EGFR is a transmembrane tyrosine kinase receptor from the HER family which, upon ligand stimulation, activates different signaling pathways involved in tumorogenesis. EGFR can be targeted by monoclonal antibodies, as cetuximab and panitumumab, which bind to EGFR preventing ligand stimulation of the receptor. Cetuximab and panitumumab are approved for colorectal cancer treatment. However, its clinical success is uniformily limited by the development of acquired drug resistance. We describe a new mechanism of acquired resistance to cetuximab in colorectal cancer that was due to a missense mutation in the EGFR ectodomain (S492R mutation). Upon chronic exposure to cetuximab, colorectal cancer cell lines acquired S492R mutation and became resistant to the treatment. We observed that cetuximab was not able to bind mutant EGFR. Notably, this amino acid change did not affect the ability of panitumumab to bind to EGFR, and panitumumab effectively suppressed growth of mutant cells. EGFRS492R mutation was detected in 2 out of 10 tumor specimens from patients following progression on cetuximab. One of these patients was subsequently treated with single agent panitumumab yielding a partial response. The S492R mutation defines a novel biomarker of resistance to cetuximab but not to panitumumab in colorectal cancer / EGFR és un receptor transmembrana tirosina cinasa de la família HER el qual, després de l’estimulació mitjançant lligands, activa vies de senyalització involucrades en processos tumorogènics. L’EGFR es pot inhibir amb anticossos monoclonals, com cetuximab i panitumumab, que s’uneixen al receptor prevenint-ne l’activació per part dels lligands. Cetuximab i panitumumab estan aprovats per al tractament del càncer colorectal, però el seu ús es veu limitat per el desenvolupament de resistència adquirida al tractament. Nosaltres describim un mecanisme de resistència adquirida a cetuximab en càncer colorectal degut a l’adquisió d’una mutació en el domini extracel•lular de l’EGFR, la mutació S492R. Durant l’exposició crònica a cetuximab, linies cel•lulars de càncer colorectal van adquirir la mutació S492R tornat-se resistents al tractament. Cetuximab no era capaç d’unir-se a l’EGFR mutat. Aquests canvi d’aminoàcid no afectava a l’habilitat que té panitumumab a unir-se al EGFR, pertant, panitumumab suprimia el creixement de les cèl•lules tumorals mutades. Vam detectar la mutació EGFRS492R en 2 de 10 mostres tumorals de pacients que havien recaigut al tractament amb cetuximab. Un d’aquest pacients va ser posteriorment tractat amb panitumumab obtenint-ne una resposta tumoral parcial. La mutació S492R defineix un nou mecanisme de resistència a cetuximab però no a panitumumab en el tractament del càncer colorectal.

The diploma thesis is focused on the Protected Landscape Areas in the Czech Republic and on the limitations resulting from the statute of conservation. The existence of protected areas is often seen as a barrier to any development of the area by public and stakeholders, as is evidenced by resistance to declaration of PLA Soutok. Thesis deals with verification of the arguments that have been raised during negotiations on the declaration of a protected area near to the confluence of the Morava and Thaya rivers. These objections were verified mainly based on interviews with representatives of municipalities, which are located in already existing protected areas, the managers of the PLA administrations, representatives of forest managers and with the help of a questionnaire survey in the affected municipalities. Through a few questions, it was investigated whether the nature and landscape conservation really means certain restrictions, or whether it is merely a relic handed down.

This diploma thesis evaluate tourism in Karlštejnsko and Křivoklátsko regions. These questions were firstly evaluated from sight of quantity which shown offer range of touristic activities and activities in selected regions. The points of the next research are estimations of government, promoter subjects and information centres in question of importance of case country-side tourist traffic and level of its support from public domain.

