisher's version ) (Closed access)PURPOSE OF REVIEW: Palliative sedation, the conscious induction of sleep in patients with a very short life expectancy who suffer intractable physical and existential distress, may offer the patient and his or her relatives a more peaceful dying. This technique is still subject to several ethical and medical controversies justifying a review of the recent literature on this subject. RECENT FINDINGS: The available evidence consists of few prospective trials and mainly retrospectively collected case reports. Two guidelines are published in the period under review. The most important points stressed in these reviews are the careful information exchange with the patient, if possible, and his or her proxies, a gradually increased sedation allowing respite if possible to evaluate the effect of the sedation and the need for consultation with colleagues, preferentially physicians experienced in palliative care. Stopping artificial nutrition and hydration is a medical decision that should be taken after evaluation of the potential side effects and consultation with the patient and relatives. SUMMARY: Palliative sedation may be considered for terminally ill patients who suffer intractable symptoms. Ideally it should be included in the patient's trajectory that has been described and discussed earlier when the disease was judged to be incurable. The main goal is to offer comfort

Relations between fatigue, neuropsychological functioning, and physical activity after treatment for breast carcinoma: daily self-report and objective behavior.

BACKGROUND: Previous research indicates that disease free breast carcinoma survivors who experienced severe fatigue also had many problems with regard to neuropsychological functioning and physical activity, measured with general self-report questionnaires. Both neuropsychological functioning and physical activity can be measured with daily self-report measures in addition to measures of objective behavior. The main objective of this study was to examine the relations between 1) fatigue and 2) daily self-reported and objective measures of neuropsychological functioning and physical activity. METHODS: Disease free breast carcinoma survivors and age-matched women with no history of breast carcinoma filled out a daily self-observation list and wore an actometer during a period of 12 days. Furthermore, they performed two standardized tests to assess neuropsychological functioning. RESULTS: No differences were found between severely fatigued disease free breast carcinoma survivors, nonseverely fatigued disease free breast carcinoma survivors, and women in a control group with regard to daily self-reported and objective physical activity. The severely fatigued disease free patients reported more impairment in neuropsychological functioning on daily questionnaires compared with nonseverely fatigued disease free patients and women in the control group. However, no differences were found between these three groups on a standardized concentration task. On a standardized reaction time task, no significant differences were found between the two groups of disease free breast carcinoma survivors: However, women in the severely fatigued group had a significantly longer reaction time compared with women in the control group. CONCLUSIONS: Fatigue is correlated strongly with daily self-reported neuropsychological functioning, but not with objective neuropsychological functioning, in a laboratory setting. In the current study, fatigue was not correlated with daily self-reported and objective physical activity

Paediatric palliative care.

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