Heavy chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region which will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and the degree to which the regulation of germline transcripts correlates with the regulation of switching, we studied this process in the murine B-lymphoma cell line I.29μ, which in the presence of bacterial lipopolysaccharide (LPS) switches primarily to IgA and less frequently to IgE. Levels of α-germline transcripts initiating upstream of α switch (Sα) sequences are elevated in clones of this line which switch well as compared to clones which switch less frequently. TGFβ1 has been shown to increase α-germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B-cells. We now demonstrate in I.29μ cells that TGFβ also increases switching to IgA and increases the level of α-germline transcripts 5 to 9 fold. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGFβ appears to direct switching to IgA by inducing transcription from the unrearranged Sα- CαDNA segment. Germline α RNA is quite stable in I.29μ cells, having a half life of about 3 to 5 hours, and we find only slight stabilization in the presence of TGFβ. Levels of ε-germline transcripts are not increased by TGFβ . IL-4, which modestly increases switching to IgA in I.29μ cells, slightly increases trancription of α-germline RNA. However, we present evidence suggesting that endogenously produced IL-4 may also act at additional levels to increase switching to IgA. IFNγ, which reduces IgA expression in these cells, also reduces the level of α-germline transcripts. IFNγ also reduces the level of ε-germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines in turn regulates the specificity of recombination. In studies aimed at identifying other signalling pathways that promote class switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase lipopolysaccharide (LPS)-induced switching to IgA in the B cell lymphoma I.29μ and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In I.29μ cells, PARP inhibitors increase IgA switching by day 2 and cause a 5-fold average increase in switching on day 3 as assayed by immunofluorescence microscopy. The PARP inhibitor, nicotinamide, also causes a reduced intensity of hybridization of Cμ and Cα specific probes to genomic DNA fragments containing the expressed VDJ-Cμ and the unrearranged Sα - Cα segments, respectively, indicating that PARP inhibition increases rearrangment of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogs or reduced by inhibitors of protein kinase A (PKA). Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged Cα gene, although TGFβ is required for optimal induction by PARP inhibitors, consistent with a requirement for transcription of the unrearranged CH gene. PARP inhibitors do not overcome the requirement for endogenously produced IL-4.

Sammanfattning Bakgrund: Lymfödem är en kronisk symtomdiagnos som kräver livslång behandling där egenvård är en viktig del. Bristande egenvård leder till försämrat lymfödem och större benägenhet för erysipelasinfektioner. Problemet är att patienterna känner sig osäkra på egenvård av lymfödem och tvivlar på dess effekt. Syfte: Förbättringsarbetets syfte var att skapa förutsättningar och bättre följsamhet till patienters egenvårdsbehandling för att bidra till bibehållet eller förbättrat stadie av lymfödem och uppleva en god hälso-relaterad livskvalitet. Syftet med studien var att beskriva patienters erfarenheter av att leva med lymfödem, erfarenhet av den lymfterapeutiska vårdtjänsten och redogöra för patienters förbättringsförslag för att förbättra egenvård. Metod: Förbättringsarbetet genomförs med inspiration från tjänstedesign och förbättringsmodellerna 5P, Nolans förbättringsmodell och PGSA.Studien utförs med tematisk analys och beskrevs utifrån delar av Kanomodellen av Noriaki Kano; bas-, uttalade- och omedvetna behov. Resultat: Patienter blir säkrare på att göra egenvård om de har en egenvårdsplan och lymfterapeuterna blir tydligare i sin kommunikation med checklistan och i samskapande med patienten. Resultat från studien visar att lymfterapeuterna behöver bättre kommunicera egenvård och anpassa informationen individuellt. Slutsatser: Genom att patienterna delar med sig av sina erfarenheter och förbättringsförslag kan vi skapa ett lärande och öppna upp för mer patientinvolvering i utvecklingsarbetet. / Summary Background: Lymphedema is a chronic diagnosis that requires lifelong treatment where self-care is an important part. Lack of self-care leads to impaired lymphedema and greater tendency for erysipelas infections. The problem is that patients feel insecure about self-care of lymphedema and doubt its effect.  Purpose: The purpose of the improvement work was to create better compliance to the patients self-care treatment. The purpose of the study was to describe patients 'experiences of the lymphatic care service and living with lymphedema and also the patients' improvement suggestions for improving self-care. Method: With inspiration from service design and the improvement models 5P, Nolan's improvement model and PGSA. The study with thematic analysis method was described from parts of the Kano model by Noriaki Kano; basic-, expected- and  unexpected needs. Results: Patients become more confident in self-care if they have a self-care plan and the Lymphatic Therapists become clearer in their communication by a checklist and in collaboration with the patient. Results from the study show that the lymphatic therapists need to better communicate self-care and adapt the information individually.  Conclusions: By sharing patients' experiences and suggestions for improvement, we can create learning and more patient involvement in development work.

