HINTERGRUND: Gastrointestinale Nebenwirkungen unter NSAR, ASS und Phenprocoumon spielen eine bedeutende Rolle im klinischen Alltag. Im Rahmen der Einführung der COX-2 Inhibitoren sollten relevanten Ursachen für Ulcera und Blutungen im Bereich des oberen Gastrointestinaltraktes dargestellt und die Behandlungsstrategien verschieden ausgerichteter Krankenhäuser im klinischen Alltag verglichen werden. METHODEN: Es wurden stationär gastroduodenoskopierte Patienten einer Rheumaklinik und von Krankenhäusern der Grund- und Regelversorgung unter der Therapie mit NSAR, ASS, Phenprocoumon, Glukokortikoiden und COX-2 Inhibitoren im Rahmen einer offenen, multizentrischen Fallkontrollstudie verglichen. Des Weiteren wurden alle Patienten mit einer oberen gastrointestinalen Blutung oder einem Ulcus im Bereich des oberen Gastrointestinaltraktes in die Studie eingeschlossen. Der Einfluss einer Helicobacter pylori Infektion und einer Magenschutzprophylaxe wurde überprüft. ERGEBNISSE: Die höchsten Ulcusraten traten unter der Kombinationstherapie von NSAR und ASS auf. Patienten mit NSAR oder ASS hatten eine Ulcusrate von 35% bzw. 31%. Die Blutungsrate der Patienten mit ASS lag jedoch um 15% höher. Die COX-2 Inhibitoren zeigten mit einer Ulcusrate von 10% deutlich bessere Ergebnisse. Patienten mit einer Helicobacter pylori Besiedlung hatten eine signifikant erhöhte Ulcusrate (48% zu 34%), zeigten aber keine Unterschiede in der Blutungsrate. Der Anteil der Patienten mit einer Magenschutzprophylaxe betrug nur 17%. Die Blutungsrate der Patienten ohne Magenschutz lag bei 87%. SCHLUSSFOLGERUNGEN: Im klinischen Alltag einer rheumatologischen Fachklinik treten gehäuft Ulcera unter NSAR auf. Unter den COX-2 Inhibitoren zeigte sich eine geringere Ulcusrate. Im Unterschied dazu haben in den Krankenhäusern der Regel -und Grundversorgung die Patienten mit einem Ulcus an erster Stelle eine Therapie mit ASS. Eine konsequent durchgeführte Magenschutzprophylaxe mit Protonenpumpeninhibitoren verhindert das Auftreten von gastrointestinalen Ulcera und Blutungen. / BACKGROUND: Gastrointestinal side effects under NSAR, ASS and Phenprocoumon play an important role in the clinical everyday life. In the context of the introduction of the COX-2 inhibitors relevant causes for ulcera and bleedings within the range of the upper gastrointestinal section should be represented and the treatment strategies of differently aligned hospitals in the clinical everyday life be compared. METHODS: Gastro duodenal syncopated in-patients of a hospital for rheumatic diseases and from state hospitals under the therapy with NSAR, ASS, Phenprocoumon, Glukokortikoiden and COX-2 inhibitors were compared within the context of an open, multi-centric drop control study. All patients with an upper gastrointestinal bleeding or an ulcus were included in the area of the upper gastrointestinal section of the study. The influence of a Helicobacter pylori infection and a stomach protection prophylaxis was examined. RESULTS: The highest ulcus rate arose under the combination therapy of NSAR and ASS. Patients with NSAR or ASS had a ulcus rate of 35% and 31% respectively. The bleeding rate of the patients with ASS was however around 15% higher. The COX-2 inhibitors showed clearly better results with an ulcus rate of 10 percent. Patients with a Helicobacter pylori colonization had a significantly increased ulcus rate (48% to 34%), however they showed no differences in the bleeding rate. The proportion of the patients with a stomach protection prophylaxis amounted to only 17 percent. The bleeding rate of the patients without stomach protection was 87%. CONCLUSIONS: In the clinical everyday life of a rheumatological specialized clinic ulcera under NSAR arise. Under the COX-2 inhibitors a smaller ulcus rate shows itself. In contrast to it, patients with an ulcus receive a therapy with ASS in the state hospitals. A consistently implemented stomach protection prophylaxis with proton pump inhibitors prevented the appearance of gastrointestinal ulcera and bleedings.

Diversos estudos têm demonstrado que a atividade coordenada das células epiteliais com o estroma é fundamental no crescimento e diferenciação em situações fisiológicas e patológicas, inclusive no câncer. Vários relatos acentuam a importância do compartimento estromal nos tumores malignos e indicam fortemente que interações contínuas entre o carcinoma e as células estromais (resultando em regulamento e modulação recíproca) são condições prévias para desenvolvimento e progressão de carcinomas. Comparativamente, pouca informação está disponível sobre as características e o papel do estroma durante o processo carcinogênico e a maioria dos dados são baseados em estudos isolados. Nos animais tratados com o carcinógeno Dimetilhidrazina foi identificado na mucosa colônica o aparececimento de “Focos de Estroma Ativado” (FEA) que diferem do foco inflamatório esporádico encontrado na mucosa normal dos animais controles devido à imuno-expressão aumentada de células CD34, CD68, α-actina de músculo liso (ASMA), COX-2 positivas e densidade microvascular. Além disso, o FEA cercou um número aumentado de criptas colônicas em fissão que freqüentemente apresentavam células epiteliais com núcleos hipercromáticos. Este último achado pode sugerir correlação entre as alterações estromais e epiteliais dentro dos FEA. Embora esses achados sejam novos, são consistentes com observações prévias que o estroma tem um papel significante na carcinogênese. Juntamente com dados da literatura, este trabalho sugere que, no cólon, a “field cancerization” epitelial pode ser acompanhada através de alterações estromais e isto pode apontar novos marcadores de transformação neoplásica. / There has been considerable that the activity of epithelial cells with their stroma is fundamental in controlling growth and differentiation in normal and pathological situations, including cancer. A number of reports stress the importance of the stromal compartment in malignant tumors and strongly indicate that continuous interactions between the carcinoma and stromal cells (resulting in their reciprocal regulation and modulation) are prerequisites for carcinoma development and progression. Comparatively, less information is available about the features and role of the stroma for the carcinogenic process. In animals treated with the carcinogen Dimethyl-hydrazine we identified the appearing of mucosal “Activated Stromal Foci” (ASF) that differ from the sporadic inflammatory foci found in the normal mucosa of the control animals because of the presence of increased immune-expression of CD34, CD68, α-smooth muscle actin (ASMA), COX-2 positive cells and microvessel density. Furthermore, the ASF surrounded a increased number of colonic crypts in fission when compared to areas of normal stroma. This last finding suggests that stromal activation and epithelial changes may be correlated. These findings are novel but expected and consistent with previous observations that the stroma has a significant role in carcinogenesis. Taken together with literature data, our findings suggest that in the colon, the epithelial field cancerization may be accompanied by stromal changes and this may point to the finding of new markers of neoplastic transformation.