Tesis sobre el tema "Diagnosis and monitoring of bladder cancer"

Mestrado em Bioquímica
O Carcinoma da Bexiga é uma doença maligna com extremas implicações físicas e psicológicas para os pacientes e de elevadas repercussões socioeconómicas. A falta de procedimentos de diagnóstico precoce não-invasivos tem permitido que a sobrevivência destes pacientes tenha permanecido inalterada nos últimos 30 anos. Desta forma, biomarcadores para diagnóstico não-invasivo são urgentemente necessários, e amostras de urina representam o meio mais promissor para alcançar este fim. Contudo, apesar de várias tentativas, ensaios imunológicos realizados em amostras de urina demonstram fraca performance clínica e analítica. Single/Multiple Reaction Monitoring (SRM/MRM) é uma técnica de espectrometria de massa para quantificação exata e absoluta. SRM/MRM representa a alternativa mais promissora para efeitos de quantificação, sendo altamente reprodutível, sensível e robusta. Desta forma, objetivou-se o desenvolvimento de ensaios por SRM/MRM para quantificação de biomarcadores de cancro da bexiga na urina, combinando múltiplos marcadores num classificador unificador. O ensaio MRM desenvolvido demonstrou em exatidão e especificidade equiparável ou superior aos ensaios imunológicos até á data disponível. Combinando SLIT2, PROF1, SPRC e NMP22 num classificador baseado em 4 marcadores resultou em performance clínica comparável (~70% sensibilidade e ~100% especificidade ou ~80% sensibilidade e ~57% especificidade) quando comparado com os ensaios convencionais. Contudo, a quantificação livre de interferências não pode ser assegurada devido a efeitos da matriz. Um método eficiente e reprodutível para remover substâncias contaminantes presentes na urina sem comprometer a deteção dos marcadores em causa é necessária para atenuar os efeitos de matriz.
Bladder cancer is a malignant disease with extreme physical and psychological implications for the patients together with major economic societal costs. The lack of early non-­‐invasive diagnostic procedures has allowed survival outcomes to remain unaltered for the past 30 years. Accordingly, non-­‐invasive diagnostic biomarkers are urgently needed, and urine samples represent the most promising means for non-­‐invasive bladder cancer diagnosis. However, despite several encouraging claims, available immuno-­‐based molecular assays display poor analytical and clinical performance in urine samples. Single/Multiple Reaction Monitoring (SRM/MRM) is a high-­‐performance mass spectrometry scanning mode for precise targeted quantification. SRM/MRM represents the most promising approach for biomarker quantification purposes, as it is highly reproducible, sensitive and robust. The main aim of this thesis was thus to develop a SRM/MRM-­‐based assay for bladder cancer urinary biomarker quantification, combining multiple markers into a unifying classifier. In addition, two independent chapters have been dedicated to i) the value of urine proteomics for disease diagnostics and to ii) the burden of the disease together with available tools for its diagnosis in the form of a literature meta-­‐analysis and book chapter, respectively. At the individual biomarker level, the MRM assay herein developed for urine profiling provided comparable-­‐to-­‐superior accuracy and specificity as comparedwhen to ELISA assays. The combination of SLIT2, PROF1, SPRC and NMP22 in a 4-­‐marker classifier resulted in comparable-­‐to-­‐superior clinical performance (~70% sensitivity with ~100% specificity ~80% sensitivity with ~57% specificity) over conventional immuno-­‐based assays. However, interference-­‐free measurements still could not be assured due to urinary matrix effects. A cost-­‐efficient and reproducible method for the removal of unidentified urinary contaminating substances without compromising the signal for the sought biomarkers is required in order to counteract urinary matrix effects.

Le diagnostic initial et la surveillance du cancer de la vessie reposent essentiellement sur la cystoscopie, un examen invasif, combinée à une cytologie urinaire dont la sensibilité est faible pour les stades précoces de ce cancer. La recherche de biomarqueurs à partir de l'urine répond au besoin de mettre au point des tests non invasifs, avec une sensibilité améliorée par rapport à la pratique actuelle. Malgré de nombreux développements, aucun test urinaire n'est actuellement recommandé pour une utilisation en routine clinique, pour des raisons de complexité d’utilisation, de performances ou de coût. L’analyse vibrationnelle des urines, par spectroscopie d’absorption infrarouge, est une approche intéressante pour la mise en place d’un test urinaire simple d’utilisation, relativement peu couteux et applicable en pratique clinique. Dans ce travail de thèse, les performances diagnostiques de cette technique, combinée à des outils d’apprentissage automatique, ont été évaluées à partir de prélèvements urinaires provenant de patients consultant dans le service d'Urologie du CHU de Reims. Malgré la forte variabilité spectrale des échantillons d'urine, l'optimisation combinée des prétraitements spectraux et des paramètres propres aux modèles de classification permet d'obtenir des résultats encourageants. Les développements algorithmiques ont été conçus pour pourvoir prendre en compte les données cliniques ; ce qui pourrait constituer une voie potentielle d'amélioration dans le cadre de futures investigations
Initial diagnosis and monitoring of bladder cancer is mainly based on cystoscopy, an invasive examination combined with urine cytology, which has limited sensitivity, especially in the early stages of this cancer. The need for non-invasive tests with improved sensitivity has led to the exploration of urine-based biomarker testing. Despite numerous advancements, no urine-based test is currently recommended for routine clinical use due to the complexity of use, performance, or cost. Vibrational analysis of urine using infrared absorption spectroscopy is an interesting approach for developing an easy-to-use, relatively inexpensive, and clinically applicable urine test. In this thesis, the diagnostic performances of this technique, combined with machine learning tools, were evaluated using urine samples from patients consulting the Urology Department of the Reims University Hospital. Despite the high spectral variability of urine samples, the combined optimization of spectral pretreatments and classification model parameters yielded promising results. Meanwhile, algorithmic developments have been developed to include clinical data, offering a way to improve the performance of these techniques in future investigations

