In this thesis, a machine learning approach was used to develop a predictive model for residual methanol concentration in industrial formalin produced at the Akzo Nobel factory in Kristinehamn, Sweden. The MATLABTM computational environment supplemented with the Statistics and Machine LearningTM toolbox from the MathWorks were used to test various machine learning algorithms on the formalin production data from Akzo Nobel. As a result, the Gaussian Process Regression algorithm was found to provide the best results and was used to create the predictive model. The model was compiled to a stand-alone application with a graphical user interface using the MATLAB CompilerTM.

Pain is often associated with depression of behavior and mood, and relief of pain-related depression is a common goal of treatment. This goal of this dissertation was to conduct preclinical research experiments designed to address a set of three inter-related aims that examine the expression, mechanisms and treatment of pain-related depression of Intracranial Self-Stimulation (ICSS) in rats. First, studies evaluated the hypothesis that acute acid-induced depression of ICSS was mediated by a kappa opioid receptor mediated decrease in mesolimbic dopamine release in the nucleus accumbens. Results support a role for depressed mesolimbic dopamine release in pain-related depression of ICSS; however, a role for kappa opioid receptors is not supported. Second, studies evaluated the effectiveness of a more sustained inflammatory noxious stimulus (intraplantar CFA) and a sustained neuropathic stimulus (intraplantar formalin) to produce a long-term pain-related depression of ICSS, and the role of kappa opioid receptors in mediating this sustained pain-related depression of ICSS. Results indicated that only the neuropathic stimulus (formalin) was sufficient to produce sustained depression of ICSS, and as in the initial studies, our data did not support a role for kappa receptors in mediating this effect. Given the poor effectiveness of a kappa receptor antagonist to block acute or chronic pain-related depression of ICSS, the final set of studies evaluated the pharmacology of representative drugs from five different classes of established or candidate analgesics (mu opioid agonists, non-steroidal anti-inflammatory drugs, monoamine uptake inhibitors, anticonvulsants, and cannabinoid agonists) to reverse the sustained depression of ICSS produced by formalin as a neuropathic stimulus. Results demonstrate the mu agonist morphine, the monoamine uptake inhibitor bupropion, the anticonvulsant gabapentin, and the cannabinoid agonist THC were able to reverse formalin-induced mechanical allodynia as a pain-stimulated behavior, but only the mu agonist morphine and the monoamine uptake inhibitor bupropion were effective to reverse formalin-induced depression of ICSS. These results provided additional evidence for dissociable drug effects in preclinical assays of pain-stimulated and pain-depressed behavior and also support further studies with monoamine uptake inhibitors with a dopaminergic component (like bupropion) for treatment of neuropathic pain.

Les C-low threshold mechanoreceptors ou C-LTMRs forment une sous-classe bien particulière de fibres C non-nociceptives innervant exclusivement la peau poilue. En temps normal, leur rôle est de transduire la caresse et détecter le refroidissement. Mais de nouvelles données montrent que les C-LTMRs jouent aussi un rôle important dans la modulation de la douleur, aussi bien inflammatoire que neuropathique. Cependant, les mécanismes moléculaires et cellulaires qui sous-tendent cette dualité sont encore méconnus.Notre équipe a récemment publié des données, issues d'une expérience de séquençage ARN à haut débit, qui précisent l'identité moléculaire des C-LTMRs. A ajouter au trois marqueurs moléculaires déjà connus Tafa4, Tyrosine Hydroxylase et Vglut3, nous avons découvert neuf autres gènes dont l'expression est fortement enrichie dans ces neurones. L'un d'eux est le facteur de transcription bhlha9. Le but de ma thèse étant d'améliorer notre connaissance des C-LTMRs, j'ai notamment étudié le rôle de ce gène dans la somatosensation et la physiologie des C-LTMRs en effectuant un grand nombre de tests comportementaux sur des souris chez qui bhlha9 a été inactivé. De façon intéressante, l'inactivation de bhlha9 entraine un large défaut d'adaptation aux variations de température. De plus, ces souris mutantes présentent une réponse exacerbée à la douleur inflammatoire induite par la formaline. Enfin, cette réponse exacerbée est insensible à l'effet normalement analgésique d'un modulateur positif du système GABAergique ionotropique.En conclusion, bhlha9 est un nouveau marqueur des C-LTMRs nécessaire pour une perception normale de la température et de la douleur inflammatoire. / C-Low Threshold MechanoReceptors (C-LTMRs) are a unique subset of non-nociceptive C-fibers that innervate exclusively hairy skin. At steady-state, these fibers convey low intensity mechanical stimuli (caress and gentle touch) and detect cooling temperatures.Recent data showed C-LTMRs have an important modulatory role