In der vorliegenden Arbeit wurde eine Beeinflussung der Aktivität der humanen neutrophilen Elastase (EC 3.4.21.37) durch wässrige und Dichlormethan-Extrakte von 15 Basidiomyceten festgestellt. Durch aktivitätsgeleitete Fraktionierung (mehrfache SC, GC-MS) der Dichlormethan-Extrakte von Heterobasidion annosum (Fr.) Bref. und Lactarius deterrimus Grög. wurden Fraktionen freier langkettiger Fettsäuren als ein wirksames Prinzip der Elastase-Hemmung und auch der Kollagenase-Hemmung (Clostridium histolyticum Kollagenase, EC 3.4.24.3) isoliert und identifiziert. Das Screening von 17 freien langkettigen Fettsäuren zeigte, dass einfach ungesättigte Fettsäuren eine stärkere Hemmung der Elastase-Aktivität bewirkten als ihre gesättigten bzw. mehrfach ungesättigten Homologa: Ölsäure (C18:1 cis-9): IC50 5µM; Stearin-(C18:0), Linolsäure (C18:2 cis-9,12): IC50 10µM; alpha- (C18:3 cis-9,12,15), gamma-Linolensäure (C18:3 cis-6,9,12): IC50 15µM. Inhibitorisch am stärksten wirksam war Erucasäur! e (C22:1 cis-13): IC50 450nM. Für Kollagenase wurde hingegen gezeigt, dass die gesättigten Fettsäuren eine erheblich stärkere Hemmaktivität als ihre ungesättigten Homologa aufwiesen. Aktivste Verbindungen waren Palmitin- (C16:0), Heptadecan- (C17:0), Stearin- und Nonadecansäure (C19:0) mit IC50-Werten von 20-45µM. Die Untersuchung von 9 ausgewählten Fettsäuren bezüglich der Hemmung der Aktivität der MMP-9 (EC 3.4.24.35) zeigte als aktivste Verbindungen Palmitolein- (16:1 cis-9), alpha- und gamma-Linolensäure. Die wirksamen Konzentrationen (250µM) lagen jedoch sehr hoch. Zytotoxizitätsuntersuchungen (ECV-304) der Extrakte von H. annosum und L. deterrimus sowie der freien Fettsäuren schlossen sich ebenso wie Untersuchungen zur Proteaseaktivität der Zelllinien ECV-304, MCF-7 und MDA-MB 231 an. Die Proteaseaktivität der Zellen nahm in der Reihenfolge ECV-304 < MCF-7 < MDA-MB 231 zu. Die einzig untersuchte Fettsäure gamma-Linolensäure zeigte keine reproduzierbare Beeinflussung d! er Proteaseaktivität. / In the present paper it was established that the activity of humane neutrophil elastase (EC 3.4.21.37) is affected by aqueous and dichloromethane extracts of 15 basidiomycetes. Bioassay-guided fractionation (repeated CC, GC-MS) of dichloromethane extracts of Heterobasidion annosum (Fr.) Bref. and Lactarius deterrimus Grög. led to isolation and identification of fractions of free fatty acids as one active principle of elastase inhibition as well as collagenase inhibition (Clostridium histolyticum collagenase, EC 3.4.24.3). By testing 17 free fatty acids for elastase inhibition it was shown that the inhibition rate of unsaturated acids was much higher than the rate of the saturated ones: oleic acid (C18:1 cis-9): IC50 5µM; stearic acid (C18:0), linoleic acid (C18:2 cis-9,12): IC50 10µM; linolenic acid (C18:3 cis-9,12,15), gamma-linolenic acid (C18:3 cis-6,9,12): IC50 15µM. The highly active erucic acid with an IC50 value of 450nM is remarkable. As a result for collagenase we can assume that the saturated fatty acids were more potent than the unsaturated ones. Palmitic acid (C16:0), heptadecanoic acid (C17:0), stearic acid, and nonadecanoic acid (C19:0) were the most potent fatty acids with IC50 values of 20-45µM. 9 selected fatty acids were investigated for their ability to inhibit the activity of MMP-9 (EC 3.4.24.35). Palmitoleic acid (16:1 cis-9), linolenic acid, and gamma-linolenic acid were the most potent fatty acids but their inhibiting concentrations were very high (250µM). Investigation of cytotoxicity of the extracts of H. annosum, L. deterrimus, and free fatty acids as well as investigation of protease activity of ECV-304, MCF-7, and MDA-MB 231 cells followed. Protease activity of cells increased in the following manner: ECV-304 < MCF-7 < MDA-MB 231. The only investigated fatty acid gamma-linolenic acid did not influence protease activity reproducibly.