Purpose: The purpose of this study was to determine the usefulness of SBAR-cued web-based communication skills training and address study participants' perceptions of the training. Specific Aims: Evaluate the usefulness and accuracy of participants to answer prompts of SBAR-cued communication responses. Describe individuals' perspectives of the acceptability of using SBAR patient-HCP communication simulation to better prepare for ED visits during a SCC. Framework: This study was guided by The Theory of Self-Care Management for Sickle Cell Disease (SCMSCD). Design: A secondary analysis was conducted using a qualitative descriptive approach. Inter-rater reliability (IRR) of qualitative data was used to evaluate the usefulness and accuracy of participants to answer prompts of SBAR-cued communication responses. Content analysis was also utilized to describe individuals' perspectives of the acceptability of using SBAR patient-HCP communication simulation to better prepare for ED visits during a SCC. Results: IRR between raters ranged from 64%-94% with predominant themes of (1) Patient-Provider Communication and Interaction, (2) Patients want to be Heard and Believed, (3) Accuracy of the ED Experience and Incorporating the Uniqueness of each Patient and (4) Overall Usefulness of the Video Trainer emerging. Conclusions: This secondary analysis supported how SBAR can be effectively used to assist patients in a SCC to communicate with their HCP. Participants' responses indicated the training module facilitated communication between patients and HCPs.

L’espérance de vie des patients atteints de mélanome à haut risque ne peut être prédite d’une façon<p>fiable en se basant sur les analyses d’histopathologies de la lésion primitive et est souvent ajustée<p>durant la progression de la maladie. Notre étude vise à élargir nos observations initiales au niveau<p>des métastases cutanées et d’évaluer la valeur pronostique de tyrosinase related protein 1 (TYRP1)<p>dans les métastases ganglionnaires des patients atteints de mélanome de stades III et IV. TYRP1 est<p>une enzyme mélanosomale qui partage des similitudes structurelles avec la tyrosinase, l'enzyme clé<p>de la mélanogenèse.<p>L’expression de l'ARNm de TYRP1 a été quantifiée dans 104 métastases ganglionnaires par PCR<p>en temps réel et normalisée par rapport à l’expression de l’ARNm de S100B (marqueur reconnu du<p>mélanome) pour corriger l’expression de TYRP1 suivant la charge tumorale de l’échantillon. Le<p>rapport TYRP1/S100B a été calculé et la médiane a été utilisée en tant que valeur seuil. Ensuite<p>nous avons étudié la relation entre les valeurs de TYRP1/S100B, le suivi clinique et les<p>caractéristiques histopathologiques de la tumeur primitive.<p>Un rapport élevé de l’ARNm TYRP1/S100B corrélait significativement avec une survie sans<p>récidive et une survie globale plus courtes, avec une épaisseur de Breslow plus élevée et avec la<p>présence d'une ulcération au niveau de la tumeur primitive. En outre, une expression élevée de<p>TYRP1/S100B était de meilleure valeur pronostique pour la survie globale que l'épaisseur de<p>Breslow et l'ulcération des primitifs. De plus, cette expression est bien conservée au cours de la<p>progression de la maladie par rapport aux groupes de TYRP1 bas/élevé.<p>Nous avons constaté qu’une expression élevée de TYRP1/S100B dans les métastases de patients<p>atteints de mélanome est associée à un résultat clinique défavorable et une survie courte. Menée sur<p>des patients atteints d'un mélanome à haut risque de récidive, cette première étude a suggéré que<p>l'ARNm de TYRP1 dans les métastases pourrait servir de biomarqueur pour affiner le pronostic<p>initial des patients surtout ceux ayant des lésions primitives de localisation inconnues ou non<p>évaluables et peut permettre une gestion différente des deux groupes de patients. Son expression<p>conservée au cours de la progression de la maladie est en faveur de son utilisation comme cible<p>thérapeutique.