in pain processing following tissue injury, both of inflammatory and neuropathic origin. Yet, their molecular and cellular modes of action are for the most part unknown.To provide a better understanding of these C-LTMRs, our team recently published RNA-seq based data that considerably broadens their molecular repertoire. Besides Tyrosine Hydroxylase, V-GluT3 and Tafa4, already known as restricted to C-LTMRs in DRG, we found nine other genes highly enriched in this subset. One of them encodes for the basic Helix-Loop-Helix (bHLH) family transcription factor bHLHa9.To understand the role of bHLHa9 in somatic sensory biology and in C-LTMRs in particular, I have performed a large panel of behavioural tests on mice lacking its expression, from mechanical to thermal stimulation under acute conditions. Interestingly, bHLHa9 knock-out (KO) mice exhibit a broad defect in temperature sensation. Furthermore, these mice present an enhanced inflammatory pain following hindpaw injection of formalin, along with an impaired analgesic response to GABAA receptor positive modulation.In conclusion, bHLHa9 is highly enriched in C-LTMRs and it is required for proper temperature and inflammatory pain perception.

Cyclin-dependent kinase-5 (Cdk5) was identified as a serine/threonine kinase that plays an important role in neuronal development. Association with one of the neuronal activators, p35 or p39, is required for Cdk5 to elicit its diverse effects in the nervous system, such as neurite outgrowth. In addition to these, increasing evidence suggests that Cdk5 also plays an important role in cocaine addiction, neurotransmitter release, NMDA receptor phosphorylation. This thesis is divided into three parts which deals with the effects of Cdk5 inhibitor¡Ðroscovitine on the morphine tolerance development, acute inflammatory pain, and pilocarpine-induced seizure respectively. The first part explored the effect of Cdk5 inhibitior¡Ðroscovitine on the morphine antinociceptive tolerance development. Delta FosB activation is involved in morphine tolerance. Cyclin-dependent kinase- 5 (Cdk5) is found to be the downstream target of delta FosB. We examined the effects of the potent selective Cdk5 inhibitor¡Ðroscovitine on the development of antinociceptive tolerance of morphine. Tolerance was induced by continuous infusion of morphine 5 µg/hr intrathecally (i.t.) for 5 days. The effect of co-administration of roscovitine 1 µg/hr i.t. for 5 days was also examined. Roscovitine co-administration enhanced the antinociceptive effect of morphine in morphine tolerant rats. It also shift the morphine antinociceptive dose¡Ðresponse curve to the left during morphine tolerance induction, and reduced the increase in the ED50 of morphine two-fold. Collectively, these findings suggest Cdk5 modulation may be involved in the development of morphine tolerance and its inhibitor will enhance antinociceptive effect. The second part discussed the roscovitine effect on acute inflammatory pain. Formalin injected into the rat hind paw will evoke flinching (consisting of an elevation and shrinking back of the injected paw), a reliable parameter of pain behavior. The nociceptive response to formalin occurs in a biphasic pattern: there isan initial acute period (phase 1), and after a short period of remission, phase 2 begins and consists of a longer period (1 hour) of sustained activity. The initial response was initially attributed to a direct algogenic effect of formalin, whereas phase 2 was associated with the central sensitization. In this study, the Cdk5 inhibitor¡Ðroscovitine was injected intrathecally to elucidate the mechanism of Cdk5 activation during formalin-induced hyperalgesia. The 50 ul of 5% formalin solution was used as the noxious stimulant. The rats were injected with 0, 50, 100, and 200ug roscovitine intrathecally thirty minutes before hind paw formalin injection. Intrathecal 200ug roscovitine injection attenuates the phase I flinch response. And intrathecal 50, 100, and 200ug roscovitine injection suppress phase II flinch response effectively. Roscovitine administration could effectively suppress the formalin-induced flinch behavior. This implies the activation of Cdk5 plays an important role in the sensitization after nociceptive stimulation. The third part focus on the roscovitine effect on the pilocarpine induced seizure. Pilocarpine temporal lobe epilepsy model is widely used. Chronic electroconvulsive therapy could upregulate Cdk5 activity. Cdk5 inhibitor¡Ðroscovitine could suppress NMDA induced long-term potentiation in hippocampal slice. Intracerebroventricular injection of 100£gg roscovitine 30 min before pilocarpine-induced epilepsy could significantly decrease the seizure-induced mortality ( 11% in roscovitine group VS 77% in control group). The escape latency, spatial memory impairment, in the pilocarpine-induced seizure group is significant longer than the roscovitine pretreatment group in the Morris water maze test after one month (p¡Õ0.05). It is concluded Cdk5 may play an important in the pathogenesis of epilepsy. Therefore, Cdk5 inhibition may become another way for the epilepsy treatment.