Bladder cancer is an important global disease. The gold standard for diagnosis is cystoscopy and biopsy; both are invasive and require highly trained personnel. In a majority of cases, treated patients are followed up by frequent cystoscopies lasting several years. The discovery of biomarkers indicating which individuals should proceed to cystoscopy would be an important addition to bladder cancer management. The odour of urine is produced by volatile organic compounds, (VOCs), detectable by gas chromatography and mass spectrometry (GC-MS). An analysis of the VOCs in urine from various groups of individuals including patients with bladder cancer is undertaken in search of possible discriminating compounds, which could be harnessed in future as a potential screening tool or adjunct in bladder cancer management. Methods First void urine was obtained from 64 patients with new non-muscle invasive bladder cancer, 71 cancer free patients with haematuria and 51 asymptomatic volunteers. After equilibration, the headspace above these pH adjusted urine samples was extracted for 20minutes, using a carboxen / polydimethylsiloxane solid phase micro-extraction fibre (SPME). This was followed by desorption and VOC identification by GC-MS. Results Urine headspace VOCs under acidic conditions, (pH of modified urine 2), were found to be discriminating. Identified compounds were analyzed using forward stepwise discriminant analysis: 9 VOCs when used together, gave 84.7% correct classification of samples (Haematuria control v Bladder cancer) with no change on cross validation of results. The calculated sensitivity and specificity of this model is 76.6% and 92.9% respectively, with Positive predictive value of 90.7% and Negative predictive value of 92.9%. These results are comparable, and in some cases better than those obtained using commercially available urinary bladder cancer biomarkers. Conclusion Volatile organic compounds in urinary head space change with the development of bladder cancer. Urinary VOCs are exciting novel potential biomarkers in the detection of bladder cancer.

The purpose of this study is to investigate whether a prolonged delay in diagnosis of bladder cancer will result in worse outcomes for those patients, compared to those patients with a shorter diagnostic time interval. Data was collected on 247 patients newly diagnosed with transitional cell carcinoma of the bladder from January 1996 to December 2006 (10 years). The medical records of these patients were reviewed for demographics, pathological stage, date of consultation to the genitourinary (GU) service, and date of diagnosis by transurethral resection of bladder tumor (TURBT). The specialty delay was calculated as the time between the date of consultation to the GU service to the establishment of a diagnosis by TURBT. Univariate analyses were performed to test the association of specialty delay with clinical features and all-cause mortality. The median specialty delay in this study was 100 days. There was a trend towards a longer specialty delay for muscle-invasive disease (T2-T4) in comparison to superficial disease (Ta and T1). There was a significant correlation between all-cause mortality and increasing clinical stage (p=0.01). There was a paradoxical finding that patients with a specialty delay greater than 100 days had a significant reduction in all-cause death in comparison to patients with a specialty delay of 100 days or less (relative risk=0.59; 95% CI 0.36-0.90; p=0.01). In conclusion, this study did not confirm the hypothesis that a prolonged specialty delay in patients diagnosed with bladder cancer would result in a worse prognosis. In fact, there was a paradoxical finding that patients with a specialty delay greater than the median delay of 100 days had a better prognosis.

Bladder cancer is among the most common cancers in the UK, responsible for significant patient morbidity. Current techniques for detection suffer from low sensitivity, particularly for early stage disease, therefore new techniques are urgently sought. Among the suggested techniques to augment bladder cancer detection is the use of autofluorescence spectroscopy. Autofluorescence arises from a number of molecules in human tissue, giving a wealth of structural and metabolic information. Autofluorescence spectroscopy has previously been applied to the detection of a wide range of cancers, however clinical implementation of the technique to bladder cancer diagnosis is inhibited by a poor understanding of the contributions of individual fluorophores to autofluorescence. I sought primarily to use the multi-functional laser based diagnostic system “LAKK-M” to study the autofluorescence profiles of bladder cancer at the cell and tissue level, with the aim of developing a better understanding of bladder autofluorescence characteristics in health and disease. The significant findings of this research are threefold: 1. Autofluorescence flow cytometry of cell optical redox ratio reveals metabolic abnormalities in bladder cancer cells, specifically a glycolytic switch in bladder cancer cells culminating in an increased optical redox (NADH/flavin, ex360em425-475/ex488em515/545) ratio relative to healthy bladder cells. 2. Lab grown bladder cancer organoids show progressive changes in autofluorescence ratios relative to control samples – specifically reductions in the NADH/flavin (ex365em490/ex365em550), elastin/NADH (ex365em450/ex365em490) and elastin/flavin ratio (ex365em450/ex365em550), suggestive of structural and metabolic changes in developing cancer. 3. Analysis of human bladder tissue reveals significant differences in key fluorophores and diagnostic ratios between healthy and cancer tissue, amounting to increased porphyrin fluorescence and a decreased optical redox ratio (ex365em490/ex365em550) in cancer tissue compared to healthy control. These findings better inform our understanding of the autofluorescence properties of the bladder in health and disease at both the cell and tissue level, contributing to future development of diagnostic techniques. Additionally, in this thesis, I discuss the diagnostic worth of collagen analysis in bladder cancer using second harmonic generation imaging, the application of bladder tissue computer simulation to better elucidate fluorophore properties, and progress in novel laser therapy techniques for bladder cancer. The ultimate goal of this research is the development of a combined laser-based system for bladder cancer diagnosis and therapy.

