Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-04-26T15:54:34Z No. of bitstreams: 1 klausrubackbertges.pdf: 2812002 bytes, checksum: 85312f3aa11372affe21388fe45dce43 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-04-27T11:52:01Z (GMT) No. of bitstreams: 1 klausrubackbertges.pdf: 2812002 bytes, checksum: 85312f3aa11372affe21388fe45dce43 (MD5) / Made available in DSpace on 2018-04-27T11:52:01Z (GMT). No. of bitstreams: 1 klausrubackbertges.pdf: 2812002 bytes, checksum: 85312f3aa11372affe21388fe45dce43 (MD5) Previous issue date: 2018-03-21 / A esofagite eosinofílica vem tendo uma importância crescente na literatura médica. É uma doença inflamatória crônica, imunoantígeno mediada, caracterizada por sintomas de disfunção esofágica e uma infiltração predominantemente eosinofílica na mucosa do esôfago. O diagnóstico histopatológico é definido pela presença de 15 ou mais eosinófilos por campo de grande aumento e o tratamento de cada paciente deve ser individualizado. O processo de remodelamento do esôfago na esofagite eosinofílica ainda não está completamente esclarecido, mas parece envolver a presença de fibrose na lâmina própria, sendo a grande responsável pelos sintomas de disfagia e impactação alimentar. A IL-13 contrarregula a expressão de filagrina nas células epiteliais, promovendo um mecanismo pelo qual antígenos alimentares ativam o sistema imunológico. Este estudo objetivou avaliar a prevalência das características histopatológicas de remodelamento tecidual e a expressão da filagrina em biópsias esofágicas de pacientes com esofagite eosinofílica. Foram avaliadas, retrospectivamente, 50 pacientes com suspeita clínica e/ou endoscópica de esofagite eosinofílica. Deste total, 27 foram selecionados e distribuídos em dois grupos: GI, com 15 a 24 eosinófilos por campo de grande aumento e GII, contendo mais de 24. Após a histomorfometria e imuno-histoquímica para filagrina, comparou-se os dois grupos. Nos parâmetros de fibrose, houve diferença estatisticamente significante, com maior prevalência no GII (p < 0,05). Também houve mais espessamento da camada basal no GII (p < 0,001). No estudo de correlação entre o número de eosinófilos e o percentual de fibrose, encontrou-se correlação positiva: 50,8% (p = 0,016), ou seja, mais fibrose nos casos do GII. Na correlação entre o número de eosinófilos e a espessura da camada basal, o resultado também foi positivo: 52,1% (p = 0,08), ou seja, membrana basal mais espessa no GII. A filagrina mostrou-se reduzida nos pacientes com esofagite eosinofílica. / Eosinophilic esophagitis is of increasing importance in the medical literature. It is a chronic inflammatory disease, mediated immunoantigen, characterized by symptoms of esophageal dysfunction and a predominantly eosinophilic infiltration in the esophagic mucosa. The histopathological diagnosis is defined by the presence of 15 or more eosinophils per large increase field and the treatment of each patient should be individualized. The process of esophageal remodeling into eosinophilic esophagitis is still not fully understood, but it seems to involve a presence of fibrosis in the lamina propria, being a major cause of the symptoms of dysphagia and food impaction. IL-13 counteracts the expression of filaggrin in epithelial cells, promoting a mechanism by which food antigens activate the immune system. This study aimed to evaluate a prevalence of the histopathological characteristics of tissue remodeling and the filaggrin expression in esophageal biopsies of patients with eosinophilic esophagitis. Fifty patients with clinical and/or endoscopic suspicion of eosinophilic esophagitis were retrospectively evaluated. Of these, 27 were selected and distributed in two groups: GI, with 15 to 24 eosinophils per large increase field and GII, containing more than 24. After a histomorphometry and immunohistochemistry for filaggrin, the two groups were compared. In the fibrosis parameters, there was a statistically significant difference, with a higher prevalence in GII (p < 0.05). There was also more thickening of the basal layer in GII (p < 0.001). The correlation study between the number of eosinophils and the percentage of fibrosis found a positive correlation: 50.8% (p = 0.016), that means, more fibrosis in cases of GII. In the correlation between the number of eosinophils and the thickness of the basal layer, the result was also positive: 52.1% (p = 0.08), that means, thicker basal layer in GII. Filaggrin was reduced in patients with eosinophilic esophagitis.