Formalin är ett ämne som finns naturligt både i naturen och i människokroppen. Formalin syntetiseras i dagsläget industriellt bland annat på grund utav sina goda egenskaper för desinficering. Det används till exempel i autoklaver på sjukhus för sterilisering av verktyg. Formalin är ett ämne som klassas som cancerogent och som i större mängder är giftigt. Därmed är det viktigt att observera och mäta upp de mängder formalin som blir till utsläpp i naturen, eller som återfinns på sjukhusverktyg. Företaget Ak Lab AB, som är ett ackrediterat laboratorium, genomför idag analyser av filter som funnits i formalinautoklaver för att kvantifiera dessa värden. Den nuvarande metoden för detta är manuell och använder sig av en spektrofotometer. Det önskas att automatisera delar av processen, samt att minska ner på beredningstiden för proverna. I detta examensarbete har en litteraturstudie med avseende på formalin, HPLC och lämplig metod utförts. Därefter har en metod modifierats och sedan har valideringsarbetet påbörjats. Denna metod är baserad på den amerikanska metoden EPA-8315a. Prover har analyserats medd en nuvarande spektrofotometermetoden men även med den nya framtagna HPLC-metoden för att jämföra metoderna, samt för att fastställa om korrekta värden uppmäts. Framkommande av arbetet är att en metod för att påvisa formalin har utvecklats. Det accepterade koncentrationsintervallet för formalinanalys är fastställt av arbetet att vara från 5mg/l till 150 mg/l. I jämförelse med nuvarande spektrofotometermetoden skilde sig kvantifigeringsmängden av formalin. En valideringsprocess för metoden har påbörjats och en arbetsbeskrivning har framtagits. Metoden kan påvisa förekomsten av formalin, men ytterligare beräkningsarbete och optimering behöver göras innan metoden kan implementeras. Förslag för förbättring samt diskussion kring arbetets svagheter respektive styrkor har genomförts. / Formalin is a substance that is found naturally both in nature and in the human body. Formalin is currently synthesized industrially due to its good disinfectant properties, among other things. It is used, for example, in autoclaves in hospitals for sterilizing tools. Formalin is a substance that is classified as carcinogenic and in large quantities is toxic. Thus, it is important to observe and measure the amounts of formalin that are released into nature, or that are found on hospital tools. The company Ak Lab AB, which is an accredited laboratory, is currently carrying out analyzes of filters that have been in formalin autoclaves to quantify these values. The current method  for this is manual and uses a spectrophotometer. The desire is to automate parts of the process, as well as to reduce the preparation time for the samples. In this thesis, a literature study with regards to formalin, HPLC and a suitable method has been performed. Subsequently, a method has been modified and then the validation work has begun. This method is based on the American method EPA-8315a. Samples have been analyzed with the current spectrofotometer method but also with the newly developed HPLC method to compare the methods, and to determine whether correct values are measured. The conclusion of the work is that a method for demonstrating the presence of formalin has been developed. The accepted concentration range for formalin analysis is determined by the work to be from 5 mg/l to 150 mg/l. In comparison to the current spectrophotometer method, the quantification amount of formalin differed. A validation process for the method has begun and practical instructions have been developed. The method can detect the presence of formalin, but further calculation work and optimization need to be done before the method can be implemented. Suggestions for improvement and discussion about the work’s weaknesses and strengths have been implemented.

No description available.