Os procedimentos para uso clínico de células-tronco derivadas de tecido adiposo (CT-TA) exigem grandes quantidades de células, por isso, em geral os protocolos envolvem a expansão e cultura celular in vitro. No entanto, as metodologias utilizadas rotineiramente para o cultivo de CT-TA envolvem a utilização de componentes xenobióticos, como a colagenase e o soro fetal bovino (SFB), que representam riscos potencias de reações imunológicas e transmissão de doenças infecciosas. Sendo assim, pretendeu-se no presente estudo analisar diferentes parâmetros metodológicos para isolamento e expansão de CT-TA, na ausência de componentes xenobióticos. Para tanto, as células-tronco foram isoladas por digestão enzimática ou dissociação mecânica e submetidas à expansão na presença de SFB ou lisado de plaquetas humano (LP). Os resultados mostraram que a metodologia de dissociação mecânica representa uma alternativa viável e eficiente para cultivo de CT-TA, e que o emprego de LP como suplemento para o meio de cultura aumentou de forma significativa a proliferação celular. Em função desses resultados, pode-se concluir que é possível a implementação de técnicas de isolamento e expansão de CT-TA, prescindindo-se de componentes xenobióticos. / The procedures for the clinical use of adipose-derived stem cells (ASC) require large amounts of cells, so in general protocols involve culture and cell expansion in vitro.However, the methods routinely used for the culture of ASC involves the use of xenobiotic components, such as collagenase and fetal bovine serum (FBS), that may representing potential risk of immunological reactions and the risk of transmission of infectious diseases. Thus, it was intended in this study to analyze different methodological parameters for the isolation and expansion of ASC in the absence of xenobiotic components. For this, stem cells were isolated by enzymatic digestion and mechanical dissociation and were submitted to expansion in the presence of FBS or human platelet lysate (PL). The results showed that the mechanical dissociation method represents an effective alternative to growing ASC, and that the use of PL as a supplement to the culture medium significantly increased cellular proliferation. In view of these results, we can conclude that it is possible to implement techniques for isolation and expansion of ASC, dispensing xenobiotic components.

It is known that muscles that lack KATP channel activity generate much greater unstimulated [Ca2+]i and force than normal muscles during fatigue. The increase in unstimulated force in KATP channel deficient muscles is abolished by a partial inhibition of L-type Ca2+ channels, suggesting that it is due to a Ca2+ influx through L-type Ca2+ channels and a subsequent increased myoplasmic Ca2+. However, there is also evidence that the increase in resting force is abolished by NAC, a ROS scavenger. The objective of this study was to reconcile these observations by studying the hypothesis that “the increase in resting [Ca2+]i during fatigue in KATP channel deficient muscles starts with an excess Ca2+ influx through L-type Ca2+ channels, followed by an excess ROS production that causes a further increase in resting [Ca2+]i”. To test the hypothesis, single FDB fibres were fatigued with one tetanic contraction/sec for 180 sec. KATP channel deficient fibres were obtained i) by exposing wild type muscle fibers to glibenclamide, a KATP channel blocker and ii) by using fibres from Kir6.2-/- mice, which are null mice for the Kir6.2 gene that encodes for the protein forming the channel pore. Verapamil, a L-type Ca2+ channel blocker, applied at 1 μM, significantly reduced resting [Ca2+]i during fatigue in glibenclamide-exposed wild type fibres. NAC (1 mM) also reduced resting [Ca2+]i in glibenclamide-exposed muscles. The results suggest that the increase in resting [Ca2+]i during fatigue in KATP channel deficient FDB fibres is due to an influx through L-type Ca2+ channels, and an excess ROS production.

