Dissertations / Theses on the topic 'Adenokarcinom'

Adenokarcinom prostate (PCa) je najčešći karcinom u muškaraca. Intraepitelne prostatične neoplazme visokog gradusa (HGPIN) su lezije koje prethode nastanku invazivnog karcinoma i podrazumevaju kompletno odsustvo bazalnih ćelija i invaziju strome malignim acinusima. Estrogeni receptor β (ERβ) se nalazi u jedrima bazalnih i sekretornih ćelija acinusa i delimično u stromalnim ćelijama. Cilj istraživanja je da prikaže i lokalizuje ERβ u različitim morfološkim lezijama prostate: hiperplaziji (BHP), PINu i u PCa sa različitim Gleason scorom. Pretpostavlja se da prekancerozne lezije u svojim različitim fazama evolucije ne koreliraju u potpunosti sa ekspresijom ERβ. LGPIN pokazuje ekspresiju, dok u HGPINu nema ekspresije. Takođe je pretpostavka da ekspresija ERβ postoji u većine srednje diferentovanih PCa, te da se ekspresija posmatranog receptora gubi sa porastom Gleason scora. Ispitivano je pet grupa bolesnika:  kontrolna grupa sa BHP i četiri eksperimentalne grupe (PIN i 3 različite grupe PCa). Studija je sprovedena na muškarcima različite starosti u periodu 2010–2012. Nijedan pacijent nije prethodno primio hormonsku terapiju. Sekstant biopsije prostate su bojene na ERβ (Novocastra). Lokalizacija i intenzitet ERβ ekspresije prikazani su kroz skor: 0 = nula; 1 = <1%; 2 = 1–10%; 3 = 11–33%; 4 = 34–66%; 5 = > 66%. Pozitivni fibroblasti i endotelne ćelije su korišćene za poređenje. Smanjena ekspresija ERβ primećena je kod malignih i premalignih lezija prostate naspram BHP. Ekspresija ERβ u epitelnim ćelijama acinusa bila je najslabija u dobro diferentovanim PCa. Kod BHP i dobro diferentovanih PCa bila je veća ekspresija ERβ u bazalnim ćelijama nego u sekretornim. Loše diferentovani PCa prikazali su smanjenje ekspresije ERβ u bazalnim ćelijama. Ukupna ćelijska ekspresija ERβ predstavlja složen i ponekad moguće paradoksalan nalaz, na osnovu čega primarni PCa zadržava ekspresiju ovog receptora, ali ipak značajno nižu u poređenju sa benignim epitelom i premalignim lezijama. Ovaj nalaz podupire stanovište o antiproliferativnoj ulozi ERβ u tkivu prostate.
Adenocarcinoma of the prostate (PCa) is the most common cancer in men. High-grade prostatic intraepithelial neoplasia (HGPIN) are lesions that precede to invasive carcinomas and include complete absence of basal cells and stromal invasion by malignant acini. Estrogen receptor ß(ERß) is located in the nuclei of basal and secretory cells and partly in stromal ones.The aim of the research is to describe and localize ERß in different morphological lesions: prostate hyperplasia (BPH), PIN and PCa with different Gleason score. It is assumed that pre-cancerous lesions in different stages of their evolution not correlate completely with the expression ERß. LGPIN shows expression, while there is no expression in HGPIN. It is also an assumption that the expression ERß exists in most medium differentiated PCa, and that the expression of this receptor loses with increasing of Gleason score. Five groups of patient were investigated: control group with BPH and four experimental groups (PIN and 3 different groups of PCa). The study was conducted on men of different ages in the period 2010-2012. None of the patients received prior hormonal therapy. Sextant prostate biopsy were stained on ERß (Novocastra). ERß expression is shown through the score: 0 = zero; 1 = <1%; 2 = 1-10%; 3 = 11-33%; 4 = 34-66%; 5 => 66%. Positive fibroblasts and endothelial cells were used for comparison. Reduced expression was observed in malignant and premalignant lesions of the prostate versus BPH. ERß expression in the epithelial cells of acini was the weakest in well-differentiated PCa. In BPH and well differentiated PCa was greater expression in the basal cells than in secretory ones. Poorly differentiated PCa showed a decreased ERß expression in basal cells. Total cellular expression of ERß is a complex and sometimes paradoxical finding on the basis of which the primary PCa retains expression of this receptor, but significantly lower compared to benign epithelium and premalignant lesions. This finding supports the antiproliferative role of ERß in prostate tissue.