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A LesÃo Central de CÃlulas Gigantes dos maxilares (LCCG) à intra-Ãssea, nÃo tem predileÃÃo por sexo, classifica-se em agressivas e nÃo-agressivas, histologicamente consistem tecido fi-broso e celularizado fusiforme associado a cÃlulas gigantes multinucleadas (CGM), focos de hemorragia e neovascularizaÃÃo, tendo na cirurgia seu habitual tratamento. Novas abordagens terapÃuticas foram propostas, sendo a principal delas o uso de corticÃides intralesionais. Este trabalho analisa retrospectivamente 21 pacientes portadores de LCCG que foram tratados por hexacetonido de triancinolona intralesional, atravÃs do seguinte protocolo: injeÃÃo de hexace-tonido de triancinolona 20mg/ml diluÃdo na soluÃÃo anestÃsica de lidocaÃna 2%/epinefrina 1:200.000 numa proporÃÃo de 1:1; infiltrando 1ml de soluÃÃo para cada 1cm3 de lesÃo, totali-zando 06 aplicaÃÃes em intervalos quinzenais. Estabeleceu-se 04 critÃrios clÃnicos para classi-ficar a resposta ao tratamento: 1- estabilizaÃÃo ou regressÃo clÃnica da lesÃo 2- ausÃncia de sintomas 3- aumento da densidade nos controles radiogrÃficos 4- aumento da resistÃncia a infiltraÃÃo intralesional da droga, bem como, fez-se uma anÃlise imunohistoquÃmica quanto à expressÃo dos Receptores de corticÃides (GCR) e Calcitonina (CTR), Cox-2, proteÃna p16 e amplificaÃÃo gÃnica da Ciclina D1 por CISH, comparando quanto a agressividade e a resposta terapÃutica a corticoterapia intralesional. Dos 21 pacientes incluÃdos neste estudo, 11 eram homens e 10 mulheres, 09 tinham lesÃo em maxila, 12 em mandÃbula. Dez eram lesÃes agres-sivas e 11 nÃo-agressivas, 15 (71,4%) apresentaram uma boa resposta ao tratamento, 04(19%) moderada e 02(9,1%) negativa. Das 11 nÃo agressivas, 10(90,9%) apresentaram boa resposta e 01 (9,1%) resposta moderada, das 10 agressivas 05(50%), 03(30%) e 02(20%) apresentaram boa, moderada e negativa resposta respectivamente, nenhuma apresentou recidiva apÃs o tra-tamento, com preservaÃÃo que variou entre 04 a 08 anos. Os achados histopatolÃgicos mos-traram uma reduÃÃo da densidade e do tamanho das CG, e um estroma fibro-colagenoso das lesÃes. Dentre os marcadores pesquisados, apenas GCR em CG antes do tratamento mostrou significÃncia estatÃstica (p<0,004) com relaÃÃo a uma boa resposta terapÃutica. O CTR ex-pressou-se em cÃlulas gigantes e mononucleares de forma variada. A p16 apresentou-se ex-pressa em 30% da amostra, COX2 nÃo apresentou expressÃo na lesÃo e 33% da amostra apre-sentou amplificaÃÃo gÃnica da ciclina D1. NÃo mostraram significÃncia estatÃstica nem quanto à agressividade, nem quanto resposta ao tratamento, nenhum dos marcadores, exceto o GCR. O estudo mostrou que a corticoterapia intralesional à efetiva e segura para o tratamento das LCCG, com tendÃncia a melhor resposta nas lesÃes nÃo-agressivas do que nas agressivas. Mostrou ainda que a marcaÃÃo para GCR em CG demonstrou ser um parÃmetro confiÃvel para prever a resposta à terapÃutica com a corticoterapia intralesional e que 33% das LCCG tÃm comportamento neoplÃsico pela amplificaÃÃo gÃnica da ciclina D1. / Central Giant Cells Lesion (CGCL) of the jaws is an intra-bone lesion with no predilection for sex and clinically divided into aggressive and non-aggressive subtypes. Histological, it shows as fibrous tissue with fusiform cells, as well as multinucleated giant cells (GC) clusters, he-morrhagic foci and neovascularization. Surgery is the regular treatment option. As new the-rapeutic approaches have been proposed, intralesional glucocorticoid injection is the main option. This paper assesses retrospectively 21 patients presenting CGCL, treated with intrale-sional triamcinolone hexacetonide by using the following protocol: intralesional injection of triamcinolone hexacetonide 20mg/mL, diluted in a solution of lidocain 2% plus epinephrine 1:200000, at a 1:1 proportion; 1mL of this final solution for each 1cm3 of lesion volume was the injected, with a total of 06 injections, one in every 15 days. Four clinical criteria were sta-bilished to evaluate treatment outcome: 1- Clinical regression or stabilization of the lesion; 2- Absence of symptoms; 3- Raising in density on radiographic controls; 4-Increased resistence when injecting the drug intralesionally. It was also performed immunohistochemical assess-ment for glucocorticoid receptor (GCR) expression, calcitonin receptor (CTR) expression, COX-2 expression, p16 expression and Ciclin D1 gene amplification by CISH, making com-parisons related to aggressivity and to therapeutic outcome. Eleven out of 21 patients of this study were women, and 10 were men. Nine of the patients had lesion located in the maxilla, 12 in the mandible. Ten patients showed aggressive lesions and 11 non-aggressive lesions. Fifteen patients showed good treatment outcome, four patients showed moderate outcome, and two patients showed negative answer to the treatment. Among the 11 patients with non-aggressive lesions, ten showed good outcome and the other, moderate outcome. Among the ten aggressive lesions, five patients showed good outcome, three patients showed moderate outcome and the remaining two patients showed negative answer to the treatment. None of them showed reicidive in a four to eight years follow-up period. Morphologic analysis found positive correlation between volume density of GC/mm2 and lesion aggressiveness, as well as significant reduction in number of GC/mm2 after treatment. Among the markers, only GCR in GC showed statistical relevance associated to the treatment. CTR was espresse in GC and in mononuclear cells in a varying way; p16 was expressed in 30% of the sample; COX-2 was not expressed at all in lesion samples and 33% of the sample showed gene amplification in Ciclin D1. None of the markers showed any statistical significant difference related to aggres-siveness nor to treatment outcome, except for GCR. The study showed the feasibility of the adopted treatment, with tendency to better outcomes in non-aggressive lesion, if compared to the aggressive ones. It also showed evidence pointing to GCR expression in GC as a reliable parameter to predict therapeutic responsiveness to glucocorticoids; and it showed that 33% of CGCL have neoplastic behaviour by Ciclin D1 gene amplification.

