Yes / Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness. / This study was supported by Belgian Science Policy Office (TRIT, P7/41), Flemish Fund for Scientific Research (G.0.B81.12), and Department of Economy, Science and Innovation in Flanders ITM-SOFIB (SINGLE project, to J.C.D.). G.D. and B.C. were supported by the Research Foundation—Flanders (FWO) (grants 12Q8115N and 11O1614N, respectively). V.S., J.M. and P.V. were supported by Czech Science Foundation (project no. 13-07500S) and Charles University (UNCE 204017/2012). J.R.V. was supported by research grants from the KU Leuven (SymBioSys [PFV/10/016]) and the Hercules Foundation (ZW11-14). M.S., M.B., and J.A.C. were supported by the Wellcome Trust through the core support for the Wellcome Trust Sanger Institute (grant no. 098051). G.B., P.P., and G.F.S. were supported by Institut Pasteur strategic fund for the LeiSHield project (to G.F.S.).

With the proliferation of plant genomes being sequenced, assembled, and annotated, duplicate gene loss from whole genome duplication events, also known in plants as frac- tionation, has shown to have a different pattern from the classic gene duplication models described by Ohno in 1970. Models proposed more recently, the Gene Balance and Gene Dosage hypotheses, try to model this pattern. These models, however, disagree with each other on the relative importance of gene function and gene expression. In this thesis we explore the effects of gene function and gene expression on duplicate gene loss and retention. We use gene sequence similarity and gene order conservation to construct our gene fam- ilies. We applied multiple whole genome comparison methods across various plants in rosids, asterids, and Poaceae in looking for a general pattern. We found that there is great consistency across different plant lineages. Genes categorized as metabolic genes with low level of expression have relatively low fractionation resistance, losing duplicate genes readily, while genes categorized as regulation and response genes with high level of expression have relatively high fractionation resistance, retaining more duplicate gene pairs or triples. Though both gene function and gene expression have important effects on retention pattern, we found that gene function has a bigger effect than gene expression. Our results suggest that both the Gene Balance and Gene Dosage models account to some extent for fractionation resistance.

In the investigation of radiation accidents, it is of great importance to estimate the dose absorbed by exposed persons in order to plan their therapy. Although occasionally in these situations physical dose measurements are possible, most often biological methods are required for dose estimation. The aim of this investigation was to assess the suitability of the cytokinesis blocked (CB) micronucleus assay as a biodosimetric method using lymphocytes irradiated in vivo. The approach adopted to achieve this was to estimate whole body doses by relating micronuclei yields in patients undergoing radiotherapy treatment with an in vitro radiation dose-response curve. These biologically derived estimates were then compared with the corresponding doses obtained by physical measurement and calculation. As a first approach a study was performed of the in vitro dose-response of gamma-ray induced micronuclei following cytokinesis-block in the lymphocytes of peripheral blood samples obtained from 4 healthy donors. The results indicated that the distribution of the induced micronuclei were overdispersed. Furthermore, a linear dose-response relationship was established when a curve was fitted to the data by an iteratively reweighted least squares method. By means of an analysis of covariance it was demonstrated that this result is in agreement with the dose-response relationships found by various other workers (Fenech et al., 1985; Fenech et al., 1986; Fenech et al., 1989; Balasem et al., 1992, and Slabbert, 1993). To assess the suitability and accuracy of dose assessment using the CB micronucleus assay for in vivo exposure of lymphocytes, blood samples obtained from 8 patients undergoing radiotherapy before, during and after treatment were examined. The physical doses of these patients were determined according to conventional radiation treatment plans and cumulative dose-volume histograms. The dose-volume histograms permitted calculation of integral doses and subsequently the estimate of equivalent whole-body doses. The results of the CB micronucleus assay applied to peripheral blood lymphocytes of 6 patients undergoing fractionated partial-body irradiation showed a dose-related increase in micronucleus frequency in each of the patients studied. This demonstrated that micronuclei analysis may serve as a quantitative biological measure of such exposures. The pooled data of these patients compared to the pooled data of the healthy donors show that there was no statistically significant difference between in vitro and in vivo results, however a slightly lower induced micronuclei frequency was observed after in vivo exposure. When the biological dose estimates for equivalent whole-body doses obtained from the in vitro dose response curve were compared with calculated physical doses, it was found that: biologically estimated dose = 0.936 physical dose. However, there was inadequate statistical evidence to discard the hypothesis that the gradient of the equation was equal to one. Therefore, the analysis of micronuclei induced in lymphocytes in vivo yields highly quantitative information on the equivalent whole-body dose. The negative binomial method was used for analysing the micronucleus data from two patients who received single, relatively larger tumour doses of 10 Gy each, with the objective to obtain estimates of the exposed body fraction and the dose to this fraction. The dose estimates to the irradiated volume were found to be within 30% of the physical tumour dose. The irradiated volume estimates seemed to be higher than the physically calculated volumes but by discarding the correction for the loss of cells due to interphase death the agreement was good between the physically and biologically determined integral doses. This study has revealed that the CB micronucleus assay appears to offer a reliable, consistent and relatively rapid biological method of whole body dose estimation. It is recognised that further corroborative work using the techniques described in this thesis is required for estimating localized exposure.

