Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Healthcare Bioanalytics Author: Bc. Dominika Polová Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Galleria mellonella in vivo model for the study of antimicrobial drug vs. pathogen interaction Background: This study focuses on establishment of the in vivo invertebrate mo- del Galleria mellonella primarily for the study of in vivo efficacy and toxicity of newly synthetized compounds with proved antiinfective potential. Major part of the study is devoted to optimization of conditions for laboratory insect rearing, determination of lethal infectious doses after infection of Galleria mellonella larvae by methicillin re- sistant Staphylococcus aureus, implementation of methodical approach for assessing the relative acute toxicity of candidate antiinfective compounds, and establishment of laboratory manuals for routine histopathological studies. Methods: In the study Galleria mellonella larvae were reared in stable laboratory con- ditions (37 ◦ C) and in the presence of rich nutrition. Sowing method was used in or- der to obtain infectious LD50. In the preliminary study we focused on acute toxicity of tested compounds and on methodology optimization leading to histopathological...

acase@tulane.edu / Introduction: High-Intensity Focused Ultrasound (HIFU) is the only noninvasive method available today for thermal ablation of tumors. HIFU-induced rapid heating and mechanical disruption of tissue, not only has a direct destructive effect on tumors, but also provides a noninvasive way for targeted release of chemotherapeutic drugs from drug delivery vehicles such as temperature sensitive liposomes (SfTSLs). The objective of this work was to evaluate the synergistic treatment of Sorafenib-loaded TSLs (SfTSLs) and HIFU via in vitro analysis of cell viability and proliferation using an aggressive human liver cancer cell line and corresponding in vivo analysis of tumor growth and survival using a human xenograft mouse model. Materials and Methods: Liposomes were developed using 70% Dipalmitoylphosphatidylcholine, 20% L-a-Phosphatidylcholinehydrogenated Soy, and 10% Cholesterol using thin film hydration method to encapsulate Sorafenib at 10μM. Pellets of Hep3B human liver cancer cells (100 μl, 2.7 million cells/ml) were placed in a 0.2 ml thin-wall PCR tube to mimic dense tumor aggregation. Cell pellets were then inoculated with HIFU alone, SfTSLs, or exposed to a combination of HIFU and SfTSLs. The focused ultrasound signal was generated by a 1.1 MHz transducer with acoustic power ranging from 4.1 W to 12.0 W. Cell viability and proliferation experiments were conducted to measure cancer cell damage at 24, 48, 72, and 96 h post treatment via Annexin V/PI and WST-8 staining. In our in vivo study, 1.0×106 Hep3B cells in Matrigel were injected into left and right flanks of athymic nude mice. Tumors were allowed to grow to 8-10 mm size and then separated into the following treatment groups: HIFU alone, SfTSLs (50 μl) alone, SfTSLs + HIFU, and sham. Tumor sizes were measured by caliper every day and a diagnostic ultrasound system was used pre-treatment, 5 days, 14 days, and prior to sacrificing. Tumors were grouped and processed at 5 days, 14 days, or placed in a survival study to evaluate whether treatment facilitated longer lifespans. Tumor tissues were collected for H&E staining and evaluated by a blinded pathologist post euthanasia. Results and Discussion: Our in vitro data indicate that Hep3B cells exposed to both SfTSLs and HIFU have a significantly lower viability and proliferation rate than untreated cells or the cells treated with only SfTSLs or HIFU. According to our in vivo study, tumor growth in the SfTSLs + HIFU group was reduced as compared to Sham, SfTSLs only, or HIFU only groups. Conclusions: The results of our in vitro and in vivo experiments clearly indicate that chemotherapeutic drug-loaded SfTSLs and HIFU can be an effective therapy for locally aggressive liver cancer. This combination treatment leads to more cellular damage, reduction in tumor growth, and better survival. / 1 / Gray Halliburton