Subklinische entzündliche Veränderungen des Endometriums können nur durch die histopathologische Untersuchung eines Bioptates diagnostiziert werden und sind von besonderer Bedeutung bei Fertilitätsstörungen. Die nicht-eitrige (NE) Endometritis ist gekennzeichnet durch eine Infiltration des Endometriums mit Lymphozyten, Plasmazellen und/oder Makrophagen. Die genaue Pathogenese ist unklar. Die immunhistologische Phänotypisierung dieser Zell-(sub-)populationen kann möglicherweise pathogenetische Hinweise geben. Der immunhistologische Nachweis equiner Immunzellen ist jedoch fast ausschließlich an Gefrierschnitten etabliert. Dies ist für die Routinediagnostik ungeeignet. Demzufolge ergaben sich die folgenden Ziele: 1. Eine alternative Methode zur Gewebsfixierung zu etablieren, die in der Routinediagnostik praktikabel einsetzbar ist, die einen guten Strukturerhalt, eine geeignete Anfärbbarkeit und ideale Auswertbarkeit gewährleistet sowie umfangreiche immunhistochemische Untersuchungen ermöglicht; 2. Die immunhistochemische Phänotypisierung von Lymphozyten- und Makrophagen-(sub-)populationen an fixierten equinen Gewebeproben zu entwickeln; 3. Eine semiquantitative Bestimmung der endometrialen Entzündungszellen ohne und mit einer NE Endometritis durchzuführen. Tiere, Material & Methoden: Gewebeproben (Lymphknoten als Referenzgewebe und endometriales Gewebe) wurden von 5 Stuten im Rahmen der Sektion entnommen, in Formalin (F), HOPE® (H) oder IHC Zinc Fixative (Z) fixiert und zusätzlich als natives Gefriermaterial aufgearbeitet. Es erfolgte eine vergleichende Beurteilung der Gewebemorphologie, des Färbeverhaltens und der Artefakte am equinen Endometrium in HE gefärbten Gefrierschnitten bzw. im fixierten, Paraffin-eingebetteten Material (F, H und Z). Equine Lymphknoten dienten als Referenzgewebe für die immunhistochemische Etablierung der Lymphozytenmarker (anti-CD3, -CD4, -CD8) an Gefrierschnitten und am fixierten, Paraffin-eingebetteten Material (F, H und Z). F- und Z-fixiertes Referenzgewebe (Leber, Dünndarm, Darmlymphknoten) von 4 Stuten wurde für die Etablierung der Makrophagenmarker (anti-CD172a, -CD14, -CD206) verwendet. Die Immunreaktionen der Primärantikörper wurden verglichen. Als Resultat aus den vergleichenden Analysen wurde die Z-Fixierung als effektivste alternative Fixierungsmethode ausgewählt. Im abschließenden Teil der Untersuchungen wurden 28 Z-fixierte Endometriumbioptate hinsichtlich der Entzündungszellen semiquantitativ ausgewertet. 4 Stuten zeigten keine Entzündungsreaktion und dienten als Kontrolle. 24 Stuten wiesen eine oberflächliche NE Endometritis auf. Die Anzahl CD3+, CD4+, CD8+, CD20+, CD172a+, CD14+ und CD206+ Zellen sowie die Anzahl von Plasmazellen mittels Methylgrün-Pyronin-Färbung wurde an Serienschnitten in 5 zufällig ausgewählten Gesichtsfeldern (400x) jeweils für das Stratum compactum, Stratum spongiosum und das luminale sowie glanduläre Epithel erhoben. Ergebnisse: 1. Die Z-Fixierung zeigt mit der F-Fixierung vergleichbare, exzellente Eigenschaften hinsichtlich des Strukturerhalts, der Anfärbbarkeit und Auswertbarkeit. Sie übersteigt insgesamt die Eigenschaften der H-Fixierung: die Z-Fixierung erweist sich einfacher in der Anwendung und zeigt eine geringere Artefaktbildung. 2. Die immunhistochemische Charakterisierung von T- und B-Lymphozyten, Helfer-T-Lymphozyten sowie zytotoxischen T-Lymphozyten und Makrophagenpopulationen sowie der Nachweis von Plasmazellen mittels Methylgrün-Pyronin-Färbung an Z-fixierten equinen Gewebeproben ist möglich. 3. Im entzündlich veränderten Endometrium sind mehr CD3+, CD4+, CD8+, CD20+, CD172a+, CD206+ Zellen sowie Plasmazellen nachweisbar als in unveränderten Gewebeproben bezogen auf das Stratum compactum und Stratum spongiosum. Die durchschnittliche Anzahl CD3+ Zellen ist im Stratum compactum NE Endometritiden knapp 3x höher als im unveränderten Endometrium. Wenige CD20+ B-Zellen und CD172a+ Makrophagen sowie eine unterschiedliche Anzahl an Plasmazellen sind innerhalb des Stratum compactum entzündlich veränderter Endometrien nachweisbar. Es existieren große individuelle Unterschiede in der Anzahl CD4+ und CD8+ T-Zellen in Endometrien ohne sowie mit einer Entzündung. Schlussfolgerungen: Eine Integration der Z-Fixierung in die Routinediagnostik mit zusätzlichen Anwendungsmöglichkeiten ist problemlos möglich. Die immunhistochemische Entzündungszelldifferenzierung im Endometrium der Stute kann an fixierten equinen Endometriumbioptaten durchgeführt