Abstract Background and aims: Stage and grade are the most widely used prognostic parameters for renal cell carcinoma (RCC). The clinical course of this disease is not, however, always predictable by traditional prognostic factors. In the era of new molecular targeted therapies a more accurate prognostication of RCC patient survival is important for the individualization of treatment and follow-up of patients. Despite exhaustive research there are still no prognostic biomarkers for RCC in clinical practice. In order to find novel prognostic tissue markers for RCC, we examined the expression of 14 biomarkers involved in carcinogenesis and clarified their prognostic significance in RCC. Material and methods: Out of 189 consecutive patients who underwent surgery for kidney cancer at Oulu University Hospital in the 1990s, 152 patients with histologically verified RCC were included in this study. The stage distribution was 70 (46%), 12 (8%), 51 (34%) and 19 (12%) patients with stages I-IV, respectively. The majority of the tumours (83 tumours, 55%) were nuclear grade II and 5 (3%), 40 (27%) and 22 (15%) of the tumours were grades I, III and IV, respectively. Clinical and follow-up data were obtained from patient records, the Finnish Cancer Registry and on demand from the Population Register Centre of Finland. The biomarkers studied included markers of the oxidative and neuroendocrine systems as well as proteins related to cell adhesion and migration, invasion, metastasis, inflammation and immune responses. The expression of various biomarkers was characterized via immunohistochemical tests of archival tumour material. The staining intensity was compared to clinicopathological parameters and patient RCC-specific survival. Results: The 5-year RCC-specific survival was 77%. The expression of Toll-like receptor 9 (TLR9) was an independent marker of favourable RCC-specific survival whereas cytoplasmic myosin VI expression was found to be an independent prognostic factor of poor RCC-specific survival. Cell culture experiments showed how cyclooxygenase-2 (COX-2) expression is regulated by HuR in RCC. HuR and COX-2 immunoexpression were also related to decreased RCC-specific survival. Immunostaining of Keap1 was associated with advanced RCC and a marker of a poorer RCC-specific prognosis. The expression of different neuroendocrine markers was evaluated but we could not establish any prognostic value for them. Conclusions: In particular, TLR9, HuR and myosin VI can be regarded as promising novel prognostic biomarkers in RCC. Stage, however, is the most important single prognostic factor for RCC. / Tiivistelmä Munuaissyöpä on vuosikymmenten ajan jatkuvasti yleistynyt. Vaikka se diagnosoidaan nykyisin useimmiten sattumalöydöksenä vatsan alueen kuvantamistutkimuksissa ja hoitomenetelmät ovat viime vuosikymmenten aikana kehittyneet, munuaissyöpäkuolleisuus ei ole laskenut. Munuaissyövän ennusteen määrittäminen voi olla haasteellista. Perinteiset ennustetekijät, levinneisyys ja erilaistumisaste, eivät riitä selittämään kaikkien potilaiden taudinkulkua, eikä munuaissyövälle vielä ole kliinisessä käytössä ennusteellista merkkiainetta. Munuaissyöpähoitojen kehittyessä taudinkulun ennustaminen on yhä tärkeämpää, jotta potilaiden hoito ja seuranta voidaan yksilöidä. Tämän väitöskirjatyön tarkoituksena oli etsiä uusia ennusteellisia kudosmerkkiaineita munuaissyöpäkasvaimille. Väitöskirjatutkimus perustuu 1990-luvulla Oulun yliopistollisessa sairaalassa leikatun 152 munuaissyöpäpotilaan aineistoon. Lähes puolet aineiston kasvaimista edusti levinneisyysluokkaa I, ja yli puolet munuaissyöpäkasvaimista oli hyvin erilaistuneita (tumagradus I ja II). Tutkimuspotilaista kerättiin kattavat seurantatiedot. Leikkauksessa poistettujen munuaissyöpäkasvainten arkistomateriaalista tutkittiin eri merkkiaineiden ilmenemistä. Tutkitut merkkiaineet käsittivät oksidatiivisen ja neuroendokriinisen järjestelmän merkkiaineita sekä valkuaisaineita, jotka liittyvät keskeisiin syövän ominaisuuksiin, kuten solujen välisiin liitoksiin ja solujen liikkumiseen sekä etäpesäkkeiden syntymiseen. Lisäksi tutkittiin merkkiaineita, jotka liittyvät tulehdusreaktioihin ja immuunipuolustukseen. Väitöskirjatutkimus paljasti useita uusia kudosmerkkiaineita, joiden ilmeneminen munuaissyöpäkasvaimessa on yhteydessä potilaan ennusteeseen. Näistä merkittävimpiä ovat myosiini VI, joka liittyy syöpäkasvainten metastasointiin, sekä immuunipuolustuksessa vaikuttava Tollin kaltainen reseptori 9 (Toll-like receptor 9, TLR9). Molemmat merkkiaineet osoittautuivat itsenäisiksi ennustetekijöiksi munuaissyövässä. Muita ennusteeseen vaikuttavia merkkiaineita ovat tutkimuksen mukaan oksidatiivista stressiä aistiva Keap1 sekä immunologisiin reaktioihin liittyvä syklo-oksigenaasi 2 (COX-2) ja sen ilmenemistä säätelevä HuR.