Multipotent mesenchymal stromal cells (MSC) were first discovered in bone marrow and can be isolated from “virtually all organs”. They could participate in tissue maintenance and self-renewing process. They are able to adhere to plastic surfaces and acquire a fibroblastic shape when isolated. They are characterized by a particular phenotype and are able to differentiate into several cell types if cultivated in a specific induction medium. These characteristics were defined on MSC in culture and do not represent how they may be in situ.MSC present particular properties. They can secrete growth factors and several cytokines that give them a trophic activity on one hand and the ability to modulate the immune system on the other hand. They are also able to differentiate. These different properties make them an attractive candidate for cell therapy.MSC are already the focus of several pre-clinical and clinical studies. Nevertheless, the results of these studies are difficult to interpret due to limited understanding of their basic biology. MSC are poorly defined in situ and are heterogeneous. Their heterogeneity is dictated by their tissue of origin and cell preparation. To date, there is no standard protocol for MSC isolation and culture. This leads to numerous questions regarding patient safety, and these questions require answers.The first part of the work deals with the methods used to optimize the extraction of MSC and purification from adipose tissue, one of the main sources of autologous MSC with bone marrow. Classical methods require an enzymatic digestion step. The enzyme used and the duration of adipose tissue digestion time can induce cellular alterations and modify cell functions. Moreover, the addition of a xenobiotic increases the risk of contamination and complicates the monitoring of good manufacturing practices (GMP). We propose a method that does not require this enzymatic digestion step while being easier, safer, faster, gentler and less expensive. Compared to the classical enzymatic method, our method yields an equivalent number of MSC from adipose tissue while preserving their properties.The second part of this work focuses on the characterization of the MSC subpopulations from adipose tissue and compares them to those from bone marrow, which are the historical gold standard. The study made it possible to deepen the knowledge of MSC surface markers in situ from these 2 sources. It also evaluated the various properties of the isolated subpopulations thanks to the cell surface markers CD271, SUSD2, MSCA-1, CD44 and CD34. We showed that MSC from bone marrow express MSCA-1, CD271 and SUSD2 markers in situ. We also found that a population clearly positive for the CD34 does exist in situ with different properties compared to those of the unselected populations or the negative counterpart. 2 populations that are negative and positive for CD44 also exist with similar properties.In contrast to bone marrow MSC, only one selection was able to effectively isolate MSC from adipose tissue by a positive selection based on the expression of CD34. We also isolated a CD271+ population but only from lipoaspirate samples and not from abdominoplasty samples. Collectively, our results suggest that MSCA-1 seems to be the best marker through which to isolate MSC from bone marrow and that CD34 is the only marker able to positively isolate cells from adipose tissue. Thus, we show that the MSC from the different sources share similar properties although they have specific characteristics. The choice of the source and of the marker with which to isolate a particular subpopulation is important depending on their intended clinical use. / Les cellules stromales mésenchymateuses multipotentes (CSM) ont été mises en évidence dans la moelle osseuse et peuvent être isolées de « virtuellement tous les organes ». Elles participeraient à la maintenance et au renouvellement des tissus. Une fois isolées, elles sont capables d’adhérer à des surfaces en plastique en prenant une forme fibroblastique. Elles sont caractérisées par un phénotype particulier et peuvent se différencier en divers types cellulaires lorsque cultivées dans un milieu d’induction spécifique. Ces caractéristiques ont été définies sur les CSM en culture et ne reflètent pas forcément ce qui se passe in situ.Les CSM présentent des propriétés particulières. Elles peuvent sécréter des facteurs de croissance ainsi que de nombreuses cytokines qui leur permettent d’une part d’avoir une activité trophique et d’autre part de moduler le système immunitaire. Elles sont aussi capables de se différencier. Ces différentes propriétés les rendent particulièrement attractives pour la thérapie cellulaire.Les CSM font déjà l’objet de nombreuses études pré-cliniques et cliniques dont les résultats sont difficilement interprétables car nous n’avons à l’heure actuelle qu’une compréhension limitée de leur biologie de base. Les CSM sont encore mal définies in situ et sont hétérogènes. Cette hétérogénéité provient de leur différence d’origine et de leur préparation cellulaire :il n’existe aucune standardisation des protocoles d’isolation et de culture. Cette hétérogénéité entraine de nombreuses questions relatives à la sécurité du patient qui doivent être élucidées.La première partie de ce travail cherche à optimiser les méthodes d’extraction et de purification des CSM du tissu adipeux humain, la principale source de CSM autologues avec la moelle osseuse. Les méthodes classiques requièrent une étape de digestion enzymatique dont l’enzyme utilisée et le temps de digestion du tissu adipeux peuvent induire des altérations cellulaires et modifier leurs fonctions. De plus, l’adjonction de xénobiotiques augmente le risque de contamination et complique le suivi des bonnes pratiques de fabrication (BPF). Nous proposons une méthode qui s’affranchit de cette étape de digestion enzymatique tout