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

L'articulation trapézométacarpienne est fréquemment l'objet de consultations en pratique clinique courante. En effet, c'est une articulation régulièrement atteinte par l'arthrose responsable de douleurs et d'une diminution de la mobilité. Néanmoins, elle demeure difficile à analyser cliniquement, sur le plan cinématique, du fait de sa complexité anatomique et biomécanique. Nous avons donc développé et validé un protocole d'examen cinématique de l'articulation trapézométacarpienne in vivo. Pour ceci, nous avons utilisé un système de mesure optoélectronique. La précision du système de mesure a été déterminée, puis, nous avons quantifié la reproductibilité du protocole d'analyse cinématique. Un logiciel a été développé pour quantifier les amplitudes de mobilités, en utilisant une séquence d'axes mobiles. La détermination des axes hélicoïdaux finis, au cours de deux mouvements purs a permis d'analyser les axes du cardan qui peut modéliser cette articulation. La cinématique de l'articulation trapézométacarpienne de 101 sujets sains a été étudiée afin de constituer une base de données. La méthodologie proposée étant portable, il a été possible d'étudier la cinématique articulaire de patients au cours de leur consultation à l'hôpital. 14 patients présentant une arthrose trapézométacarpienne, puis 9 patients opérés pour une arthrose trapézométacarpienne par ablation du trapèze (trapézectomie) ont été analysés. Ce protocole permettra d'évaluer le retentissement d'une atteinte trapézométacarpienne et l'efficacité de son traitement. Il pourra, de plus, apporter une aide à la conception d'implants trapézométacarpiens.

<p>Transcriptional regulation largely determines which proteins and the protein levels that are found in a cell, and this is crucial in development, differentiation and responses to environmental stimuli. The major effectors of transcriptional regulation are a group of proteins known as transcription factors, which importance is supported by their frequent involvement in mendelian and complex diseases.</p><p>In paper I, we attempted to establish the importance of DNA sequence variation in transcriptional control, by analyzing the potential functionality of polymorphic short repetitive elements as cis-regulatory elements. However, the relevance of this study was constrained by the limited number of analyzed sequences and the <i>in vitro</i> nature of the experiments. To overcome these limitations, (paper II) we optimized an <i>in vivo</i> large-scale technology named ChIP-chip, which couples chromatin immunoprecipitation and microarray hybridization. We successfully identified the binding profiles of metabolic-disease associated transcription factors in 1% of the human genome, using a liver cellular model, and inferred the binding sites at base pair resolution.</p><p>Another important characteristic of transcriptional regulation is its plasticity, which allows adjusting the cellular transcriptome to cellular and environmental stimuli. In paper III, we investigated such plasticity by treating HepG2 cells with butyrate, a histone deacetylase inhibitor (HDACi) and interrogating the changes in histone H3 and H4 acetylation levels in 1% of the genome. Observation of frequent deacetylation around transcription start sites and hyperacetylation at the nuclear periphery challenges pre-assumed HDACi mechanisms of action.</p><p>Finally, in paper IV we extended the DNA binding profiles of the medically relevant transcription factors, USF1 and USF2, and H3 acetylation to the whole non-repetitive fraction of the human genome. Using motif finding tools and chromatin profiling, we uncovered the major determinants of USF-DNA interactions. Furthermore, USFs and H3ac were clearly localized around transcription start sites, frequently in the context of bidirectional promoters.</p>

The purpose of this project is to develop a retinal imaging system suitable for routine examination or screening of mouse models that acquires fundus and Optical Coherence Tomography (OCT) images. The imaging system is composed of a digital camera with an objective for biomicroscopic examination of the fundus, an OCT interferometer, an OCT beam delivery system designed for the mouse eye, and a mouse positioning stage. The image acquisition is controlled with software that displays the fundus and OCT images in real-time, and allows the user to control the position of the OCT beam spot on the fundus image display. The system was used to image healthy mice and a mouse model of glaucoma. Fundus images and OCT scans were successfully acquired in both eyes of all mice with eyes that had clear optics. The study demonstrates the feasibility of acquiring simultaneous fundus and OCT images of the mouse retina, by a single operator, in a manner suitable for rapid evaluation of mouse models of retinal disease.

A model with human melanoma xenografts transplanted to athymic mice has been adopted for in vivo studies of 5-S-cysteinyldopa (an intermediate pigment metabolite), glutathione, and cysteine. L-Dopa is an intermediate metabolite in pigment formation and is also important in the treatment of Parkinson's disease, and therefore 1 have also studied the pharmacokinetics of this compound. We were first to describe in vivo microdialysis in melanoma tissue and showed that dialysis membranes of cuprophane or polyamide are suitable for studies of interstitial 5-S-cysteinyldopa and selected thiols. Analytical procedures were also improved for quantitation of 5-S-cysteinyldopa, L-dopa, glutathione, cysteine, and N-acetylcysteine (NAC). In the melanoma xenografts the interstitial concentration of 5-S-cysteinyldopa reflected the high intracellular production of this intermediate metabolite. For in vivo manipulation of glutathione in the melanoma tissue we gave intraperitoneal injection of buthionine sulphoximine to the animals and thus reduced the glutathione concentrations substantially. We showed that restitution of glutathione in melanoma tissue occurs spontaneously and is not much improved by treatment with the cysteine deliverers NAC and L-2-oxothiazolidine-4-carboxylate (OTC). 5-S-Cysteinyldopa was not substantially affected by great variations in glutathione concentrations. Transport of NAC from intraperitoneal injection to melanoma tissue occurred rapidly and deacetylation to cysteine in vivo could be detected soon after NAC injection. In vivo formation of cysteine was slower from OTC than from NAC. Pharmacokinetic studies of L-dopa in human subjects indicated a slight to moderate protein binding. Plasma free L-dopa had similar elimination T½ as interstitial L-dopa, but in some cases the elimination of total L-dopa was slower. Difficulties in intestinal absorption of L-dopa were revealed by microdialysis in blood and subcutaneous tissue. Studies showed that this was due to delayed emptying of the stomach. L-Dopa intake increased 5-S-cysteinyldopa concentrations in blood within 30 min in patients with Parkinson's disease and a history of melanoma. No melanoma activation occurred during long-term treatment with L-dopa. Microdialysis is thus a safe and easily applied method for in vivo studies of both pigment metabolites from human melanoma tissue transplanted to nude mice and for pharmacokinetic studies of L-dopa. / On the day of the public defence the status of the articles IV, V and VI was: Submitted.

Recent approaches in depression therapeutics include triple reuptake inhibitors, drugs that target three monoamine systems. Using in vivo electrophysiological and microdialysis techniques, the effects of 2- and 14-day treatments of escitalopram, nomifensine and the co-administration of these two drugs (TRI) were examined in male Sprague-Dawley rats. Short- and long-term TRI administration decreased NE firing and had no effect on DA neurons. Normal 5-HT firing rates were maintained after 2-day TRI administration compared to the robust inhibitory action of selective serotonin reuptake inhibitors (SSRIs). Escitalopram treatment enhanced the tonic activation of the 5-HT1A receptors given the increase in firing observed following WAY100635 administration. Nomifensine treatment enhanced tonic activation of the α2–adrenoceptors following idazoxan administration. TRI treatment caused a robust increase in extracellular DA levels that was in part mediated by a serotonergic contribution. Therapeutic effects of the drugs examined in this study may be due to the enhancement of 5-HT, NE and/or DA neurotransmission.

Calcium phosphate cements are used in medicine to fill bone defects or give support to screws and plates in fracture fixation. The cements are formed via mixing a powder with water and the mixture harden through a dissolution-precipitation reaction. Today the cement mixing is performed in the operating room and consists of several complicated steps that need to be performed under sterile conditions. This renders the mixing a risk factor, potentially leading to harm for the patient e.g. unsatisfactory healing or infection. To reduce this risk, premixed cements have been developed using glycerol as mixing liquid. The premixed cement sets when it is exposed to body liquids. Therefore, premixed cement can be delivered to the operating room in prefilled syringes ready for use, thus eliminating the mixing step. The aim of this thesis is to describe differences between premixed and water-mixed cements and their advantages and drawbacks. The differences will be discussed based on results obtained from bench testing of specific cement properties as function of cement formulations as well as in vitro and in vivo studies. Several cement formulations were evaluated e.g. the influence of powder to liquid ratio (P/L), powder particle size and addition of water on key properties. The results showed that premixed cements have excellent handling properties and have mechanical properties similar to water-based cements. Both P/L and particle size can be used to control these properties. It was shown that small amounts of water improve certain cement properties while dry raw materials were important for long shelf life. To better understand the setting of premixed cements new methods for evaluating working time and setting of premixed cements were developed. In vivo studies showed that the formulations developed in this thesis are biocompatible, resorbable and show good tissue response in bone. This thesis concludes, that the premixed cements are a promising biomaterial with excellent handling properties and good biological response. The most important challenge for the premixed cements, in order to become commercially successful, is to obtain clinically relevant setting time and shelf life simultaneously. An increasing use of premixed cements in the clinics should shorten operation times and reduce infection rates to the benefit of both patients and medical staff.

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.