Einleitung: Nasenhöhlentumoren stellen mit bis zu 47 % die Hauptursache für Nasenausfluss beim Hund dar. Sie sind überwiegend maligne, zu 60 % Karzinome und zu 34 % Sarkome. Die mediane Überlebenszeit (MÜZ) liegt ohne Therapie bei etwa drei Monaten. Nach einer Bestrahlungstherapie beträgt sie etwa 8-20 Monate. Eine Therapie mit klassischen Chemotherapeutika oder eine Tumorablation über eine offen-chirurgische Rhinotomie führen nicht zu einer Verlängerung der Überlebenszeit. Durch ein neues Therapieverfahren, die endoskopisch interventionelle Zytoreduktion (EIZ), werden bei deutlich weniger Nebenwirkungen und Sitzungen in Allgemeinanästhesie ähnliche Überlebenszeiten erreicht wie durch eine Radiotherapie. Da es sich bei der EIZ um eine Zytoreduktion handelt, bei der die Nasenhöhlentumoren nicht mit Sicherheitsabstand im gesunden Gewebe entfernt werden können, stellt sich die Frage, ob die Überlebenszeit zusätzlich durch adjuvante Therapeutika verlängert werden kann. Als solche kommen beispielsweise Tyrosinkinase-Inhibitoren (TKI) und Cyclooxygenase-2 (COX-2)-Inhibitoren infrage, deren Zielstruktur-Expression besonders in kaninen nasalen Sarkomen noch unbekannt ist. Ziele der Untersuchungen: Ziel dieser Arbeit ist, anhand von Bioptaten von kaninen nasalen Karzinomen und Sarkomen eine immunhistochemische Charakterisierung durchzuführen. Hierzu wurden 10 Marker ausgewählt. Besonders im Fokus standen die Wachstumsfaktorrezeptoren vascular endothelial growth factor receptor-2 (VEGFR-2) und epidermal growth factor receptor (EGFR) sowie COX-2, die ersten beiden als Zielstrukturen von TKI und der letztere als Zielstruktur von COX-2-Inhibitoren. So soll eine Wirksamkeit dieser Medikamente bei kaninen nasalen Karzinomen und Sarkomen evaluiert werden. Weiterhin soll eine Korrelation zwischen der Expression von bestimmten Markern (p53, Ki-67, aktivierte Caspase-3, Survivin, E-Cadherin) mit den klinischen Daten zur Tumorkategorie und Überlebenszeit der Patienten untersucht werden. Außerdem soll als Grundlage für den Einsatz neuartiger Medikamente analysiert werden, ob EGFR, VEGFR-2 oder COX-2 in Karzinomen und Sarkomen unterschiedlich stark exprimiert werden. Tiere, Material und Methoden: Es wurden 19 Karzinome, sieben Sarkome und drei andere Tumorarten (ein malignes Melanom, zwei undifferenzierte maligne Tumoren unklarer Histogenese) retrospektiv immunhistochemisch auf die Expression von EGFR, VEGFR-2, COX-2, p53, Ki-67, aktivierter Caspase-3, Survivin, E-Cadherin, Zytokeratinen und Vimentin untersucht. Von drei Patienten wurden insgesamt vier Rezidivbioptate entnommen und ebenfalls immunhistochemisch untersucht. Beidseitige Nasenschleim-hautbioptate von neun gesunden Beagles dienten als Kontrollgruppe (genehmigungspflichtiger Tierversuch: TVV 02/18, Landesdirektion Sachsen). Alle Bioptate wurden während einer standardisierten Diagnostik mit computer-/ magnetresonanztomographischer Untersuchung - bei der auch ein Staging in vier Tumor-Kategorien (T1-T4) durchgeführt wurde - und Rhinoskopie zwischen Jan. 2015 und Dez. 2018 entnommen. Die immunhistochemische Untersuchung der in Formalin fixierten und in Paraffin eingebetteten Gewebeschnitte wurde mit der Avidin-Biotin-Komplex-Methode durchgeführt, nachdem die histopathologischen Diagnosen an Hämatoxylin-Eosin-gefärbten Schnitten gestellt wurden. Die immun-histochemischen Färbungen wurden entweder quantitativ oder semiquantitativ ausgewertet. Die Ergebnisse wurden auf Normalverteilung getestet und u.a. mit One-way Anova oder Kruskal-Wallis Test analysiert. Die MÜZ wurde mit der Kaplan Meier Methode berechnet und mit Log-Rank Test und Gehan-Breslow-Wilcoxon Test verglichen (Signifikanzniveau alpha = 5 %). Ergebnisse: 29 Hunde haben die Einschlusskriterien erfüllt. 14 Hunde wurden unmittelbar nach der Diagnostik euthanasiert; 15 Hunde wurden mit einer EIZ behandelt. Die MÜZ der Patienten in T1 (n = 3) nach EIZ betrug 1362 Tage und war signifikant länger als die MÜZ der Patienten in T2 (n = 1) mit 379 Tagen, in T3 (n = 8) mit 250 Tagen und in T4 (n = 1) mit 75 Tagen (p = 0,0062). Von den nasalen Karzinomen zeigten 68 % für EGFR, 100 % für VEGFR-2, 63 % für COX-2, 100 % für Survivin und 100 % für E-Cadherin eine immunhistochemisch positive Reaktion. Von den nasalen Sarkomen reagierten 100 % für VEGFR-2, 57 % für COX-2 und 86 % für Survivin positiv. Die Proteine EGFR und E-Cadherin werden ausschließlich von epithelialen Zellen exprimiert. Die Expression lag somit bei den vorliegenden Sarkomen bei 0 %. Unter den anderen Tumoren waren 33 % für EGFR, 100 % für VEGFR-2, 67 % für COX-2, 67 % für Survivin und 67 % für E-Cadherin positiv. Die mediane Expression von p53 lag bei 0,9 %, von Ki-67 bei 25 % und von aktivierter Caspase-3 bei 0,7 %. Die Unterschiede in der Expression von EGFR, VEGFR-2, COX-2, p53, Ki-67, aktivierter Caspase-3, Survivin und E-Cadherin zwischen den einzelnen histogenetischen Gruppen sowie zwischen den vier Tumor-Kategorien und in der MÜZ waren nicht signifikant. Eine Korrelation der VEGFR-2-Expression mit der MÜZ oder den T-Kategorien konnte nicht untersucht werden, da alle Tumoren der drei histogenetischen Gruppen VEGFR-2-positiv waren. 