Zielsetzung dieser Arbeit ist die Vorstellung des klinischen Aufbaus und des Arbeitsablaufs eines robotischen Assistenzsystems für bildgeführte Interventionen in einem konventionellen Magnetresonanztomographen (MRT), sowie die Beurteilung der Genauigkeit und der klinischen Erfahrungen bei perkutanen Biopsien in verschieden Körperregionen. Material und Methoden: Das MR- kompatible, servopneumatische robotische Assistenzsystem lässt sich mit dem Patienten in die 60- cm Gantry eines Standard- MR- Scanners fahren. Die Genauigkeit des Systems wurde anhand von Nadelpunktionen (n= 25) in einem Phantommodell ermittelt. Perkutane diagnostische Biopsien wurden bei sechs Patienten durchgeführt. Ergebnisse: Für eine Interventionstiefe zwischen 29 und 95 mm wurde eine 3-DGenauigkeit von 2,2 +/- 0,7 mm (Intervall 0,9- 3,8 mm) bestimmt. Patienten mit einem BMI bis zu ≈30 kg/m2 konnten mit dem System punktiert werden. Die klinischen Arbeitsschritte werden anhand der Fallbeispiele dargestellt. Die mittlere Interventionszeit betrug 44 Minuten (Intervall 36 – 68 Minuten). Zusammenfassung: Die Punktion verschiedener Körperregionen ist mit Hilfe des robotischen Assistenzsystems in einem geschlossenen MRT erfolgreich und sicher möglich. Die Genauigkeit des Systems ist vergleichbar mit anderen Assistenzsystemen in der Literatur und genügt den klinischen Anforderungen. Eine kürzere Interventionszeit ist mittels einer Optimierung der einzelnen Arbeitsschritte möglich.:Einleitung 1 Publikation 5 Titelseite mit Abstract 6 Einleitung Publikation (Introduction) 7 Material und Methoden (Materials and Methods) 8 Robotisches Assistenzsystem (Robotic Assistance System) 8 Phantommessung (Phantom Experiment) 9 Patientenauswahlkriterien (Patient Selection Criteria) 11 Magnetresonanztomographie (MRI) 11 Biopsiedurchführung (Biopsy Procedure) 12 Ergebnisse (Results) 13 Phantommessung (Phantom Experiment) 13 Klinische Fälle (Clinical Experience) 14 Weichteilbiopsie kleines Becken (Soft-Tissue Biopsy in the Lesser Pelvis) 14 Weichteilbiopsie iliakaler Lymphknoten (Soft-Tissue Biopsy in Iliac Lymph Node) 15 Knochenbiopsie rechtes Femur (Bone Biopsy in Right Femur) 16 Knochenbiopsie Beckenkamm (Bone Biopsy in Iliac Crest) 17 Abszessaspiration (Abscess Aspiration) 18 Weichteilbiopsie Leber (Soft-Tissue Biopsy in the Liver) 19 Diskussion (Discussion) 22 Danksagungen (Acknowledgments) 25 Literaturverzeichnis Publikation (References) 25 Interpretation und Bewertung 30 Zusammenfassung 35 Literaturverzeichnis 39 Tabellenverzeichnis 46 Abbildungsverzeichnis 46 Erklärung über die eigenständige Abfassung der Arbeit 47 Lebenslauf und wissenschaftlicher Werdegang 48 Danksagung 49 Anlagen Anlage 1 Aufklärungsbogen zur Studie 50 Anlage 2 Einverständniserklärung 54 Anlage 3 Zertifikat des Assistenzsystems 57 durch TÜV- Süd

Glioblastomas are the most common malignant brain tumors in adults. Despite resection of the tumor, combined radio- and chemotherapy and new-targeted therapy approaches, the average life expectancy is only 15 months with a 3-year survival rate of less than 5%. Invasive growth of tumor cells into the surrounding brain tissue complicates treatment and causes recurrence. Imaging techniques such as magnetic resonance imaging (MRI) are conventionally used for diagnosis and for monitoring after surgery in order to detect tumor lesions. However, next to tumor progression, contrast enhancement could also result from pseudoprogression or radiation necrosis. Similarly, next to therapy response, a reduction of contrast enhancement could also mimic a success in therapy (pseudoresponse). Furthermore, the use of targeted therapies requires the molecular detection of biomarkers, which is often performed on tissue biopsies. During therapy, resistance mechanisms to therapy may develop, which have a significant impact on the success of the therapy. Repeated biopsies are needed to distinguish tumor progress from therapy-associated tissue changes and simultaneously detect therapy resistance. Often they are not feasible due to the high risk for the patient. Currently, there are methods to improve monitoring in glioblastoma patients. Besides improved imaging techniques, liquid biopsies are a promising method to detect tumor progression. At the same time, liquid biopsies also allow