en étant plus facile, plus sûre, plus rapide, moins chère et moins traumatisante pour les cellules. Elle permet d’obtenir un nombre tout aussi important de CSM du tissu adipeux que la méthode enzymatique classique en préservant leurs propriétés.La deuxième partie de ce travail vise à caractériser les sous populations de CSM du tissu adipeux humain en les comparant à celles de la moelle osseuse, source de référence historique. Cette étude a permis d’approfondir la connaissance des marqueurs de surface des CSM de ces 2 sources in situ, tout en évaluant les différentes propriétés des sous-populations isolées grâce aux marqueurs de surface CD271, SUSD2, MSCA-1, CD44 et CD34. Nous avons montré que les CSM de la moelle osseuse expriment les marqueurs MSCA-1, CD271 et SUSD2 in situ et qu’il existait une sous-population clairement positive pour le CD34 avec des propriétés différentes de celles de la population non sélectionnée ou négative pour ce marqueur. Il existe aussi 2 sous-populations positive et négative pour le CD44 avec des propriétés similaires.Contrairement aux CSM de la moelle osseuse, une seule sélection a permis d’isoler efficacement les CSM du tissu adipeux par une sélection positive sur base de l’expression du CD34. Nous avons pu aussi isoler une population CD271+ mais seulement des prélèvements de lipoaspirations et non des abdominoplasties.Au vu de nos résultats, MSCA-1 semble le meilleur marqueur pour isoler les CSM de la moelle osseuse tandis que le CD34 est le seul marqueur capable d’isoler positivement celles du tissu adipeux. Ainsi, nous montrons que les CSM issues de différentes sources partagent des propriétés similaires avec cependant des caractéristiques propres. Le choix de la source et du marqueur pour isoler une sous-population sont donc importants en fonction de leur utilité clinique envisagée. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

It is known that muscles that lack KATP channel activity generate much greater unstimulated [Ca2+]i and force than normal muscles during fatigue. The increase in unstimulated force in KATP channel deficient muscles is abolished by a partial inhibition of L-type Ca2+ channels, suggesting that it is due to a Ca2+ influx through L-type Ca2+ channels and a subsequent increased myoplasmic Ca2+. However, there is also evidence that the increase in resting force is abolished by NAC, a ROS scavenger. The objective of this study was to reconcile these observations by studying the hypothesis that “the increase in resting [Ca2+]i during fatigue in KATP channel deficient muscles starts with an excess Ca2+ influx through L-type Ca2+ channels, followed by an excess ROS production that causes a further increase in resting [Ca2+]i”. To test the hypothesis, single FDB fibres were fatigued with one tetanic contraction/sec for 180 sec. KATP channel deficient fibres were obtained i) by exposing wild type muscle fibers to glibenclamide, a KATP channel blocker and ii) by using fibres from Kir6.2-/- mice, which are null mice for the Kir6.2 gene that encodes for the protein forming the channel pore. Verapamil, a L-type Ca2+ channel blocker, applied at 1 μM, significantly reduced resting [Ca2+]i during fatigue in glibenclamide-exposed wild type fibres. NAC (1 mM) also reduced resting [Ca2+]i in glibenclamide-exposed muscles. The results suggest that the increase in resting [Ca2+]i during fatigue in KATP channel deficient FDB fibres is due to an influx through L-type Ca2+ channels, and an excess ROS production.

Os procedimentos para uso clínico de células-tronco derivadas de tecido adiposo (CT-TA) exigem grandes quantidades de células, por isso, em geral os protocolos envolvem a expansão e cultura celular in vitro. No entanto, as metodologias utilizadas rotineiramente para o cultivo de CT-TA envolvem a utilização de componentes xenobióticos, como a colagenase e o soro fetal bovino (SFB), que representam riscos potencias de reações imunológicas e transmissão de doenças infecciosas. Sendo assim, pretendeu-se no presente estudo analisar diferentes parâmetros metodológicos para isolamento e expansão de CT-TA, na ausência de componentes xenobióticos. Para tanto, as células-tronco foram isoladas por digestão enzimática ou dissociação mecânica e submetidas à expansão na presença de SFB ou lisado de plaquetas humano (LP). Os resultados mostraram que a metodologia de dissociação mecânica representa uma alternativa viável e eficiente para cultivo de CT-TA, e que o emprego de LP como suplemento para o meio de cultura aumentou de forma significativa a proliferação celular. Em função desses resultados, pode-se concluir que é possível a implementação de técnicas de isolamento e expansão de CT-TA, prescindindo-se de componentes xenobióticos. / The procedures for the clinical use of adipose-derived stem cells (ASC) require large amounts of cells, so in general protocols involve culture and cell expansion in vitro.However, the methods routinely used for the culture of ASC involves the use of xenobiotic components, such as collagenase and fetal bovine serum (FBS), that may representing potential risk of immunological reactions and the risk of transmission of infectious diseases. Thus, it was intended in this study to analyze different methodological parameters for the isolation and expansion of ASC in the absence of xenobiotic components. For this, stem cells were isolated by enzymatic digestion and mechanical dissociation and were submitted to expansion in the presence of FBS or human platelet lysate (PL). The results showed that the mechanical dissociation method represents an effective alternative to growing ASC, and that the use of PL as a supplement to the culture medium significantly increased cellular proliferation. In view of these results, we can conclude that it is possible to implement techniques for isolation and expansion of ASC, dispensing xenobiotic components.