Background: At the time of diagnosis, approximately 75% of all bladder cancers are Non-Muscle Invasive Bladder Cancers (NMIBC) - the standard treatment for these cancers is a Transurethral Resection of the Bladder Tumour (TURBT). Although, the vast majority of these cancers are not life-threatening, they have a high risk of recurrence (and progression, particularly in higher risk NMIBC), despite the use of adjuvant intravesical chemotherapy. Consequently, patients are kept on long term cystoscopic surveillance with endoscopic removal if recurrences are detected - this impacts on patients' quality of life and contributes to the high cost for the healthcare provider. Aims: The fundamental aim of this series of clinical studies, spanning 12 years, was to identify and implement, means of improving the efficiency in both processing and operating on patients with NMIBC to not only reduce recurrence, but also to reduce the duration of follow up and repeat operations. It was an evolutionary process where the findings in the preceding studies formed the basis of the subsequent one - while the aim of the individual studies were different, there was a clear link to the essential principles, thus forming a coherent collection of studies. Methods and results: The project was carried out in 3 phases (with 2 or 3 main studies in each phase, augmented by 1 to 2 linked studies - making the entire submission for PhD by publications a series of 12 studies, to date): Phase 1 (5 studies in this phase): The aim was to demonstrate the natural history of non-invasive bladder cancer and identify sub-categories of patients who could be discharged from surveillance at 5 years. This was initially achieved by evaluating a prospectively maintained cohort of non-invasive bladder cancer patients diagnosed between 1978 and 1984 at the Western General Hospital, Edinburgh. This study identified the importance of the recurrence rate at the first follow up cystoscopy (RRFFC) as an essential prognostic marker. This finding was further validated using 2 separate cohorts from a different Centre (the Royal Infirmary, Edinburgh) managed in the 80s and the 90s, respectively. The data confirmed that over the decades, recurrence patterns do change, possibly as a result of differing techniques and improvements in optics and instruments; however, what remained the same was the prognostic value of the RRFFC. Phase 2 (3 studies in this phase): The early recurrence was deemed to be the result of missed and tumours left behind at the initial TURBT, i.e. a marker of quality. However, RRFFC was only known 3 months after the initial surgery. Since the RRFFC was such an important prognostic factor, the aim of this phase was to determine the surgical factors contributing to the quality of TURBT and subsequently implement changes to the principles in carrying out the surgery to improve this quality. This was achieved by prospective collection of information regarding all patients undergoing TURBT for new bladder cancers, recording the tumour features, surgeon experience, if the resection was deemed to have been complete or not, and the pathological results. We identified that the detrusor muscle in the resected specimen and the experience of the surgeon were independent determinants of TURBT quality. This finding was validated in a further study using cohorts from another time period and another Centre - this allowed me to develop the concept of Good Quality White Light TURBT (GQWLTURBT) as the benchmark for the white light TURBT. Phase 3 (4 studies in this phase): Photodynamic Diagnosis assisted TURBT (PDDTURBT) was demonstrated in randomised controlled trials as a technique that reduces the recurrences in NMIBC. In the absence of evidence with this technique in the 'real life' setting nor comparisons with standardised, benchmarked white light TURBT technique, we performed a prospective controlled study comparing PDD-TURBT and GQ-WLTURBT, evaluating early and delayed recurrence rates in 2 separate studies. I also performed a multicentre UK study on the outcomes with PDD-TURBT and collaborated with other experts in Europe in producing a review article around Photodynamic Diagnosis and the cost effectiveness of this technique. Summary: This coherent series of studies has contributed to knowledge in bladder cancer surgery by, among others: (a) mapping the individual patient natural history of non-invasive bladder cancer; (b) confirming the importance of early recurrence as a strong prognostic indicator; (c) identifying predictors of this early recurrence and the quality of TURBT; (d) introducing the concept of the benchmark Good Quality White Light TURBT and (e) demonstrating the benefits of photodynamic diagnosis within a 'real life' setting.

L'objectif de cette thèse est d'évaluer les performances d'une méthode d'imagerie optique non-invasive pour la détection de précancers et cancers précoces de la vessie, à l'aide d'une analyse de lumière laser rétro-diffusée. L'analyse de la distribution spatiale de la lumière à la surface de fantômes multi-couches imitant l'épithelium de vessie avec différentes propriétés d'absorption et de diffusion nous a permis de montrer les modifications de ces propriétés optiques entraînent des changements de la taille de la surface du spot de lumière rétro-diffusée, mesurables par une caméra vidéo. La méthode développée est également sensible à l'accumulation d'un photosensibilisateur et est applicable aussi bien pour des études en réflectance diffuse qu'en fluorescence induite. Les paramètres optiques des fantômes synthétiques tri-couches imitant différents états des épithéliums de vessie ont été calculés à partir de la théorie des ondes électromagnétiques appliquée aux diffuseurs sphériques sans et avec une couche. Ces paramètres ont servi comme entrées aux simulations de Monte Carlo qui ont permis d'obtenir les matrices des distributions d'intensité de réflectance diffuse. Notre étude démontre que les mesures en imagerie de réflectance diffuse non-polarisée permettent de fournir des informations utiles au diagnostic tissulaire
The present thesis aimed to evaluate the performance of non-invasive optical method for bladder pre- and early- cancer detection by means of diffuse-reflected laser light analysis. The analysis of light distribution at the surface of multi-layered bladder phantoms with different scattering and absorption properties showed that the changes in the optical properties lead to increase or decrease of the diffuse-reflected light spot area, detectable by a video camera. It was also determined, that the presented method is capable of detection of the photosensitizer accumulation, and can be applied for both (diffuse-reflected laser and fluorescence) studies simultaneously. The calculations for spherical and ?coated?-spherical tissue scatterers, based on the electromagnetic wave theory, allowed for obtaining optical parameters of three-layered biological phantoms and of bladder tissues at different states. These parameters served as inputs for Monte Carlo simulations, which provided us with matrices of diffuse-reflected light distributions. The study showed that the measurements of non-polarized back-scattered laser light can provide useful information on the tissue state