molecular characterization of the lesions. A key advantage over tissue biopsies is that they are minimally invasive, making them well suited for longitudinal tumor monitoring. For clinical application of liquid biopsies in the form of blood and CSF, components such as circulating tumor cells, circulating tumor DNA, extracellular vesicles, or tumor-educated platelets are used, with the former two probably being the most common tumor markers at present. In this study, a highly sensitive next generation sequencing-based method was established to detect tumor mutations in plasma and CSF of glioblastoma patients. To this end, crucial sample preparation steps were initially optimized. Therefore, four isolation kits extracting cell-free DNA from plasma were compared and the best-performing kit was chosen for the analysis of patient material. Furthermore, a multi-gene next generation sequencing panel (cfDNA-GBM panel) was designed specifically for use in liquid biopsies from glioblastoma patients and tested on a reference standard for cell-free DNA with mutations of different allele frequencies. The newly designed cfDNA-GBM panel, which uses hybrid-capture based enrichment, was compared to a primer-extension based panel. Due to the more consistent coverage of target regions, superior sensitivity, and better clinical applicability due to its smaller size, the cfDNA-GBM panel was used for subsequent clinical investigations. In both enrichments, unique molecular barcodes were used to label all original fragments and thus identify and eliminate errors in subsequent data analysis that occur during amplification in library preparation This increases the sensitivity of the method. During the establishment of the methodology, the use of molecular barcodes clarified the limitation of sequence information due to the small input amount of cell-free DNA. For tumor tissue analysis, an already established larger panel was adapted and extended based on the results of a proof of concept study. After the establishment of the sensitive method, clinical samples of glioblastoma patients were analyzed. Isolation and analysis of cell-free DNA from plasma and CSF resulted in highly variable amounts of cell-free DNA with characteristic oligonucleosomal fragment lengths. Furthermore, no difference in the amount or fragmentation of cell-free DNA from pre- or post-surgery plasma was determined. To increase the amount of cell-free DNA for sequencing, the amount of blood samples was increased from 10 ml (5 patients) to 50 ml (9 patients). Detection of circulating tumor DNA in plasma and CSF from glioblastoma patients was performed using two strategies. In a first approach, somatic tumor mutations were detected in genomic DNA from tumor tissue, which were searched for in the sequence data of liquid biopsies in a second step. In this approach, an increased detection rate (in 56% of patients compared to 25% in 10 ml blood samples) of circulating tumor DNA was detected in blood samples with a volume of 50 ml. Almost all tumor mutations lying in the target region of the smaller cfDNA-GBM panel could be detected in CSF (92%) with similar allele frequencies to those detected in the tumor. In a second strategy, somatic variants were detected in plasma and CSF without prior knowledge of variants in tumor tissue. Interestingly, three variants were detected in the CSF of one patient, which occurred with an allele frequency over 5% in the CSF, while they were not detectable in the analyzed tumor tissue. These variants may be indicators for tumor heterogeneity that was not accessible by analyzing sections of tumor tissue. Finally, mutation-specific probes were designed for three tumor-somatic variants detected in the patients' plasma to validate them by digital PCR. A pair of probes for a mutation in PTEN was successfully established to robustly detect minimal allele frequencies down to 0.1% but could not validate the mutation in cell-free DNA from plasma of the respective patient. Overall, it was shown that analysis of tumor somatic mutations using the cfDNA-GBM sequencing panel designed and established in this study is limited in the analysis of cell-free DNA from plasma of patients with glioblastomas, whereas the detection of circulating tumor DNA from CSF is promising.