100 % der Karzinome zeigten eine Zytokeratin-Expression, 0 % eine Vimentin-Expression. Sarkome verhielten sich dazu konträr. In den anderen Tumoren konnten weder Zytokeratine noch Vimentin immunhistochemisch nachgewiesen werden. In den Rezidivbioptaten war ein Anstieg der COX-2 und aktivierte-Caspase-3-Expression zu beobachten, der aufgrund der geringen Fallzahl nicht statistisch untersucht werden konnte. Schlussfolgerungen: In der vorliegenden Studie wurden erstmalig kanine nasale Karzinome und Sarkome vergleichend immunhistochemisch untersucht. Weiterhin konnte erstmalig gezeigt werden, dass auch mesenchymale und andere Tumoren in vergleichbarer Häufigkeit wie Karzinome der Nase COX-2 exprimieren, wodurch ein Einsatz von COX-2-Inhibitoren nach einer Zytoreduktion bei Nasenhöhlentumoren allgemein von Nutzen sein könnte. Da alle Tumoren VEGFR-2 und die Mehrzahl der Karzinome (68 %) EGFR exprimierten, könnte eine adjuvante Therapie nach EIZ durch einen TKI mit VEGFR-2 oder EGFR als Zielstruktur einen positiven Einfluss auf die Überlebenszeit der erkrankten Hunde haben. Durch die Expression von E-Cadherin und Zytokeratinen in 100 % der Karzinome und 0 % der Sarkome sowie der Expression von Vimentin in 0 % der Karzinome und 100 % der Sarkome konnte die histopathologische Diagnose im Hinblick auf die Histogenese der Tumoren bestätigt werden. Auf der Grundlage der Ergebnisse der vorliegenden Studie sollte eine klinische Studie zur Anwendung von TKI und COX-2-Inhibitoren zur Untersuchung der klinischen Wirksamkeit und Sicherheit bei nasalen Tumoren von Hunden durchgeführt werden.:1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Physiologie der Nasenhöhle 2 2.1.1 Anatomischer und histologischer Aufbau 2 2.1.2 Funktionen der Nasenhöhle und Nasenschleimhaut 3 2.2 Tumoren der Nase und der Nasennebenhöhlen 3 2.2.1 Prävalenz und Signalement von Hunden mit Nasentumoren 3 2.2.2 Biologisches Verhalten der Tumoren 3 2.3 Klinische Symptome 4 2.4 Diagnostik 5 2.4.1 Laboruntersuchungen 5 2.4.2 Bildgebende Verfahren 6 2.4.3 Rhinoskopie 9 2.4.4 Histopathologische Untersuchung 10 2.5 Therapieoptionen 10 2.5.1 Radiotherapie 10 2.5.2 Rhinotomie 11 2.5.3 Chemotherapie 11 2.5.4 Endoskopisch interventionelle Zytoreduktion (EIZ) 12 2.5.5 Tyrosinkinase-Inhibitoren 13 2.5.6 Antikörper gegen Rezeptortyrosinkinasen 15 2.5.7 Cyclooxigenase-Inhibitoren 16 2.6 Prognose 16 2.7 Zielantigene für die Immunhistochemie 17 2.7.1 Epidermal growth factor receptor (EGFR) 17 2.7.2 Vascular endothelial growth factor receptor-2 (VEGFR-2) 18 2.7.3 Cyclooxygenase-2 (COX-2) 18 2.7.4 p53 19 2.7.5 Ki-67 19 2.7.6 Aktivierte Caspase-3 20 2.7.7 Survivin 20 2.7.8 E-Cadherin 21 2.7.9 Zytokeratine 21 2.7.10 Vimentin 21 3 HUNDE, MATERIAL UND METHODEN 22 3.1 Patienten 22 3.2 Bioptate und Einschlusskriterien 23 3.3 Kontrolltiere 24 3.4 Immunhistochemische Untersuchungen 25 3.5 Auswertung der immunhistochemischen Reaktionen 28 3.6 Statistische Auswertung 29 4 ERGEBNISSE 31 4.1 Patienten 31 4.1.1 Signalement und Anamnese 31 4.1.2 Einteilung der Patienten in T-Kategorien 33 4.1.3 Histopathologische Befunde 34 4.1.4 Mediane Überlebenszeit nach endoskopisch interventioneller Zytoreduktion 34 4.1.5 Gesunde Kontrollgruppe 35 4.2 Ergebnisse der immunhistochemischen Untersuchungen 37 4.2.1 Kontrollen und Absorptionsreaktionen 37 4.2.2 Epidermal growth factor receptor (EGFR) 37 4.2.3 Vascular endothelial growth factor receptor-2 (VEGFR-2) 40 4.2.4 Cyclooxygenase-2 (COX-2) 42 4.2.5 p53 46 4.2.6 Ki-67 48 4.2.7 Aktivierte Caspase-3 50 4.2.8 Survivin 52 4.2.9 E-Cadherin 55 4.2.10 Zytokeratine 58 4.2.11 Vimentin 58 4.2.12 Bioptate von Tumorrezidiven 60 5 DISKUSSION 62 6 ZUSAMMENFASSUNG 84 7 SUMMARY 86 8 LITERATURVERZEICHNIS 88 9 ANHANG 105 9.1 Übersicht über die Hunde und Bioptate 105 9.2 Ergebnistabellen 107 9.3 Immunhistochemisches Reaktionsprotokoll 120 9.4 Ansatz der Lösungen und Puffer für die Immunhistochemie 122 9.5 Bezugsquellen für Geräte, Einmalartikel, Reagenzien und Chemikalien 123 9.6 Abbildungs- und Tabellenverzeichnis 125 / Introduction: Tumours of the nasal cavity are the main cause of nasal discharge in dogs (up to 47 %). They are almost always malignant, 60 % are carcinomas and 34 % are sarcomas. Without performing any treatment, the median survival time (MST) is about three months. After radiation therapy, the MST is about 8-20 months. A therapy with conventional chemotherapeutics or tumour ablation via open surgical rhinotomy does not prolong the survival time. A new treatment method, the endoscopic interventional cytoreduction (EIC), achieves survival times similar to radiotherapy with considerably fewer sessions under general anaesthesia and fewer potential adverse events. As EIC is a cytoreduction procedure in which the intranasal tumours cannot be removed with safety margins in surrounding healthy tissue, the question arises whether survival could be prolonged by an additional application of adjuvant therapeutics. As such, for example, tyrosine kinase inhibitors (TKIs) and cyclooxygenase-2 (COX-2) inhibitors may be considered, whose target expression is still unknown, especially in canine nasal sarcomas. Aims of the study: One aim of this study is to perform an immunohistochemical characterisation on the basis of biopsy specimens from canine nasal carcinomas and sarcomas. For this purpose, 10 markers were selected. We particularly focused on the growth factor receptors vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) as well as COX-2, the first two being targets of TKIs and the latter one being the target of COX-2-inhibitors. Thus, a possible efficacy of these drugs in canine nasal carcinomas and sarcomas should be evaluated. Furthermore, a correlation between the expression of specific markers (p53, Ki-67, cleaved