Genotoxic effects such as mutations and chromosome abnormalities can augment the risk of adverse health effects such as cancer and heritable genetic diseases; chemicals in commerce must be screened for genotoxic activity. To this end, Toxys B.V. developed the in vitro ToxTracker® assay, which detects (geno)toxicity by monitoring the activity of six reporter genes in cultured mES cells (murine embryonic stem cells), i.e., Rtkn, Bscl2, Btg2, Srxn1, Blvrb and Ddit3. The reporters respond to genotoxic stress, oxidative stress, and endoplasmic reticulum stress characterized by protein unfolding; reporter induction is monitored using flow cytometry. The ToxTracker® assay generates large amounts of multivariate concentration-response data; this study employed innovative quantitative methods to scrutinize ToxTracker® assay results. The work (i) defined a fold-change threshold for identification of a significant positive response, (ii) used two analytical approaches to define endpoint-specific Benchmark Response (BMR) values, (iii) used the BMD (Benchmark Dose) combined-covariate approach for potency ranking of assay validation compounds, and (iv) used PCA (Principal Component Analysis) to investigate functional and statistical relationships between the reporters. The results revealed fold-change cut-offs of 1.5 and 1.7 for identification of weak and strong positive responses, respectively. 1.5-fold is consistent with the value advocated by Toxys B.V.; 1.7-fold is more conservative than the Toxys-advocated 2-fold value. Potency ranking of the validation compounds permitted comparative identification of the most potent inducers of each reporter. The most potent compounds consistently included clastogens used for cancer chemotherapy. BMR values determined using the Zeller et al. (2017) approach ranged from 2.2% for Blvrb and Rtkn, to 7.0% for Ddit3, with an average of 3.9% across all the reporters. The Slob (2016) approach yielded values that ranged from 30% for Ddit3, to 52% for Rtkn, with an average of 43%. The PCA results indicated the Rtkn, Bscl2 and Btg2 reporters are functionally redundant; collectively indicative of genotoxic stress. The Blvrb and Ddit3 reporters are orthogonal indicators of oxidative stress and protein unfolding, respectively; they are essential for toxicological profiling using the ToxTracker® assay. PCA axis scores reflect the toxicological MOA (Mode of Action) of the tested compounds; hitherto unknown MOAs can be inferred using PCA axis-plot proximity to well-studied compounds. Like most in vitro (geno)toxicity assessment assays, ToxTracker® employs a material known as S9 to simulate mammalian hepatic metabolism. S9 is prepared from the livers of rats exposed to an inducer of microsomal CYP (Cytochrome P450) isozymes; the most common CYP inducer is the PCB (polychlorinated biphenyl) mixture known as Aroclor-1254. Due to restrictions in the availability of Aroclor-1254, this study also evaluated the utility of Phenobarbital (PB)/β-Naphthoflavone (BNF)-induced S9, a proposed substitute for Aroclor-induced S9. The results indicate that, despite differences in enzymatic profiles, a 24-hr protocol using 0.40% v/v PB/BNF-induced S9 yields results that are comparable to those obtained using 0.25% v/v Aroclor-induced S9. This study constitutes a significant step towards augmenting the utility of the ToxTracker® assay; it provides a foundation for eventual adoption of high-throughput reporter assays for routine regulatory screening of new and existing chemicals.