This diploma thesis is focused on the study of bioactive wound dressings. During the thesis, hydrogel, lyophilized and nanofiber wound dressings were prepared. Hydrogel and lyophilized wound dressings were prepared on basis of two polysaccharides – alginate and chitosan. Nanofiber wound dressings were prepared by spinning polyhydroxybutyrate. All prepared wound dressings were enriched with bioactive substances, which represented analgesics (ibuprofen), antibiotics (ampicillin) and enzymes (collagenase). Into hydrogel and lyophilized wound dressings were all the mentioned active substances incorporated, whereas nanofiber wound dressings were only with ibuprofen and ampicillin prepared. The theoretical part deals with the anatomy and function of human skin. There was explained the process of wound healing and also there were introduced available modern wound dressings. The next chapter of the theoretical part deals with materials for preparing wound dressings (alginate, chitosan, polyhydroxybutyrate) and with active substances, which were used during the experimental part of this thesis. In the theoretical part, the methods of preparation of nanofiber wound dressings and also the methods of cytotoxicity testing used in this work were presented. The first part of the experimental part of this thesis was focused on preparing already mentioned wound dressings. Then, their morphological changes over time and also the gradual release of incorporated active substances into the model environment were monitored. The gradual release of ampicillin was monitored not only spectrophotometrically, but also by ultra-high-performance chromatography. In wound dressings, in which collagenase was incorporated, was also the final proteolytic activity of this enzyme monitored. The effect of the active substances was observed on three selected microorganisms: Escherichia coli, Staphylococcus epidermidis and Candida glabrata. The cytotoxic effect of the active substances on the human keratinocyte cell line was monitored by MTT test and LDH test. A test for monitoring the rate of wound healing – a scratch test – was also performed.

Einleitung: Die oberflächliche Beugesehne (OBS) ist die am häufigsten verletzte Struktur des Bewegungsapparates von Pferden. Verletzungen der OBS verursachen ökonomische Einbußen im Pferdesport, sind unter Tierschutzaspekten hochrelevant und sind gekennzeichnet von langen Rekonvaleszenzzeiten von 9--18 Monaten und einer Wiederverletzungsrate von bis zu 80 %. Um Sehnenverletzungen, deren Heilung und den Therapieerfolg zu detektieren, bedarf es grundlegender Kenntnisse der Sehnenbiomechanik. Zur Anwendung kommende Messmethoden sollten idealerweise hochpräzise, minimalinvasiv und über einen längeren Zeitraum einsetzbar sein. Bislang etablierte Methoden genügen diesen Ansprüchen nur teilweise. Daher soll biplanare Hochfrequenz-Fluoreszenz-Kinematografie (FluoKin) als aktueller Goldstandard für Bewegungsstudien auf ihren Einsatz an equinen Sehnengewebe geprüft werden. Ziele der Untersuchungen waren es daher, 1) die Messgenauigkeit von FluoKin zu bestimmen und hinsichtlich Dehnbarkeitsmessungen equiner OBS zu evaluieren, 2) eine Technik zur Nutzung von FluoKin (3D-Röntgenmethode) zu finden, um die Bewegung eines Weichteilgewebes im Röntgenvideo zu visualisieren, 3) diese Methodik in zyklischen Zugprüfversuchen mit gesunden und Kollagenase-geschädigten OBS ex vivo zu prüfen und dafür eine geeignete Haltevorrichtung für die Sehnen zu entwickeln, 4) in einem Pilotversuch die Technik in vivo zu übertragen und in wiederkehrenden Messserien die Dehnung von gesundem, verletztem und heilendem Sehnengewebe zu messen. Tiere, Material und Methoden: Die Präzisionsmessungen wurden mit einem maßgefertigten Testplättchen und einer gefrorenen distalen Vordergliedmaße (VGlm) jeweils statisch (1976 und 5473 Bilder) und in Bewegung (2816 und 5021 Bilder) durchgeführt. Die Sehnenhaltevorrichtung inkl. Kryo-Klemme wurde neben dem Einsatz in der Ex-vivo-Studie zusätzlich Langzeittests (bis 50 min) und Rupturversuchen bis 10 kN (Maschinenlimit) unterzogen. Im Rahmen der Ex-vivo-Zugprüfversuche wurden 13 OBS von VGlm in Schritt (2 %)- und Trab (4 %)-simulierender Dehnung zyklisch getestet. Vier weitere Proben wurden mit Kollagenase inkubiert und inkl. einer Kontrollgruppe (n=4) bei 6 % getestet. Die biomechanischen Kenngrößen wurden von der Zugprüfmaschine erfasst und anhand der Bewegung von implantierten, röntgendichten Markern in zeitgleich aufgenommenen FluoKin-Videos errechnet. In der In-vivo-Langzeitstudie (37 Wochen) wurde in vier FluoKin-Messungen das Dehnverhalten der OBS beider VGlm eines Shetland Ponys in Schritt und Trab ermittelt. Vor der zweiten