<p>En second lieu, en évaluant l’expression de la protéine TYRP1 par immunohistochimie dans les<p>métastases cutanées et ganglionnaires, nous avons observé qu’elle n'était pas détectée dans la moitié <p>7<p>des tissus exprimant bel et bien l'ARNm correspondant et qu’elle, contrairement à l'ARNm, n’était<p>pas associée à la survie.<p>Des données récentes ont indiqué que le 3'-UTR de l’ARNm de TYRP1 contient trois sites de<p>liaison putatifs de miR-155 dont deux présentant un polymorphisme d'un seul nucléotide (SNPs:<p>rs683 et rs910) qui favorisent la dégradation en cas d’hybridation miARN-ARNm parfaite de<p>l’ARNm ou non en cas d’hybridation imparfaite. Nous avons cherché à examiner si miR-155 peut<p>affecter l’expression de l’ARNm et de la protéine TYRP1 en fonction de ces SNPs. Tout d'abord,<p>nous avons transfecté deux lignées de mélanome ayant chacune l’une ou l’autre de l’allèle (au<p>niveau rs683 et rs910) avec différentes concentrations de pré-miR-155 et nous avons évalué<p>l’expression du miR-155 et l’ARNm TYRP1 par PCR en temps réel ainsi que l’expression de la<p>protéine TYRP1 par western blot. Nous avons constaté qu’une surexpression de miR-155 a induit<p>une dégradation importante des ARNm TYRP1 et a perturbé sa traduction en protéine dans la lignée<p>avec le génotype “hybridation parfaite”. Ensuite, nous avons examiné l'expression des ARNm et<p>protéines de TYRP1, le niveau de miR-155 et les SNPs rs683 et rs910 dans 192 échantillons de<p>métastases cutanées et ganglionnaires de mélanome. Nous avons trouvé que le groupe d'échantillons<p>avec le génotype “hybridation parfaite” était significativement associé à un niveau de protéine de<p>TYRP1 plus bas alors qu'aucune différence de niveau d’expression n'a été trouvée pour l’ARNm de<p>TYRP1 ou miR-155 entre les deux groupes de génotype, confirmant que les SNPs au niveau de 3’-<p>UTR de TYRP1 peuvent spécifiquement affecter l'expression de la protéine TYRP1. En outre, nous<p>avons montré que l’ARNm de TYRP1 est inversement corrélé avec l’expression miR-155, mais pas<p>avec la protéine TYRP1 dans le groupe " hybridation parfaite", alors qu'il corrèle positivement avec<p>la protéine mais pas avec miR-155 dans le groupe "hybridation imparfaite" où la protéine corrélait<p>inversement à la survie. Cela montre que les SNPs dans le 3'-UTR de l'ARNm TYRP1 affectent la<p>régulation de l’ARNm par miR-155 et la traduction en protéine. Ces SNPs rendent la régulation de<p>l’ARNm et la protéine de TYRP1 indépendante de miR-155 et confèrent une valeur pronostique à<p>la protéine TYRP1 / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Knapp 25 Jahre nach dem ersten Auftreten der bovinen spongiformen Enzephalopathie (BSE) in Europa, bleibt die Entfernung und unschädliche Beseitigung des sogenannten spezifizierten Risikomaterials (SRM) eine der wichtigsten Maßnahmen zur Reduzierung des humanen oralen BSE-Expositionsrisikos. Angesichts des Rückgangs der BSE-Inzidenzen in Europa hat sich die Europäische Kommission (EC) in ihrer „TSE Roadmap“ das Ziel gesetzt, die Auflistung und/oder die Alters-grenzen für das SRM schrittweise zu modifizieren. In diesem Sinne hat die EC die Europäische Behörde für Lebensmittelsicherheit (EFSA) mehrfach aufgefordert, das BSE-Risiko anhand aktueller Daten neu zu bewerten, insbesondere das, welches von Rindernaturdärmen für den Verbraucher ausgeht. Die wissenschaftliche Bewertung des humanen BSE-Expositionsrisikos ist jedoch bis heute mit großen Unsicherheiten verbunden und deshalb sollten erfassbare Varianzeinflüsse so weit wie möglich reduziert werden. Nachdem experimentell BSE-Infektiosität im distalen Ileum demonstriert wurde, erfolgte vorsorglich die Einstufung des gesamten bovinen Darmes (Duodenum bis Rektum, einschließlich des Mesenteriums) als SRM. Eine der wesentlichen Eingangsgrößen