En radiothérapie, le niveau de précision de la dose délivrée au patient au cours de son traitement est d’une importance essentielle dans l’évolution vers une amélioration de la qualité et de la cohérence des données de suivi. L’une des premières étapes vers un système de support à la décision clinique (Clinical-Decision Support System CDSS) est la reconstruction précise de cette dose délivrée, en prenant en compte les nombreux facteurs pouvant générer des déviations significatives entre la dose planifiée visualisée à l’écran par l’utilisateur et la dose réellement accumulée lors des séances de traitement. Ces facteurs incluent les variations de débit de l’accélérateur, les incertitudes d’étalonnage, de calcul de dose, les mouvements du patient et des organes, etc.L’objectif de cette étude est d’implémenter et tester une plate-forme de calcul Monte Carlo pour la validation des systèmes Cyberknife et Tomothérapie installés au Centre Oscar Lambret. L’étude d’un détecteur dédié aux petits faisceaux (la chambre d’ionisation microLion) est également incluse, ce détecteur étant particulièrement adapté aux mesures sur le système Cyberknife.Le contexte et les concepts théoriques sont introduits dans les deux premiers chapitres. Dans le troisième chapitre, la modélisation Monte Carlo du Cyberknife et du détecteur microLion est détaillée. La quatrième partie inclut la description de la plate-forme Moderato et de son module d’évaluation. Dans le dernier chapitre, la modélisation du dernier modèle de Cyberknife (M6) équipé d’un collimateur multi-lames est décrite. Une nouvelle technique est également introduite dans le but d’accélérer la recherche des paramètres du faisceau d’électrons pour un modèle Monte Carlo, permettant une intégration plus simple et automatisée de nouveaux appareils dans Moderato. / In radiation therapy, the accuracy of the dose delivered to the patient during the course of treatment is of great importance to progress towards improved quality and coherence of the outcome data. One of the first steps to evolve towards a Clinical-Decision Support System (CDSS) is to be able to accurately reconstruct that delivered dose, taking into account the range of factors that can potentially generate significant differences between the planned dose visualized on the screen of the dosimetrist, and the actually delivered dose accumulated during the treatment sessions. These factors include accelerator output variations, commissioning uncertainties, dose computation errors, patient and organ movement, etc.The objective of this work is to implement and test a Monte Carlo platform for the validation of the Cyberknife and Tomotherapy systems installed at Centre Oscar Lambret. A study of a small field-dedicated detector (the microLion ionization chamber) is also included, this detector being particularly suited for measurements on the Cyberknife system.The context and theoretical concepts are introduced in the first two chapters. In the third chapter, the Monte Carlo modelling of the Cyberknife and microLion detector is detailed. The fourth part includes the description of the Monte Carlo platform Moderato and its evaluation module. In the final chapter, the modelling of the latest MLC-equipped Cyberknife model (the M6) is described. A new technique is also introduced to accelerate the optimization of the beam electron parameters of a Monte Carlo model, thus allowing for an easier and more automated use of the Moderato system. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Metronomic cyclophosphamide (CPA) treatment activates robust anti-tumor immunity and induces regression of implanted tumors in mouse models of brain cancer when administered on an intermittent, every 6-day schedule (CPA/6d), but not on a daily low-dose or a maximum-tolerated dose schedule. Five intermittent metronomic CPA schedules were investigated in GL261 gliomas implanted in scid mice. Metronomic CPA treatments spaced 9 or 12 days apart induced extensive tumor regression, however, tumor-infiltrating natural killer cell responses were not sustained, and tumor growth rapidly resumed after treatment day 24. Increasing the CPA dose prolonged the period of tumor regression on the every 9-day schedule, but natural killer cell activation was markedly decreased. Thus, sustained immune and anti-tumor responses were only achieved on the CPA/6d schedule. Furthermore, CPA/6d treatment eradicated GL261 tumors implanted in immunocompetent C57BL/6 mice by activating anti-tumor CD8-T cell responses and immune memory, which provides proof-of-concept that single agent chemotherapy delivered on an optimized metronomic schedule can cure large established cancers. Transcriptomic profiling, KEGG pathway, and upstream regulator analysis were employed to compare CPA/6d-induced gene expression changes between: immune-responsive GL261 tumors and immune-unresponsive Lewis lung carcinoma (LLC) and B16F10 melanoma tumors; between GL261 tumors implanted in immunocompetent mice versus in scid immunodeficient mice; and between GL261 tumors in scid mice treated with CPA every 6-days or every 9-days. CPA-treated LLC tumors were associated with inhibited VEGFA-targeted genes, down-regulated cell adhesion and transendothelial migration genes, and up-regulated drug metabolism pathways. In B16F10 tumors, CPA activated genes in chemokine signaling and antigen processing and presentation pathways, but no NK cell and T cell effector pathways were activated. GL261 tumors in scid mice were deficient in CPA activation of a subset of cytokine and cytokine receptor genes and T cell receptor signaling genes seen in immunocompetent mice. Cytokine gene expression was lower and drug metabolism gene expression was higher in every 9-day CPA-treated tumors versus CPA/6d-treated tumors. Together, these studies elucidate the dose, schedule, and adaptive immune-dependence of CPA-induced anti-tumor immune responses, giving new insight into the molecular signaling events underlying the deficiencies in immune responses seen in intermittent metronomic CPA-unresponsive tumor models. / 2017-05-31T00:00:00Z

