Made available in DSpace on 2014-07-29T15:07:47Z (GMT). No. of bitstreams: 1 Dissertacao_Gabriela_Uchoa.pdf: 897498 bytes, checksum: 9761f9b1b5b6c7f028d1d5674bbaa4d8 (MD5) Previous issue date: 2009-02-06 / Urinary disorders are an important discovery in the ileocystoplasties, especially uroliths and bladder crystals, and are partially related to the kind of material used the in suture itself. The purpose of this study is to verify if there are differences regarding the formation of uroliths or bladder crystals after dogs ileocystoplasties practices involving sutures using unabsorbed titanium staples and sutures with polyglatine 910, observing the impact of the procedure in the renal function, modifications in the urinary constituents and differences in the surgery time duration between the groups. For that, two experimental groups of animals, each one with six, have been considered. In one group it has been used a polyglactin 910 suture thread (group A) and in the other, a titanium stapler (group B). In each animal of both groups there was selected a terminal ileac segment with approximately 5 cm for bladder augmentation. In group A, a suture of the detubularized ileum segment in bladder was executed using a polyglactin 910. In group B, the bladder augmentation with the selected ileac segment was done by linear cutter stapler using titanium staples. It was observed the presence of struvite crystals in both groups, in 11 animals of the experiment. In group A the operation duration was longer if compared to group B . It was also observed the formation of urinary mucus in great quantity in all animals in the post-operation first days. It was confirmed the formation of calculi in two animals, one in each experimental group, but in the group A animal the calculi was free from lumen and in the group B animal the stone was adhered to the stapling zone, attached to a staple that got exposed to direct contact with the urine. In the parameters verified in the urinalysis, urea and seric creatinine there was no sign of renal alteration and in the verification of blood count, no alterations were noticed or considered significant. It was possible to conclude that no significant differences were observed between the groups as for the formation of urinary stones and crystals in ileocystoplasties after 100 days. If compared to group B , Group A presented a longer operation and there were not evidences of alteration in the renal function in any phase of the experiment in both groups / Alterações urinárias são um importante achado nas ileocistoplastias, sobretudo os urólitos e a cristalúria, e em parte estão relacionados ao tipo de material de sutura envolvido neste procedimento. O objetivo deste estudo foi verificar se existe diferença na formação de urólitos ou cristalóides urinários após ileocistoplastias em cães, realizadas por meio de sutura com grampos inabsorvíveis de titânio e sutura com poliglactina 910, observando o impacto do procedimento na função renal, alterações nos constituintes urinários e a diferença de tempo cirúrgico entre os grupos. Utilizou-se dois grupos experimentais com seis animais cada, um com fio de poliglactina 910 para a sutura da ileocistoplastia (grupo A), e outro usando grampos de titânio (grupo B). Em cada animal foi selecionado um segmento de aproximadamente 5 cm de íleo terminal para ampliação vesical. No grupo A realizou-se a sutura do segmento ileal detubulizado na bexiga com fio de poliglactina 910. No grupo B foi realizada a ampliação vesical com o segmento ileal selecionado com auxílio de grampeador linear cortante para detubulização e sutura grampos de titânio. Observou-se a presença de cristais de estruvita em 11 animais de ambos os grupos do experimento. No grupo A o tempo operatório foi maior se comparado ao grupo B . Foi possível observar a formação de grande quantidade de muco na urina de todos os animais já nos primeiros dias de pós-operatório. Verificou-se a formação de cálculos em dois animais, um de cada grupo experimental, sendo que no grupo A o cálculo estava livre no lúmen e no grupo B o cálculo estava aderido à zona de grampeamento, ligado a um grampo em contato direto com a urina. Dentro dos parâmetros avaliados na urinálise, uréia e creatina séricas, não houve qualquer sinal de alteração renal e nas avaliações de hemograma não foram percebidas alterações significativas. Foi possível concluir que não houve diferenças significativas entre os grupos quanto à formação de cristais urinários e urólitos após 100 dias das ileocistoplastias, o grupo A apresentou maior tempo cirúrgico se comparado ao grupo B e não houve evidências de alteração na função renal em nenhuma fase do experimento em ambos os grupos.