bei der wissenschaftlichen Bewertung des Expositionsrisikos ist die Masse des in die Nahrungskette eingebrachten potentiell infektiösen Materials. Alle bis zum heutigen Zeitpunkt durchgeführten Risikobewertungen legten eine Masse von 800 g für das bovine Ileum zugrunde. Die eigenen Untersuchungen hatten das Ziel die Gewichtsangabe des bovinen Ileums zu validieren, die Effizienz des SRM-Verbotes zur Reduktion des humanen oralen BSE-Infektionsrisikos sowie das Infektionspotential nach der Bearbeitung des Darmes im Hinblick auf seine Einstufung als SRM einzuschätzen. Von 129 Schlachtrindern wurde das Ileum unter kontrollierten Bedingungen entnommen und in der Folge der technologischen Bearbeitung vermessen. Zudem wurden 13 bovine Ilea und 11 Jejuna jeweils vor und nach der manuellen Bearbeitung histologisch auf das Vorkommen von lymphatischem Gewebe untersucht. Das lymphatische Gewebe des Darmes stellt wahrscheinlich den Ort der Erregeraufnahme nach einer oralen BSE-Infektion dar. Wesentliches Ergebnis ist ein mittleres Gewicht für das bovine Ileum im entleerten und bearbeiteten Zustand von 57 g (Spannweite: 23,1 bis 135,8 g). Dieser Wert liegt im Vergleich zu der bisher verwendeten Eingangsgröße um eine Größenordnung niedriger. Im Hinblick auf die Effizienz des SRM-Verbots zur Risikoreduktion in der Lebensmittelkette ergibt sich mit den neuen Daten ein Anstieg von 95% auf 99% für das zentrale Nervensystem (ZNS) und das ZNS-nahe periphere Nervensystem. In den histologischen Untersuchungen konnte lymphatisches Gewebe nur bei einer der bearbeiteten Ileumproben (7.7%) und zwei der bearbeite-ten Jejunumproben (18,2%) gefunden werden. Dies zeigt, dass durch die Bearbeitung der Rinderdärme der größte Teil des lymphatischen Gewebes entfernt wird. Die vorliegenden Ergebnisse stützen die Aussage anderer Studien, dass durch die Reinigung der Därme signifikante Mengen an lymphatischem Gewebe entfernt werden. Aufgrund des niedrigen Ileumgewichtes und der effektiven Entfernung des lymphatischen Gewebes kann der Anteil des Ileums an der gesamten Infektiosität, bei einem klinisch an BSE erkrankten Rind, als sehr gering eingestuft werden. Das bovine Ileum ist nach vorliegender Untersuchung lediglich mit 1 % an der Gesamtinfektiosität beteiligt und nicht, wie bisher angenommenen, mit 3,3 % bzw. 9,6 %. Angesichts dieser Ergebnisse, der aktuellen BSE-Inzidenzen in Europa, der Verteilung und Höhe der BSE-Infektiosität im bovinen Darm wird, wie auch von einer Reihe anderer Autoren, die Übertragung von BSE durch Rinderdärme als ein vernachlässigbares Risiko angesehen. Vor diesem Hintergrund sollte der SRM-Status der Rinderdärme erneut geprüft und bewertet werden.:INHALTSVERZEICHNIS 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 4 2.1 Transmissible spongiforme Enzephalopathien 4 2.1.1 Transmissible spongiforme Enzephalopathien beim Tier 5 2.1.1.1 Scrapie 6 2.1.1.2 Bovine spongiforme Enzephalopathie 9 2.1.2 Transmissible spongiforme Enzephalopathien beim Menschen 16 2.1.2.1 Klassische Formen der CJD 16 2.1.2.2 Neue Variante der CJD 19 2.2 Lymphatisches Gewebe des Rinderdarms 24 2.2.1 Bestandteile 25 2.2.2 Embryonale Entwicklung 26 2.2.3 Morphologie der Peyer’schen-Platten 27 2.2.3.1 Lymphfollikel 28 2.2.3.2 \"dome\" 29 2.2.3.3 Follikelassoziiertes Epithel 29 2.2.3.4 M-Zellen 29 2.2.3.5 Interfollikuläre Zone 31 2.2.4 Verteilung und Lage 32 2.2.4.1 Solitärfollikel 32 2.2.4.2 Peyer’sche-Platten 33 2.2.4.3 Lymphoglanduläre Komplexe 34 2.2.5 Involution 34 2.2.5.1 Involution der Peyer’schen-Platten 35 2.2.5.2 Involution der Solitärfollikel und des lymphatischen Gewebes des Dickdarms 36 2.3 Naturdarm 36 2.3.1 Gewinnung und Bearbeitung 38 2.3.1.1 Manuelle Bearbeitung 39 2.3.1.2 Maschinelle Bearbeitung 40 2.3.2 Einsatzmöglichkeiten 41 2.3.3 Wirtschaftliche Bedeutung 42 2.4 Risikoanalyse 43 2.4.1 Risikobewertung 44 2.4.1.1 BSE-Risikobewertung 45 2.4.2 Risikomanagement 49 