MicroRNAs are a class of small non-coding RNAs involved in post-transcriptional regulation. Their role in almost all processes of the cell, make microRNAs ubiquitary players of cell development, growth, differentiation, cell to cell communication and cell death. Thus, cells’ physiological or pathological conditions are reflected by variations in the levels of expression of microRNAs, enabling them to be used as biomarkers of such states. In the past decade, there has been an exponential increase of studies using microRNAs as potential biomarkers for cancer, neurodegenerative diseases, inflammation and cardiac diseases, from tissues and liquid biopsies. However, none of them has reached the clinics yet, due to inconsistency of results through the literature and lack of assay standardization and reproducibility. Technological limitations of microRNAs detection have been, to date, the biggest challenge for using these molecules in clinical settings. In fact, although microarrays, RT-qPCR and RNA-seq are well-established technologies, they all require complex procedures and trained personnel, for performing RNA extraction, labelling of the target and PCR amplification. All these steps introduce variability and, in addition, since no universally standardized protocol – from sample extraction to analyte detection - has been produced yet, methodological procedures are difficult to reproduce. For this reason, we developed a new platform for the rapid detection of microRNAs in biofluids composed of an innovative silicon-photomultiplier (SiPM) based detector and a new chemistry for nucleic acid testing (Chem-NAT). Chem-NAT exploits a dynamic labelling chemistry which allows the sensitive detection of nucleic acids till single base level. On the other hand, SiPM-based device, compared to normal vacuum photomultipliers, grants miniaturization and higher capacity of fitting in a bench-top solution for clinical settings, among other advantages. The new platform – ODG – has been validated for the direct detection – neither RNA extraction nor PCR amplification needed - of microRNA-21 in plasma of lung cancer patients. In this work, we also explored the use of microRNAs as biomarkers in metastatic castration resistant prostate cancer (mCRPC). We collected plasma samples from mCRPC patients before and after abiraterone acetate treatment – androgen deprivation type of drug – and performed a miRnome analysis for discovering microRNAs predicting the efficacy of the drug. We chose miR-103a-3p and miR-378a-5p and we validated them via TaqMan RT-qPCR. We discovered that the ratio between the two microRNAs is able to predict the efficacy of abiraterone acetate and follow the responsiveness in time. In liquid biopsies, extracellular vesicles are getting increasing importance for diagnostic and prognostic purposes. Therefore, in this work we also explored the expression of some microRNAs in extracellular vesicles from plasma, isolated via nickel-based method. We discovered that microRNA-21 and microRNA-223 are not enriched in vesicles from healthy individuals.

Prostatos vėžys yra dažniausia vyrų onkologinė liga JAV, Vakarų Europoje bei Lietuvoje. Dėl senstančios visuomenės ateityje bus nustatoma dar daugiau naujų prostatos vėžio atvejų. Lietuvos vėžio registro duomenimis 1995 – 2005 metais vidutinis metinis prostatos vėžio sergamumo didėjimas - 14,5 proc. per metus. Vyrų sergamumas prostatos vėžiu Lietuvoje 2005 metais siekė 125,9/100000 atvejų, o mirtingumas nuo šios ligos siekė 31/100000 atvejų. Vyrų mirtingumas nuo prostatos vėžio antras pagal dažnį po plaučių vėžio su vėžiu susijusio mirtingumo grupėje. Todėl daugelis tyrėjų pabrėžia, kad norint mažinti mirtingumą, reikia ankstinti prostatos vėžio nustatymo laiką. Pradėta Lietuvos vyrų ankstyvosios prostatos vėžio diagnostikos programa ir dažnas prostatos specifinio antigeno nustatymas lėmė padidėjusį apsilankymų pas urologus skaičių ir padidino prostatos biopsijų kiekį. Norint efektyviai ir optimaliai ištirti šiuos pacientus, reikia daug materialinių išteklių ir laiko.Šio darbo tikslas buvo optimizuoti pacientų su padidėjusia prostatos vėžio rizika ištyrimą ir stebėjimą bei nustatyti ryšį tarp prostatos vėžio rizikos veiksnių ir prostatos vėžio diagnozavimo padidėjusios rizikos grupėje. Darbo uždaviniai: 1. Išanalizuoti prostatos vėžio nustatymo dažnį pirmąja ir kartotinėmis lateralinėmis sekstantinėmis prostatos biopsijomis ir įvertinti jų efektyvumą. 2. Nustatyti amžiaus, rūkymo, alkoholio vartojimo, prostatos vėžio šeiminės anamnezės, viršsvorio ir padidėjusio cholesterolio... [toliau žr. visą tekstą] / Prostate cancer is the most frequent malignant disease in men in United States, Western Europe and in Lithuania. Due to ageing population incidence of prostate cancer will rise even more in the future. Since the year 2003 prostate cancer became the most common form of cancer diagnosed in men in Lithuania (more than 1500 new prostate cancer cases a year). There were 2005 of new prostate cancer cases diagnosed in the year 2005. According to Lithuanian Cancer Registry data during the years 1995-2005 the prevalence of prostate cancer was increasing 14.5 percent annually. Prostate cancer was detected in 24.3 percent of all cancer cases in men in the year 2005 in Lithuania and in 48.3 percent of them disease was detected in the stages I and II. In the year 2005 the prevalence of prostate cancer in Lithuanian men was 125.9 per 100000 population and mortality was 31 per 100000 population. Prostate cancer is a second common form of death after lung cancer in cancer-associated mortality group in Lithuania. Prostate cancer mortality ranged between 19 and 55 per 100000 in Europe and it was 23.2 per 100000 populations in the year 2006 in European Union. Many authors stress that it is important to diagnose prostate cancer in the early stages in order to reduce prostate cancer mortality rate. The aim of the study was to optimize investigation and follow-up of the high prostate cancer risk patients, and to define the relation between prostate cancer risk factors and prostate cancer... [to full text]

A l'heure actuelle, la procédure clinique standard de prélèvement de biopsies de la prostate est effectuée sous contrôle échographique 2D en utilisant un protocole systématique. Il est difficile pour le clinicien de localiser les cibles de biopsie avec précision, et il est impossible de connaître la position exacte des tissus échantillonnés après l'intervention. Dans ce mémoire, nous proposons une méthode permettant de localiser la position des tissus prélevés avec une précision millimétrique par rapport à une image 3D de la prostate de référence. Elle combine des techniques de recalage rigide et élastique basées sur les intensités (recalage iconique) avec des modèles a priori des contraintes biomécaniques. Ce travail permet la mise en œuvre d'applications telles que la validation postopératoire de la distribution des biopsies et l'établissement de cartographies précises des tissus cancéreux, ce qui permettrait éventuellement un traitement localisé du cancer de la prostate. L'approche proposée permet également de guider le clinicien vers des cibles définies sur l'image de référence, provenant par exemple d'une autre modalité d'imagerie telle que l'IRM ou le SpectroIRM.