werden und bietet darüber hinaus die Möglichkeit, diese Methode an anderen equinen Organen anzuwenden. Die Ergebnisse der semiquantitiven Auswertung deuten auf das mögliche Vorliegen unterschiedlicher Formen der nicht-eitrigen Entzündung hin. Das etablierte Verfahren gibt möglicherweise nähere Hinweise auf immunologische Konstellationen, die das Auftreten einer nicht-eitrigen Endometritis begünstigen.:INHALTSVERZEICHNIS I ABKÜRZUNGSVERZEICHNIS II 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Das Endometrium der Stute 2 2.1.1 Endometritiden 4 2.1.1.1 Eitrige Endometritis 4 2.1.1.2 Nicht-eitrige Endometritis 5 2.1.1.3 Sonderformen 6 2.1.2 Resistant und susceptible mares 8 2.1.3 Entzündungszelltypisierung im Endometrium der Stute 10 2.1.4 Diagnostische Bedeutung des Endometriumbioptates 13 2.2 Immunologie 14 2.2.1 T-Lymphozyten 14 2.2.2 B-Lymphozyten 17 2.2.3 Makrophagen 18 2.2.4 Immunhistochemische Charakterisierung von equinen Entzündungszellen 21 2.3 Fixierungsmethoden 24 2.4 Fazit aus der Literatur bezüglich der Fragestellung dieser Arbeit 26 3 PUBLIKATIONEN 28 3.1 Publikation 1 inkl. Stellungnahme zum Eigenanteil 28 3.2 Publikation 2 inkl. Stellungnahme zum Eigenanteil 39 4 DISKUSSION 53 4.1 Etablierung alternativer Methoden zur Gewebefixierung 54 4.2 Immunhistochemische Detektion von Immunzell- (sub-)populationen 59 4.3 Erkenntnisse im Hinblick auf die Pathogenese nicht-eitriger Endometritiden 60 5 ZUSAMMENFASSUNG 63 6 SUMMARY 65 7 LITERATURVERZEICHNIS 67 8 DANKSAGUNG 81 / Clinically unapparent inflammatory alterations of the equine endometrium can only be diagnosed by the histopathological examination of a biopsy and are the main cause of subfertility in mares. The non-suppurative endometritis is characterised by infiltration of the endometrium with lymphocytes, plasma cells and/or macrophages. So far, the cause and pathogenesis of non-suppurative endometritis are unclear. The subclassification of the immune cell populations involved will likely provide important information on the etiology and pathogenesis of this disease. Available antibodies are often established exclusively for immunohistochemistry on cryostat sections of native frozen equine tissue. However, this method is impractical for the routine diagnostic work-up. The aim of the present study was: 1. To reveal the method most suitable for a routine diagnostic work-up in combination with proper tissue preservation, staining results as well as histological and immunohistochemical analysis; 2. To establish an immunohistochemical method for the detection of lymphocytes and macrophages including their subpopulations in fixed equine tissue samples; 3. To characterize the immune cell populations in fixed endometria of mares without and with non-suppurative endometritis. Material & methods: Equine endometrial tissue and intestinal lymph node were obtained from five mares during post mortem examination and were either fixed in formalin, HOPE® or IHC Zinc Fixative. Aditionally snap frozen tissue samples were processed for cryostat sectioning. HE stained cryostat sections and fixed paraffin embedded tissue samples (formalin, HOPE®, IHC Zinc Fixative) of the equine endometrium were analysed regarding tissue preservation, staining results and artefacts. To establish lymphocyte markers (anti-CD3, anti-CD4 and anti-CD8) immunohistochemically on cryostat sections and fixed paraffin embedded tissue samples (formalin, HOPE®, IHC Zinc Fixative) equine lymph node was used as reference. Formalin fixed and zinc fixed tissue samples with macrophage populations (liver, small intestine, intestinal lymph node) from four mares were used for the immunohistochemical establishment of the macrophage markers (anti-CD172a, anti-CD14, anti-CD206). The immunoreactivity of the primary antibodies was compared. The results of the comparative analysis revealed the most suitable alternative fixation method (zinc fixation). Finally, 28 zinc fixed endometrial biopsies were evaluated semiquantitatively with regard to the numbers of CD3+, CD4+, CD8+, CD20+ lymphocytes, CD172+, CD14+, CD206+ macrophages as well as plasma cells. Four mares had no evidence of endometritis and represent the control group. The remaining 24 mares showed a mild superficial non-suppurative endometritis. The comparative analysis was performed in serial sections within five randomly selected high power fields (400x). Positive cells were counted separately within the luminal and glandular epithelium, the stratum compactum and the stratum spongiosum. Results: 1. The zinc fixation provides excellent staining results, tissue preservation and allows histological and immunohistochemical analysis. It has even some advantages compared to the HOPE® fixation regarding the routine diagnostic work-up. The zinc fixation produces less artefacts. 