2.4.2.1 Überwachungsmaßnahmen 50 2.4.2.2 Verfütterungsverbote 53 2.4.2.3 Spezifizierte Risikomaterialien 54 2.4.3 Risikokommunikation 56 3 TIERE, MATERIAL UND METHODEN 57 3.1 Gewichts- und Längenbestimmung der Ilea 57 3.1.1 Auswahl der Tiere 57 3.1.2 Entnahme der Ilea 58 3.1.3 Bearbeitung der Ilea 59 3.1.4 Bestimmung des Gewichtes und der Länge 60 3.2 Histologische Untersuchungen 60 3.2.1 Ileum 60 3.2.1.1 Auswahl der Tiere 61 3.2.1.2 Entnahme der Ilea 61 3.2.1.3 Bearbeitung der Ileumabschnitte 61 3.2.1.4 Vorbereitung und Anfertigung der Schnitte 63 3.2.1.5 Auswertung der Schnitte und Dokumentation 64 3.2.2 Jejunum 65 3.2.2.1 Auswahl der Tiere 65 3.2.2.2 Entnahme der Jejuna 65 3.2.2.3 Bearbeitung der Jejunumabschnitte 65 3.2.2.4 Vorbereitung und Anfertigung der Schnitte 66 3.2.2.5 Auswertung der Schnitte und Dokumentation 67 3.3 Untersuchung auf Peyer’sche-Platten 67 3.4 Gewichtsreduktion durch die Bearbeitung 68 4 ERGEBNISSE 69 4.1 Gewicht und der Länge der Ilea 69 4.2 Histologische Untersuchung der Ilea 73 4.3 Histologische Untersuchung der Jejuna 79 4.4 Untersuchung des Rinderdünndarmes auf sichtbares lymphatisches Gewebe 82 4.5 Gewichtsreduktion durch die Bearbeitung 84 5 DISKUSSION 85 5.1 Gewicht und Länge der Ilea 85 5.2 Histologische Untersuchung der Ilea und Jejuna 88 6 ZUSAMMENFASSUNG 95 7 SUMMARY 97 8 LITERATURVERZEICHNIS 99 ANHANG 135 DANKSAGUNG 148 / Nearly two and a half decades after the emergence of a new transmissible spongiform encephalopathy (TSE) in bovines (BSE) in Europe the ban on so called specified risk material (SRM) remains the most important measure to reduce any potential oral human BSE exposition risk from the food chain. In view of the overall and constant reduction of the frequency of bovine TSE cases in Europe the European Commission expressed in its “TSE-road map” the need for a future lifting of the SRM-ban and the European Food Safety Authority has been repeatedly asked by the European Commission to re-assess the BSE risk for the consumer on the basis of current data, amongst others the risk associated with natural bovine sausage casings. However, the scientific evaluation of the human BSE exposition risk is impeded by great uncertainties and ascertainable variances should be reduced as far as possible. After BSE-infectivity was demonstrated experimentally for the distal bovine ileum, the whole bovine intestine from duodenum to rectum including mesentery was categorized as specified risk material (SRM).The weight of potential infective material brought into the food chain is a major influencing variable in this context. All subsequent studies concerning the human BSE exposure risk have been based on an ileum weight of 800 g per adult cattle. The aim of the own studies was to validate the existing measurement of bovine ileum and its infectivity after processing with respect to the efficiency of the SRM ban for a risk reduction in the food chain. 129 ilea were removed from slaughtered cattle and weighted during subsequent technical processing. In parallel, 13 bovine ileum and 11 jejunum samples were analyzed histologically before and after manual processing in order to detect lymphatic tissue which is presumed to be the point of entry for the infective agent. The mean weight for the whole empty and processed ileum was 57 g ranging from 23.1 g to 135.8 g. This is one order of magnitude lower than the input level used to date. Residues of lymphatic tissue were detected in one of the processed ileum samples (7.7%) and two of the jejunum samples (18.2%). This means that the major part of lymphatic tissue is removed from the bovine intestine during the different processing steps. Our results are backed up by a number of studies made by other researchers who showed that significant quantities of lymphatic tissues are removed from the bovine intestine by cleaning.The ileum has to be recognized as only a minor fraction of