Les essais cliniques de phase I en cancérologie permettent d’identifier la dose optimale (DO), définie comme la dose maximale tolérée (DMT). Les approches conventionnelles de recherche de dose reposent uniquement sur les événements de toxicité observés au cours du premier cycle de traitement. Le développement des thérapies moléculaires ciblées (TMC), habituellement administrées sur de longues périodes, a remis en question cet objectif. Considérer uniquement le premier cycle de traitement n’est pas suffisant. De plus, comme l'activité n'augmente pas nécessairement de façon monotone avec la dose, la toxicité et l'activité doivent être prises en compte pour identifier la DO. Récemment, les biomarqueurs continus sont de plus en plus utilisés pour mesurer l'activité.L’objectif de cette thèse était de proposer et d'évaluer des designs adaptatifs pour identifier la DO. Nous avons développé deux designs de recherche de dose, basés sur une modélisation conjointe des mesures longitudinales de l'activité des biomarqueurs et de la première toxicité dose-limitante (DLT), avec un effet aléatoire partagé. En utilisant des propriétés de distribution normales asymétriques, l'estimation reposait sur la vraisemblance sans approximation ce qui est une propriété importante dans le cas de petits échantillons qui sont souvent disponibles dans ces essais. La DMT est associée à un certain risque cumulé de DLT sur un nombre prédéfini de cycles de traitement. La DO a été définie comme la dose la moins toxique parmi les doses actives, sous la contrainte de ne pas dépasser la DMT. Le second design étendait cette approche pour les cas d’une relation dose-activité qui pouvait atteindre un plateau. Un modèle à changement de pente a été implémenté. Nous avons évalué les performances des designs avec des études de simulations en étudiant plusieurs scénarios et divers degrés d'erreur de spécification des modèles.Finalement, nous avons effectué une analyse de 27 études des TMCs de phase I, en tant que monothérapie. Les études ont été réalisées par l'Institut National du Cancer. L'objectif principal était d'estimer le risque par cycle et l’incidence cumulative de la toxicité sévère, jusqu’à six cycles. Les analyses ont été effectuées séparément pour différents sous-groupes de doses, ainsi que pour les toxicités hématologiques et non-hématologiques. / Conventional dose-finding approaches in oncology of phase I clinical trials aim to identify the optimal dose (OD) defined as the maximum tolerated dose (MTD), based on the toxicity events observed during the first treatment cycle. The constant development of molecularly targeted agents (MTAs), usually administered in chronic schedules, has challenged this objective. Not only, the outcomes after the first cycle are of importance, but also activity does not necessarily increase monotonically with dose. Therefore, both toxicity and activity should be considered for the identification of the OD. Lately, continuous biomarkers are used more and more to monitor activity. The aim of this thesis was to propose and evaluate adaptive designs for the identification of the OD. We developed two dose-finding designs, based on a joint modeling of longitudinal continuous biomarker activity measurements and time to first dose limiting toxicity (DLT), with a shared random effect, using skewed normal distribution properties. Estimation relied on likelihood that did not require approximation, an important property in the context of small sample sizes, typical of phase I/II trials. We addressed the important case of missing at random data that stem from unacceptable toxicity, lack of activity and rapid deterioration of phase I patients. The MTD was associated to some cumulative risk of DLT over a predefined number of treatment cycles. The OD was defined as the lowest dose within a range of active doses, under the constraint of not exceeding the MTD. The second design extended this approach for cases of a dose-activity relationship that could reach a plateau. A change point model was implemented. The performance of the approaches was evaluated through simulation studies, investigating a wide range of scenarios and various degrees of data misspecification. As a last part, we performed an analysis of 27 phase I studies of MTAs, as monotherapy, conducted by the National Cancer Institut. The primary focus was to estimate the per-cycle risk and the cumulative incidence function of severe toxicity, over up to six cycles. Analyses were performed separately for different dose subgroups, as well as for hematologic and non-hematologic toxicities.