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The purpose of this study was to examine the pharmacokinetics of d-xylose in man as a function of age with particular emphasis on its absorption characteristics. This study required the development of a specific and sensitive method for the quantitation of xylose from plasma and urine. Following a clean-up procedure, plasma or urine samples are concentrated and undergo two sequential derivatization steps and then are quantitated by capillary column gas liquid chromatography (GLC). D-Xylose is frequently quantitated by a tedious colorimetric assay involving the use of thiourea, a proven animal carcinogen. We have evaluated a more expedient colorimetric assay employing less toxic reagents. Based upon these comparisons the "phloroglucinol" method has been recommended as a replacement for the currently used clinical method for quantitating d-xylose. The human studies revealed age related changes in some but not all d-xylose disposition parameters. Systemic, renal, and nonrenal clearances all declined with advancing age. The terminal elimination half life increased with age. Age had very little influence on the various volumes of distribution. In general, parameters relating to oral absorption showed no age-related dependence. In contrast to what is generally believed, the bioavailability of d-xylose did not decline with age. Lastly, this dissertation addresses the problem of how infusion data may best be fit. Concentration-time data may be fit by a nonlinear regression algorithm in two ways; (1) concentration-time data may be collected and fit both during infusion and after infusion is terminated, (2) concentration-time data may be collected only after the infusion is terminated and be fit as a bolus. Concentration-time data were computer simulated with random error and we found that fitting the entire curve gave the most accurate estimates of disposition parameters.

1 Abstract Charles University in Prague, Faculty of Pharmacy in Hradci Králové Department of: Pharmaceutical Technology Consultant: doc. PharmDr. Zdeňka Šklubalová, Ph.D. Student: Lucie Pospěchová Title of Thesis: Dosage forms in therapy of posterior segments of the eye. Therapy of the posterior eye segments is very difficult. Because most of diseases of the posterior eye segments could lead to complete blindness, the research of dosage forms for the drug delivery into this eye part is intensively developing at this moment. The aim of this thesis is to provide a review of anatomical and physiological specifics of both- anterior and posterior segments of the eye. The most common diseases of the posterior eye are referred to, for example the age related macular degeneration and/or the cytomegalovirus retinitis. There are various routes of application and drug dosage forms that are used for the therapy and which are mentioned in this work, including the drug delivery and targeting systems.

Warfarin is the most widely prescribed oral anticoagulant used for the long-term treatment and prevention of thromboembolic events. Its administration is challenging as it may result in bleeding-related deaths, inadequate anticoagulation and fetal teratogenesis, including fetal warfarin syndrome. A number of environmental and genetic factors contribute to interindividual warfarin dosage variability. The CYP2C9 and VKORC1 genes explain 40- 50% of this variability. The aim of this study was to determine the frequency of known and any new variants in these genes in the SA black population, and correlate these variants and a small subset of environmental factors to dosage variability and pregnancy outcomes. I sequenced the exons and intron/exon boundaries of the CYP2C9 and VKORC1 genes in 100 random black control and 113 patient samples that had at least one pregnancy on warfarin. I observed six previously described CYP2C9 variants, 27 novel CYP2C9 variants, and three previously described VKORC1 variants. 14 of these variants were observed at an allele frequency of 0.02. Of these 14, six appear to decrease (all of which are CYP2C9 variants) and four increase (2 CYP2C9 variants and two VKORC1 variants) warfarin dosage requirement. These 14 CYP2C9 and VKORC1 variants along with a small subset of environmental factors account for 45.3% of warfarin dosage variability in the SA population. I observed an increase in the number of poor pregnancy outcomes in patients on high doses of warfarin. These results allow us to predict the maintenance dose of warfarin in SA black patients better, thereby reducing the risk of adverse effects, and identify those at risk of having a poor pregnancy outcome.