the total infective load per animal due to its weight and the removal of most lymphatic tissue during processing. Contrary to previous assumptions that ileum amounts to 3.3 and 9.6% respectively of the total infective tissues this study clearly shows that this premise has to be corrected to 1%. Regarding these results in combination with the decreasing BSE incidences in Europe, the distribution and the amount of the BSE infectiosity the potential BSE-infectivity represented by the bovine intestine can be considered negligible. This assessment is shared by numerous authors. A possible amendment of the current SRM status of bovine intestine should be re-assessed.:INHALTSVERZEICHNIS 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 4 2.1 Transmissible spongiforme Enzephalopathien 4 2.1.1 Transmissible spongiforme Enzephalopathien beim Tier 5 2.1.1.1 Scrapie 6 2.1.1.2 Bovine spongiforme Enzephalopathie 9 2.1.2 Transmissible spongiforme Enzephalopathien beim Menschen 16 2.1.2.1 Klassische Formen der CJD 16 2.1.2.2 Neue Variante der CJD 19 2.2 Lymphatisches Gewebe des Rinderdarms 24 2.2.1 Bestandteile 25 2.2.2 Embryonale Entwicklung 26 2.2.3 Morphologie der Peyer’schen-Platten 27 2.2.3.1 Lymphfollikel 28 2.2.3.2 \"dome\" 29 2.2.3.3 Follikelassoziiertes Epithel 29 2.2.3.4 M-Zellen 29 2.2.3.5 Interfollikuläre Zone 31 2.2.4 Verteilung und Lage 32 2.2.4.1 Solitärfollikel 32 2.2.4.2 Peyer’sche-Platten 33 2.2.4.3 Lymphoglanduläre Komplexe 34 2.2.5 Involution 34 2.2.5.1 Involution der Peyer’schen-Platten 35 2.2.5.2 Involution der Solitärfollikel und des lymphatischen Gewebes des Dickdarms 36 2.3 Naturdarm 36 2.3.1 Gewinnung und Bearbeitung 38 2.3.1.1 Manuelle Bearbeitung 39 2.3.1.2 Maschinelle Bearbeitung 40 2.3.2 Einsatzmöglichkeiten 41 2.3.3 Wirtschaftliche Bedeutung 42 2.4 Risikoanalyse 43 2.4.1 Risikobewertung 44 2.4.1.1 BSE-Risikobewertung 45 2.4.2 Risikomanagement 49 2.4.2.1 Überwachungsmaßnahmen 50 2.4.2.2 Verfütterungsverbote 53 2.4.2.3 Spezifizierte Risikomaterialien 54 2.4.3 Risikokommunikation 56 3 TIERE, MATERIAL UND METHODEN 57 3.1 Gewichts- und Längenbestimmung der Ilea 57 3.1.1 Auswahl der Tiere 57 3.1.2 Entnahme der Ilea 58 3.1.3 Bearbeitung der Ilea 59 3.1.4 Bestimmung des Gewichtes und der Länge 60 3.2 Histologische Untersuchungen 60 3.2.1 Ileum 60 3.2.1.1 Auswahl der Tiere 61 3.2.1.2 Entnahme der Ilea 61 3.2.1.3 Bearbeitung der Ileumabschnitte 61 3.2.1.4 Vorbereitung und Anfertigung der Schnitte 63 3.2.1.5 Auswertung der Schnitte und Dokumentation 64 3.2.2 Jejunum 65 3.2.2.1 Auswahl der Tiere 65 3.2.2.2 Entnahme der Jejuna 65 3.2.2.3 Bearbeitung der Jejunumabschnitte 65 3.2.2.4 Vorbereitung und Anfertigung der Schnitte 66 3.2.2.5 Auswertung der Schnitte und Dokumentation 67 3.3 Untersuchung auf Peyer’sche-Platten 67 3.4 Gewichtsreduktion durch die Bearbeitung 68 4 ERGEBNISSE 69 4.1 Gewicht und der Länge der Ilea 69 4.2 Histologische Untersuchung der Ilea 73 4.3 Histologische Untersuchung der Jejuna 79 4.4 Untersuchung des Rinderdünndarmes auf sichtbares lymphatisches Gewebe 82 4.5 Gewichtsreduktion durch die Bearbeitung 84 5 DISKUSSION 85 5.1 Gewicht und Länge der Ilea 85 5.2 Histologische Untersuchung der Ilea und Jejuna 88 6 ZUSAMMENFASSUNG 95 7 SUMMARY 97 8 LITERATURVERZEICHNIS 99 ANHANG 135 DANKSAGUNG 148

The thesis evaluates the results of experimental protocol involving the fertility sparing treatment procedure in early stage cervical carcinoma (LAP I protocol). Sentinel lymph node detection and experimental extirpation of afferent channels using laparoscopy and its technical aspects were analysed in prospective group of 85 women. The oncologic results and early/late morbidity show that established surgical procedures can be considered safe with minimal morbidity, provided that the indication criteria are met. The second part analyses the results of 148 women with no further pregnancy plans suffering from cervical tumors less than 