Concerns arise in radiation therapy for head and neck cancers when dental prostheses are involved. These prostheses are high-density materials that induce image artifacts in computed tomography (CT) scans used for dose calculation. Two approaches are utilized in mitigating the impact of these artifacts on the accuracy of dose calculation. First, metal artifact reduction (MAR) algorithms or dual-energy CT scans are used to recover image quality. Second, a planning protocol is adopted whereby residual artifacts are manually contoured and assigned appropriate densities. This study evaluated the current planning process using a holistic approach. In this work, an axial section of a head phantom containing dental implants at the level of the oral cavity was constructed and scanned using various protocols on two different commercial scanners; Philips and Siemens, to assess the appearance of artifacts. An MVCT image set was merged with the corresponding kVCT image to improve visualization of the dental implants for use in density overrides. Three ion chamber measurement points in the simulated mouth facilitated the determination of measured dose which was compared to calculated dose at various single beam irradiation geometries. The influence of density override values on agreement between calculation and measurement was investigated for each geometry and imaging modality. Percent error was computed, and initial results compared to results manipulated by use of; a CT density table (Head); density overrides of walls and wax; and density overrides of walls, wax, and effective density of saturation regions. The study established that normal tissue doses are not significantly affected by metal artifact reduction (MAR) algorithms, and improvements in dose calculation compared to uncorrected CT images are small. Furthermore, the inclusion of a MVCT image set improved implant visualization reducing the treatment planning time while providing more information. Evidence led to the deduction that manual overrides of effective density for clipped OMAR CT pixels reduce dose calculation errors. When the phantom was configured with amalgam and Co-Cr-Mo alloy dental implants the effective density of these implants was found to be 4.5 g/cm3. When the phantom was configured with implants containing amalgam and gold, the effective density of amalgam in the presence of gold was 5.5 g/cm3 while gold had an effective density of 6.5 g/cm3. The median and maximum range of errors for the uncorrected images were ± 0.6 % and 7.4% respectively for the phantom configured with amalgam and Co-Cr-Mo (tray one) and ± 0.5 % and 18.1 % respectively for the phantom containing amalgam and gold (tray two). The median and maximum range of errors for the corrected images after applying overrides of effective densities were ± 0.5 % and 4.7% respectively for tray one and ± 0.3 % and 7.7 % respectively for tray two. In conclusion, introduction of density overrides of walls, wax and effective density of high-density materials can reduce the errors induced by metal artifacts and improve the accuracy of dose calculations in treatment planning systems to deliver the relevant dose to a target organ. / Thesis / Master of Science (MSc)

The severity and number of people affected by cancer is a worldwide problem with millions of people affected annually. The search for treatment and cures of cancer continues to be a global effort. As part of this global effort, many natural products have been tested against cancer cell lines, most from plants located in tropical regions. However, this study reports that extracts of Atriplex confertifolia, a native North American plant, has significant bioactivity against human breast cancer cell lines MCF-7, 435, and 231, and HeLa cells (cervical cancer cells). The bioactivity of A. confertifolia extracts of these cells lines was compared to an FDA-approved cancer drug and an industry-standard leukocyte control cell line. Active portions of the extracts were found primarily in the polar fractions of the plant. A dose-response curve of the extracts clearly showed significant cell death similar to the FDA-approved drug. The plant extracts did not inhibit the viability of the leukocyte cell line. Cancer cell death was followed as a function of time and concentration. Cell death appears to be a result of apoptosis.

No description available.