caspase-3, survivin, E-cadherin) with clinical data of the tumour stage and patient survival time should be investigated. Moreover, it will be analysed as a basis for the use of novel drugs whether EGFR, VEGFR-2 or COX-2 are differentially expressed in carcinomas and sarcomas. Animals, Material and Methods: A total of 19 carcinomas, seven sarcomas and three other tumour types (one malignant melanoma, two undifferentiated malignant tumorus of unclear histogenesis) were retrospectively examined by immunohistochemistry for the expression of EGFR, VEGFR-2, COX-2, p53, Ki-67, cleaved caspase-3, survivin, E-cadherin, cytokeratins and vimentin. A total of four biopsies from recurrent tumours were obtained from three patients and also examined by immunohistochemistry. Bilateral nasal mucosal samples from nine healthy beagles served as a control group (animal experiment requiring approval: TVV 02/18, Directorate of the Federal State of Saxony, Germany). All biopsy specimens were collected during a standardised diagnostic procedure with computer /magnetic resonance tomography, which also included a staging into four tumour stages (T1-T4) and rhinoscopy between Jan 2015 and Dec 2018. The immunohistochemical examination of tissue sections fixed in formalin and embedded in paraffin was performed using the avidin-biotin complex method after histopathological diagnoses had been made on haematoxylin-eosin stained sections. The immunohistochemically stained sections were evaluated either quantitatively or semiquantitatively. Results were tested for normal distribution and analysed by the one-way anova or Kruskal-Wallis test, among others. MST was calculated using the Kaplan Meier method and compared with the log-rank test and Gehan-Breslow-Wilcoxon test (significance level alpha = 5 %). Results: A total of 29 dogs met the inclusion criteria. A total of 14 dogs were euthanised immediately after diagnosis; 15 dogs were treated with an EIC. The MST of patients in T1 (n = 3) after EIC was 1362 days and was significantly longer than the MST of those patients in T2 (n = 1) at 379 days, T3 (n = 8) at 250 days and T4 (n = 1) at 75 days (p = 0.0062). Of the nasal carcinomas, 68 % were immunohistochemically positive for EGFR, 100 % for VEGFR-2, 63 % for COX-2, 100 % for survivin and 100 % for E-cadherin. Of the nasal sarcomas, 100 % reacted positively for VEGFR-2, 57 % for COX-2 and 86 % for survivin. The proteins EGFR and E-cadherin were expressed exclusively by epithelial cells and, therefore, the expression was 0 % in the present sarcomas. Amongst the other tumours, 33 % were positive for EGFR, 100 % for VEGFR-2, 67 % for COX-2, 67 % for survivin and 67 % for E-cadherin. The median expression of p53 was 0.9 %, that of Ki-67 25 % and that of cleaved caspase-3 0.7 %. Differences in expression of EGFR, VEGFR-2, COX-2, p53, Ki-67, cleaved caspase-3, survivin and E-cadherin among the histogenetic groups, the four tumour stages and in MST were not significant. However, a correlation of VEGFR-2 expression with MST or the tumour stages could not be investigated because all tumours in the three histogenetic groups were VEGFR-2 positive. A total of 100 % of carcinomas showed cytokeratin expression, and 0 % showed vimentin expression. Sarcomas behaved in a contrary manner. In the other tumours, neither cytokeratins nor vimentin could be detected by immunohistochemistry. An increase in COX-2 and cleaved caspase-3 expression was observed in the recurrent tumour biopsies, which could not be statistically investigated due to the small number of cases. Conclusions: In the present study, canine nasal carcinomas and sarcomas were investigated comparatively by immunohistochemistry for the first time. Again, it was shown for the first time that mesenchymal and other tumours also express COX-2 at comparable frequencies to carcinomas of the nose, suggesting that the use of COX-2 inhibitors after cytoreduction may be of general benefit in nasal cavity tumours. As all tumours expressed VEGFR-2 and the majority of carcinomas (68 %) expressed EGFR, the adjuvant therapy after EIC by a TKI targeting VEGFR-2 or EGFR could have a beneficial effect on the survival of diseased dogs. The expression of E-cadherin and cytokeratins in 100 % of the carcinomas and 0 % of the sarcomas as well as the expression of vimentin in 0 % of the carcinomas and 100 % of the sarcomas confirmed the histopathological diagnosis with regard to the histogenesis of the tumours. Based on these results of the present study, a clinical trial should be performed on the use of TKIs and COX-2 inhibitors to investigate the clinical efficacy and safety of canine nasal tumours.:1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 Physiologie der Nasenhöhle 2 2.1.1 Anatomischer und histologischer Aufbau 2 2.1.2 Funktionen der Nasenhöhle und Nasenschleimhaut 3 2.2 Tumoren der Nase und der Nasennebenhöhlen 3 2.2.1 Prävalenz und Signalement von Hunden mit Nasentumoren 3 2.2.2 Biologisches Verhalten der Tumoren 3 2.3 Klinische Symptome 4 2.4 Diagnostik 5 2.4.1 Laboruntersuchungen 5 2.4.2 Bildgebende Verfahren 6 2.4.3 Rhinoskopie 9 2.4.4 Histopathologische Untersuchung 10 2.5 Therapieoptionen 10 2.5.1 Radiotherapie 10 2.5.2 Rhinotomie 11 2.5.3 Chemotherapie 11 2.5.4 Endoskopisch interventionelle Zytoreduktion (EIZ) 12 2.5.5 Tyrosinkinase-Inhibitoren 13 2.5.6 Antikörper gegen Rezeptortyrosinkinasen 15 2.5.7 Cyclooxigenase-Inhibitoren 16 2.6 Prognose 16 2.7 Zielantigene für die Immunhistochemie 