2 cm in size with invasion less than half of the stroma (LAP II protocol). The oncological results in our defined group are very good and comparable to 'standard' procedure of modified radical hysterectomy type B or C with lower morbidity. In the separate section the thesis analyses the possibilities of laparoscopy in endometrial cancer treatment including the potentials of use of sentinel lymph node detection and technical aspects of laparoscopy in obese women. Currently the biggest controversy is the use of laparoscopy in malignant ovarian tumors. Our oncogynaecological study group at FN Motol prefers the laparotomic approach and we chose to include the set of advanced...

The leading cause of breast cancer-associated death is metastasis. During metastasis, tumor cells metastasize from primary tumors to distant organs via the circulatory and lymphatic systems. However, in 80% of solid tumors, metastasis via the lymphatic system precedes metastasis via the vascular system. There is a lot of information about metastasis through the circulatory system. However, not much information is available about the tumor cell dissemination through the lymphatic system or the lymphatic microenvironment that aids in this process in breast cancer metastasis. In addition, the molecular properties of tumor cells as they exit the primary tumor into the afferent lymphatics en route to the sentinel lymph nodes (SLNs) are not yet known.<br><div><br></div><div>This project aims to determine why and how tumor cells metastasize to the lymphatic system. The proposal is based on the hypothesis that active migration is needed for tumor cells to spread via the lymphatic vessels. Thus, finding and understanding the molecules that contribute to this can be a breakthrough for breast cancer metastasis therapy.<br></div><div><br></div><div>The goals of this thesis are to 1) Examine the molecular, genetic, and proteomic characteristics of circulatory tumor cells and compare these to the primary tumor and lung metastasis, 2) Examine the role of Toll-like receptors in tumor cell migration to the lymph node, and 3) Identify the difference in protein expression among two different types of breast cancer (Triple-Negative and Luminal A) and understand their aggressive biology.<br></div>

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Pre-surgical diffusion weighted imaging (DWI) is increasingly important in the context of thyroid cancer for identification of the optimal treatment strategy. It has exemplarily been shown that DWI at 3T can distinguish undifferentiated from well-differentiated thyroid carcinoma, which has decisive implications for the magnitude of surgery. This study used DWI histogram analysis of whole tumor apparent diffusion coefficient (ADC) maps. The primary aim was to discriminate thyroid carcinomas which had already gained the capacity to metastasize lymphatically from those not yet being able to spread via the lymphatic system. The secondary aim was to reflect prognostically important tumor-biological features like cellularity and proliferative activity with ADC histogram analysis. Fifteen patients with follicular-cell derived thyroid cancer were enrolled. Lymph node status, extent of infiltration of surrounding tissue, and Ki-67 and p53 expression were assessed in these patients. DWI was obtained in a 3T system using b values of 0, 400, and 800 s/mm2 . Whole tumor ADC volumes were analyzed using a histogram-based approach. Several ADC parameters showed significant correlations with immunohistopathological parameters. Most importantly, ADC histogram skewness and ADC histogram kurtosis were able to differentiate between nodal negative and nodal positive thyroid carcinoma. Conclusions: histogram analysis of whole ADC tumor volumes has the potential to provide valuable information on tumor biology in thyroid carcinoma. However, further studies are warranted.