FluoKin-Messung wurde im mittleren metakarpalen Segment einer OBS eine Sehnenläsion mit Kollagenase induziert, deren Heilungsverlauf verfolgt wurde. Die Ergebnisbeschreibung erfolgte sowohl deskriptiv als auch bei entsprechender Stichprobengröße mit t-Tests (p<0,05). Ergebnisse: In Präzisionsmessungen wurden sowohl die Messgenauigkeit der FluoKin-Anlage (max. Exaktheit von 0,0287 mm ± 0,0377 mm), als auch die zu erwartende Standardabweichung in der studienrelevanten Region (Os metacarpale III, OBS) ermittelt. Im Rahmen der Ex-vivo-Zugprüfversuche wurde eine Haltevorrichtung inkl. Kryo-Klemme für equine OBS entwickelt, welche Probleme herkömmlicher Einspanntechniken zuverlässig löst. Erstmals konnten biomechanische Kenngrößen für die OBS von Shetland Ponys ermittelt werden. Im Mittel konnten abhängig von der Dehnungsrate (simulierter Schritt und Trab) eine Maximalkraft von 325 bzw. 953 N, eine Zugfestigkeit von 1649 bzw. 4820 N/cm² und ein Elastizitätsmodul von 828 bzw. 1212 MPa verzeichnet werden. Nach Inkubation mit Kollagenase stieg die Längenänderung im Mittel um 4,87 %. In vivo konnte die Dehnungszunahme der geschädigten OBS im Schritt bestätigt werden (2,86 % auf 3,38 %); im Trab zeigte sich hingegen ein Abfall (6,78 % auf 5,96 %). Schlussfolgerungen: FluoKin wurde als hochpräzise und minimalinvasive Messtechnik zur Ermittlung der Sehnendehnung equiner OBS ex vivo und erstmals in vivo erfolgreich eingesetzt. Dank der neu entwickelten Haltevorrichtung inkl. Kryo-Klemme konnten langandauernde Ex-vivo-Zugprüfversuche durchgeführt werden. Hierbei zeigten sich Änderungen des Dehnverhaltens der OBS (Konditionierung, Hysterese, Kriechphänomen), was die Notwendigkeit solcher Versuche für die Beschreibung des biomechanischen Verhaltens von Sehnen verdeutlicht. Der In-vivo-Pilotversuch unterstreicht die Bedeutung von FluoKin für innovatives und hochpräzises Monitoring von Sehnenläsionen in Langzeitstudien.:1 Einleitung 2 Literaturübersicht 2.1 Sehnengewebe 2.1.1 Histologischer und molekularer Aufbau 2.1.2 Biomechanik 2.1.3 Einflüsse auf die Sehnenstruktur und -zusammensetzung 2.2 Tendinopathien der oberflächlichen Beugesehne 2.2.1 Ätiologie 2.2.2 Heilung 2.3 Möglichkeiten zur Bestimmung der Sehnendehnbarkeit 2.3.1 Ex-vivo-Zugprüfversuche mit Sehnen 2.3.2 In-vivo-Ermittlung der Sehnendehnbarkeit 2.4 Zentrale Fragestellungen 3 Publikation 1 Zyklische Zugprüfversuche mit einer neuartigen Kryo-Klemme an oberflächlichen Beugesehnen von Shetland Ponys kombiniert mit biplanarer Hochfrequenz-Fluoreszenz-Kinematografie 4 Publikation 2 Biplanare Hochfrequenz-Fluoreszenz-Kinematografie an der oberflächlichen Beugesehne eines Shetland Ponys – eine In-vivo-Pilotstudie 5 Diskussion 5.1 Diskussion von Material und Methoden 5.1.1 Ermittlung der Messgenauigkeit von FluoKin 5.1.2 Verwendetes Tiermaterial 5.1.3 Implantationstechnik für Sehnenmarker 5.1.4 Aufbau der Ex-vivo-Zugprüfversuche 5.2 Bestimmung der Sehnendehnbarkeit mit FluoKin 5.2.1 Sehnendehnung ex vivo und in vivo in nativem Sehnengewebe 5.2.2 Sehnendehnung ex vivo und in vivo in Kollagenase-geschädigtem Sehnengewebe 5.3 Schlussfolgerungen und Ausblick 5.3.1 Praktische und klinische Relevanz 5.3.2 Fazit und Perspektiven 6 Zusammenfassung 7 Summary 8 Literaturverzeichnis 9 Anhang 9.1 Bauplan des Messplättchens zur Genauigkeitsbestimmung von FluoKin 9.2 Baupläne der Einspanntechnik für Zugprüfversuche mit equinen OBS 9.2.1 Kronbeinhalterung 9.2.2 Kryo-Klemmen 9.3 Genauigkeit von biplanaren Fluoroskopie-Systemen in Abhängigkeit des Versuchsaufbaus 9.4 Lagerungseinflüsse auf biomechanische Eigenschaften von Sehnengewebe 9.5 Vorträge und Präsentationen während der Doktorarbeitszeit / Introduction: The superficial digital flexor tendon (SDFT) is the most frequently injured structure of the musculoskeletal system of horses. Injuries of the SDFT are relevant under animal welfare aspects, cause substantial economic losses in equestrian sport and are associated by long convalescence times of 9 to 18 months and a re-injury rate of up to 80 %. In order to detect tendon injuries, improve their healing and the success of therapy, fundamental knowledge of tendon biomechanics is required. The measurement methods to be used should ideally be highly precise, minimally invasive and applicable over a long period of time. So far, established methods only partially meet these requirements. Therefore, biplanar high-speed fluoroscopic kinematography (FluoKin) as the current gold standard for movement studies will be tested for its use on equine tendon tissue Aims of the study were therefore 1) to determine the measurement accuracy and precision of FluoKin and to evaluate it with regard to measurements on the equine SDFT, 