L'objectif de cette thèse est d'évaluer les performances d'une méthode d'imagerie optique non-invasive pour la détection de précancers et cancers précoces de la vessie, à l'aide d'une analyse de lumière laser rétro-diffusée. L'analyse de la distribution spatiale de la lumière à la surface de fantômes multi-couches imitant l'épithelium de vessie avec différentes propriétés d'absorption et de diffusion nous a permis de montrer les modifications de ces propriétés optiques entraînent des changements de la taille de la surface du spot de lumière rétro-diffusée, mesurables par une caméra vidéo. La méthode développée est également sensible à l'accumulation d'un photosensibilisateur et est applicable aussi bien pour des études en réflectance diffuse qu'en fluorescence induite. Les paramètres optiques des fantômes synthétiques tri-couches imitant différents états des épithéliums de vessie ont été calculés à partir de la théorie des ondes électromagnétiques appliquée aux diffuseurs sphériques sans et avec une couche. Ces paramètres ont servi comme entrées aux simulations de Monte Carlo qui ont permis d'obtenir les matrices des distributions d'intensité de réflectance diffuse. Notre étude démontre que les mesures en imagerie de réflectance diffuse non-polarisée permettent de fournir des informations utiles au diagnostic tissulaire
The present thesis aimed to evaluate the performance of non-invasive optical method for bladder pre- and early- cancer detection by means of diffuse-reflected laser light analysis. The analysis of light distribution at the surface of multi-layered bladder phantoms with different scattering and absorption properties showed that the changes in the optical properties lead to increase or decrease of the diffuse-reflected light spot area, detectable by a video camera. It was also determined, that the presented method is capable of detection of the photosensitizer accumulation, and can be applied for both (diffuse-reflected laser and fluorescence) studies simultaneously. The calculations for spherical and ?coated?-spherical tissue scatterers, based on the electromagnetic wave theory, allowed for obtaining optical parameters of three-layered biological phantoms and of bladder tissues at different states. These parameters served as inputs for Monte Carlo simulations, which provided us with matrices of diffuse-reflected light distributions. The study showed that the measurements of non-polarized back-scattered laser light can provide useful information on the tissue state

There is an urgent need to improve the performance of urine cytology for the diagnosis of bladder cancer. In preliminary studies, telomerase activity evaluated by telomeric repeat amplification protocol (TRAP) assay and chromosomal aneuploidy detected by fluorescence in situ hybridization (FISH) in the diagnosis of bladder cancer have produced important results. Urine cell-free (UCF) DNA has also been proposed as a potential marker for early bladder cancer diagnosis. In the first study the diagnostic performance of TRAP assay and FISH analysis was assessed, while the second study evaluated the potential role of UCF DNA integrity in early bladder cancer diagnosis. In the first cross-sectional study, 289 consecutive patients who presented with urinary symptoms underwent cystoscopy and cytology evaluation. In the second study, UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. c-Myc, BCAS1 and HER2 gene sequences longer than 250 bp were quantified by real time PCR to verify UCF DNA integrity. In the first study, sensitivity and specificity were 0.39 and 0.83, respectively, for cytology; 0.66 and 0.72 for TRAP; 0.78 and 0.60 for the cytology and TRAP combination; 0.78 and 0.78 for the cytology, TRAP and FISH combination; and 0.65 and 0.93 for the TRAP and FISH combination. In the second study, at the best cutoff of 0.1 ng/µl, UCF DNA integrity analysis showed a sensitivity of 0.73 and a specificity of 0.84 in healthy individuals and 0.83 in symptomatic patients. The preliminary results suggest that these biomarkers could potentially be used for the early diagnosis of bladder cancer, especially in high-risk populations (e.g, symptomatic individuals exposed to occupational risk) who may benefit from the use of noninvasive diagnostic tests in terms of cost-benefit.

There is an urgent need to improve the performance of urine cytology for the diagnosis of bladder cancer. In preliminary studies, telomerase activity evaluated by telomeric repeat amplification protocol (TRAP) assay and chromosomal aneuploidy detected by fluorescence in situ hybridization (FISH) in the diagnosis of bladder cancer have produced important results. Urine cell-free (UCF) DNA has also been proposed as a potential marker for early bladder cancer diagnosis. In the first study the diagnostic performance of TRAP assay and FISH analysis was assessed, while the second study evaluated the potential role of UCF DNA integrity in early bladder cancer diagnosis. In the first cross-sectional study, 289 consecutive patients who presented with urinary symptoms underwent cystoscopy and cytology evaluation. In the second study, UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. c-Myc, BCAS1 and HER2 gene sequences longer than 250 bp were quantified by real time PCR to verify UCF DNA integrity. In the first study, sensitivity and specificity were 0.39 and 0.83, respectively, for cytology; 0.66 and 0.72 for TRAP; 0.78 and 0.60 for the cytology and TRAP combination; 0.78 and 0.78 for the cytology, TRAP and FISH combination; and 0.65 and 0.93 for the TRAP and FISH combination. In the second study, at the best cutoff of 0.1 ng/µl, UCF DNA integrity analysis showed a sensitivity of 0.73 and a specificity of 0.84 in healthy individuals and 0.83 in symptomatic patients. The preliminary results suggest that these biomarkers could potentially be used for the early diagnosis of bladder cancer, especially in high-risk populations (e.g, symptomatic individuals exposed to occupational risk) who may benefit from the use of noninvasive diagnostic tests in terms of cost-benefit.

Real-time multi-circle detection has been a challenging problem in the field of biomedical image processing, due to the variable sizes and non-ideal shapes of cells in microscopic images. In this study, two new multi-circle detection algorithms are developed to facilitate an automatic bladder cancer diagnosis system: one is a modified circular Hough Transform algorithm integrated with edge gradient information; and the other one is a stochastic search approach based on real valued artificial immune systems. Computer simulation results show both algorithms outperform traditional methods such as the Hough Transform and the geometric feature based method, in terms of both precision and speed.