ABSTRACT: UTILIZING NOVEL DOSE EQUIVALENCE METHODOLOGIES TO EXAMINE COCAINE'S EFFECTS ON THE VASCULATURE Neil S. Lamarre Doctor of Philosophy Temple University School of Medicine, 2013 Doctoral Advisory Committee Chair: Ronald J. Tallarida, Ph.D. Cocaine abuse and addiction is a serious health problem, resulting in thousands of emergency room visits and deaths each year in the United States. It is particularly toxic to the cardiovascular system, including deleterious effects on the peripheral vasculature. These effects are not well understood, but evidence suggests chronic cocaine use may lead to endothelial dysfunction, thereby increasing relative risk of a number of other cardiovascular diseases including stroke, aneurysm, myocardial infarction, hypertension, etc. Data from our lab, and others, suggest that the presence of a functional endothelium has a dramatic effect on the contractility of the rat aorta that is agonist-specific. Attenuation of this endothelium-dependent vasodilatory component of agonist action is a primary feature of endothelial dysfunction. We have utilized dose equivalence theory to calculate the dose response relationship for the endothelium-dependent vasodilatory component of an agonist causing overt vasoconstriction. This component cannot be measured directly, but our novel methodology allows us to quantitate agonist-specific impairment of vasodilation, and describe it using the familiar parameters of the dose response curve. Another strength of this method, relative to currently used in vitro methods, is that it also avoids the confounding variable of a second agonist used to produce the initial vasoconstriction. To validate the methodology, a pilot study was performed examining the endothelial dysfunction in STZ-induced diabetic rats, as a positive control for endothelial dysfunction. Interestingly, this treatment showed impairment in the endothelium-dependent vasodilatory component of action of norepinephrine, but not of angiotensin-II. Thus, our initial hypothesis was confirmed - that disruption of the vasodilatory components of various agonists are independent, and that agonist-specific information may prove useful. Next, we employed our new methodology utilizing the rat aorta as our vascular model to test the hypothesis that chronic cocaine administration causes endothelial dysfunction. We first examined the endothelium-dependent vasodilation component of a number of physiologically important vasoconstrictors, and attempted to determine which vasodilatory mediators contributed to the effect. We found the endothelium to have a profound effect on the dose response curve to three important endogenous agonists. These data suggest that under conditions of endothelial dysfunction exaggerated vasoconstriction could occur, even within normal plasma concentration ranges of these vasoconstrictors, resulting in elevated blood pressure and further damage to the endothelium over time. No endothelial dysfunction was observed with this treatment paradigm, using our methodology or the standard approach. This may be a result of insufficient duration of cocaine treatment, or a result of our selection of the rat aorta as a model. We wanted to further investigate which vasodilatory mechanisms were involved in this vasodilatory component of action. We inhibiting various endothelium-derived mediators of this vasodilatory component of action (such as nitric oxide or prostacyclin), which revealed differential activation of these mediators by the agonists examined. For example, inhibition of nitric oxide synthesis abolished the endothelium-dependent vasodilatory component of endothelin-1, but only partially attenuated that of angiotensin-II. Thus, the agonist-specific pattern of impairment may also prove useful in examining the underlying mechanisms of impaired vasodilation. Endothelial dysfunction is one reported consequence of long term cocaine abuse; however, there are conflicting reports on the acute vascular effects of cocaine, with some reports concluding that cocaine is a vasoconstrictor, and some reporting its action as a vasodilator. There are in vitro reports of cocaine causing release of vasoconstrictors from the endothelium, which supports the longstanding notion of cocaine as a vasoconstrictor. However, one recent report demonstrates a dose-dependent vasodilatory effect of cocaine in rat aorta that is independent of the endothelium. This complexity is perhaps due, in part, to cocaine's affinity for a number of molecular targets, acting in combination. In examining the acute action of cocaine in our preparation, we observed an "inverted-U" shaped dose response, also referred to as a hormetic dose response curve. We then applied dose equivalence methodology in order to derive the "unknown" second component contributing the vasodilatory action of cocaine at higher doses. This methodology lets us calculate