2) to find a technique for using FluoKin (a 3D X-ray method) for visualizing the movement of a soft tissue in X-ray video, 3) to test this methodology in cyclic tensile tests with healthy and collagenase-incubated SDFT ex vivo and to develop a suitable holding device for the tendons in the testing machine, 4) to transfer the technique in an in vivo pilot study on a pony and to measure the elongation of healthy, injured and healing tendon tissue in measurement series. Materials and Methods: Precision measurements were performed with a custom-made test sheet and a frozen distal forelimb static (1976 and 5473 frames) and in motion (2816 and 5021 frames) resp. The tendon holding device with a cryo-clamp was subjected to long-term cyclic tests (up to 50 min) and rupture tests up to 10 kN (machine limit) in addition to its use in the ex vivo study. As part of the ex vivo tensile testing, 13 SDFT of forelimbs were cyclically tested in walk (2 % strain) and trot (4 % strain) simulated elongation. Four additional specimens were incubated with collagenase and tested including a control group (n=4) at 6 % strain. Biomechanical parameters were recorded by the testing machine and calculated from the movement of implanted radiopaque markers in simultaneously recorded FluoKin videos. In the in vivo long-term study (37 weeks) the strain behaviour of the SDFT of both forelimbs of a Shetland pony at walk and trot were determined in four FluoKin measurements. Before the second FluoKin measurement, a tendon lesion was induced with collagenase in the mid-metacarpal segment of one SDFT, and its healing process was monitored. The description of the results was done both descriptively and, with an appropriate sample size, with t-tests (p<0.05). Results: Measurements to assess the precision of both the FluoKin system (max. accuracy of 0.0287 mm ± 0.0377 mm) and the standard deviation to be expected in the region relevant to the study (Os metacarpale III, SDFT). For the ex vivo tensile tests, a holding device with a cryo-clamp for equine SDFT was developed that reliably solved problems of conventional clamping techniques. For the first time, biomechanical parameters for the SDFT of Shetland ponies could be determined. On average, depending on the strain rate (simulated walk and trot), a maximum force of 325 N and 953 N resp., a tensile strength of 1649 N/cm² and 4820 N/cm² resp. and a modulus of elasticity of 828 MPa and 1212 MPa resp. was recorded. After incubation with collagenase, the change in length increased by an average of 4.87 %. In vivo, this increased elongation of the collagenase-injured SDFT could be confirmed at walk (2.86 % to 3.38 %); in contrast, a decrease in strain was observed at trot (6.78 % to 5.96 %). Conclusions: FluoKin was successfully used as a highly precise and minimally invasive measurement technique to determine the tendon strain of equine SDFT ex vivo and for the first time also in vivo. Thanks to the newly developed holding device with a cryo-clamp, long-term ex vivo tensile tests could be carried out and changes in the strain behaviour of the SDFT became apparent (conditioning, hysteresis, creep). This shows the necessity of such tests for the description of the biomechanical behaviour of tendons. The in vivo pilot study underlines the importance of FluoKin for innovative and highly accurate monitoring of tendon lesions in long-term studies.:1 Einleitung 2 Literaturübersicht 2.1 Sehnengewebe 2.1.1 Histologischer und molekularer Aufbau 2.1.2 Biomechanik 2.1.3 Einflüsse auf die Sehnenstruktur und -zusammensetzung 2.2 Tendinopathien der oberflächlichen Beugesehne 2.2.1 Ätiologie 2.2.2 Heilung 2.3 Möglichkeiten zur Bestimmung der Sehnendehnbarkeit 2.3.1 Ex-vivo-Zugprüfversuche mit Sehnen 2.3.2 In-vivo-Ermittlung der Sehnendehnbarkeit 2.4 Zentrale Fragestellungen 3 Publikation 1 Zyklische Zugprüfversuche mit einer neuartigen Kryo-Klemme an oberflächlichen Beugesehnen von Shetland Ponys kombiniert mit biplanarer Hochfrequenz-Fluoreszenz-Kinematografie 4 Publikation 2 Biplanare Hochfrequenz-Fluoreszenz-Kinematografie an der oberflächlichen Beugesehne eines Shetland Ponys – eine In-vivo-Pilotstudie 5 Diskussion 5.1 Diskussion von Material und Methoden 5.1.1 Ermittlung der Messgenauigkeit von FluoKin 5.1.2 Verwendetes Tiermaterial 5.1.3 Implantationstechnik für Sehnenmarker 5.1.4 Aufbau der Ex-vivo-Zugprüfversuche 5.2 Bestimmung der Sehnendehnbarkeit mit FluoKin 5.2.1 Sehnendehnung ex vivo und in vivo in nativem Sehnengewebe 5.2.2 Sehnendehnung ex vivo und in vivo in Kollagenase-geschädigtem Sehnengewebe 5.3 Schlussfolgerungen und Ausblick 5.3.1 Praktische und klinische Relevanz 5.3.2 Fazit und Perspektiven 6 Zusammenfassung 7 Summary 8 Literaturverzeichnis 9 Anhang 9.1 Bauplan des Messplättchens zur Genauigkeitsbestimmung von FluoKin 9.2 Baupläne der Einspanntechnik für Zugprüfversuche mit equinen OBS 9.2.1 Kronbeinhalterung 9.2.2 Kryo-Klemmen 9.3 Genauigkeit von biplanaren Fluoroskopie-Systemen in Abhängigkeit des Versuchsaufbaus 9.4 Lagerungseinflüsse auf biomechanische Eigenschaften von Sehnengewebe 9.5 Vorträge und Präsentationen während der Doktorarbeitszeit