The lymphatic and blood vascular systems are two important vessel networks that serve different roles in healthy states and in cancer. In breast cancer the most common cancer amongst women, mortality remains high despite increased treatment response due to metastatic spread, preferentially through the lymphatics. One aggressive subtype, triple negative breast cancer (TNBC) contributing to 15 to 30 percent of cases and is characterized by the absence of expression of three therapeutic biomarkers. As targeted therapy is limited, treatment relies on standard of care via surgery, radiotherapy, and chemotherapy with limited efficacy and increase in survival. Chemotherapies negatively alter the lymphatic vasculature benefiting the tumor, through lymphangiogenesis. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics, like carboplatin, and a novel 2nd generation ablative therapy called High Frequency Irreversible Electroporation (H-FIRE), which utilizes electric pulses to ablate tumor cells, affect the lymphatic and blood microvasculature in the tumor, surrounding fat pad, tumor draining lymph node (TDLN) using multiple analysis methods. This occurred through three main methods 1) identification of oxidative stress effects of chemotherapeutic application of carboplatin on lymphatic endothelial cells in vitro, 2) characterization of lymphatic and blood microvascular dynamics in a 4T1 breast cancer mouse model treated with sub-ablative H-FIRE, 3) through the development of a novel habitat imaging method to identify treatment specific changes in the tumor draining lymph node, and the development of a hybrid agent-based model (ABM) to test cancer cell flow mediated invasion in brain cancer. Herin the work showed that carboplatin induced lymphatic phenotypic changes occurred through generation of reactive oxygen species dependent on VEGFR3 and was reversed through treatment with the antioxidant N-acetylcysteine. In the 4T1 model, sub ablation with H-FIRE induced temporal remodeling of the lymphatic and blood vasculature within the viable tumor, in the surrounding fat pad, and in the tumor draining lymph node over seven days, suggesting an optimal time of application of adjuvant therapy. The development of a habitat imaging analysis method to identify TDLN vascular habitats and the perturbation to treatment in a retrospective analysis of prior work. Lastly, the development of a hybrid ABM through the incorporation of experimentally measured fluid flow fields from dynamic contrast enhanced MRI imaging building upon existing work, and showing the usefulness in comparing mechanisms of cancer cell invasion mediated fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within various treatments contexts and new methods of quantifying changes due to treatment. Hopefully, these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer. / Doctor of Philosophy / The lymphatic and blood vascular systems are two important vessel networks that serve different purposes in healthy states and in the disease called cancer. In breast cancer , a common form of cancer in women , spread of this cancer tends towards the lymphatic vasculature and eventually to other parts of the body. Triple negative breast cancer (TNBC) a less common, but more aggressive form, relies on clinical standard treatments with anti-tumor drugs called chemotherapies. These chemotherapies negatively alter the lymphatic vasculature to the tumors benefit, leaving a lack new methods of treatment. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics and a new promising pulsed electric field therapy , High frequency Irreversible Electroporation (H-FIRE), change the lymphatic and blood vessels over time and in different locations using different tools. This occurred through three main methods 1) the effects on lymphatic vascular cells treated with chemotherapy, 2) in a breast cancer mouse model treated with H-FIRE, 3) in math models of the draining lymphatic organ, called the lymph node and an agent-based math model (ABM) of cancer cell movement due to fluid flow. The work showed that in the lymphatic cells, carboplatin a type of chemotherapeutic used to treat breast cancer, changed lymphatic vasculature through generating stress through oxidation and was reversed through treatment with an anti-oxidant. In the breast cancer mouse model, incomplete ablation with H-FIRE caused time dependent changes to the lymphatic and blood vasculature in the tumor, in the surrounding tissue, and in the lymph node over seven days. This work shows the novel findings of pulsed electric field therapy causing changes to the lymphatic vasculature. The creation of a new method of identifying habitats of the lymph node was used to compare changes to the lymphatic and blood vasculature to treatment. Lastly, the creation of an ABM added measured fluid flow maps from medical imaging methods to build upon existing work, and showed the usefulness in comparing mechanisms of cancer cell invasion due to fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within after various treatments are applied and new methods of measuring these changes because of treatment using multiple methods. It is our hope that these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer.