Freshwater ecosystems have been recognized as important components of the global carbon cycle, and the flux of organic matter (OM) from freshwater to marine environments can significantly affect estuarine and coastal productivity. The focus of this study was the assessment of carbon dynamics in two aquatic environments, namely the Florida Everglades and small prairie streams in Kansas, with the aim of characterizing the biogeochemistry of OM. In the Everglades, particulate OM (POM) is mostly found as a layer of flocculent material (“floc”). While floc is believed to be the main energy source driving trophic dynamics in this oligotrophic wetland, not much is known about its biogeochemistry. The objective of this study was to determine the origin/sources of OM in floc using biomarkers and pigment-based chemotaxonomy to assess specific biomass contributions to this material, on a spatial (freshwater marshes vs. mangrove fringe) and seasonal (wet vs. dry) scales. It was found that floc OM is derived from the local vegetation (mainly algal components and macrophyte litter) and its composition is controlled by seasonal drivers of hydrology and local biomass productivity. Photo-reactivity experiments showed that light exposure on floc resulted in photo-dissolution of POC with the generation of significant amounts of both dissolved OM (DOM) and nutrients (N & P), potentially influencing nutrient dynamics in this ecosystem. The bio-reactivity experiments determined as the amount and rate of CO2 evolution during incubation were found to vary on seasonal and spatial scales and were highly influenced by phosphorus limitation. Not much is known on OM dynamics in small headwater streams. The objective of this study was to determine carbon dynamics in sediments from intermittent prairie streams, characterized by different vegetation cover for their watershed (C4 grasses) vs. riparian zone (C3 plants). In this study sedimentary OM was characterized using a biomarker and compound specific carbon stable isotope approach. It was found that the biomarker composition of these sediments is dominated by higher plant inputs from the riparian zone, although inputs from adjacent prairie grasses were also apparent. Conflicting to some extent with the River Continuum Concept, sediments of the upper reaches contained more degraded OM, while the lower reaches were enriched in fresh material deriving from higher plants and plankton sources as a result of hydrological regimes and particle sorting.

Advancements in the micro-and nano-scale fabrication techniques have opened up new avenues for the development of portable, scalable and easier-to-use biosensors. Over the last few years, electrodes made of carbon have been widely used as sensing units in biosensors due to their attractive physiochemical properties. The aim of this research is to investigate different strategies to develop functionalized high surface carbon micro/nano-structures for electrochemical and biosensing devices. High aspect ratio three-dimensional carbon microarrays were fabricated via carbon microelectromechanical systems (C-MEMS) technique, which is based on pyrolyzing pre-patterned organic photoresist polymers. To further increase the surface area of the carbon microstructures, surface porosity was introduced by two strategies, i.e. (i) using F127 as porogen and (ii) oxygen reactive ion etch (RIE) treatment. Electrochemical characterization showed that porous carbon thin film electrodes prepared by using F127 as porogen had an effective surface area (Aeff 185%) compared to the conventional carbon electrode. To achieve enhanced electrochemical sensitivity for C-MEMS based functional devices, graphene was conformally coated onto high aspect ratio three-dimensional (3D) carbon micropillar arrays using electrostatic spray deposition (ESD) technique. The amperometric response of graphene/carbon micropillar electrode arrays exhibited higher electrochemical activity, improved charge transfer and a linear response towards H2O2 detection between 250μM to 5.5mM. Furthermore, carbon structures with dimensions from 50 nano-to micrometer level have been fabricated by pyrolyzing photo-nanoimprint lithography patterned organic resist polymer. Microstructure, elemental composition and resistivity characterization of the carbon nanostructures produced by this process were very similar to conventional photoresist derived carbon. Surface functionalization of the carbon nanostructures was performed using direct amination technique. Considering the need for requisite functional groups to covalently attach bioreceptors on the carbon surface for biomolecule detection, different oxidation techniques were compared to study the types of carbon–oxygen groups formed on the surface and their percentages with respect to different oxidation pretreatment times. Finally, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor oncoprotein detection on functionalized three-dimensional carbon microarrays platform was demonstrated. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5 pmol.

Surface Plasmon Resonance (SPR) and localized surface plasmon resonance (LSPR) biosensors have brought a revolutionary change to in vitro study of biological and biochemical processes due to its ability to measure extremely small changes in surface refractive index (RI), binding equilibrium and kinetics. Strategies based on LSPR have been employed to enhance the sensitivity for a variety of applications, such as diagnosis of diseases, environmental analysis, food safety, and chemical threat detection. In LSPR spectroscopy, absorption and scattering of light are greatly enhanced at frequencies that excite the LSPR, resulting in a characteristic extinction spectrum that depends on the RI of the surrounding medium. Compositional and conformational change within the surrounding medium near the sensing surface could therefore be detected as shifts in the extinction spectrum. This dissertation specifically focuses on the development and evaluation of highly sensitive LSPR biosensors for in situ study of biomolecular binding process by incorporating nanotechnology. Compared to traditional methods for biomolecular binding studies, LSPR-based biosensors offer real-time, label free detection. First, we modified the gold sensing surface of LSPR-based biosensors using nanomaterials such as gold nanoparticles (AuNPs) and polymer to enhance surface absorption and sensitivity. The performance of this type of biosensors was evaluated on the application of small heavy metal molecule binding affinity study. This biosensor exhibited ~7 fold sensitivity enhancement and binding kinetics measurement capability comparing to traditional biosensors. Second, a miniaturized cell culture system was integrated into the LSPR-based biosensor system for the purpose of real-time biomarker signaling pathway studies and drug efficacy studies with living cells. To the best of our knowledge, this is the first LSPR-based sensing platform with the capability of living cell studies. We demonstrated the living cell measurement ability by studying the VEGF signaling pathway in living SKOV-3 cells. Results have shown that the VEGF secretion level from SKOV-3 cells is 0.0137 ± 0.0012 pg per cell. Moreover, we have demonstrated bevacizumab drug regulation to the VEGF signaling pathway using this biosensor. This sensing platform could potentially help studying biomolecular binding kinetics which elucidates the underlying mechanisms of biotransportation and drug delivery.

Environmental change in the Arctic proceeds at an unprecedented rate. The Pliocene epoch (5-2.65 million years ago) represents an analog for future climate conditions, with pCO2 and continental configurations similar to present. Yet conditions in the Pliocene Arctic are poorly characterized because of sparse sampling. The records that do exist indicate periods of extreme warmth, as well as the first expansion of large ice-sheets in the Northern Hemisphere, took place from the end of the Pliocene into the early Pleistocene. Understanding these deposits and their implications for our future requires developing a sense of climatic evolution across the Plio-Pleistocene transition and especially during the intensification of Northern Hemisphere Glaciation (iNHG) ~2.7 million years ago. Here we reconstruct environmental change in the Arctic using a suite of organic geochemical proxies in a sedimentary archive recovered from Lake El'gygytgyn, Arctic Northeast Russia. We use the distribution of branched glycerol dialkyl glycerol tetraethers (brGDGTs) and the hydrogen isotopic composition (δD) of plant leaf-waxes (n-alkanes) to reconstruct relative temperature change across the interval spanning 2.8 to 2.4 million years ago. Our work demonstrates that, following the first major glaciation of the Northern Hemisphere, it took multiple glacial cycles for the Arctic to become synchronized with the climatic changes recorded in the deep ocean. This work has implications for understanding the role of sea-level, sea-ice, vegetation and carbon-cycle feedbacks in a changing Arctic.

Background: MicroRNAs (miRNAs) are small non-coding single-stranded RNAs involved in the posttranscriptional inhibition of gene expression and thereby regulating all cellular functions. Their dysregulation contributes to the pathophysiology of many diseases, including rheumatic diseases. MiRNAs can also be found extracellularly in body fluids and represent promising diagnostic and prognostic biomarkers. Our study aimed to investigate miRNAs as biomarkers of stage and activity and predictors of therapeutic response of two most common inflammatory rheumatic diseases: spondyloarthritis (SpA) and rheumatic arthritis (RA). Results: We found several circulating miRNAs differentially expressed in SpA patients reflecting the severity of axial involvement and/or disease activity. The decrease in circulating miR-145 in plasma of patients with ankylosing spondylitis 3 months of anti-TNF therapy predicted a good therapeutic response and low disease activity after a year of therapy. Circulating and intracellular expression of miR-125b in peripheral blood mononuclear cells (PBMC) was lower in treatment-naïve patients with early RA than in healthy controls. Baseline expression of miR-125 in PBMC predicted a (non)adequate therapeutic response. We also found the increased expression of miR-451 in PBMC in...

Patienten mit arterieller Hypertonie haben ein erhöhtes Risiko eine Tumorerkrankung, insbesondere Nierenzellkarzinome, zu entwickeln. Die arterielle Hypertonie ist über die Entstehung von oxidativem Stress mit der Entwicklung von DNA-Schäden verknüpft, wobei ein hochreguliertes Renin-Angiotensin-Aldosteron-System (RAAS) eine entscheidende Rolle einnimmt. Das Ziel dieser Arbeit war es zum einen Hypertoniker (HypAll) und gesunde Kontrollen und zum anderen gut (HypGut) und schlecht (HypSch) eingestellte Hypertoniker unter Berücksichtigung der eingenommenen Antihypertensiva bezüglich ihrer Level an oxidativem Stress und DNA-Schäden zu vergleichen. Zusätzlich erfolgte im Rahmen einer Längsschnittanalyse der intraindividuelle Vergleich unter den Hypertonikern. Hierfür erfolgte die Bestimmung von SHp, D-ROM und 3-Nitrotyrosin als Marker für oxidativen Stress im Plasma, von 8-oxodG, 15-F2t-Isoprostan und Malondialdehyd als Marker für oxidativen Stress im Urin und von γ-H2AX und Mikrokernen als Marker für DNA-Schäden in Lymphozyten. Dabei konnte ein erhöhter oxidativer Stress in der HypAll-Gruppe verglichen zu den Kontrollen anhand aller Marker für oxidativen Stress mit Ausnahme von Malondialdehyd festgestellt werden. Nach Altersadjustierung zeigte sich dieser Gruppenunterschied nur noch für die Proteinstressmarker SHp und 3-Nitrotyrosin signifikant. Bezüglich der Marker für DNA-Schäden ergab sich kein Unterschied zwischen HypAll und Kontrollen. Ebenso zeigte sich kein signifikanter Unterschied in den Leveln für oxidativen Stress und DNA-Schäden zwischen der HypGut- und HypSch-Gruppe. Zuletzt konnte im Rahmen der Längsschnittstudie ein positiver Zusammenhang zwischen der Entwicklung des Blutdrucks und des oxidativen Stresses anhand der Veränderung von D-ROM und des systolischen Blutdrucks beobachtet werden. Die teils nicht-signifikanten und teils mangelnden Unterschiede zwischen HypAll und Kontrollen sowie zwischen HypGut und HypSch sind am ehesten durch das besondere Patientengut, welches sich auch grundlegend von dem anderer vergleichbarer Studien unterscheidet, erklärbar. Die Patienten mit therapieresistenter Hypertonie (TRH) zeichnen sich durch eine langjährige Einnahme zahlreicher Antihypertensiva aus. Diese, insbesondere die RAAS-wirksamen, besitzen eine über die reine Blutdrucksenkung hinausgehende antioxidative und antigenotoxische Wirkung, welche vermutlich zu einer Angleichung der Level für oxidativen Stress und DNA-Schäden geführt hat. Um die Dynamik der Biomarker und den Einfluss der Antihypertensiva auf oxidativen Stress und DNA-Schäden besser zu verstehen, sind weitere Studien über einen längeren Beobachtungszeitraum sowie mit zusätzlich therapienaiven Hypertonikern sinnvoll. Die weitere Erforschung von Biomarkern, um sie im klinischen Alltag zur Verbesserung der Patientenbehandlung einsetzen zu können, ist notwendig. / Patients with arterial hypertension are at an increased risk of developing tumors, especially renal cell carcinoma. Arterial hypertension is linked to the development of DNA damage through the development of oxidative stress, with an upregulated renin-angiotensin-aldosterone system (RAAS) playing a decisive role. The aim of this work was to compare 1. hypertensive patients (HypAll) and healthy controls and 2. hypertensive patients with good (HypGut) and poorly (HypSch) adjusted hypertension with regard to their level of oxidative