Islet transplantation has found its niche in diabetes treatment. It has contributed to a better quality of life and better glycemic control of patients with diabetes suffering from severe hypoglycemia that are not eligible for vascularized pancreas transplantation. Islet isolation is a technically challenging procedure. The different studies within this doctoral thesis aim to improve and standardize different steps in the isolation procedure. They are in particular looking to improve human pancreas preservation during cold storage, to optimize islet release from the exocrine tissue and to assess whether the isolated islet yield can be predicted from a biopsy. We found that pancreas preservation with pre-oxygenated perfluorodecalin (two-layer method) did not improve the ischemic tolerance of the human pancreas as compared to cold storage with the University of Wisconsin (UW) solution. Furthermore, in pancreas with long cold ischemia time (CIT) (&gt;10 hours), Histidine-Tryptophan-Ketoglutarate (HTK) had a limited preservation capacity as compared with the UW solution with respect to isolation outcome. We also found that during enzymatic pancreas digestion, Vitacyte HA was able to provide a similar islet yield and quality as Serva NB1 with less collagenase activity and shorter digestion time. We further describe the first experience with a new GMP manufactured enzyme called Liberase MTF-S for successful human islet isolation. Finally, we found that the isolated islet yield could not be predicted from a biopsy taken from the head of the pancreas concerning solely morphological parameters of the islets tissue. The improvement of pancreas preservation will allow for marginal organs with prolonged cold ischemia time to expand the donor pool. Better knowledge of how the pancreatic extracellular matrix is digested by collagenase will lead to a fast and predictable islet release from the exocrine tissue. By standardizing the isolation procedure and improving organ selection we will increase the success rate in human islet isolation, thereby making islet transplantation available for more patients.

L'étude phytochimique d'Armeria maritima et d'Helichrysum stoechas a permis d'isoler pour la première fois 31 molécules dans le genre Armeria dont 4 nouveaux flavonols diglycosylés, ainsi que le développement d'une stratégie de déréplication pour l'étude d'H. stoechas. Dans les deux espèces, nous avons relevé une richesse en polyphénols, qui pourraient être extraits par des techniques respectueuses de l'environnement comme la SFE. En parallèle, ces deux halophytes ont montré un fort potentiel biologique avec des extraits et des molécules dotés d'activités anti-oxydante, anti-collagénase, anti-inflammatoire ou encore cicatrisante. De plus, nous avons initié pour la première fois des suspensions cellulaires d'A. maritima et identifié des éliciteurs comme le méthyl jasmonate permettant d’augmenter dans les cellules d'H. stoechas la teneur en acide 3,5-dicaféoylquinique, un bio-marqueur de l'activité anti-inflammatoire. La production de molécules bioactives dans des cultures végétales in vitro pourrait par la suite être transposée à plus grande échelle, afin d’amplifier le potentiel de valorisation de ces deux halophytes en dermo-cosmétique. / The phytochemical study of Armeria maritima and Helichrysum stoechas led to the isolation of 31 molecules never reported before in the genus Armeria, 4 of which being new flavonol diglycosides, and to the development of a dereplication strategy for the study of H. stoechas. In both species, an abundance of polyphenols was observed, which could be extracted with eco-friendly methods like SFE. Both halophytes showed a strong biological potential as their extracts and molecules demonstrated antioxidant, anti-collagenase, anti-inflammatory and wound healing activities. Moreover, we initiated for the first time cell suspensions of A. maritima, and identified elicitors, such as methyl-jasmonate, which led to H. stoechas cell suspensions with an increased content in 3,5-dicaffeoylquinic acid, a bio-marker of anti-inflammatory activity. The production of bioactive molecules in "plant cell factories" could be scaled-up to enhance the valorization potential of both halophytes in dermocosmetics.