L’immunoterapia basata sull’utilizzo di anticorpi non coniugati, coniugati a tossine o radiomarcati, che riconoscono antigeni associati a tumore, è promettente per la cura di tumori solidi o ematici. Un possibile target per l’immunoterapia potrebbe essere il prostate stem cell antigen (PSCA), un antigene appartenente alla famiglia delle “GPIanchored protein”. Il PSCA è un antigene di superficie espresso a bassi livelli nel tessuto prostatico sano ed over espresso nel tumore prostatico, pancreatico e della vescica. L’espressione di PSCA è inoltre correlata positivamente a “Gleason score” e allo stadio della patologia nel tumore prostatico. Il presente lavoro di tesi descrive la generazione e caratterizzazione di un anticorpo monoclonale murino anti PSCA (mAb), ottenuta tramite la tecnologia dell’ibridoma, e del suo frammento anticorpale a singola catena (scFv), generato clonando la regione variabile della catena leggera (VL) e della catena pesante (VH) nel vettore di espressione pHEN-2. Tramite citofluorimetria è stato dimostrato che l’anticorpo monoclinale possiede una buona affinità e specificità di legame all’antigene. Il potenziale diagnostico dell’anticorpo è stato dimostrato tramite Western Blot su lisati di tessuti neoplastici di prostrata e pancreas, in cui l’anticorpo è in grado di legare l’antigene denaturato e glicosilato, e tramite ELISA, in cui l’anticorpo si lega all’antigene espresso da cellule precedentemente fissate. Il potenziale terapeutico dell’anticorpo è stato valutato tramite saggio di proliferazione: l’anticorpo da solo non è in grado di indurre morte cellulare tramite un meccanismo diretto, mentre in seguito a coniugazione chimica con la catena A della ricina (RTA) rivela effetto citotossico su cellule PC-3 hPSCA con IC50 (concentrazione in grado di inibire la massima proliferazione cellulare del 50%) pari a 1.3x10-9, valore 100 volte più piccolo di quello ottenuto con la sola tossina RTA. Il frammento scFv è stato prodotto nel ceppo batterico E. Coli. Mediante analisi citofluorimetrica su cellule PSCA positive e saggio immunoenzimatico sull’antigene ricombinante è stato verificato che il frammento anticorpale mantiene le stesse caratteristiche di specificità di legame all’antigene dell’anticorpo monoclinale parentale, ma possiede affinità minore. Quando l’ scFv viene reso bivalente, tramite il cross-linking dei monomeri utilizzando un anticorpo anti-myc, l’affinità raggiunge quasi quella dell’anticorpo parentale. Successivamente l’scFv è stato unito attraverso fusione genetica al dominio enzimatico della tossina batterica Pseudomonas aeruginosa exotoxin A (PE40). L’immunotossina risultante è espressa nel ceppo batterico E. Coli e si accumula nei corpi d’inclusione. L’analisi citofluorimetrica su cellule PSCA positive fatta utilizzando i corpi d’inclusione rinaturati e contenenti l’immunotossina di fusione ha confermato che l’interazione tra l’scFv e l’antigene viene conservata in seguito alla fusione con la tossina PE40. L’effetto citotossico dell’immunotossina purificata scFv-PE40 verrà valutata prima in vitro su linee cellulari PSCA positive e negative e poi in modelli in vivo che permetteranno di valutare anche eventuali effetti collaterali.
Antibody-based therapy using unconjugated, toxin-conjugated or radiolabeled immunoglobulins recognizing tumor-associated antigens has proven beneficial for solid and hematolymphoid neoplasms. A suitable target could be prostate stem cell antigen (PSCA), a member of the “GPI-anchored protein”. PSCA is a cell surface-antigen expressed at low levels in normal prostate tissue and over expressed in prostate, pancreatic and bladder carcinomas. Moreover PSCA expression is positively correlated with Gleason score and with pathologic stage in prostate cancer. The present thesis describes the generation and characterization of the murine anti PSCA monoclonal antibody (mAb), obtained by hybridoma technology, and its fragment single chain (scFv), generated by cloning the variable heavy (VH) and light (VL) chain sequences in the expression vector pHEN-2. The mAb showed the ability to recognize with good affinity and specificity the native PSCA by flow cytometry. The diagnostic potential of the mAb was demonstrated by Western Blot performed with prostate and pancreatic neoplastic tissue lysates, showing the binding to denaturated and glycosylated PSCA, and by ELISA performed with fixed cells. The mAb was also assessed for its possible use in the therapeutic approach: the cell-proliferation assay demonstrated that the antibody alone is not able to induce cell death through a direct mechanism, while when it is conjugated to the ricin A chain toxin (RTA) by chemical linkage it can poison PC-3 hPSCA cells with an IC50 (i.e. concentration inhibiting 50% of the maximal cell proliferation) of 1.3x10-9 M, value 100 fold lower than the IC50 of the RTA toxin alone. The scFv was produced in E. Coli bacteria; flow cytometric analysis on PSCA-positive cells and immunoenzymatic assay on the recombinant antigen proved that the antibody fragment maintains the binding specificity of the parental monoclonal antibody. The affinity of the scFv is lower than the affinity of mAb but it is partially recovered making the scFv divalent by cross-linking the scFv monomers via an antibody-mediated myc- Tag interaction. To create a fusion immunotoxin (IT) the scFv was later genetically fused to the enzymatic domain of Pseudomonas aeruginosa exotoxin A (PE40). The resulting IT was expressed in E. Coli bacteria and it is accumulated in the inclusion bodies. The flow cytometric analysis on PSCA-positive cells performed with the whole refolded inclusion bodies extract containing the fusion IT confirmed that the interaction of scFv with the PSCA is preserved after fusion to PE40. The efficacy of purified scFv-PE40 will be analyse in vitro on positive and negative cell lines and subsequently in vivo models which also will be useful to study the side effects of this new drug.