stress and DNA damage. For this purpose, SHp, D-ROM and 3-nitrotyrosine were determined as markers for oxidative stress in plasma, 8-oxodG, 15-F2t-isoprostane and malondialdehyde as markers for oxidative stress in urine and γ-H2AX and micronuclei as markers for DNA damage in lymphocytes. An increased oxidative stress was found in the HypAll group compared to the controls as measured by all markers for oxidative stress with the exception of malondialdehyde. After adjusting for age, this group difference was only significant for the protein stress markers SHp and 3-nitrotyrosine. With regard to the markers for DNA damage, there was no difference between HypAll and controls. Also there was no significant difference in the levels of oxidative stress and DNA damage between the HypGut and HypSch groups. The partly insignificant and partly lacking differences between HypAll and controls as well as between HypGut and HypSch can best be explained by the special patient population, which is also fundamentally different from that of other comparable studies. The patients with therapy-resistant hypertension are characterized by long-term use of numerous antihypertensive drugs. These, especially the RAAS-effective ones, have an antioxidant and antigenotoxic effect that goes beyond the pure lowering of blood pressure. This has presumably led to an equalization of the levels for oxidative stress and DNA damage. In order to better understand the dynamics of the biomarkers and the influence of antihypertensive drugs on oxidative stress and DNA damage, further studies over a longer observation period and with additional therapy-naive hypertensive patients are useful. Further research into biomarkers is necessary so that they can be used in everyday clinical practice to improve patient treatment.

Diagnosing post-traumatic stress disorder (PTSD) is challenging and is currently, diagnosis through self-administered checklists. Because a diagnosis of PTSD can open up significant benefits to compensation, education, and medical care, people can tailor their responses to the checklist to help ensure a diagnosis of PTSD. The purpose of the study was to examine the utility of the quantitative electroencephalograph for diagnosing PTSD. Frequency and presence of biomarkers and alpha brain wave symmetry in the frontal and parietal lobes were examined. Research questions involved examining the presence of alpha wave imbalance across the frontal lobe and between the right and left parietal lobes. A secondary data analysis was conducted using data from 108 subjects; these data included records from those with and without a PTSD diagnosis. The results of logistic regression showed that 63% of the clients diagnosed with PTSD were correctly identified and between 7% and 8% of the variance in PTSD was accounted for by frontal lobe asymmetry. The parietal lobe imbalance correctly classified PTSD in 59% of the patients and it identified 3.5–4.9% of the variance, suggesting that asymmetry in the frontal and parietal lobes should not be used as the primary method for diagnosing PTSD. Implications for social change include identifying an objective diagnostic tool that can potentially decrease the possibility of inaccurate diagnoses based on self-reported symptoms. This could lead to eliminating some of the shame and embarrassment veterans and first responders feel toward seeking help for PTSD.

Le cancer du sein est l’un des cancers le plus souvent diagnostiqué chez les femmes en Europe et dans le monde. Pour certaines formes agressives de cancer du sein, la chimiothérapie est le seul traitement systémique efficace. L'identification des nouveaux biomarqueurs potentiels de progression du cancer, ainsi que leur caractérisation et validation est cruciale, et peut permettre une meilleure compréhension de la biologie du cancer du sein. Nous avons réalisé le séquençage à haut débit de l'exome sur une cohorte de 29, 23 et 27 patients atteints de cancer du sein micropapillaire, métaplasique, et lobulaire pléomorphe respectivement. Outre le taux de mutation élevé dans les gènes déjà étudiés dans la progression du cancer, les carcinomes lobulaires pléomorphiques ont présenté des mutations du gène PYGM (8 patients sur 27, 30%), impliqué dans le métabolisme du glycogène. D'autres analyses de bases de données publiques montrent que PYGM est considérablement sous-exprimé dans les cancers de manière générale par rapport aux tissus normaux et que la faible expression dans les tumeurs est corrélée avec une faible survie sans rechute. Le marquage immunohistochimique sur les tissus inclus en paraffine et fixés au formol de notre cohorte de patients, a confirmé une diminution de l'expression de PYGM dans la zone tumorale comparé aux tissus non-cancéreux adjacents. Pour étudier l'effet de la dérégulation du PYGM sur le métabolisme de la cellule cancéreuse et sa fonction potentielle dans la progression du cancer, nous avons surexprimé PYGM dans des lignées cellulaires cancéreuses. Nous avons observé que la surexpression du PYGM a diminué la quantité du glycogène et modulé certains produits finaux du métabolisme du glycogène. L’analyse métabolomique a révélé une activation métabolique due à la surexpression de PYGM; la modulation de la mort cellulaire dans les conditions de privation de glucose a été observée, mais ces deux effets étaient dépendants de la lignée cellulaire. Les analyses biochimiques et moléculaires nous ont aidés à étudier le comportement des cellules en fonction du niveau d'expression de PYGM. Ces analyses pourraient nous informer sur la pertinence du ciblage du PYGM dans le développement de nouveaux traitements basés sur les biomarqueurs métaboliques utilisés pour le traitement du cancer du sein. L'impact de plusieurs autres biomarqueurs potentiels, identifiés par des analyses de génome, a été testé au cours de ce travail et les gènes étudiés sont mentionnés à la fin de la section résultats. En conclusion, les technologies haut débit jouent un rôle important dans l’identification des nouvelles voies qui peuvent être impliquées dans le développement des cancers. / Breast cancer is the most common cancer diagnosed in women in Europe and worldwide. For some aggressive forms of breast cancer, chemotherapy represents the only effective systemic treatment option. The identification of new potential cancer progression biomarkers, as well as their characterization and validation, is crucial, and may lead to a better understanding of breast cancer biology. We performed whole exome and targeted sequencing on a cohort of 29 micropapillary, 23 metaplastic, and 27 pleomorphic lobular breast cancer patients samples. Apart from high mutational rate in genes already known in cancer progression, pleomorphic lobular carcinoma presented mutations in PYGM, a gene involved in glycogen metabolism, in 8 out of 27 samples (30 %). Further analyses of publicly available datasets showed that PYGM is dramatically underexpressed in common cancers as compared to normal tissues and that low expression in tumors is correlated with poor relapse-free survival. Immunohistochemical staining on formalin-fixed paraffin-embedded tissues available in our cohort of patients confirmed higher PYGM expression in normal breast tissue compared to equivalent tumoral zone. We thus overexpressed PYGM in different cancer cell lines in order to investigate the effect of PYGM dysregulation on cancer cell metabolism and its potential function in cancer progression. We observed that PYGM overexpression decreased glycogen content and modulated final glycogen metabolism products content. Metabolomics analysis revealed a metabolic activation due to PYGM overexpression; and modulation of cell death under glucose deprivation was observed, however these effects were cell line dependent. Biochemical and molecular analyses help us to investigate cell behavior according to PYGM expression level and could inform about a potential efficacy of PYGM targeting in new therapy development based on metabolic biomarkers applied to breast cancer treatment. The impact of several other potential biomarkers, identified by broad genome analyses, was tested during this thesis and these genes are mentioned in the end of result section. Altogether, high-throughput technologies play an important role in novel cancer pathways identification that may be involved in the pathogenesis of common malignancies.

Charles University University of Porto Faculty of Pharmacy in Hradec Králové Faculty of Pharmacy Department of Pharmacology and Toxicology Department of Biological Sciences Student: Karolína Zubatá Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Consultants: Susana Rocha, Ph.D., prof. Alice Santos-Silva, Ph.D. Title of diploma thesis: Oxidative stress biomarkers of the erythrocyte in the newborn - a follow-up study Increased levels of oxidative stress (OS) have been described in healthy, full-term newborns as a consequence of the drastic changes introduced by birth and by the exposure to extrauterine environment. Our intention was to examine the OS levels in red blood cells (RBCs) of neonates and to further understand the changes that the newborn organism undergoes with its newly- acquired autonomy as this knowledge is limited and there are no reference values. Umbilical cord blood samples were collected from a small population of newborns (n = 8) and several hematological and biochemistry parameters were evaluated. Our experimental data consist of OS biomarkers measurements performed in different fractions of blood (RBC membrane, total RBCs and plasma): membrane bound hemoglobin (MBH), lipid peroxidation (LPO), quantification of catalase (CAT) and glutathione peroxidase (GPx) activities,...

Die Akute Myeloische Leukämie (AML) ist eine sowohl zytogenetisch als auch molekulargenetisch äußerst heterogene Erkrankung, die durch die klonale Proliferation myeloider Vorläuferzellen sowie eine Ausreifungsblockade charakterisiert ist. Trotz des in den letzten Jahren zugenommenen Wissens über die Biologie dieser Erkrankung und Weiterentwicklung von Therapiemethoden bleibt das Gesamtüberleben der Patienten mit AML überwiegend schlecht. Damit für mehr Patienten eine Heilung der AML möglich werden kann, sind ein tieferes Verständnis über die funktionellen Zusammenhänge in der Leukämogenese, eine bessere Risikostratifizierung und neue Therapieoptionen erforderlich. Diese Habilitationsschrift fasst Publikationen zusammen, die neue molekulare Biomarker und deren klinischen Einfluss in der AML untersucht haben. Der Fokus liegt auf der Erfassung molekularbiologischer Veränderungen bei Diagnose einer AML oder im Krankheitsverlauf, die die Risikostratifizierung der Patienten verbessern kann. Außerdem gestattet die Arbeit Einblicke in die mit diesen molekularen Markern verbundene Biologie der AML, sowie mögliche neue Therapieoptionen. Der erste bis dritte Abschnitt der Arbeit fokussiert sich auf Genmutationen und Genexpressionen in der AML. Es wird dargelegt, wie das Vorhandensein bestimmter Fusionstranskripte (hier CBFB-MYH11), rekurrenter Mutationen allein (hier im DNMT3A Gen) sowie als Teil genetischer Risikoklassifikationssysteme (hier die genetischen Risikogruppen des European LeukemiaNet) und die abberrante Expression AML-assoziierter Gene (hier BAALC, ERG und MN1) Beiträge zur Prognoseabschätzung der AML bieten. Darüber hinaus hat sich in den letzten Jahren die entscheidende Rolle von MicroRNAs in der Pathophysiologie der AML herausgestellt. Heute weiß man, dass für die Leukämieentstehung und die Aggressivität der Erkankung schon die Dysregulation einer einzelnen microRNA entscheidend sein kann. Im vierten Abschnitt wird auf den progostischen Einfluss der Expressionslevel zweier MicroRNAs – miR-181a und miR-29b – und deren klinische und biologische Konsequenzen eingegangen. Außerdem wird dargestellt, wie verschiedene therapeutische Interventionen zu günstigen Änderungen des Expressionsniveaus dieser beiden neuen Biomarker und so zu potentiell neuen Therapiestrategien in der AML führen können. Weiterhin wächst die Erkenntnis, dass in der AML so gennannte Leukämie-initiierende Zellen für Therapieresistenz und Rezidive verantwortlich zu sein scheinen. Der letzte Abschnitt dieser Habilitationsschrift fokussiert sich auf das CD34+/CD38- Zellkompartiment, welches einen Großteil der Leukämie-initiiernden Zellen enthält. Es wird gezeigt, dass die Bestimmung der Größe des CD34+/CD38- Zellkompartiments bei Diagnose geeignet ist, AML Patienten mit einem erhöhten Rezidivrisiko nach allogener Stammzelltransplantation zu identifizieren. Zusammenfassend zeigt die Arbeit verschiedene Ansätze, wie neue molekulare Biomarker zu einer besseren Risikostratifizierung und einem tieferen Verständnis der AML zugrunde liegenden Biologie führen können. Des Weiteren beschreibt sie Möglichkeiten der therapeutischen Intervention und weist insgesamt auf die klinischen Implikationen dieser neuen Biomarker hin.