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Necrotizing enterocolitis (NEC), a severe inflammatory disorder of the gastrointestinal tract with high morbidity and mortality, affects primarily preterm infants. The diagnosis represents a challenging task, and no biomarker has been found to aid early diagnosis with high accuracy. Microdialysis has been widely used to detect metabolites of anaerobic metabolism, enabling a local and early detection of ischemia. This thesis aims to evaluate the possibility of detecting intestinal ischemic stress in experimental and clinical  NEC, by use of rectal intraluminal microdialysis. Intraluminal rectal microdialysis was performed on rats subjected to total intestinal ischemia. Metabolites of ischemia were detectable in both ileum and rectum, with raised glycerol concentrations and lactate/pyruvate ratios. Elevated concentrations of glycerol correlated to increasing intestinal histopathological injury. Experimental early NEC was induced in newborn rat pups, by hypoxia/re-oxygenation treatment. Development of NEC was confirmed by histopathology. Elevated glycerol concentrations were detected by rectal microdialysis. The genetic alterations following experimental NEC in rat pups were studied with microarray. Immunohistochemistry staining was performed for tight junction proteins claudin-1 and claudin-8. Several genes were altered in experimental NEC, mainly genes regulating tight junctions and cell adhesion. Immunohistochemistry revealed reduced expression of claudin-1. A prospective study was conducted on preterm infants with a gestational age of less than 28 weeks. The infants were admitted to a neonatal intensive care unit, and observed during a 4-week period. Rectal microdialysis was performed twice a week, and blood was drawn for analysis of I-FABP. A total of 15 infants were included in the study, whereof four infants developed NEC, and 11 served as controls. Rectal glycerol and I-FABP displayed high concentrations, which varied considerably during the observation periods, both in NEC and controls. No differences in either glycerol or I-FABP concentrations were seen in the NEC-group vs. the controls. In conclusion, rectal microdialysis can detect metabolites of intestinal ischemia, both in experimental and clinical NEC. Rectal microdialysis is safe and could provide a valuable non-invasive aid to detect hypoxia-induced intestinal damage or ischemic stress in extremely preterm infants. In this study however, it was not possible to predict the development of clinical NEC using microdialysis or I-FABP.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A successful immune response depends on prompt and sufficient recruitment of leukocytes from the circulation to infected or injured sites. Mobilization of leukocytes to hypoxic tissues is vital for angiogenesis, i.e. the formation of new blood vessels from preexisting vasculature, and thus crucial for tissue growth and regeneration. Deviations from normal leukocyte recruitment drive a variety of pathologies, including chronic inflammation, autoimmune diseases and cancer, for which therapeutic options are limited or unspecific. Understanding the mechanisms by which the body controls leukocyte recruitment is therefore critical for the development of novel therapeutic strategies. The present investigations focused on delineating the mechanisms behind leukocyte mobilization from the bloodstream to afflicted sites, by means of in vivo imaging techniques and in vitro assays. We demonstrate that, in response to inflammation, increased vascular permeability enhances transendothelial transport of tissue-released chemokines. Within the vasculature, chemokines form a chemotactic gradient sequestered on heparan sulfate, which directs crawling neutrophils and expedites their extravasation to the inflamed tissue. Consequently, gradient formation grants efficient bacterial clearance. Citrullination of chemokines by leukocyte-derived PAD enzymes in the inflamed tissue prevents chemokine transport into blood vessels, which dampens further neutrophil recruitment and thereby controls the amplitude of the inflammatory response. Moreover, the mechanisms of neutrophil recruitment in response to proangiogenic factors released during hypoxia are revealed to differ from those observed during classical inflammation. Particularly, VLA-4 integrin and VEGFR1 expressed on a defined subset of neutrophils, along with endothelial VEGFR2, are required for efficient neutrophil recruitment to hypoxia. Rather than stimulus-induced phenotypic changes on neutrophils, specific neutrophil subtypes with innate proinflammatory or proangiogenic functions (respectively, CD49d-VEGFR1lowCXCR4low and CD49d+VEGFR1highCXCR4high) coexist in the circulation of humans and mice. In summary, this dissertation provides relevant information on specific steps of neutrophil recruitment to inflamed or hypoxic tissues, which may represent future means to down-regulate aberrant immune responses during chronic inflammation and autoimmune diseases; to increase angiogenesis during ischemia; or to limit pathological angiogenesis, a characteristic of tumor growth and of several chronic inflammatory disorders.

Over half of patients with gastroesophageal reflux disease (GERD) report nocturnal symptoms. Proton pump inhibitors (PPIs) are the main medications used to treat GERD. Multichannel intraluminal impedance with pH (MII-pH) monitoring is the most sensitive method for detection and characterization of GERD. The aim of this study was to assess and compare reflux frequency in patients with refractory GERD symptoms on and off PPI therapy during the nocturnal recumbent period, as assessed by MII-pH testing. We analyzed 24-hour MII-pH studies performed in 200 patients monitored either on twice-daily (n=100) or off (n=100) PPI therapy. Demographic analysis of the on-therapy group revealed a mean age of 52 years (24-78 years) with 37% males, and the off-therapy group revealed a mean age of 49 years (18-84 years) with 40% males. All studies were interpreted to assess and characterize the number of acid and nonacid reflux episodes in the nocturnal recumbent period identified by each patient on an overnight recorder (Zephyr, Sandhill Scientific, Inc., Highlands Ranch, CO, USA). The nocturnal recumbent period was the period documented by patients during which they lie in the recumbent period at night to sleep with average periods lasting 456 and 453 minutes for patients on and off PPI therapy. There were more mean recumbent reflux episodes in the on-therapy group in comparison with the off-therapy group (3.76 mean reflux episodes [mre] per patient in the recumbent vs. 2.82 mre); the difference was not statistically significant (P=0.187). When the reflux events are classified into acid and non-acid reflux episodes, the relative occurrence of acid reflux events is less in the on-therapy group (P=0.047), while the off-therapy group have fewer nonacid reflux episodes (P=0.003). PPIs decrease the acidity of esophageal refluxate but do not decrease the relative frequency of reflux episodes in the recumbent position in patients with refractory GERD despite twice-a-day treatment with PPI therapy. The explanation for the finding of numerically increased, although not statistically significant, amount of reflux episodes in the PPI treatment group in this study, and previous studies is unclear and warrants further evaluation.

The aim of this thesis is the problematic of abdominal aortic aneurysm wall stress in relation to a different material behavior of intraluminal thrombus, which is in most cases present in the aneurysmal volume. In this thesis, the influence of neglecting the patient-specific material properties of the intraluminal thrombus, on the aneurysmal wall stress, obtained from finite element stress-strain analysis, is investigated. In terms of solution method selection, a system approach was applied so that the solution method was selected in order to respect a system of essential variables as much as possible. The first part of this thesis is focused on a description of the problematic and the human cardiovascular system with important aspects contributing to development and growth of the abdominal aortic aneurysm. Next, this part of the thesis includes chapters devoted to the intraluminal thrombus in terms of its basic characteristics (anatomy, physiology, pathology), structure and its influence on processes within the abdominal aneurysm. The second part of this work is devoted to the accomplishment of the first and second goal of this thesis, which is analyzing the available literature to obtain mean population stiffness values of the intraluminal thrombus and conducting biaxial experimental tests of provided samples of intraluminal thrombus. The experimental testing was conducted in order to obtain the patient-specific mechanical properties, which are used as the inputs in the finite element analysis. The experimental testing confirmed the stiffness negligibility of the intraluminal thrombus’s outer layer, which is mentioned is several studies, however, the influence of this layer on resulting aneurysmal wall stress has been to this date not tested. The dominant part of this thesis is focused on the third goal of this work, which is a comparison of aneurysmal wall stress obtained from the finite element computation that included mechanical properties of intraluminal thrombus obtained either from the literature analysis of experimental testing. This part includes discerption of idealized geometry model development, which was used to analyze the sensibility of computed stresses on a number of ILT layers representing different material properties. In order to obtain this analysis, a macro was created prescribing each element of the intraluminal thrombus finite element mesh with material properties derived from its distance from the lumen. Next, this chapter contains description of patient-specific geometry models development, material models, and boundary conditions selection. In the end of this part, results of the finite element computations are presented together with their statistical analysis. Within the last part of this thesis, discussion of results and conclusions of this thesis is included. Also, an overview of important aspects entering computational modeling of abdominal aortic aneurysms is presented.

This thesis deals with stress strain analysis of an aortic abdominal aneurysm (AAA) and the influence of its contact with the spine on the extreme wall stress. The influence was tested on the idealized geometry, as well as on ten patient specific geometries obtained from computer tomography (CT-A) scans. Hyperelastic constitutive models were used for the AAA wall and intraluminal thrombus (ILT) tissue description. The prestress algorithm was used for reconstruction of the unloaded geometry to get more trustworthy results against the geometry from CT which was obtained under the blood pressure. Statistical analysis was used for the results evaluation. The maximal increase of peak wall stress was as high as 81 %.

This paper deals with the problem of abdominal aortic aneurysms (AAA), taking into account the possibility of using mechanical tests of aortic tissues for improvement of level of their constitutive models. First part of thesis deals with the introduction into the problem, description of the structure of the wall of the healthy aorta, its main components and the degenerative changes which lead to formation of AAA. This is followed by a brief excursion into constitutive modeling, which focuses closely on the description of the models used to describe the mechanical behavior of soft tissues. The theoretical part is then supplemented by a narrower selection of constitutive models used for modeling aortic wall and intraluminal thrombus, together with published results, which are reviewed and discussed at the end of this section. The main part of this thesis is devoted to tests of mechanical properties of arterial tissues. First, the methodology is presented together with the description of the customizations of the laboratory equipments together with the test rig. In addition, attention is focused on the results of mechanical tests of intraluminal thrombus, where the results of both uniaxial tensile tests and equbiaxial testing are presented. Also the influence of distance ILT from the lumen on the mechanical properties of the thrombus is examined. Another area of interest is the investigation of the effect of elastase on the chnage of mechanical properties of pig aorta. In this case, porcine aortas are experimentally tested only by biaxial testing, and the time of elastase action to alter the mechanical properties is analyzed so that the resulting tissue has a similar stress-strain response as aneurysmal tissue. Finally, the results of experimental measurements, limitations and other possible ways of research are summarized.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Subject of study: Specialist in laboratory methods Student: Bc. Miroslava Hošková Supervisor: RNDr. Klára Konečná, Ph.D. Title: Microbiological diagnostics of causative agents of infectious diseases associated with central venous catheter insertion Background: The purpose of the thesis is to provide a comprehensive insight into the issue of microbiological diagnosis of agents that cause central venous catheter related infectious processes. The primary objective of the thesis is to evaluate the frequency of occurrence of individual microbiological agents and draw a comparison between published reports and empirical findings. The secondary objective of the thesis is to consider the extent of central venous catheter microbial colonization, i.e., in how many cases of positive culture cultivation, their contamination or significant colonization have been determined, and eventually whether system catheter infection has been proved in correlation with the results of blood culture. Methods: One hundred and seventy-one central venous catheters from different inpatient departments were delivered to the microbiological section of hospital Nemocnice Boskovice s. r. o. for culture examination have been...