17 2.7.1 Epidermal growth factor receptor (EGFR) 17 2.7.2 Vascular endothelial growth factor receptor-2 (VEGFR-2) 18 2.7.3 Cyclooxygenase-2 (COX-2) 18 2.7.4 p53 19 2.7.5 Ki-67 19 2.7.6 Aktivierte Caspase-3 20 2.7.7 Survivin 20 2.7.8 E-Cadherin 21 2.7.9 Zytokeratine 21 2.7.10 Vimentin 21 3 HUNDE, MATERIAL UND METHODEN 22 3.1 Patienten 22 3.2 Bioptate und Einschlusskriterien 23 3.3 Kontrolltiere 24 3.4 Immunhistochemische Untersuchungen 25 3.5 Auswertung der immunhistochemischen Reaktionen 28 3.6 Statistische Auswertung 29 4 ERGEBNISSE 31 4.1 Patienten 31 4.1.1 Signalement und Anamnese 31 4.1.2 Einteilung der Patienten in T-Kategorien 33 4.1.3 Histopathologische Befunde 34 4.1.4 Mediane Überlebenszeit nach endoskopisch interventioneller Zytoreduktion 34 4.1.5 Gesunde Kontrollgruppe 35 4.2 Ergebnisse der immunhistochemischen Untersuchungen 37 4.2.1 Kontrollen und Absorptionsreaktionen 37 4.2.2 Epidermal growth factor receptor (EGFR) 37 4.2.3 Vascular endothelial growth factor receptor-2 (VEGFR-2) 40 4.2.4 Cyclooxygenase-2 (COX-2) 42 4.2.5 p53 46 4.2.6 Ki-67 48 4.2.7 Aktivierte Caspase-3 50 4.2.8 Survivin 52 4.2.9 E-Cadherin 55 4.2.10 Zytokeratine 58 4.2.11 Vimentin 58 4.2.12 Bioptate von Tumorrezidiven 60 5 DISKUSSION 62 6 ZUSAMMENFASSUNG 84 7 SUMMARY 86 8 LITERATURVERZEICHNIS 88 9 ANHANG 105 9.1 Übersicht über die Hunde und Bioptate 105 9.2 Ergebnistabellen 107 9.3 Immunhistochemisches Reaktionsprotokoll 120 9.4 Ansatz der Lösungen und Puffer für die Immunhistochemie 122 9.5 Bezugsquellen für Geräte, Einmalartikel, Reagenzien und Chemikalien 123 9.6 Abbildungs- und Tabellenverzeichnis 125

Epilepsy affects 3.4 million patients in the USA and is characterized by recurring seizures. The blood-brain barrier is leaky in epilepsy and may contribute to seizure progression but the mechanisms which cause this leakage are not fully understood. We hypothesized that seizures trigger LOX- and COX-mediated blood-brain barrier leakage and that dual LOX/COX inhibition prevents barrier leakage in vivo. To test this hypothesis, we administered either the dual LOX/COX inhibitor licofelone or a combination of the 5-LOX inhibitor zileuton and the COX-2 inhibitor celecoxib to rats that experienced status epilepticus (SE). Serum and brain capillaries were isolated 48 hours after SE and serum S100β levels were measured and Texas Red™ leakage rates were determined. Dual inhibition of 5-LOX and COX prevented serum S100β elevations observed in SE rats in a dose-dependent manner with licofelone. Inhibition of 5-LOX and COX-2 with zileuton and celecoxib completely prevented serum S100β elevation. Texas Red™ leakage rates for SE rats were also reduced in a dose-depended manner with licofelone and reduced to control rates with zileuton and celecoxib. These data support our hypothesis that seizure-induced blood-brain barrier leakage is mediated by LOX and COX, and inhibition of these enzymes prevents barrier leakage.

Mouse models of human disease are valuable tools for studying pathogenesis and for evaluating novel therapies. T-cell lymphoma is a relatively rare disease in humans, affecting 100-150 persons yearly in Sweden. It exists in both aggressive and more indolent forms. We have established a mouse model for an aggressive T-cell lymphoma, the T-cell lymphoma/leukemia (TLL) mouse. In the present thesis, the TLL mouse model was characterized and used for experimental therapeutic and primary prevention studies. The TLL mouse was established unintentionally in our laboratory during work on VH-gene replacement in a “knock-in” mouse experimental setting. The generated chimeras all developed aggressive T-cell lymphomas affecting the lymphoid organs, lungs, kidneys and liver. The lymphoma phenotype segregated from the targeted locus and we could demonstrate the presence of Moloney murine leukemia virus (MMLV) in the germline of the affected mice. MMLV is a retrovirus known to induce T-cell lymphomas when inoculated in newborn mice.  We further characterized two TLL substrains; TLL-2 and TLL-14 carrying the proviral integrations on chromosomes 2 and 14 respectively. Significant differences were found between the substrains regarding lymphoma frequency and immunophenotype, the TLL-14 substrain developing tumors with higher frequency than TLL-2 and with a more mature immunophenotype. A transfer model was developed in which TLL cells could be readily transferred intravenously to syngenic recipients causing aggressive lymphomas. The transfer model was used in a therapeutic study where the selective COX-2 inhibitor celecoxib was evaluated as a single agent and in combination with the established anti-tumor agent cyclophosphamide. The study was based on results from other tumor types that have indicated celecoxib, originally an anti-inflammatory and analgetic drug, to have possible anti-tumor effects. In our TLL model, however, we could not demonstrate any benefit of celecoxib monotherapy or any additive effect to cyclophosphamide. Dietary fatty acids, in particular omega-3 fatty acids, have been a focus of public and scientific interest due to observed effects on the prevention of cardiovascular disease, cancer and inflammatory conditions. In addition, omega-3 fatty acids inhibit T-cell proliferation in vitro. We supplemented the diet of TLL mice with omega-3 and omega-6 fatty acids respectively and could demonstrate a significant delay in lymphoma onset between 5-8 months of age in the group receiving an omega-3 rich diet.