The topic of my master’s thesis was to create an architectural study of Hospital with Speleotheraphy in Ostrov u Macochy. Sanatorium provides expert care to children suffering diseases of the respiratory tract, particular allergic and infections. Operation of the building is intended primary for children od preschool age. The building was divided into two main section. It ensures a conflict-free, natural traffic - two interconnected buildings. These buildings were carefully planted in the landscape of the Moravian Karst and contribute to teh development of Ostrov u Macochy.

Tato práce je zaměřena na návrh terénní bezdrátové senzorové sítě pro mikroklimatologická měření, zejména výronů juvenilního oxidu uhličitho v oblasti hydrotermílního krasu, založené na LoRaWAN. V teoretické části práce je předložen přehled cílové oblasti, možností řešení měření, získávání a přenosu dat. Praktická část se pak věnuje kompletnímu návrhu celého systému, to znamená od měřicího zařízení přes koncentrátor a gateway až po backend odesílání dat do cloudového úložiště. Výsledky tohoto komplexního problému jsou prezentovány průběžně a přílohy obsahují detailnější a názornější ukázky.

This master‘s thesis deals with impacts on prices of sites which are meant by planning for residential housing in the landscape park Moravian Karst. In the research section of the thesis, there are defined fundamental concepts that concern themselves with the given issues and there are portrayed impacts on prices of sites according to the accessible literature. In the analytical section of the thesis, there is conducted an analysis of the real estate market of the given segment. Furthermore, there are examined and statistically evaluated impacts on prices of sites based on a multiple linear regression modelled in a statistical tool gretl. Impacts are evaluated both within a scope of the whole area and within particular municipalities. In the discussion section of the thesis, the manifested questions are answered, the stated hypotheses are tested and the obtained results are compared to the findings that were described in the research section.

L'ADN tumoral circulant (ADNtumc) est apparu depuis plusieurs années comme un biomarqueur prometteur susceptible d'apporter des informations permettant l'optimisation de la prise en charge du patient en oncologie. L'objectif de cette thèse était double et s'articule autour de deux axes : i) évaluer différentes conditions préanalytiques et analytiques (digitale PCR (dPCR) principalement) pour la détection de ce biomarqueur ii) évaluer l'intérêt clinique potentiel de ce biomarqueur en oncologie digestive. La première partie rapporte 3 travaux (3 articles originaux dont une collaboration nationale (équipe parisienne dirigée par J. Tost)). Dans le travail n°1, nous avons montré la faisabilité de détecter l'ADNtumc par dPCR directement à partir du plasma de 43 prélèvements de patients avec cancer colorectal métastatique (CCRm). Il n'y avait pas de différence significative pour le taux de détection des mutations KRAS circulantes entre les groupes avec et sans extraction d'ADN (93 % (40/43) versus 88 °A) (38/43), respectivement). Dans le travail n°2, nous avons mis au point une méthode basée sur l'apport d'héparinase pour la détection d'ADNtumc à partir de 194 prélèvements héparinés de patients suivis en oncologie. Ce traitement des échantillons par l'héparinase permettait l'analyse de l'ADNtumc pour 117/194 (60 %) patients avec inhibition Préalable de la dPCR par l'héparine. Enfin, dans le travail n°3, nous avons comparé plusieurs plate-formes de détection d'ADNtumc et montré que la dPCR affichait des résultats de détection comparables sur le plan qualitatif et quantitatif avec une plateforme ultrasensible d'Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) pour les échantillons avec une fréquence allélique d'ADNtumc >0,4 °A La deuxième partie rapporte 3 travaux (3 articles originaux) sur l'intérêt clinique de la détection d'ADNtumc par dPCR en oncologie digestive. Nous avons ainsi montré que ce biomarqueur conférait un intérêt diagnostique (travail n°4), Pronostique (travail n 4 à 6) et prédictif de la réponse aux traitements (travail n°6) chez les Patients avec adénocarcinome pancréatique (AP) (travail n°4) et CCRm (travail n°5 à 6). / For several years, circulating tumor DNA (ctDNA) has emerged as a promising biomarker providing relevant information to optimize patient care in oncology. The aim of this thesis was both: (i) to evaluate different preanalytical and analytical conditions (digital PCR (dPCR) mainly) for the detection of this biomarker; (ii) to evaluate the potential clinical interest of this biomarker in digestive oncology. The first part reports 3 works (3 original articles including a national collaboration (Parisian team led by J. lost)). In work no. 1, we have shown the feasibility of ctDNA detection by dPCR directly from the plasma of 43 samples from patients with metastatic colorectal cancer (mCRC). There was no significant difference in the detection rate of circulating KRAS mutations between groups with and without DNA extraction (93% (40/43) versus 88% (38/43), respectively). In work no. 2, we developed a method based on the heparinase addition for the ctDNA detection from 194 heparinized samples of patients followed in oncology. This treatment of samples by heparinase allowed the ctDNA analysis of 117/194 (60%) patients with prior inhibition of dPCR by heparin. Finally, in work no. 3, we compared several ctDNA detection platforms and snowed that dPCR displayed qualitatively and quantitatively comparable detection results with an ultrasensitive platform of E-ice-COLD-PCR for the samples with ctDNA allelic fraction ?.0 4%. The second part reports 3 works (3 original articles) on the clinical interest of the ctDNA detection by dPCR in digestive oncology. We have thus shown that this biomarker had a diagnostic (work no. 4). prognostic (works no. 4 to 6) and predictive response to treatments (work no. 6) interest in patients with pancreatic adenocarcinoma (work no. 4) and mCRC (works no. 5 to 6).