2. The zinc fixation allows the immunohistochemical detection of all applied T- and B-lymphocyte-, helper- and cytotoxic-T-cell- and macrophage-markers and the histochemical detection of plasma cells (methyl green-pyronin stain) within equine fixed tissue. 3. Endometria with non-suppurative endometritis have higher numbers of CD3+, CD4+, CD8+, CD20+, CD172a+, CD206+ as well as plasma cells within the stratum compactum and stratum spongiosum than endometria without inflammation. The average cell count of CD3+ lymphocytes within the stratum compactum was approximately 3 x higher within inflamed endometria than this value obtained from endometria without endometritis. Few CD20+ B cells and CD172+ macrophages as well as variable numbers of plasma cells could be detected within the stratum compactum of mares with non-suppurative endometritis. Endometria with and without inflammation showed marked differences in the numbers of CD4+ and CD8+ T cells. Conclusion: The zinc fixation can be easily incoorporated into the routine diagnostic work-up and offers additional application possibilities. Immunohistochemical phenotyping of immune cells in the equine endometrium can be performed on fixed endometrial biopsies of the mare. Furthermore, a widespread applicability is possible, e. g. on other equine organs. These findings suggest the existence of different forms of non-suppurative endometritis. The established method will likely assist to reveal possible etiologies and predisposing conditions for non-suppurative endometritis.:INHALTSVERZEICHNIS I ABKÜRZUNGSVERZEICHNIS II 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Das Endometrium der Stute 2 2.1.1 Endometritiden 4 2.1.1.1 Eitrige Endometritis 4 2.1.1.2 Nicht-eitrige Endometritis 5 2.1.1.3 Sonderformen 6 2.1.2 Resistant und susceptible mares 8 2.1.3 Entzündungszelltypisierung im Endometrium der Stute 10 2.1.4 Diagnostische Bedeutung des Endometriumbioptates 13 2.2 Immunologie 14 2.2.1 T-Lymphozyten 14 2.2.2 B-Lymphozyten 17 2.2.3 Makrophagen 18 2.2.4 Immunhistochemische Charakterisierung von equinen Entzündungszellen 21 2.3 Fixierungsmethoden 24 2.4 Fazit aus der Literatur bezüglich der Fragestellung dieser Arbeit 26 3 PUBLIKATIONEN 28 3.1 Publikation 1 inkl. Stellungnahme zum Eigenanteil 28 3.2 Publikation 2 inkl. Stellungnahme zum Eigenanteil 39 4 DISKUSSION 53 4.1 Etablierung alternativer Methoden zur Gewebefixierung 54 4.2 Immunhistochemische Detektion von Immunzell- (sub-)populationen 59 4.3 Erkenntnisse im Hinblick auf die Pathogenese nicht-eitriger Endometritiden 60 5 ZUSAMMENFASSUNG 63 6 SUMMARY 65 7 LITERATURVERZEICHNIS 67 8 DANKSAGUNG 81

This study determined if any of six endodontic solutions would have a softening effect on resorcinol-formalin paste in extracted teeth, and if there were any differences in the solvent action between these solutions. Forty-nine single-rooted extracted teeth were decoronated two mm coronal to the CEJ, and the roots sectioned apically to a standard length of 15 mm. Canals were prepared to a 12mm WL and a uniform size with a #7 Parapost drill. Teeth were then mounted in a cylinder ring with acrylic. The resorcinol-formalin mixture was placed into the canals and was allowed to set for 60 days in a humidor. The solutions tested were 0.9% sodium chloride, 5.25% sodium hypochlorite, chloroform, Endosolv R®, 3% hydrogen peroxide, and 70% isopropyl alcohol. Seven samples per solution were tested and seven samples using water served as controls. One drop of the solution was placed over the set mixture in the canal, and the depth of penetration of a 1.5 mm probe was measured at 2, 5, 10, and 20 min using a dial micrometer gauge. A repeated-measures ANOVA showed a difference in penetration between the solutions at 10 min (p=0.04) and at 20 min (p=0.0004). At 20 min, Endosolv R®, had significantly greater penetration than 5.25% sodium hypochlorite (p=0.0033) and chloroform (p=0.0018); however, it was not significantly better than the control (p=0.0812). Although Endosolv R®, had statistically superior probe penetration at 20 min, the softening effect could not be detected clinically at this time.