Receptor humanog epidermalnog faktora rasta 2 (HER2) pripada porodici receptora protein-tirozin kinaze čija je aktivacija povezana sa proliferacijom malignih ćelija, inhibicijom apoptoze, tumorskom angiogenezom i sposobnosti invazije i metastaziranja. Povećana proteinska ekspresija HER2 receptora može nastati kao posledica amplifikacije gena i/ili transkripcijskih promena. Ekspresija HER2 receptora u humanim tumorima povezuje se sa agresivnijim ponašanjem i lošijom prognozom. Učestalost povećane proteinske ekspresije HER2 receptora u nesitnoćelijskim karcinomima pluća (NSCLC) je najviše zastupljena u adenokarcinomu u odnosu na druge histološke tipove. Identifikacija HER2 pozitivnih NSCLC omogućava određivanje grupe pacijenata koji bi bili kandidati za specifičnu terapiju. Problem predstavlja izbor metode detekcije HER2 receptora i nepostojanje utvrđenog protokola za očitavanje rezultata kao što postoji kod karcinoma dojke i želuca. Osnovni ciljevi ove doktorske disertacije su bili: da se odredi učestalost povećane proteinske ekspresije HER2 receptora u adenokarcinomu pluća; da se uporede rezultati povećane proteinske ekspresije HER2 receptora dobijene korišćenjem HER2 antitela „Hercep Test Dako“ i „Ventana anti-HER2/neu (4B5)“ antitela; da se uporedi prisustvo amplifikacije HER2 gena pomoću in situ hibridizacije (ISH) (Dual IHC HER2 kit;Ventana Medical Systems) retestiranjem uzoraka kod kojih je povećana proteinska ekspresija HER2 receptora ocenjena sa 2+ i 3+ dobijena „Hercep Test Dako“ sa prisustnom amplifikacijom HER2 gena na uzorcima koji su pomoću „Ventana anti-HER2/neu (4B5)“ ocenjeni sa 2+ i 3+; da se uporedi učestalost povećane proteinske ekspresije HER2 receptora i prisustva HER2 genske amplifikacije kod različitih histoloških podtipova adenokarcinoma pluća; da se utvrdi da li je povećana proteinska ekspresija HER2 receptora u adenokarcinomu pluća i/ili prisustvo genske amplifikacije povezano sa demografskim (starost i pol pacijenta) parametrima, pušačkim statusom, pojavom metastaza u regionalnim limfnim čvorovima i udaljenim organima, infiltracijom pleure i okolnih struktura, odnosno stadijumom bolesti. Povećana proteinska ekspresija HER2 receptora u adenokarcinomu pluća iznosi 7,4% za Hercep Test Dako i 2,7% za Ventana anti-HER2/neu (4B5) antitelo. Kod pozitivne ekspresije slažu se u 2%, dok se kod negativne ekspresije slažu u 91,9% slučajeva, što je ukupno 93,9%. Učestalost amplifikacije HER2 gena kod adenokarcinoma pluća je 17,6%, od toga je kod 2,7% slučajeva prisutna high grade amplifikacija. Postoji statistički značajna povezanost između povećane proteinske ekspresije HER2 receptora dobijene upotrebom HercepTest Dako i Ventana anti-HER2/neu (4B5) antitela i amplifikacije HER2 gena. Amplifikacija HER2 gena prisutna je kod 90,9% pacijenata sa povećanom proteinskom ekspresijom HER2 receptora koja se dobije upotrebom HercepTest Dako i kod 75% upotrebom Ventana anti-HER2/neu (4B5) antitela. Povećana proteinska ekspresija HER2 receptora dobijena pomoću HercepTest Dako i Ventana anti-HER2/neu (4B5) antitela je najčešća kod solidnog predominantnog tipa adenokarcinoma u patološkom T2a deskriptoru i IB stadijumu i acinarnog predominantnog tipa adenokarcinoma u patološkom T1b deskriptoru i IA stadijumu. Amplifikacija HER2 gena je najčešća kod solidnog a zatim kod acinarnog i papilarnog predominantnog tipa adenokarcinoma. Povećana proteinska ekspresija HER2 receptora dobijena pomoću HercepTest Dako i Ventana anti-HER2/neu (4B5) antitela i amplifikacija HER2 gena se najčešće javljaju kod muškaraca, pušača, u starosnoj dobi od 61-70 godina, tumora veličine 31-50 mm, N0 i M0 statusu bolesti, bez prisustva tumorske infiltracije pleure i okolnih struktura.