Abstract Colorectal carcinoma (CRC) is an important cause of cancer-associated deaths. About 30–50% of CRCs show KRAS or BRAF mutation. In many cancers, anoikis, i.e. apoptosis induced by loss of extracellular matrix (ECM) contact, is disturbed. Anoikis resistance is essential for the formation of metastases, and since anoikis resistance assessment is based on in vitro cell cultures, the prognostic value of anoikis resistance is largely unknown. We aimed to identify the histopathological features indicating anoikis resistance in CRC and analyze their prognostic value. The roles of BRAF and KRAS mutations and survivin in anoikis resistance were analyzed and 3-D cell culture was used to model the histopathology of anoikis resistant (AR) structures. The two cohorts of CRC cases used in the study consisted of 62 (series 1) and 137 patients (series 2). Immunohistochemistry for ECM proteins enabled identification of tumor cells with and without ECM contact, and in both populations, apoptosis was determined with staining for caspase-cleaved keratin 18. Based on absence of ECM contact and decreased apoptosis rate, we identified micropapillary (MIP), cribriform and solid structures to represent the putative AR populations. High areal density of AR structures associated independently with short survival and was an independent prognostic factor. MIPs showed lower survivin expression, proliferation and apoptosis rates than non-MIP cells, and low apoptosis rate was associated with poor prognosis in stage I and II cases. For 3-D in vitro model of AR structures, we transfected Caco-2 cells with mutated KRAS or BRAF genes; both induced anoikis resistance as measured with Annexin V test in suspension culture. In 3-D cultures, native Caco-2 cells formed polarized cysts. In contrast, mutated cell lines formed partially filled cysts or solid structures, and inverted polarity in KRAS mutant cells. In conclusion, it is possible to identify putative AR structures by conventional histopathology and their number is associated with poor prognosis. MIPs represent a distinct subpopulation of CRC cells with features of quiescence. KRAS and BRAF mutations induce anoikis resistance in Caco-2 cells. In 3-D cultures, oncogenes KRAS and BRAF induce solid structures and cell piling, with structural resemblance to putative AR structures observed by histopathology. The mutated Caco-2 cells thus serve as a model to study the manifestation of anoikis resistance as a distinct histological feature with oncological significance. / Tiivistelmä Paksu- ja peräsuolisyöpä on yleinen syöpäkuoleman aiheuttaja. KRAS- tai BRAF-geenien mutaatio todetaan 30–50 prosentissa suolisyövistä. Anoikis tarkoittaa apoptoosia, jonka käynnistää solun irtoaminen soluväliaineesta. Anoikisresistenssi on etäpesäkkeen synnyn edellytys. Anoikisresistenssiä voidaan todeta vain soluviljelyssä, joten sen merkitystä syövässä in vivo ei ole aiemmin arvioitu. Tässä työssä pyrittiin tunnistamaan anoikisresistenssiin viittaavat muutokset histopatologisista suolisyöpänäytteistä ja selvittämään niiden vaikutusta potilaan