A dor é um fenômeno multidimensional, que geralmente desencadeia reações emocionais desconfortáveis quando identificada. Sua relação com injúria tecidual pode ser interpretado como um mecanismo adaptativo de defesa à integridade do organismo, tendo em vista sua preservação evolutiva. Porém, o substrato neurobiológico do organismo parece determinar a complexificação do repertório comportamental em diferentes espécies. Nesse sentido, o córtex cingulado anterior (CCA) tem sido amplamente descrito em mamíferos modulando diferentes aspectos da dor. O presente trabalho utilizou os testes algesimétricos de vocalização e da formalina em cobaias, para se avaliar o decurso temporal do efeito da microinjeção de agonistas e antagonistas GABAérgico (muscimol e bicuculina); colinérgico (carbacol e atropina) e opioidérgico (morfina e naloxona). A microinjeção de bicuculina (1 nmol / 0,2 µl) exacerbou as respostas nociceptivas em ambos os testes, porém diferentes doses de muscimol (0,5, 1 e 2 nmol / 0,2 µl), não modificaram as respostas. O efeito da bicuculina foi bloqueado em ambos os testes pela microinjeção prévia de muscimol (1 nmol/ 0,2 µl) no CCA. A microinjeção de carbacol (2,7 nmol /0,2 µl) neste substrato promoveu antinocicepção, evidenciada por meio da atenuação da amplitude das vocalizações, mas não pelo teste da formalina. Esse efeito foi bloqueado pela administração prévia de atropina (0,7 nmol /0,2 µl) e de naloxona (2,7 nmol /0,2 µl). A microinjeção de morfina (4,4 nmol /0,2 µl) promoveu antinocicepção em ambos os testes. Concluímos que a inibição do tônus GABAérgico no CCA exacerba os comportamentos nociceptivos e que a antinocicepção promovida por carbacol pode ter sido mediada pelo sistema de opióides endógenos, tendo em 9 vista o bloqueio do seu efeito com naloxona. Além disso, a estimulação opióide promove uma contundente antinocicepção. / Pain is a multidimensional phenomenon which usually triggers uncomfortable emotional reactions when identified. Its relation injury can be interpreted as an adaptive mechanism to defend the integrity of the body given its evolutionary conservation. However the neurobiological substrate of the body seems to determine the complexification of behavioral repertoire in different species. Thus, the anterior cingulated cortex (ACC) has been widely described in mammals by modulating different cognitive aspects of pain. This study used algesimetric tests of vocalization and formalin in guinea pigs to evaluate the time course of the effect of microinjection of GABA agonists and antagonists (bicuculline and muscimol) and cholinergic (carbachol and atropine) beyond the opioid antagonist naloxone. The microinjection of bicuculline (1 nmol / 0,2 µl) exacerbated the nociceptive behavior in both tests but different doses of muscimol (0,5; 1 e 2 nmol / 0,2 µl) did not change the responses. The effect of bicuculline was blocked in both tests by prior microinjection of muscimol (1 nmol / 0,2 µl) in the ACC. The microinjection of carbachol (2,7 nmol / 0,2 µl) on this substrate promoted antinociception as evidenced by attenuation of the amplitude of the vocalizations, but not by the formalin test. This effect was blocked by prior administration of atropine (0,7 / 0,2 µl) and naloxone (0,7 nmol / 0, 2 µl). The microinjection of morphine (4,4 nmol / 0,2 µl) promoted antinociception in both tests. We conclude that inhibition of GABAergic tone in the ACC exacerbates nociceptive behaviors and that the antinociception promoted by carbachol may 11 have been mediated by endogenous opioid system in order blocking its effect with naloxone. In addition opioid stimulation promotes a striking antinociception.