Σκοπός της μελέτης ήταν η ανάδειξη της νεοαγγειογένεσης και ο προσδιορισμός της έκφρασης των VEGF-A, FGF-2, COX-2, AR και BCL-2 στην καλοήθη υπερπλασία και το αδενοκαρκίνωμα του προστάτη. Το υλικό αφορούσε σε δείγματα προστατικού ιστού μονιμοποιημένα και εγκλεισμένα σε παραφίνη, από 24 περιστατικά καλοήθους υπερπλασίας και 139 περιστατικά προστατικού αδενοκαρκινώματος. Τα τελευταία χωρίστηκαν περαιτέρω σε 3 υποομάδες (Grade I, II και ΙΙΙ) ανάλογα με το βαθμό διαφοροποίησης του νεοπλάσματος κατά Gleason (2-4, 5-7 και 8-10 αντίστοιχα). Χρησιμοποιήθηκε ανοσοϊστοχημική μέθοδος Βιοτίνης-Στρεπταβιδίνης-Υπεροξειδάσης, και εφαρμόστηκε ημιποσοτική μέθοδος για την εκτίμηση της ανοσοϊστοχημικής χρώσης. Τα ευρήματά μας ανέδειξαν σαφή αύξηση της νεοαγγείωσης (MVD) στο αδενοκαρκίνωμα του προστάτη σε σχέση με την καλοήθη υπερπλασία, η οποία εμφάνισε στατιστικώς σημαντική θετική σχέση με το βαθμό κακοήθειας των προστατικών νεοπλασμάτων (ANOVA p<0.001) και με την έκφραση των VEGF-A και COX-2 (ANOVA p<0.001). Αναδείχθηκε αντίστροφη συσχέτιση της πυρηνικής έκφρασης του ανδρογονικού υποδοχέα (AR) με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.0001) και την έκφραση του VEGF-A στο προστατικό στρώμα (p<0.001 Spearman r = -0.312). Η έκφραση της BCL-2 παρουσιάστηκε αυξημένη στα προστατικά αδενοκαρκινώματα και σχετίστηκε με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.001) και την έκφραση των VEGF-A και COX-2 (p<0.001). Η έκφραση του VEGF-A σε όλα τα περιστατικά αδενοκαρκινώματος του προστάτη εμφάνισε στατιστικώς σημαντική σχέση με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.0001). Ωστόσο στα πτωχής διαφοροποίησης αδενοκαρκινώματα η μέση τιμή της έκφρασης του VEGF-A παρουσίασε πτώση. Αντίθετα, η σημασία της COX-2 στον προστατικό καρκίνο αναδείχθηκε με την έκφρασή της τόσο στην καλοήθη υπερπλασία όσο και στα αδενοκαρκινώματα του προστάτη. Η έκφραση παρουσίασε σημαντική σχέση με το βαθμό διαφοροποίησης των νεοπλασμάτων (p<0.01). Η σαφώς αυξημένη έκφραση της COX-2 σε σχέση με τη μείωση της έκφρασης του VEGF-A στα πτωχής διαφοροποίησης νεοπλάσματα πιθανόν υποδηλώνει την ύπαρξη ενός αγγειογενετικού διακόπτη στα νεοπλάσματα αυτά όπου η COX-2 φαίνεται να παίζει σημαντικότερο ρόλο από τον VEGF-A. Η σημαντική αυτή πληροφορία θα μπορούσε να βρει πιθανή θεραπευτική εφαρμογή, καθώς σε πτωχής διαφοροποίησης αδενοκαρκινώματα του προστάτη ίσως η θεραπεία με COX-2 εκλεκτικούς αναστολείς να είχε πολύ καλύτερα αποτελέσματα από τις αντι-αγγειογενετικές θεραπείες με αναστολείς του VEGF. / The aim of this study was to immunohistochemically evaluate the expression of VEGF-A, FGF-2, COX-2, AR and BCL-2 in benign prostatic hyperplasia (BPH) and prostate carcinoma in relation to microvessel density (MVD) and the Gleason grade of the neoplasms. A total of 139 cases of primary prostate carcinoma and 24 cases of benign hyperplasia were included in the study. Tumors were graded according to the Gleason grading system and further divided into 3 subgroups (GRADE I, II and III). The immunostaining was performed according to the Streptavidin-Biotin Complex Peroxidase method, in formalin fixed paraffin-embedded tissue. Mean micro vessel density (MVD) was strongly related to tumor grade, VEGF-A and COX-2 histoscore (ANOVA, p<0.001). The androgen receptor was localized in the nuclei of prostate epithelial cells in 97% of cases. The comparison of AR staining with tumor grade revealed an inverse relationship between these two parameters (ANOVA, p<0,0001). An interesting finding was the inverse relationship of stromal AR expression in relation to VEGF-A immunoreactivity. BCL-2 expression was correlated with tumor grade in prostate carcinoma cases (p<0.001) and was strongly correlated with COX-2 and VEGF-A expression (p<0.001). These findings suggest that BCL-2 may play a dual role in tumorigenesis, possibly through an angiogenetic axis. VEGF-A expression was detected in only 17% of BPH cases but all prostate cancer specimens demonstrated some degree of immunoreactivity. COX-2 immunopositivity was present in 54% of BPH specimens and in 99% of primary prostate carcinomas. The increased COX-2 expression correlated significantly with Gleason grade. In our study, high-grade neoplasms presented low to moderate VEGF staining intensity compared to COX-2 expression. These results suggest the activation of an angiogenic switch in poorly differentiated neoplasms, where COX-2 may play a crucial role compared to VEGF and the possible key role of COX-2 in poorly differentiated cancers. According to our findings, anti-VEGF therapy could prove to be more beneficial in patients with low-grade disease, while patients with high-grade prostate carcinoma are more likely to respond to selective COX-2 inhibitors. Immunohistochemical determination of VEGF-A and COX-2 content might prove a useful tool in the design of patient-tailored, anti-angiogenic treatments.

Currently, opioids are extensively used in clinical practices in order to treat pain in patients. However, prolonged administration of opioids are not feasible due to the development of side effects especially tolerance, constipation, addiction and dependence. Our drug design is mainly aimed to reduce opioid induce side effects such as development of tolerance. The first strategy examined involves design and synthesis of peptide based single molecules that have a mu agonist and delta agonist pharmacophore in combination with a COX2 inhibitory pharmacophore. A new molecule, 3-17 having good delta agonist activity, partial COX2 inhibitory activity and weak mu agonist activity was produced. Moreover, Investigation of the bioactivities of the synthesized ligands including 3-17 in terms of their ligand receptor interactions were probed using NMR conformational analysis along with docking analysis to the respective homology modeled mu and delta opioid receptors as well as the COX2 enzyme. As a further continuation of this work, instead of peptide based mu agonist and delta agonist type pharmacophore, the highly mu selective fentanyl pharmacophore was used in combination with a pyrazole based and a pyrazolone based COX pharmacophore. Based on the SAR study and docking analysis of synthesized ligands to the homology modeled mu opioid receptor, an ideal tolerant position without significant loss of mu opioid agonist activity for fentanyl were found. The second strategy involves design and synthesis of a peptide based single molecule that has a mu agonist and a delta antagonist pharmacophore in combination with a NK1 antagonist pharmacophore. A novel molecule (4-2) containing delta antagonist activity, weak mu agonist activity and NK1 antagonist activity was identified. Its homology modeled mu opioid receptor bound conformation was compared with that of reference ligands. Docking analysis of modified 4-2 to the homology modeled mu opioid receptor revealed that it can be further modified to obtain better mu agonist activity. 4-2 showed antinociception for 45 min period of time after injection in tail flick assay. In addition to studies that were directed to avoid tolerance development due to opioid administration, peptide based potential analgesics such as biphalin was modified by introducing more peptidomimetic character in order to enhance its blood brain barrier permeability and proteolytic stability. The novel molecule (6-7) was produced in this study and its antinociception lasted for 30 min period of time after injection in the tail flick assay.