this unknown component, and describe it with the familiar parameters of a dose response curve, which could potentially aid in the identification of the unknown component. The preliminary studies with acute cocaine utilized a sub-maximal dose of phenylephrine in order to observe tension changes in either direction. This prompted us to further characterize the interaction of cocaine with other alpha adrenoceptor agonists. Importantly, because cocaine alone had no effect at doses up to 100 µM, but potentiated the vasoconstriction of alpha agonists, the interaction is therefore synergistic. This constitutes evidence of a previously undescribed mechanism contributing to cocaine's vasoconstricting effect. In vivo, reuptake inhibition is a major mechanism for cocaine-induced vasoconstriction, but is excluded in this experiment by virtue of low levels of sympathetic innervation in the rat aorta, and the use of methoxamine, an alpha agonist not subject to the reuptake mechanisms. This interaction may contribute to cocaine-induced vasoconstriction in the coronary arteries, especially in circumstances of endothelial dysfunction. In summary, the work presented in this dissertation applies new methodologies utilizing dose equivalence theory to the study of cocaine's effects on peripheral vasculature, and presents novel findings of synergy with respect to cocaine's enhancement on the action of alpha adrenoceptor-mediated vasoconstriction. / Pharmacology

Recently, hyperthermia has been investigated as an alternate therapy for the treatment of tumors. The present project explored the feasibility of preferential hyperthermia as a method of treating deep seated tumors. The overall goal of this research was to determine theoretically if preferential heating could be used to attain the desired thermal dose (DTD) for a two cm diameter tumor. The simulations in this work show that, when using a single silicon carbide insert, the model cannot provide enough energy for an entire 2 cm diameter tumor to receive the DTD. However, when using an enhanced design model with multiple (4) silicon carbide inserts, the DTD could be attained in a tumor up to 3.5 cm in diameter. This study involved using the commercially available software package ANSYS 7.0 program to model a spherical 2 cm tumor, assuming the tumor is located in deep tissue with a constant perfusion rate and no major blood vessels nearby. This tumor was placed in the center of a cube of healthy tissue. To achieve the preferential heating of the tumor, a silicon carbide insert was placed in the center of the tumor and microwave energy was applied to the insert (in the form of volumetric heating). The thermal modeling of this system was based on the Pennes Bioheat equation with a maximum temperature limitation of of 80 ºC. The Thermal Dose Analyzer software program was used to evaluate the results of the thermal simulations (from ANSYS) to determine if the DTD had been attained. Additional enhanced design models were also examined. These models include 2 cm and 4 cm tumors with four silicon carbide inserts symmetrically placed about the tumor and a 4 cm tumor model using a single silicon carbide insert with antennae attached to the insert to increase energy distribution to the tumor. The simulations show that only the enhanced design cases with four silicon carbide inserts can achieve the DTD for an entire 2 cm tumor. / Master of Science

No / The dose emitted from dry powder inhalers may be inhalation flow-dependent. Using an ex vivo method, the Electronic Lung¿, we have measured the aerodynamic characteristics of the emitted dose for both active constituents from Seretide® Diskus® (salmeterol xinafoate 50 mcg; fluticasone propionate 500 mcg) and Symbicort® Turbuhaler® (formoterol 6 mcg; budesonide 200 mcg).1 Electronic inhalation profiles were collected from 20 severe asthmatics (mean PEFR 53% predicted) when they inhaled using a placebo Seretide® Diskus® and a placebo Symbicort® Turbuhaler®. These were replayed in the Electronic Lung¿ with the respective active inhaler in situ. Mean(S.D.) peak inhalation flow rates (PIFR) through the Diskus® and Turbuhaler® were 94.7(32.9) and 76.8(26.2) l min¿1, respectively. From the Electronic Lung¿ the Diskus® inhalation profiles provided a mean(S.D.) fine particle dose (FPD) for fluticasone propionate and salmeterol of 20.4(4.8) and 18.4(4.4)% labelled dose. For Turbuhaler® inhalation profiles the FPD was 23.1(12.9) and 20.7(11.1)% labelled dose for budesonide and formoterol, respectively. The linear (p < 0.001) relationships between FPD against PIFR for budesonide and formoterol were 3 (p = 0.002) and 2.8 (p = 0.007) times steeper than fluticasone propionate and salmeterol, respectively. The results highlight a more significant effect of inspiratory flow on variable dosage emission when using the Symbicort® Turbuhaler® compared with the Seretide® Diskus®.