A method of designing early clinical trials is developed for finding an optimum dose level of a new drug to be recommended for use in later phases. During the trial, the efficacious doses are allocated to the patients more often and those with a high probability of toxicity are less likely to be chosen. The method proposed is adaptive in the sense that the statistical models are updated after the data from each cohort of patients are collected and the dose level is adjusted at each stage based on the current data. Two classes of designs are presented. Although both are for efficacy and toxicity responses, one of them also considers pharmacokinetic information. The dose optimisation criteria are based on the probability of success and on the determinant of the Fisher information matrix for estimation of the dose-response parameters. They can be constrained by both acceptable levels of the probability of toxicity and desirable levels of the area under the concentration curve or the maximum concentration. The method presented is general and can be applied to various dose-response and pharmacokinetic models. To illustrate the methodology, it is applied to two different classes of models. In both cases, the pharmacokinetic model incorporates the population variability by making appropriate assumptions about the model parameters, while the dose responses are assumed to be either trinomial or bivariate binomial. Various design properties of the method are examined by simulation studies. Efficiency measures and the sensitivity of the designs to the assumed prior parameter values are presented. All of the computations are conducted in R, where the D- v optimal sampling time points are obtained by using the package PFIM. The results show that the proposed adaptive method works well and could be appropriate as a seamless phase IB/IIA trial design.

It is an FDA requirement that the“first in human" dose be based on pre-clinical animal model efficacy and safety testing to ensure a safe entry into Phase I clinical trials. Pre-clinical safety and efficacy models range from mouse to non-human primates. Interspecies scaling of pharmacokinetic parameters is therefore important for predicting drug doses in human clinical trials, although it continues to be less than optimal. Understanding the disposition of the compound in different species through in vitro and in vivo experiments is necessary to ensure appropriate species are selected for human estimates. Data for three imaging agents and a pharmacological stress agent (Oncology tumor agent (DPC-A80351), Thrombus agent (DMP-444), Infection agent (RP-517) Pharmacological stress agent (DPC-A78445-00)) that entered clinical trials and an imaging agent being developed (RP845), were assessed for scaling accuracy. Initially, pharmacokinetic data from animal models were used to extrapolate to human though body weight allometric scaling. Subsequently, the impact of adjusting for plasma protein binding and the impact of metabolic stability in the different models were examined. Allometric scaling of animal pharmacokinetic parameters (clearance (CL), half-life (t½) and volume of distribution (Vdss)) achieved a prediction of the human pharmacokinetic parameter within 13 to 109% of the observed values. This prediction was further improved by adjusting for plasma protein binding of the drug, and achieved an estimate within 5 to 57% of the clinically observed values. Since the parent compound was the dominant species (>95%) in the circulation, metabolic stability was not used as a correction factor. Weight based allometric scaling was further examined for an atherosclerotic plaque targeted radiopharmaceutical imaging agent, RP845-Tc-99m, currently in development. Pharmacokinetic parameters were determined in mouse, rat and rabbit followed by allometric scaling to predict the non-human primate values. Differences between predicted versus observed non-human primate Cl, t½ and Vdss were 40%, 52% and 8%, respectively. Correcting for plasma protein binding improved the prediction for Cl and t½ to within 12 and 3 %, respectively. The Vdss prediction, however became less accurate (38% difference). Since blood clearance is the major parameter in predicting human dose, the improvement from 40% to 12% was important. The plasma protein binding adjusted animal data was then used with allometric scaling to predict human CL, t½ and Vdss. The predicted values were 7.6 mL/min/kg, 70.6 minutes and 0.87 L/kg respectively. Based on the predicted human blood clearance and the dose required to image atherosclerosis in a rabbit model, the estimated human dose would be unacceptably high. This demonstrates how allometric scaling can be used in research projects to assess clinical feasibility. The impact of metabolism differences influencing the reliability of various species to predict for man was highlighted by DPC-A78445-00. DPC-A78445-00 is being developed as an alternative to exercise in myocardial perfusion imaging for the evaluation of coronary artery disease. DPC-A78445-00 was rapidly metabolized to the carboxylic acid by mouse and rat blood in vitro and in vivo, however longer stability was observed in the dog. In vitro human blood data was consistent with the dog, suggesting that mouse and rat would not be representative species. DPC-A78445-00 plasma protein binding was at a similar, moderate level in rat, dog and human plasma and metabolism by hepatocytes was similar in dog and human. Phase I human clinical trial testing determined the area under the blood concentration-time curve (AUC) and clearance predicted by the dog were within 32% of the human values. Overall, body weight based allometric scaling of pharmacokinetic parameters from animal models, when corrected for plasma protein binding, yielded reliable predictions of the human pharmacokinetics (within 50%) for radiopharmaceutical imaging agent. However, although predictive scaling from animal data can give insight into feasibility of compounds working in human, it is important to identify species differences with respect to metabolic stability. This allometric scaling method provides an additional tool to better predict doses in human for novel Medical Imaging agents.