Human epidermal growth factor 2 (HER2) is a member of the epidermal growth factor family having tyrosine kinase activity, which is directly linked to malignant cells proliferation, apoptosis inhibition, tumor angiogenesis and ability for invasion and metastasis. Increased protein expression of HER2 receptors can be the consequence of gene amplification and/or transcription changes. Expression of HER2 receptors in human tumors is associated with more aggressive behavior and worse prognosis. Incidence of increased protein expression of HER2 receptors in non-small-cell lung carcinoma (NSCLS) is mainly represented in adenocarcinoma, in comparison with other histological types. Identification of HER2 positive NSCLC enables determination of a group of patients who would be candidates for specific therapy. The problem occurs in choosing the method of detection of HER2 receptors and non-existence of determined protocol for reading the results, as the one ones which exist for breast and gastric carcinoma. The main objectives of this PhD dissertation were: to determine the incidence of increased protein expression of HER2 receptors in lung adenocarcinoma; to compare the results of the increased protein expression of HER2 receptors obtained by using HER2 antibodies "HercepTest Dako" and "Ventana anti-HER2/neu (4B5)" antibodies; to compare the presence of HER2 gene amplification by in situ hybridization (ISH) (Dual IHC HER2 kit: Ventana Medical Systems) by retesting the samples in which the increased protein expression of HER2 receptors was graded with 2+ and 3+, obtained by "HercepTest Dako" with present gene HER2 amplification on samples obtained by "Ventana anti-HER2/neu (4B5) and graded with 2+ and 3+; to compare the incidence of increased protein expression of HER2 receptors and presence of HER2 gene amplification in different histological subtypes of lung adenocarcinoma; to determine if the increased protein expression of HER2 receptors in lung adenocarcinoma and/or presence of gene amplification is related to demographic (age and sex of the patient) parameters, smoking status, appearance of metastases in regional lymphatic nodes, distant organs, infiltration of pleura and surrounding structures, and stage of the disease. Increased protein expression of HER2 in lung adenocarcinoma is 7.4% for HercepTest Dako and 2.7% for Ventana anti-HER2/neu (4B5) antibody. In positive expression they are correlated in 2%, while in negative expression they are correlated in 91.9% cases, which is overall 93.9%. The incidence of HER2 gene amplification in lung adenocarcinoma is 17.6%, from that in 2.7% of the cases high grade amplification is present. There is a statistically significant correlation between increased protein expression of HER2 receptors obtained by use of HercepTest Dako and Ventana anti-HER2 /neu (4B5) antibody and amplification of HER2 genes. Amplification of HER2 genes is present in 90.9% of patients with increased protein expression of HER2 receptors, which is obtained by using HercepTest Dako and in 75% patients by using Ventana anti-HER2/neu (4B5) antibody. Increased protein expression of HER2 receptors obtained by HercepTest Dako and Ventana anti-Her2/neu (4B5) antibody is most common in solid predominant type of adenocarcinoma in pathological T2a descriptor and IB stadium and acinar predominant type of adenocarcinoma in pathological T1b descriptor and IA stadium. Amplification of HER2 genes is most common in solid, and then in acinar and papillary predominant type of adenocarcinoma. Increased protein expression of HER2 receptors obtained by HercepTest Dako and Ventana anti-HER2/neu (4B5) antibody and amplification of HER2 genes most commonly occurs in men, smokers, at the age of 61-70 years, tumor size 31-50 mm, NO and MO disease status, without presence of tumor infiltration of pleura and surrounding structures.

Napredak na polju molekularne biologije omogućio je identifikaciju molekularnih markera za karcinom bronha sa vrednim prognostičkim i prediktivnim značajem i njihova uloga kod uznapredovalog, metastatskog oblika bolesti je u velikoj meri istražena, dok kod ranih stadijuma bolesti još uvek nije sasvim jasna. Cilj ovog istraživnja bio je da se utvrdi učestalost najčešćih genskih alteracija u tumorskim ćelijama bolesnika sa ranim stadijumom adenokarcinoma bronha, da se utvrdi pojedinačna zavisnost ispitivanih genskih alteracija u tumorskim ćelijama sa određenim kliničko-patološkim karakteristikama i da se utvrdi potencijalni prognostički značaj pojedinačne genske alteracije u tumorskim ćelijama na vreme preživljavanja bez povratka bolesti i ukupno vreme preživljavanja. Istraživanje je obuhvatilo 161 bolesnika sa adenokarcinomom bronha, stadijuma bolesti od I do IIIA, kod kojih je sprovedena radikalna hirurška resekcija u Institutu za plućne bolesti Vojvodine u periodu izmedju 2007 i 2014 godine. U tumorskim uzorcima fiksiranim u parafinu odredjivane su mutacije EGFR, KRAS i PIK3CA gena, ALK i ROS1 rearanžman i PD1 i PD-L1 ekspresija. Kliničkopatološke karakteristike su preuzete iz registra za karcinom bronha Instituta za plućne bolesti Vojvodine. Ukupno preživljavanje je računato od dana operacije do dana smrti, a preživljavanje bez povratka bolesti je računato od dana operacije do momenta ponovne pojave bolesti. Od 161 testiranog tumorskog uzorka, prisustvo mutacija detektovano je kod 96 uzoraka (59.6%). Prisustvo mutacije KRAS gena detektovano je kod 69 (42.9%), mutacije EGFR gena kod 10 (6.2%), a mutacije PIK3CA gena kod 7 (4.3%) tumorskih uzoraka. ALK rearanžman je detektovan kod 3 (1.9%), a ROS1 rearanžman kod 7 (4.3%) tumorskih uzoraka. PD-1 ekspresija detektovana je u 71 tumorskom uzorku (45%), dok je PD-L1 ekspresija detektovana u 59 tumorskih uzoraka (36.6%). PD-1 ekspresija nije bila značajno povezana ni sa jednim od klinčko-patoloških karakteristika (uključujući KRAS, EGFR, ALK, ROS1 i PI3KCA status). PD-L1 ekspresija je bila značajno povezana sa tipom hirurgije (P = 0.01) i sa prisustvom KRAS mutacije (P = 0.02). Mutacioni status u domenu KRAS gena je bio značajno povezan sa godinama starosti (P = 0.004), polom (P = 0.006) i pušačkim statusom (P = 0.004). Mutacioni status u domenu EGFR gena je bio značajno povezan sa pušenjem (P < 0.001) i sa godinama starosti (P = 0.013). Mutacioni statusi u domenu gena za ALK, ROS1 i PI3KCA nisu bili značajno povezani ni sa jednom od ispitivanih kliničko-patoloških karakteristika. Prisustvo PD-1 ekspresije je bilo značajno povezano sa preživljavanjem bez povratka bolesti (P = 0.03) i ukupnim preživljavanjem (P = 0.01). PD-L1 ekspresija, KRAS, EGFR, ALK, ROS1 i PIK3CA mutacioni status nisu bili značajno opvezani sa preživljavanjem bez povratka bolesti i ukupnim preživljavanjem. Najčešće detektovane genske alteracije su mutacije u domenu KRAS i EGFR gena. Prisustvo KRAS mutacije je značajno povezano sa godinama starosti ispitanika, polom i pušačkim statusom dok je prisustvo EGFR mutacije značajno povezano sa godinama starosti ispitanika i pušačkim statusom. Prisustvo PD-L1 ekspresije je značajno povezano sa vrstom hirurškog lečenja i sa prisustvom KRAS mutacija. Jedino prisustvo PD-1 ekspresije u tumorskim ćelijama predstavlja nezavistan prognostički faktor za preživljavanje bez povratka bolesti i ukupno preživljavanje bolesnika sa ranim stadijumom adenokarcinoma bronha.
Advances in the field of molecular biology gave us insight into biomarkers for lung cancer with great prognostic and predictive value and their role in advanced stage disease is well known while in early stage disease is yet to be proven. The aim of this study was to determine the frequencies of the most common gene alterations in patients with early stage lung adenocarcinoma, to determine the relationship between gene alterations in tumor cells and clinicopathologial characteristics and to determine prognostic value of each gene alteration regarding overall survival and disease free survival. One hundred sixty-one patients diagnosed with lung adenocarcinoma clinical stage I-IIIA who underwent radical surgical resection at the Institute for Pulmonary Diseases of Vojvodina between 2007 and 2014 were included in this study. Mutations in EGFR, KRAS and PIK3CA gene, ALK and ROS1 rearrangement and PD-1 and PD-L1 expression were determined in representative formalin-fixed, paraffin-embedded (FFPE) tumor block from each patient. Clinical data were extracted from the institutional lung cancer registry of the Institute for Pulmonary Diseases. Overall survival was calculated as time from the day of surgery to the day of death. Disease free survival was calculated as time from the day of surgery to the day of disease relapse. Among 161 tested tumor tissue, presence of mutation was found in 96 (59.6%) of them. There were 69 (42.9%) mutations in KRAS gene, 10 (6.2%) in EGFR gene and 7 (4.3%) in PIK3CA gene. ALK and ROS1 rearrangement were present in 3 (1.9%) and 7 (4.3%), respectively. PD-1 expression was determined in 71 (45.0%) tumor sample while PD-L1 expression was determined in 59 (36.6%). PD-1 expression was not correlated with any of the clinicopathologial characteristics (including KRAS, EGFR, ALK, ROS1 and PIK3CA mutational status). PD-L1 expression correlated with type of surgery (P = 0.01) and KRAS positivity (P = 0.02). KRAS mutation status correlated with age (P = 0.004), sex (P = 0.006) and smoking status (P = 0.004). EGFR status correlated with smoking status (P < 0.001) and age (P = 0.013). ALK, ROS1 and PIK3CA status were not correlated with any of the clinicopathologial characteristics. PD-1expression was significantly associated with disease free survival (P = 0.03) and overall survival (P = 0.01). PD-L1 expression, KRAS, EGFR, ALK, ROS1 and PIK3CA status were not associated with disease free survival and overall survival. The most frequent gene alteration are mutations in KRAS and EGFR gene. Presence of KRAS mutation is in correlation with patients age, sex and smoking status while presence of EGFR mutation is in correlation with patients age and smoking status. PD-L1 expression is in correlation with type of surgery and KRAS mutational status. Only presence of PD-1 expression represent an independent prognostic factor for disease free survival and overall survival.

Ugljenične nanocevi (UNC) imaju sve veću primenu u elektronici, kompjuterskoj i optičkoj industriji, kao i u biomedicini. Dok proizvodnja jednoslojnih UNC nanocevi beleži sve veći rast poslednjih godina, rizik koji nosi izlaganje ovom nanomaterijalu ostaje nerazjašnjen. Oskudni i često kontradiktorni podaci o toksičnim efektima jednoslojnih UNC ukazuju na potrebu za daljim ispitivanjima. U našim istraživanjima ispitivane su promene u ćelijskom odgovoru kao i morfološke promene nakon delovanja jednoslojnih ugljeničnih nanocevi na ćelijskoj liniji humanih fetalnih fibroblasta pluća MRC-5 i ćelijskoj liniji humanog adenokarcinoma pluća A549. U ovoj studiji korišćene su jednoslojne ugljenične nanocevi koje su sadržale rezidualne nečistoće poput gvožđa. Citotoksičnost jednoslojnih UNC (engl. single-walled carbon nanotubes – SWCNT) je ispitivana kolorimetrijskim MTT testom. Tokom 24 i 48h niske koncentracije jednoslojnih ugljeničnih nanocevi (<250 μg/mL) pokazale su nisku toksičnost na proliferaciju i vijabilnost u obe ispitivane ćelijske linije. Ipak, pri visokim koncentracijama UNC (250-750 μg/mL) antiproliferativni efekat je bio blizu IC50 vrednostima. Na osnovu rezultata dobijenih MTT testom može se zaključiti da su maligne A549 ćelije osetljivije na delovanje jednoslojnih UNC u odnosu na normalne MRC-5 ćelije. Kombinacija ugljeničnih nanocevi sa prirodnim polifenolima (resveratrolom i proantocijanidolima) nije značajno uticala na citotoksičnost u MRC-5 ćelijama, za razliku od A549 ćelija gde je tretman kombinacijama umanjio toksičnost ugljeničnih nanocevi. Transmisionom elektronskom mikroskopijom ispitivan je efekat jednoslojnih ugljeničnih nanocevi na ćelijsku morfologiju i preživljavanje. Intracelularni agregati ugljeničnih nanocevi primećeni su u obe ćelijske linije, čime je potvrđeno da ugljenične nanocevi ulaze u ćelije. Imajući u vidu da nanomaterijali poput ugljeničnih nanocevi indukuju oksidativni stres i njime posredovanu apoptozu, na protočnom citometru je određivano prisustvo ćelija u apoptozi i nekrozi. Tretman ćelija sa jednoslojnim ugljeničnim nanocevima nije doveo do značajnog porasta broja apoptotskih ili nekrotičnih ćelija, što ide u prilog niskoj toksičnosti ovog nanomaterijala, odnosno ukazuje na alternativne mehanizme toksičnosti. Međutim kombinacija jednoslojnih ugljeničnih nanocevi sa antioksidantima, resveratrolom i proantocijanidolima indukuje veći procenat apoptoze i nekroze u odnosu na tretman samo sa nanocevima. Promene u ekspresiji gena praćene su lančanom reakcijom polimeraza (PCR). Komparativna analiza rezultata genske ekspresije MRC-5 i A549ćelija nakon tretmana sa jednoslojnim ugljeničnim nanocevima pojedinačno i u kombinaciji sa antioksidantima ukazala je na kompleksnost i raznolikost biološkog odgovora ispitivanih ćelija. U našem istraživanju ispitivana je i promena aktivnosti enzima antioksidativne zaštite i količine glutationa u ćeliji. Primena jednoslojnih ugljeničnih nanocevi u MRC-5 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD i GR, povećava specifičnu aktivnost GPx i ne utiče na promenu specifične aktivnosti GST i količine glutationa u ćeliji Primena jednoslojnih ugljeničnih nanocevi u A549 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD, ne utiče na promenu specifične aktivnost enzima GR, GST i GPx, i dovodi do povećanja količine glutationa u ćeliji. Ćelijska vijabilnost, morfološke promene, redoks homeostaza i ekspresija ispitivanih gena bile su promenjene nakon tretmana sa jednoslojnim ugljeničnim nanocevima. Iako su dobijeni rezultati značajni za procenu toksičnosti ugljeničnih nanocevi, neophodna su dalja istraživanja koja treba da doprinesu boljem razumevanju toksičnih efekata ugljeničnih nanocevi.
Carbon nanotubes are being actively introduced in electronics, computer science, and optics as well as for various biomedical applications. While production of single-walled carbon nanotubes (SWCNT) has escalated in recent years, the knowledge on risk associated with exposure remains unclear. Contradictory data on the toxic effects of single-walled carbon nanotubes highlights the urgent need for further studies. In this study we investigated the alterations in cellular response along with morphological changes induced by single-walled carbon nanotubes in human lung fibroblast cell line MRC-5 and adenocarcinoma human alveolar basal epithelial cells A549. In this study we used SWCNT containing large amounts of residual metallic impurities such is iron, and the iron concentration increased in dose dependent manner in cells exposed to SWCNT. Cytotoxicity was evaluated by MTT assay and SWCNT showed little cytotoxic effect on the proliferation and viability of two cell lines tested at the concentrations used (<250 μg/mL) within 24 and 48h. However exposing both cell lines to high concentrations (250-750 μg/mL) resulted in near IC50 values. Based on MTT test SWCNT were more cytotoxic to A549 cell line. Cytotoxicity of SWCNT in combination with natural polyphenols (resveratrol and proanthocyanidins) did not noticeably affect the cytotoxicity of SWCNT to MRC-5 cells. However introduction of polyphenols did reduce the cytotoxicity of SWCNT to A549 cells. Transmission electron microscopy was used to complement cytotoxicity assays and to investigate the pathological effect of internalized SWCNT on cell morphology and survival. Intracellular bundles of CNTs, possibly aggregated/agglomerated were observed in both cell lines, confirming internalization after 24h exposure. Since nanoparticles like carbon nanotubes are toxic mainly because they cause oxidative stress, often associated with an increased apoptosis we checked for apoptotic and necrotic cells using flow cytometry. Incubation with SWCNT did not result in pronounced apoptosis or necrosis supporting its low toxicity and possibly alternative mechanism of cell damage. However incubation with SWCNT in combination with resveratrol and proanthocyanidins induced higher levels of both apoptosis and necrosis than SWCNT alone. Changes in gene expression following exposure to SWCNT were evaluated by polymerase chain reaction PCR array which indicated complex and diverse change in expression of genes involved in apoptosis, cell proliferation and oxidative stress. Finally we investigated the modulation of the antioxidant enzyme system and the changes in the cytosolic levels of GSH. SWCNT reduced the specific activity of SOD and GR enzymes, increased GPx activity. No changes in intracellular levels of GSH were observed in MRC-5 cell line. Same treatment in A549 cell reduced the specific activity of SOD, had no effect on GR, GST and GPx activity, but increased intracellular levels of GSH. Cell viability, morphologic changes, redox homeostasis and gene expression were affected by the presence of SWCNT. Although our findings are useful in predicting human response against SWCNT exposure, further study is needed for better understanding of the effects of SWCNT.

Receptor epidermalnog faktora rasta (EGFR) pripada porodici receptora protein-tirozin kinaze čija je aktivacija povezana sa proliferacijom malignih, invazijom, inhibicijom apoptoze, tumorskom angiogenezom i metastatskim širenjem stoga ima važnu ulogu u karcinogenezi i tumorskoj progresiji. Aktivirane mutacije se odvijaju oko katalitičkog tirozin kinaza domena. Biopsijski, citološki i hirurški uzorci se koriste u detekciji EGFR mutacija u momentu postavljanja dijagnoze adenokarcinoma ili karcinoma sa komponentom adenokarcinoma, najpouzdanije lančanom reakcijom polimeraze. Činjenica da primena ciljane molekularne terapije tirozin kinaza inhibitorima kod obolelih sa EGFR mutiranim adenokarcinomom pluća poboljšava prognozu bolesti, postoji rezistencija kod pojedinih tipova EGFR mutacija i povezanost histopatološkim i imunohistohemijskim karakteristikama tumora, da je bronhološki uzorak često jedini uzorak u kome je potrebno odrediti i molekularni profil tumora osnovni cilj ove disertacije bio je da se odredi učestalost i tip EGFR mutacija i povezanost sa karakteristikama adenokarcinoma. Da bi se taj cilj realizovao postavljeni su sekundarni ciljevi odnosno da se: izvrši histopatološka reklasifikacija adenokarcinoma pluća na osnovu kriterijuma koje je postavila internacionalna asocijacija za proučavanje carcinoma pluća, američko torakalno društvo i evropsko respiratorno društvo; odredi ekspresija TTF-1 u adenokarcinomu pluća i povezanost sa EGFR mutacionim statusom; odredi učestalost, tip i povezanost EGFR mutacija sa predominantnim tipom adenokarcinoma i utvrdi da li bronhoskopska biopsija može da bude reprezentativni uzorak za određivanje EGFR mutacionog statusa. Histopatološka građa adenokarcinoma pluća u hirurškim uzorcima je heterogenija u odnosu na biopsijske uzorke i ta razlika je statistički značajna (p<0,001). Acinarno predominantni tip je najzastupljeniji u hirurškim i biopsijskim uzorcima bez statistički značajne razlike u raspodeli predominantnih tipova u njima (p=0,65883). Predominantni tip u primarnom tumoru određuje predominantni tip u limfogenim metastazama. EGFR mutacije tipa insercija na egzonu 21 i L858R mutacija na egzonu 20 su detektovane kod tri od 60 (5%) bolesnika u pet od 120 uzoraka (tri hirurška i dva biopsijska uzorka), češće kod žena, starijih od 60 godina, pušača i u solidno predominantnom tipu. Ne postoji statistički značajna razlika u koncentraciji izolovane DNK između EGFR mutiranih i wt EGFR adenokarcinoma u biopsijskim (p=0,132) i hirurškim uzorcima (p=0,641). Procenat invalidnih rezultata prilikom određivanja EGFR mutacionog statusa u je veći u biopsijskim uzorcima u odnosu na hirurške uzorke. Postoji statistički značajna razlika izmeĐu broja TTF-1 pozitivnih i TTF-1 negativnih adenokarcinoma (p<0,001), ali ne i u raspodeli ovih bolesnika prema polovima (p=0,1231), prosečnoj starosti, pušačkim navikama (p=0,6488) i prosečnoj veličini tumora (p=0,21). Postoji pozitivna korelacija između TTF-1 pozitivne ekspresije i EGFR mutacionog statusa stoga TTF-1 pozitivna ekspresija može da bude prediktor pozitivnog EGFR mutacionog statusa. Bronhoskopska biopsija je reprezentativni uzorak za određivanje EGFR mutacionog statusa zato što: većina dijagnostičkih biopsijskih uzoraka ima više od 100 očuvanih tumorskih ćelija, nema razlike u raspodeli predominantnih tipova u odnosu na hirurške uzorke, EGFR mutacije se detektuju u uzorcima sa manje od 100 tumorskih ćelija i manje od 20% volumenske gustine tumorskog tkiva, razlika između koncentracije izolovane DNK u EGFR mutiranim i wt EGFR adenokarcinomima u biopsijskim i hirurškim uzorcima nije statistički značajna (p=0,132 i p=0,641).
Epidermal growth factor receptor (EGFR) belongs to the family of protein-tyrosin kinase family, whose activation is associated with the proliferation of malignant cells, invasion, inhibition of apoptosis, tumor angiogenesis and metastatic spread and thus plays an important role in carcinogenesis and tumor progression. Activated mutations take place around the catalytic tyrosine kinase domain. Biopsy, cytological and surgical specimens are used for the detection of EGFR mutations at the time of diagnosis of adenocarcinoma or carcinoma with an adenocarcinoma component, most reliably using a polymerase chain reaction. The fact that the application of molecular tyrosin kinase inhibitor therapy to patients with EGFR mutated lung adenocarcinoma improves the prognosis of the disease, there is resistance in certain types of EGFR mutations and connection with histopathological and immunohistochemical characteristics of tumor, that the bronchoscopic specimen is often the only specimen in which it is necessary to determine the molecular profile of the tumor, the primary objective of this thesis is to determine the frequency and type of EGFR mutations and their connection with the characteristics of adenocarcinoma. In order to realize this objective, the following secondary objectives have been set: to execute histopathological reclassification of lung adenocarcinoma based on the criteria set by the International Association for the Study of Lung Cancer, the American Thoracic Society and European Respiratory Society; determine the expression of TTF-1 in lung adenocarcinoma and connection with EGFR mutation status; determine the frequency, type and connection of EGFR mutations with predominant type of adenocarcinoma and confirm whether bronchoscopic biopsy may be a representative specimen for the determination of EGFR mutation status. Histopathological material of lung adenocarcinoma in surgical specimens is more heterogeneous in relation to biopsy specimens and such difference is statistically significant (p<0,001). Acinar predominant type is the most common in surgical and biopsy specimens with no statistically significant differences in the distribution of predominant type among them (p=0,65883). The predominant type in the primary tumor determines the predominant type in lymphatic metastases. EGFR mutations in the type of insertions on exon 21 and L858R mutations on exon 20 have been detected in three out of 60 (5%) of patients in five out of 120 specimens (three surgical and two biopsy samples), more often in women older than 60, smokers and in a solid predominant type. There are no statistically significant differences in the concentration of isolated DNA between EGFR mutated and wt EGFR adenocarcinoma in biopsy (p=0,132) and surgical specimens (p=0,641). The percentage of invalid results in determining the EGFR mutation status is higher in biopsy specimens compared to the surgical specimens. There is a statistically significant difference between the number of TTF-1 positive and TTF-1 negative adenocarcinoma (p<0,001), but not in the distribution of these patients according to gender (p=0,1231), average age, smoking habits (p=0,6488) and average tumor size (p-0,21). There is a positive correlation between TTF-1 positive expression and EGFR mutation status and therefore TTF-1 positive expression can be a predictor of positive EGFR mutation status. Bronchoscopic biopsy is a representative sample for the determination of EGFR mutation status because: most diagnostic biopsy specimens have more than 100 preserved tumor cells, there is no difference in the distribution of predominant types in relation to surgical specimens, EGFR mutations are detected in samples with less than 100 tumor cells and less than 20% of volume density of tumor tissue, the difference between the concentration of isolated DNA in EGFR mutated and wt EGFR adenocarcinoma in biopsy and surgical specimens is not statistically significant (p=0,132 and p=0,641).

Classification of adenocarcinoma prostatae is very important for the treatment. I have described the disease, its treatment and classification in the introduction. I have analysed a sample of 52 patients from the Urological Clinic of General Faculty Hospital. Simple statistics was used for comparing and analysing of relation of preoperative classification and postoperative development of PSA. I have compared preoperative and operative Gleason's score. Preoperative Gleason score underscores the operative score. PSA decrease is significantly higher in patients with high initial PSA and in patients with classification T2N0M0. PSA one month after surgery does not differ in patients with classification T1C and T2 and it does not correlate with the preoperative characteristics of patients. The decrease of PSA is perhaps dependent only on the surgical treatment. Effect of operation cannot be predicted by Gleason score and by age. These results are valid only for patients undergoing radical prostractomy

Aim of the study: The aim of this retrospective study was to determine the prognostic impact of epidermal growth factor receptor (EGFR) expression changes during neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer. Material and methods:50 patients with with locally advanced rectal cancer were evaluated. All the patients were administered the total dose of 44 Gy. Capecitabine has been concomitantly administered in the dose 825 mg/m2 in two daily oral administrations. Surgery was indicated 4-8 weeks from the chemoradiotherapy completion. EGFR expression in the pretreatment biopsies and in the resected specimens was assessed with immunohistochemistry. Results:All of 50 patients received radiotherapy without interruption up to the total planned dose. In 30 patients sphincter-saving surgery was performed, 20 patients underwent amputation of the rectum. Downstaging was described in 30 patients. 4 patients have had complete pathologic remission. 26 patients have had partial remission, the disease was stable in 15 patients. Progression was reported in 5 patients. The median disease-free survival was 64.9 months, median overall survival was 76.4 months. Increased EGFR expression was found in 12 patients (26,1 %). A statistically significantly shorter overall survival (p < 0.0001) and...

This study examines the role of innate and adaptive immunity in the immunotherapy based on the combination of the ligands stimulating phagocytosis anchored in the tumour cells membrane and the mixture of TLR agonists. This immunotherapy is primarily focused on the innate immunity activation and induces strong inflammatory infiltration, which neutrophils and NK cells are part of. Therefore, the next aim of this study was to evaluate the anti-tumour activity of neutrophils and NK cells. For examination malignant melanoma and pancreatic adenocarcinoma mouse tumour models were used.

Prostate cancer is one of the most abundant types of cancer among men and the demand for a specific treatment is very high. In this thesis, I have focused on using Glutamate Carboxypepti- dase II (GCPII), as a target for a proof-of-principle delivery system. GCPII is a transmembrane protein that internalizes after a binding of a ligand and is overexpressed in prostate cancer. Virus-like particles from Murine polyomavirus (VLPs) are a suitable nanocarrier for the delivery of imaging agents and drugs. Here I describe modifying these VLPs with inhibitors of GCPII and fluorescent dyes and characterize their binding to GCPII on surface plasmon resonance and to cells expressing GCPII on confocal microscopy. VLPs carrying a GCPII inhibitor show specific binding to GCPII on surface plasmon reso- nance, however they bind non-specifically to cells that don't express GCPII. Several approaches have been tried to avoid that. The substitution of BC loop on the exterior surface of VLPs that is partially responsible for the binding of sialic acid did not seem to affect specificity on cells. Another approach tested was coating of the wild-type VLPs with large polymer carrying a flu- orescent label and a GCPII inhibitor. After the conjugation of the polymer to the VLP, specific binding and internalization in GCPII-positive...

The aim of this thesis is to improve the therapeutic effect of the immunotherapy based on the synergy of TLR agonists with phagocytosis stimulating ligands. Furthermore, this thesis is focused on the information transfer to the specific immunity, as well as it pursues the study of the specific immunity relevance during cancer immunotherapy.

The aim of this thesis was to study how to increase effectiveness of cancer immunotherapy based on synergy of compounds stimulating phagocytosis and TLR agonist. Tumor microenvironment was modified by enzymes, which catalised conversion of lactate to pyruvate or acetate. It was monitored effect of enzymes on tumor size, survival of experimental mice and cytotoxicity on tumor cells.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Mgr. Lucie Jirásková Supervisor: doc. PharmDr. Lukáš Červený, Ph.D. Title of doctoral thesis: Interactions of membrane transporters with drugs in the placenta and pancreatic ductal adenocarcinoma Membrane transporters are found throughout the body, where they are responsible for many vital functions. Important representatives of membrane transporters are P-glycoprotein (ABCB1), Breast cancer resistance protein (ABCG2) and multidrug resistance-associated protein 2 (ABCC2) belonging to the ATP-biding Cassette (ABC) family. Nucleoside transporters belonging to Solute Carriers family (SLC) transporters represent another important group. It has been well evidenced that these transporters also affect drug disposition and contribute to tumor resistance to anticancer therapy. Over working on this dissertation thesis, we investigated the mentioned transporters (with special focus on nucleoside transporters) in complex fashion. We described the expression profile of nucleoside transporters in the placenta at different stages of gestation. We also examined whether the expression of nucleoside transporters changes depending on the degree of differentiation or can be affected epigenetically, and we...

Ductal adenocarcinoma (PDAC) is the most common primary pancreatic neoplasm. It's frequently diagnosed in late inoperable stage and has high resistance to chemotherapy; this situation contributes to its unfavourable prognosis. Therefore, there is a need for biomarkers enabling early detection of PDACs and by this way to improve patient prognosis. MicroRNAs (miRNA), short non-coding RNA molecules involved in post-transcriptional regulation of gene expression, belong to such markers. Contrary to other RNA molecules, miRNAs are stable in biological samples. Their expression is measured by several analytical methods, including real-time quantitative PCR (RT-qPCR). Normalization of methods determining miRNA levels requires adequate endogenous controls. However, variable expression of endogenous controls in tumors may cause bias in determining miRNA levels. The aim of the first study was to investigate the expression of six miRNAs isolated from formalin fixed paraffin embedded (FFPE) samples of PDACs. Four controls were chosen for RT-qPCR result normalization: artificial spike miR-39 from C. elegans, U6 snRNA, miR-16 and snoRNA U91. Expression values of all studied miRNAs in tumors were significantly different depending on selected endogenous controls. Additionally, stability of the controls varied...

Úvod: Karcinom pankreatu patří mezi nádorová onemocnění s nejhorší prognózou. Vzhledem к lokalizaci pankreatu v retroperitoneu i dalším anatomickým a patofyziologickým souvislostem se příznaky karcinomu slinivky objevují velmi pozdě a u většiny nemocných již není možné zahájit radikální léčbu. Z těchto důvodů jen 5% nemocných přežívá 5 let od stanovení diagnózy. Většinou postihuje nemocné mezi 30-70 lety, přičemž průměrný věk v době stanovení diagnózy je přibližně 56 let. V posledním desetiletí se dostává do popředí zájmu tzv. DNA microarray technologie umožňující monitorování exprese genů. Exprese genů představuje komplexní proces, kterým je genetická informace uložená ve formě deoxyribonukleové kyseliny (DNA) přeměněna v konkrétní buněčné struktury a metabolické nástroje. Produkty exprese genů jsou molekuly bílkovin, jsou jimi však i ribonukleové kyseliny (RNA). Statiny (inhibitory HMG-CoA reduktázy) patří mezi klíčové léky v léčbě hypercholesterolémie. Z výzkumných studií vyplývá, že tato skupina léků má velmi významné protinádorové vlastnosti. Inhibicí HMGCoA reduktázy, klíčového enzymu v syntéze cholesterolu, totiž dochází také к depleci jednotlivých meziproduktů v syntéze cholesterolu, z nichž nejvýznamnější je farnesyl pyrofosfát hrající důležitou roli v buněčné signalizaci ovlivňující apoptózu....

Key words: adenocarcinoma of the esophagus and esophagogastric junction, neoadjuvant chemotherapy, PET/CT, histopathological response, technique of esophagogastric anastomosis, anastomotic leak, anastomotic stricture Previous studies have shown that preoperative chemotherapy of locally advanced AEG is beneficial only for patients with a good histopathological response, the so-called responders. The aim of the first part of the thesis was to prospectively verify whether positron emission tomography (PET/CT) could be used for early identification of histopathological non- responders, who could be spared ineffective neoadjuvant treatment. Our study did not prove that the early metabolic response, expressed as the percentage change of the consumption of glucosis on PET/CT performed before (PET1) and 12 to 22 days after the start of the first cycle of preoperative chemotherapy (PET2) correlated with the histopathological response in the resection specimen in the entire population of 90 patients. In a post hoc explorative analysis we found the correlation between metabolic and histopathological response in a subgroup of patients with PET2 performed ≤16 days after the start of the therapy, but this hypothesis needs to be prospectively validated. Our study suggests that PET/CT performed after the first...

Rectum adenocarcinoma is one of the most frequent tumour diseases in the Czech Republic. Therefore any progress in therapy or prevention of this disease can have a significant impact on mortality and morbidity of major amount of oncological patients. Within therapy the neoadjuvant concomitant radiochemotherapy or the sole radiotherapy is used before surgical resection in one third to half of all newly diagnosed cases. The goal is to achieve tumor downstaging and local relaps probability decrease. There is a large amount of processes on molecular and cellular level in organism reaction to neoplasia genesis, whose understanding can have a substantial impact on therapy's success rate or on therapy's efficiency prediction. One of these processes is angiogenesis induced by high production of vascular endothelial growth factor (VEGF). As per immunohistochemical studies VEGF is not expressed in typical colorectum mucous membrane. However it is strongly expressed in case of adenocarcinoma. Another important process of organism's malignancy response is tumour tissue infiltration by CD8+ T lymphocytes (TIL), which are one of the important components of cellular immunity against tumour and which significantly contribute to tumour cells elimination. Potential of CD8+ T lymphocytes is considerable not just in...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Magdaléna Kozlíková Supervisor: RNDr. Miloslav Macháček, PhD. Title of diploma thesis: Development of 3D spheroid cultures derived from human breast adenocarcinoma cell line MCF-7 and murine colon carcinoma cell line CT-26 In recent decades, photodynamic therapy (PDT) has become a topic of intensive research in the context of experimental models of tumors, especially for the treatment of solid tumors. The study mainly focuses on research of novel photosensitizers (PS), which should provide better efficacy, higher tumor specificity and lower toxicity to non- malignant tissue. PDT is based on the interaction of PS, the presence of molecular oxygen, and light, leading to the formation of reactive oxygen species causing damage of tumor tissue. 3D tumor spheroids should provide a better tool for the study of tumor microenvironment, anticancer drugs, or therapeutic approaches. In contrast to 2D cell cultures, they are mimicking in vivo-like cell-cell and cell-matrix interactions. In our work, we studied the cytotoxicity of original phthalocyanine PSs designated P40, P44, ZIP300 and the clinically used compound PhotoSens® on 3D cell cultures derived from CT-26 and MCF-7 cell lines generated using an...