ennusteeseen. Lisäksi tutkittiin BRAF- ja KRAS-mutaatioiden yhteyttä anoikisresistenssiin ja mallinnettiin anoikisresistenttejä (AR) rakenteita kolmiulotteisessa soluviljelmässä. Potilasaineisto koostui 199 suolisyöpäpotilaasta. Kudosleikkeistä värjättiin soluväliaineen komponentteja sekä määritettiin apoptoottiset ja jakautuvat solut (M30- ja Ki-67-värjäykset). AR-solupopulaatioiden tunnistamisessa käytettiin kriteereinä soluväliainekontaktin puuttumista ja vähentynyttä apoptoositiheyttä. AR-populaatioiksi osoittautuivat mikropapillaariset (MIP), seulamaiset ja solidit rakenteet. Näiden rakenteiden korkea kokonaisesiintyvyys osoittautui itsenäiseksi huonon ennusteen tekijäksi. MIP-rakenteissa surviviinin ilmentyminen ja apoptoosi- ja proliferaatiotiheys olivat vähentyneet muihin kasvainsoluihin verrattuna. Lisäksi apoptoottisten solujen pieni määrä MIP-rakenteissa liittyi huonoon ennusteeseen paikallisessa syövässä. Mallinnusta varten Caco-2 solut transfektoitiin mutatoiduilla KRAS- tai BRAF-geeneillä. Onkogeenien transfektion todettiin indusoivan anoikisresistenssiä. Kolmiulotteisessa soluviljelyssä polarisoituneet Caco-2 solut muodostivat säännöllisiä rauhasmaisia rakenteita. Onkogeeneillä transfektoidut solut muodostivat puolestaan osittain tai kokonaan täyttyneitä rakenteita ja KRAS-transfektio aiheutti solujen polariteetin kääntymistä. Havainnot osoittavat, että anoikisresistenssiä edustavat rakenteet voidaan tunnistaa kudosleikkeestä ja niiden runsas määrä viittaa huonoon ennusteeseen. MIP-rakenteissa todettiin lepotilan (quiescence) piirteitä. KRAS- ja BRAF-mutaatiot aiheuttavat Caco-2 soluissa anoikisresistenssiä. Kolmiulotteisissa soluviljelmissä onkogeenien vaikutus näkyy solujen pinoutumisena, mikä muistuttaa syöpäkudosnäytteissä todettuja AR-rakenteita. Tulosten perusteella modifioituja Caco-2 soluja voidaan hyödyntää anoikisresistenssin mallintamiseen ja tarkempien mekanismien tutkimiseen.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21260号 / 医博第4378号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 川口 義弥, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (<b>PDAC</b>) and are considered the initiating event during the development of pancreatic intraepithelial neoplasia (<b>PanIN</b>) precursor lesions. While it is well established that KRAS mutations can drive the initiation of pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (<b>PP2A</b>) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both Cancerous Inhibitor of PP2A (<b>CIP2A</b>), an endogenous inhibitor of PP2A activity, and the PP2A target, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56a, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation <i>in vivo</i>, knockout of B56a promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (<b>ADM</b>) and the formation of PanIN lesions. The process of ADM was attenuated <i>ex vivo</i> in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (<b>SMAP</b>s). Together, the results of this study suggest that suppression of PP2A-B56a through KRAS signaling can promote Myc-driven initiation of pancreatic tumorigenesis.</p>