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Les gliomes diffus de bas grade (GBG) sont des tumeurs cérébrales primaires. Après une phase de croissance lente, ils évoluent en gliomes de haut grade, entrainant une issue fatale. Ce sont des tumeurs très diffuses donc difficiles à traiter. Une meilleure connaissance de ces tumeurs pourrait permettre de les guérir ou, à défaut, d'optimiser les traitements. Nous avons étudié la croissance des GBG grâce à un modèle mathématique simple, ce qui nous a amené à spéculer (i) qu'ils surviennent à l'adolescence, (ii) que l'âge de la tumeur au moment du diagnostic peut être calculé facilement et (iii) que la vitesse de croissance est un fac- teur pronostique important. Cette dernière prédiction concorde avec les observations cliniques. Pour vérifier ce modèle spatial, nous avons caractérisé quantitativement des tissus de biopsies étagées de GBG humains, en particulier la présence d'œdème. L'analyse de ces données micro- scopiques étaie l'idée que l'œdème est à l'origine de l'anomalie de signal IRM en séquence T2. Pour prendre en compte ce résultat nouveau, nous avons incorporé l'œdème au modèle initial comme conséquence de la présence de cellules tumorales. Ce modèle permet d'expliquer la longue décroissance du rayon tumoral pendant des dizaines de mois après la radiothérapie : les cellules tumorales désormais moins nombreuses, le drainage de l'œdème devient prédominant. Ce mo- dèle, qui ne comprend que trois paramètres libres, a été validé grâce à des données cliniques sur une vingtaine de patients.

This thesis describes two different sample preparation strategies for inductively coupled plasma atomic emission spectrometry (ICP-AES), and their ability regarding multi element quantification in complex samples. Sensitivity, repeatability, reproducibility and accuracy were investigated. The aim was to increase the over all efficiency, the speed of analysis, and/or the sensitivity of the analytical method. The intention was to measure analytes with concentrations ranging from ng/g to mg/g simultaneously. The aim was additionally to study chemical and physical processes occurring during the sample preparation, the sample transport to the plasma, and the atomization therein. In the first sample preparation strategy, a hydrophilic highly cross-linked iminodiacetate-agarose adsorbent, IDA-Novarose, was used for preconcentration of metal ions, and matrix elimination in natural water samples. The sorbent was synthesized with different binding capacities. The effect of the capacity on preconcentration, matrix elimination, and uptake capability at high flow rates was studied. For a high capacity IDA-Novarose (≥ 45 µmole/ml) quantitative uptake was seen even at high flow rates (100 ml/min) for Cu2+ with a high affinity to the adsorbent, and for Cd2+ with a moderate affinity. For lower capacities the uptake of Cd2+ was affected by the sample matrix and the flow rate. A method based on the determination of the conditional stability constant of the metal sorbent complex was suggested for the prediction of the sorbent capacity needed to obtain quantitative recovery and optimal matrix elimination. The sorbent was used in a flow system with online buffering for the analysis of a certified riverine water (SLRS-3), tap water and lake water. With few exceptions the results obtained by ICP-AES after preconcentration agreed well with the certified concentrations and results obtained by ICP-MS. The other sample preparation strategy discussed is a method for non digested biological samples from different animal organs for the multi element analysis by ICP-AES. This “mix and measure method” consists of a simple homogenization of the sample with a mixing rod in a small amount of neutral media, followed by dilution and direct measurement with ICP-AES. The total time of analysis is only a few minutes. The ability of this fast method to accurately quantify some elements of toxic, environmental, and/or physiological concern with the lowest possible sample dilution and the highest possible plasma load was evaluated. In 10 % liver slurry Cd, Co, and Sr, at concentration levels around 0.05 µg/g were quantified simultaneously with P and K around 2000 µg/g and with several other elements in between (Al, Ca, Cu, Fe, Mg, Mn, Pb, and Zn). The relative standard deviation of repeated measurements of samples was around 5 - 6 % for regardless of the concentration of the element. The method was also used for fast screening of the elemental distribution in mice organs (brain, heart, kidney, liver, lung and spleen).