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.:1. Introduction............................................................................ 1 1.1. Anatomy and histology of the human urinary bladder..................... 1 1.1.1. Anatomy of the human urinary bladder..................................... 1 1.1.2. Structure of the human urinary bladder wall............................... 2 1.2. Normal bladder function and bladder dysfunction.......................... 3 1.2.1 Normal bladder function......................................................... 3 1.2.2 Sensory aspect.................................................................... 4 1.2.3 Overactivity or hypersensitivity of bladder.................................. 5 1.3 The role of functional cell types and interaction in urinary bladder... 6 1.3.1 The role of urothelium.......................................................... 7 1.3.2Theroleofsuburotheliamyofibroblast...................................... 7 1.3.3Theroleofdetrusorsmoothmusclecells.................................. 9 1.3.4 Possible interactions in urinary bladder cell types........................ 10 1.4 ATP function and Purinergic signalling in bladder........................... 11 1.5 Spontaneous activity of bladder................................................... 13 2. Objective.................................................................................. 15 3. Material and methods............................................................... 16 3.1. Ethics Statement........................................................................ 16 3.2. Cell preparation.......................................................................... 16 3.3. Solutions and chemicals............................................................. 19 3.4. Intracellular calcium measurements............................................. 20 2.4.1. Preparing cells for Calcium Imaging.......................................... 20 2.4.2. Preparing workspace of calcium imaging................................... 20 2.4.3. Calcium imaging recording...................................................... 22 3.5 Data analysis with automated Fluorescence analysis..................... 22 3.6 Confocal Immunofluorescence.................................................... 25 3.7 Statistics................................................................................. 26 4. Results.................................................................................. 27 4.1 Spontaneous calcium activity of sMF........................................... 27 4.2 ATP effects on calcium response in sMF...................................... 27 4.3 Analysis of purinergic receptors involved.................................... 30 3.3.1 Agonist stimulation.............................................................. 30 3.3.2 Signal inhibition by specific antagonists................................... 31 4.4 Confocal immunofluorescence of purinergic receptors.................. 32 5. Discussion............................................................................. 34 5.1 Myofibroblast identification....................................................... 34 5.2 Spontaneous activity in the bladder............................................ 36 5.3 ATP modulated calcium activity in sMF....................................... 37 5.4 purinergic signalling in sMF........................................................ 39 6. Summary................................................................................ 42 7. References.............................................................................. 45 Declaration............................................................................. 50 Acknowledgements................................................................. 51

Karzinome der Gallenwege sind mit einer schlechten Prognose assoziiert. Eine potentiell kurative chirurgische Resektion ist bei der Mehrzahl der Patienten aufgrund des späten Zeitpunkts der Diagnosestellung nicht möglich, so dass derzeit vorrangig palliative Therapieansätze Anwendung finden. Das nur geringe Ansprechen auf konventionelle Radio- oder Zytostatikatherapie begründet die Notwendigkeit neuer Therapieansätze. Einen möglichen Angriffspunkt stellt hierbei die Polo-like-Kinase 1 (Plk1) dar, da ihre zentrale Rolle in der Regulation des Zellzyklus zunehmend erkannt und eine vermehrte Expression in malignem Tumorgewebe verglichen mit gesundem Gewebe nachgewiesen wurde. Das Dihydropteridinon BI 2536 ist ein poten-ter, niedermolekularer und selektiver Hemmstoff der Plk1 und sollte daher auf seine Wirksamkeit an Gallenwegskarzinomen untersucht werden. In der vorliegenden Arbeit konnte gezeigt werden, dass BI 2536 die untersuchten 14 Zelllinien von Gallenblasen- und Gallengangskarzinomen wirkungsvoll hemmt. Das Ansprechen unterschied sich zwischen den Zelllinien und ordnet sich vergleichbar zu Veröffentlichungen an anderen malignen Tumoren ein. Die Expression von Plk1 und dessen assoziierten Transkriptionsfaktor FoxM1 konnte bei Westernblot-Versuchen bei allen Zelllinien nachgewiesen werden, was eine Bedeutung in der Onkogenese vermuten lässt. Die Behandlung mit BI 2536 beeinflusste jedoch die Proteinmenge beider nicht. An für die folgenden Versuche ausgewählten drei Zelllinien zeigten sich in der reversen Transkription mit anschließender Echtzeit-Polymerase-Kettenreaktion (qRT PCR) ähnliche Ergebnisse in Bezug auf die exprimierte mRNA von Plk1. Westernblot-Analysen ermittelten keine signifikanten Veränderungen der an wichtigen intrazellulären Kaskaden beteiligten Proteine p42/44 und Akt sowie deren phosphorylierten Formen. Obwohl die Proteinmenge des Mitosemarkers Phospho-Histon H3 ebenso unverändert blieb, führte die Behandlung mit BI 2536 – dies zeigen Ergebnisse der Durchflusszytometrie – zu einer signifikanten, dosisabhängigen Zunahme der G2/M Fraktion des Zellzyklus und Zunahme der Apoptose-rate. Der maximale Hemmeffekt in der Behandlung von BI 2536 lag bei einer Inkubations-dauer von vier Tagen. Die Empfehlungen aus den klinischen Studien der Phase II von BI 2536 sowie dem Ziel der Vermeidung von Resistenzen ergibt sich die Notwendigkeit von Kombinationsversuchen mit Zytostatika, die in einer anderen Phase des Zellzyklus angreifen. Die in der Behandlung von Gallenwegskarzinomen etablierten Antimetaboliten 5-Fluorouracil und Gemcitabin wurden hierzu ausgewählt und es zeigten sich für 5 Fluorouracil synergistische, für Gemcitabin hingegen additive Kombinationseffekte. Zusätzlich wurde die Wechselwirkung mit dem IGF 1-Rezeptor-Inhibitor NVP-AEW541 untersucht, der ebenfalls einen neuen Behand-lungsansatz in der Krebstherapie darstellt und bei Gallenwegskarzinomen in vitro wirksam ist. Auch hier zeigen sich synergistische Effekte, die jedoch erst in höheren Behandlungs-dosen auftraten. Die Ergebnisse dieser Arbeit zeigen, dass die Hemmung der Plk1 bei Gallenblasen- und Gallengangskarzinomen einen wirksamen Behandlungsansatz darstellt. Auf der Grundlage der in dieser Arbeit beschriebenen Ergebnisse wird eine weitere präklinische und klinische Testung von selektiven Plk1-Hemmstoffen wie BI 2536 an Gallenwegskarzinomen empfohlen.:Vorbemerkung 1 Inhaltsverzeichnis 2 Abkürzungsverzeichnis 3 Bibliographische Beschreibung 4 1 Einleitung 5 1.1 Gallenwegskarzinome 5 1.1.1 Epidemiologie, Einteilung und Histologie 5 1.1.2 Klinische Symptomatik und Diagnostik 6 1.1.3 Therapie und Prognose 7 1.2 Polo like-Kinase 1 9 1.2.1 Entdeckung und Aufbau der Polo like-Kinasen 9 1.2.2 Funktionen 9 1.2.3 Regulation 11 1.2.4 Bedeutung in der Onkologie 12 1.2.5 Hemmung 12 1.3 BI 2536 14 1.4 Zielstellung der Arbeit 15 2 Publikation 17 3 Zusammenfassung 29 4 Summary 31 5 Literaturverzeichnis 32 6 Erklärung über die eigenständige Abfassung der Arbeit 45 7 Lebenslauf 46 8 Publikationen im Rahmen dieser Arbeit 48 9 Danksagung 49

This work presents the results ofour research of cell-free nucleic acids (cfNA). The first part shows changes in methylation patterns of immune response genes promoters that are detectable in plasma during the hemodialysis sessions and also differences in methylation between patients and healthy subjects. Alterations include genes that play their role in the regulation of hematopoiesis and these changes are in close relation with the need of anemia therapy. In the other plasma cfNA study we detected miRNA signatures in patients with acute myeloid leukemia at diagnosis (6 highly abundant miRNAs found) and in remission achieved after standard chemotherapy (trend to n01malization, lower levels ofthese miRNAs). Another part of work presents data from the study of potential non-invasive biomarker of bladder cancer. The amounts of cfDNA in urine are higher in patients than in healthy subjects and there were found 5 down-regulated miRNAs. Simultaneously it was established set of 30 miRNAs that are constantly present in urine supematants independently on sex, age and healthy status of subjects. The last part presents analysis ofcell-free fetal DNA. We analyzed differences between a new quantification method - droplet digital PCR and real-time PCR which is used routinely nowadays. Slightly more precise was...

<p> </p> <p>Learning about ‘behavior’ has always been at the heart of my research endeavors. While my undergraduate work in evolution and ecology exposed me to the science behind why a behavior exists, in my graduate work, I intended to explore how to use something’s behavior to widen its applicability. In this thesis, <em>I will present three works that utilize some of the fundamental</em></p> <p><em>behaviors (i.e., properties) of elastin-like polypeptides (ELP) to improve existing protein purification methods or explore their applicability in bladder cancer imaging and immunotherapy. </em></p> <p>Bladder cancer has high recurrence rates (60-70 % annually) that necessitate multiple follow-up therapies making it one of the costliest cancers per patient. In this work, we have attempted to address two leading causes of the recurrence. First is a low sensitivity (62-84 %) and variable specificity (43-95 %) of white light cystoscopy used to diagnose and remove tumors. We aimed to address the heart of this problem, i.e., the non-specific mode of detection using white light. Only the trained eyes can discern abnormal from normal-appearing tissues even then, leaving up to 45% of tumors unresected to colonize and spread. <em>We developed and characterized near infrared dye-peptide-ligand conjugates (NIR-ELP-ligand) that undergo receptor-mediated binding and internalization to human bladder cancer cells in vitro and tissues ex vivo.</em> By using a molecular target-based probe in combination with NIR imaging, we can aid in improving the detection limit via selective binding to the tumor and reduction in background autofluorescence.</p> <p>Bacillus-Calmette Guérin (BCG) instillation in the bladder is the gold-standard</p> <p>immunotherapy used after surgical removal of bladder tumors. This was approved as a response to the inefficiency of surgery alone in improving cancer status. It has succeeded by reducing the recurrence rate to 30-50 %. But it comes with the complications of putting a live mycobacterium</p> <p>in the human body and giving a patient a urinary tract infection right after surgical tumor resection. <em>Thus, we aimed to deliver nucleic acid as immunotherapeutic cargo in a selective manner to elicit robust anti-tumor immune responses while minimizing the side effects due to its carrier.</em> Towards</p> <p>this goal, we have developed a highly modular and adaptable ELP-ligand fusion protein-based nucleic acid delivery carrier targeted toward bladder cancer. Before developing targeted peptide-based cancer imaging and nucleic acid delivery modalities, we addressed the Achilles heel of peptide-based approaches. The peptide and protein industry suffers</p> <p>through complex, time-consuming, inconsistent, and low-yielding purification methods. <em>We have developed a scalable, facile, and reproducible protein purification method that delivers ELP and ELP fusion proteins free of host cell proteins and nucleic acids and has low lipopolysaccharide</em></p> <p><em>content in just 3 h starting from a bacterial pellet. </em>Thus, for a coherent narrative, the thesis is structured as follows:</p> <p>1. Introduction</p> <p>2. ELP as a protein purification tag: Development of a rapid purification method for ELPs and ELP fusion proteins.</p> <p>3. ELP as a cancer imaging agent: Development of NIR-ELP-Ligand imaging probe targeting bladder cancer.</p> <p>4. ELP as a drug delivery agent: Utilizing ELP-ligand fusion protein in the formulation of targeted nucleic acid delivery carrier to bladder cancer.</p>

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No / Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. (c)2012 AACR.

No / We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor alpha-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in gamma-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers.

No / Fibroblast growth factor receptors (FGFR) play key roles in proliferation, differentiation, and tumorigenesis. Many urothelial carcinomas contain activating point mutations or increased expression of FGFR3. However, little is known about the role of other FGFRs. We examined FGFR expression in telomerase-immortalized normal human urothelial cells, urothelial carcinoma cell lines, and tumor samples and showed that FGFR1 expression is increased in a high proportion of cell lines and tumors independent of stage and grade. To determine the role of FGFR1 in low-stage bladder cancer, we overexpressed FGFR1 in telomerase-immortalized normal human urothelial cells and examined changes in proliferation and cell survival in response to FGF2. FGFR1 stimulation increased proliferation and reduced apoptosis. To elucidate the mechanistic basis for these alterations, we examined the signaling cascades activated by FGFR1. FRS2alpha and PLCgamma were activated in response to FGF2, leading to activation of the mitogen-activated protein kinase pathway. The level of mitogen-activated protein kinase activation correlated with the level of cyclin D1, MCL1, and phospho-BAD, which also correlated with FGFR-induced proliferation and survival. Knockdown of FGFR1 in urothelial carcinoma cell lines revealed differential FGFR1 dependence. JMSU1 cells were dependent on FGFR1 expression for survival but three other cell lines were not. Two cell lines (JMSU1 and UMUC3) were dependent on FGFR1 for growth in soft agar. Only one of the cell lines tested (UMUC3) was frankly tumorigenic; here, FGFR1 knockdown inhibited tumor growth. Our results indicate that FGFR1 has significant effects on urothelial cell phenotype and may represent a useful therapeutic target in some cases of urothelial carcinoma.