OBJETIVO: A perda de heterozigosidade (LOH) é capaz de avaliar as alterações genéticas de lesões potencialmente malignas. Este ensaio avalia as regiões cromossômicas polimórficas que estão próximas ou na região dos oncogenes e genes supressores de tumor conhecidos. Os objetivos desta tese foram três principais: 1) Avaliar a frequência de perda de heterozigosidade de leucoplasias bucais com diferentes graus de severidade histopatológico em regiões cromossômicas próximas aos genes supressores de tumores. 2) Comparar e correlacionar o perfil de perda de heterozigosidade entre indivíduos da British Columbia (Canadá) e Rio Grande do Sul (Brasil). 3) Avaliar os danos ao DNA que podem ocorrer durante o processamento e armazenamento das amostras de tecido parafinado. MÉTODOS: Amostras de leucoplasia bucal (com e sem displasias), fixadas em formalina tamponada 10% e parafinadas, obtidas nos laboratório de patologia bucal do Canadá e do Brasil foram selecionadas e microdissectadas. Procedeu-se a extração de DNA, amplificação por PCR das seguintes regiões microssatélites: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) e 17p13.1 (tp53 e D17S786). Após o produto do PCR foi separado e visualizado em gel de poliacrilamida por autoradiografia. RESULTADOS: Observou-se uma forte correlação entre o perfil de perda de heterozigosidade entre indivíduos com leucoplasia bucal de ambos os países, independentemente da etnicidade. Além disso, pode-se notar que amostras de tecidos parafinados submetidos a mais de 24 horas de fixação em formalina tamponada 10% não serão, em sua maioria, boas amostras para análises de DNA. CONCLUSÃO: As lesões potencialmente malignas, provavelmente não são influenciadas em sua etiopatogênia pelas diferenças étnicas. O modelo de risco genético validado por Zhang e colaboradores (2012) parece ser aplicável em nossa comunidade, sendo necessário a sua validação, respeitando procedimentos técnicos padronizados. Ainda, vale ressaltar, que é imprescindível que a comunidade científica passe a adotar metodologias que preservem o material genético das peças dos bancos de tecidos parafinados, que são de inestimável valor para a pesquisa biomédica. / OBJECTIVE: Loss of heterozygosity (LOH) can evaluate genetic alterations of pre-malignant lesions. This assay evaluates the chromosomal polymorphic regions that are present in tumor suppressor genes and oncogenes. The main objectives of this thesis were: 1) Evaluate the frequency of LOH of oral leukoplakias with different histopathological degrees at chromosomal regions of tumor suppressor genes. 2) Compare the profile of LOH between individuals from British Columbia (Canada) and Rio Grande do Sul (Brazil). 3) Evaluate the DNA damage that may occur with FFPE (formalin-fixed paraffin-embedded) tissues. METHODS: FFPE samples of oral leukoplakia (with and without dysplasia), obtained in Canadian and Brazilian oral pathology laboratories were selected and microdissected. DNA extraction and PCR amplification of the following microsatellite regions were conducted: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) and 17p13.1 (tp53 and D17S786). PCR products were separated and visualized on polyacrylamide gel by autoradiography. RESULTS: A strong correlation between the LOH profile among individuals with oral leukoplakia from both countries was observed, regardless ethnicity. Furthermore, FFPE tissues subjected to more than 24 hours of fixation in 10% buffered formalin are not, generally, good samples for DNA analysis. CONCLUSION: Pre-malignant lesions etiopathogenesis may not be influenced by ethnicity. The genetic risk model validated by Zhang et al. (2012) seems to be applicable in our community, requiring its own validation, respecting standardized procedures. Still, it is important to emphasize that it is imperative that a scientific community adopts methodologies that preserve the genetic material FFPE tissues that are an invaluable resource for biomedical research.