In Africa, Prostate cancer (PCa) is the most frequently diagnosed solid organ tumour in males and use of prostate specific antigen (PSA) is presently fraught with diagnostic inaccuracies. Not least, in a multi-ethnic society like South Africa, proteome differences between African, Caucasian and Mixed-Ancestry PCa patients are largely unknown. Hence, discovery and validation of affordable, non-invasive and reliable diagnostic biomarkers of PCa would expand the frontiers of PCa management. We have employed two high-throughput proteomics technologies to identify novel urine- and blood-based biomarkers for early diagnosis and treatment monitoring of prostate cancer in a South African cohort as well as elucidate proteome differences in patients from our heterogeneous cohort. We compared the urinary proteomes of PCa, Benign Prostatic Hyperplasia (BPH), disease controls comprising patients with other uropathies (DC) and normal healthy controls (NC) both by pooling and individual discovery shotgun proteomic assessment on a nano-Liquid chromatography (nLC) coupled Hybrid Quadrupole-Orbitrap Mass Spectrometer platform. In-silico verification of identified biomarkers was performed using the Human Protein Atlas (HPA) as well as SRMAtlas; and verified potential biomarkers were experimentally prevalidated using a targeted parallel reaction monitoring (PRM) proteomics approach. Further, we employed the CT100+ antigen microarray platform to assess the differential humoral antibody response of PCa, DC and BPH patients in our cohort to a panel of 123 tumour-associated cancer antigens. Candidate antigen biomarkers were analyzed for ethnic group variation in our cohort and potential cancer diagnostic and immunotherapeutic inferences were drawn. Using these approaches, we identified 5595 and 9991 non-redundant peptides from the pooled and individual experiments respectively. While nine proteins demonstrated ethnic trend, 37 and 73 proteins were differentially expressed by pooled and individual analysis respectively. All 32 verified biomarkers were prevalidated with parallel reaction monitoring. Good PRM signals for 12 top ranking biomarker was observed, including PSA and prostatic acid phosphatase. We also identified 41 potential diagnostic and immunotherapeutic antigen biomarkers. Proteogenomic functional pathway analyses of differentially expressed antigens showed similar enrichments of biologic processes. We identified herein novel urinary and blood-based potential diagnostic biomarkers and immunotherapeutic targets of PCa in a South African PCa Cohort using multiple proteomics approaches.

Los tumores cerebrales constituyen menos del 2% de los tumores primarios, pero son uno de los peores tipos de cáncer en cuanto a “años de vida perdidos”. Los gliomas son los tumores cerebrales más prevalentes, con una esperanza de vida inferior a 15 meses para los casos de alto grado, como los glioblastomas (GBM). El método no invasivo más utlilizado para diagnóstico y seguimiento de la respuesta a la terapia en tumores cerebrales es la Resonancia Magnética (MR), en forma de imagen (MRI) y espectroscopía (MRS) o imagen espectroscópica (MRSI). Sin embargo, debido a restricciones éticas relacionadas a la participación de pacientes humanos en investigación, la optimización de los métodos de diagnóstico y seguimiento de la terapia requieren modelos preclínicos que reproduzcan las patologías humanas. Para este tipo de estudios, se utilizan principalmente modelos murinos, que pueden dividirse en modelos genéticamente modificados (GEM) con desarrollo espontáneo de tumor y los modelos de generación de tumores por inyección estereotáxica de líneas celulares. En la presente tesis, se llevó a cabo una detallada caracterización de dos colonias GEM, S100β-v-erbB/inK4a-Arf (+/-) y GFAP-V12 HA-ras B8. Se observó una baja penetrancia de desarrollo de tumores (16% y 1% respectivamente), haciendo de éstos una herramienta poco útil para estudios de respuesta a la terapia. La escasez de modelos preclínicos de grado bajo/intermedio sirvió de motivación para desarrollar un modelo de tumor glial transplantable con esas características, por disgregación de tumores provenientes de la colonia GEM. Ello nos debería permitir obtener una mayor incidencia tumoral en comparación con las colonias GEM. Se generaron gliosferas a partir de tumores GEM de grado III y se logró más de un 60% de penetrancia cuando estas células fueron inyectadas estereotácticamente en el estriado de ratones C57BL/6. Sin embargo, la aplicación de protocolos de descongelación y cultivo a éstas células ocasionó una progresión a grado IV (GBM), lo que sugiere que el modelo generado constituye, potencialmente, un modelo murino de GBM secundario. Además, este modelo transplantable fue ampliamente caracterizado por métodos de MRI/MRSI, así como por métodos de perturbación del patrón espectral con MRSI (PE-MRSI) para una posible aplicación en el desarrollo de clasificadores de respuesta a la terapia en tumores. También se llevó a cabo una evaluación genética restringida de los modelos murinos seleccionados (p.e. tumores GL261, línea celular GL261, GEM y tumores derivados de GEM), utilizando el método de secuenciación de Sanger para comprobar la presencia de mutaciones normalmente presentes en gliomas (en los genes IDH1, IDH2 y p53). Finalmente, esta tesis describe la estrategia desarrollada para estudios longitudinales de detección de respuesta temprana a la terapia/recidiva en tumores preclínicos, utilizando métodos de imagen molecular basados en MRSI. Así ratones implantados con tumores GL261 (glioblastoma) recibieron tratamiento con temozolamida (TMZ), basándonos en protocolos previamente establecidos. La detención del crecimiento tumoral (respuesta a la terapia) fue detectada por MRI. Tanto animales tratados como animales control fueron estudiados por MRSI y técnicas de reconocimiento de patrones (extracción de fuentes en sistema semi-supervisado). Las fuentes extraídas de regiones de interés fueron capaces de distinguir entre tumores GL261 proliferando activamente y tumores respondiendo a la terapia, basándose en cambios de patrón del metaboloma registrado por MRSI. Se obtuvieron mapas nosológicos codificados en tipo e intensidad de color durante y después de la terapia, lo que permitió un seguimiento de la respuesta, así como la detección de heterogeneidad intratumoral en dicha respuesta, siendo capaces de detectar la detención del crecimiento tumoral y la recidiva antes de los cambios observados en el volumen tumoral por MRI. Esta metodología fue ratificada por análisis histopatológico y cálculo de tasas de proliferación, apoptosis y de índice mitótico.
Brain tumours account for less than 2% of all primary tumours, but are one of the most lethal cancers when “life lost” years are considered. Gliomas are the most prevalent type with a median life expectancy below 15 months for the high grade ones, such as glioblastomas (GBM). The most common non-invasive medical technique used for tumour diagnosis and therapy monitoring of brain tumours patients is Magnetic Resonance (MR), in the form of imaging (MRI) and spectroscopy (MRS) or spectroscopic imaging (MRSI). However, due to the ethical restrictions regarding the use of human patients for research study, the improvement of diagnostic and therapy follow-up protocols requires reliable models that mimic human disease. In this regard, mainly murine models are used and can be divided into the genetically engineered model (GEM) of spontaneous tumour development and the engrafted tumour model. In this thesis, a comprehensive MR characterization of two GEM colonies, namely S100β-v-erbB / inK4a-Arf (+/-) and GFAP-V12 HA-ras B8, was carried out. A low tumour penetrance found (16% and 1%, respectively) together with stochastic onset of GEM tumours, made them impractical for use in therapy response studies. The latter and the scarcity of low/intermediate grade brain tumour preclinical models motivated us to attempt to develop a transplantable glial tumour model of low/intermediate grade by disaggregation of a tumour mass from GEM. This should allow us to obtain an increased tumour incidence rate in comparison to GEM animals. Gliospheres from a grade III GEM tumour were successfully generated and displayed more than 60% penetrance, when stereotactically injected into the striatum of C57BL/6 mice. However, the application of freezing and cell culture protocols produced a progression to grade IV GBM, which made the developed transplantable model qualify as potential secondary GBM model in mice. Additionally, this transplantable model was widely characterized using MRI/MRS methods, as well as perturbation-enhanced MRSI (PE-MRSI) for a possible application in the future in therapy strategies and development of tumour therapy response detection classifiers. A restricted genetic evaluation of selected murine tumour models (i.e. GL261 tumours, GL261 cell line, GEM and GEM-derived tumours) was carried out using the Sanger method to check for a possible presence of particular driver mutations commonly occurring in gliomas (IDH1, IDH2 and p53). Finally, the work describes the strategy followed for longitudinal therapy studies follow-up and early response/relapse detection in preclinical brain tumours, through molecular imaging methods based in MRSI. GL261 (glioblastoma) tumour bearing mice were treated with temozolomide (TMZ), based on previously established protocols. The expected transient growth arrest (response to therapy) was detected by MRI. Animals subjected to therapy and control animals were followed up by MRSI and pattern recognition techniques (semi-supervised source extraction) were applied. The sources extracted from the region of interest were able to discriminate between GL261 tumours actively proliferating and tumours responding to therapy, based on their metabolome pattern changes recorded by MRSI. Colour-coded nosological images produced throughout and after the course of therapy allowed convenient tracking of response changes and differentiated the intratumoural heterogeneity of response, hinting the growth arrest and relapse, before changes in tumour volume were observed by MRI. The methodology was validated with histopathological analysis and calculation of proliferation and apoptotic rates and mitotic index.

Electronic medical record databases (e.g. the Clinical Practice Research Datalink, CPRD) are increasingly used in epidemiological research. The CPRD has two formats of data: coded, which is the sole format used in almost all research; and free-text (or ‘hidden’), which may contain much clinical information but is generally unavailable to researchers. This thesis examines the ramifications of omitting free-text records from research. Cases with bladder (n=4,915) or pancreatic (n=3,635) cancer were matched to controls (n=21,718, bladder; n=16,459, pancreas) on age, sex and GP practice. Coded and text-only records of attendance for haematuria, jaundice and abdominal pain in the year before cancer diagnosis were identified. The number of patients whose entire attendance record for a symptom/sign existed solely in the text was quantified. Associations between recording method (coded or text-only) and case/control status were estimated (χ2 test). For each symptom/sign, the positive predictive value (PPV, Bayes' Theorem) and odds ratio (OR, conditional logistic regression) for cancer were estimated before and after supplementation with text-only records. Text-only recording was considerable, with 7,951/20,958 (37%) of symptom records being in that format. For individual patients, text-only recording was more likely in controls (140/336=42%) than cases (556/3,147=18%) for visible haematuria in bladder cancer (χ2 test, p<0.001), and for jaundice (21/31=67% vs 463/1,565=30%, p<0.0001) and abdominal pain (323/1,126=29% vs 397/1,789=22%, p<0.001) in pancreatic cancer. Adding text records reduced PPVs of visible haematuria for bladder cancer from 4.0% (95% CI: 3.5–4.6%) to 2.9% (2.6–3.2%) and of jaundice for pancreatic cancer from 12.8% (7.3–21.6%) to 6.3% (4.5–8.7%). Coded records suggested that non-visible haematuria occurred in 127/4,915 (2.6%) cases, a figure below that generally used for study. Supplementation with text-only records increased this to 312/4,915 (6.4%), permitting the first estimation of its OR (28.0, 95% CI: 20.7–37.9, p<0.0001) and PPV (1.60%, 1.22–2.10%, p<0.0001) for bladder cancer. The results suggest that GPs make strong clinical judgements about the probable significance of symptoms – preferentially coding clinical features they consider significant to a diagnosis, while using text to record those that they think are not.

La cistoscòpia és el mètode habitual per al diagnòstic i vigilància del tumor vesical (TV). Per realitzar una cistoscòpia es requereix personal entrenat, instruments especialitzats i un lloc adequat. Aquest procediment no és indolor i té riscos inherents. En les últimes 2 dècades s'han buscat marcadors (proteïnes, canvis cel·lulars, gens) en orina que puguin reemplaçar la cistoscòpia en el diagnòstic i la vigilància del TV. El nostre projecte es focalitza en la vigilància del TV. Primer vam realitzar una revisió exhaustiva de la literatura en marcadors urinaris utilitzats en la vigilància del TV. Un cop feta la revisió, hem escollit un assaig per al nostre estudi. Després, fent servir l'assaig que quantifica la presència de les citoqueratines 8 i 18 en orina, vam realitzar un estudi prospectiu amb l'objectiu de millorar el protocol de vigilància del TV.
La cistoscopia es el método habitual para el diagnóstico y vigilancia del tumor vesical (TV). Para realizar una cistoscopia se requiere personal entrenado, instrumentos especializados y un lugar adecuado. Este procedimiento no es indoloro y tiene riesgos inherentes. En las últimas 2 décadas se han buscado marcadores (proteínas, cambios celulares, genes) en orina que puedan reemplazar a la cistoscopia en el diagnóstico y la vigilancia del TV. Nuestro proyecto se focaliza en la vigilancia del TV. Primero realizamos una revisión exhaustiva de la literatura en marcadores urinarios utilizados en la vigilancia del TV. Una vez hecha la revisión, hemos escogido un ensayo para nuestro estudio. En la segunda parte, utilizando el ensayo que cuantifica la presencia de las citoqueratinas 8 y 18 en orina, se realiza un estudio prospectivo con el objetivo de mejorar el protocolo de vigilancia del TV
Cystoscopy is the usual method for the diagnosis and surveillance of bladder tumor (BT). Performing a cystoscopy requires trained personnel, specialized instruments, and a suitable location. This procedure is not painless and has inherent risks. In the last 2 decades, urine markers (proteins, cell changes, genes) have been tested aiming to replace cystoscopy in the diagnosis and surveillance of BT. Our project focuses on BT surveillance. We first performed a comprehensive review of the literature on urinary markers used in BT surveillance. After this review, we chose an assay for our study. In the second part, using the assay that quantifies the presence of cytokeratins 8 and 18 in urine, a prospective study was carried out with the aim of improving the TV surveillance protocol.

Les travaux présentés dans cette thèse concernent le développement de nouveaux outils d'analyse multiparamétrique de type biopuce ou puce électrochimique, appliqués au diagnostic et au contrôle environnemental. Le premier axe de recherche a pour objectif le diagnostic, par la détection de panels d'anticorps marqueurs d'un état pathologique dans le sérum de patients. Dans cette optique, deux systèmes d'immunotests ont été développés, permettant la détection multiparamétrique d'anticorps spécifiques grâce à l'analyse automatisée et haut-débit d'échantillons de sérum. Cette approche s'est basée sur la capture des anticorps cibles par des sondes antigéniques immobilisées selon une matrice de plots sur des membranes constituant les fonds de puits de micro-plaques. La détection des interactions est effectuée par colorimétrie à l'aide d'un marqueur enzymatique. Ces outils ont permis l'analyse de 96 échantillons en moins de trois heures, et ont été mis au point pour deux applications. La première consiste en le diagnostic d'allergies, et la seconde s'intéresse au diagnostic du cancer. La seconde partie des travaux est appliquée au contrôle environnemental par surveillance de l'eau. Des pesticides, toxines et explosifs ont été définis comme composés cibles du test multiparamétrique. Afin de les intégrer dans une matrice de plots, des conjugués sondes ont été synthétisés à partir de ces haptènes. Après criblage et optimisation des conjugués en fonction de leur réactivité et réactivité-croisée avec les anticorps spécifiques, l'outil développé a démontré ses performances analytiques en termes de sensibilité et de sélectivité pour la détection des cibles. Un autre capteur pour la surveillance de l'eau a été développé dans le cadre du projet Européen BONAS. Ce test électrochimique vise à détecter des précurseurs d'explosifs utilisés dans la préparation de systèmes explosifs improvisés, pour la localisation de fabriques de bombes artisanales. La puce mise au point consiste en un réseau d'électrodes sérigraphiées, modifiées par électrodépôt de différents métaux
The work reported in this thesis focuses on the development of new multiplex analytical devices on biochip or electrode microarray format, dedicated to diagnosis and environmental monitoring. The objective of the first research axis is diagnosis, thanks to the detection in patients’ serum of a panel of antibodies, biomarkers of a pathological state. For that purpose, two immunotests have been developed, enabling the multiparametric detection of specific antibodies by automated and high-throughput analysis of serum samples. This approach is based on the antibodies capture by antigens probes immobilized in a matrix of spots on a membrane surface composing the wells bottom of a micro-titer plate. Enzyme-labeled antibodies have been used, providing a colorimetric detection. This device enabled the achievement of the analysis of 96 samples in less than three hours and has been applied to different applications. The first one consists of allergy diagnosis, and the second focuses on cancer diagnosis. The second part of this work is applied to environmental monitoring, through water analysis. Different types of pollutants have been defined as targets: pesticides, toxins and explosives. In order to integrate them in a matrix of probes, different conjugates have been synthesized with these haptens. After screening and optimization of the conjugates through their reactivity and cross-reactivity with the specific antibodies, the developed device demonstrated his analytical performances in terms of sensitivity and selectivity. Finally, for the European Project BONAS, a last sensor based on water analysis has also been developed. This electrochemical microarray aims to detect explosives precursors, used in improvised explosive devices, for the localization of hidden bomb factory. The chip was designed as a screen-printed electrode network, which was modified by different metals electrodeposition

Les travaux présentés dans cette thèse concernent le développement d'un outil d'analyse multiparamétrique pour la quantification de biomarqueurs urinaires du cancer de la vessie.La première partie des travaux de recherche a pour objectif la sélection de marqueurs pour le diagnostic et la récidive des tumeurs urothéliales. Une première étude a permis l'évaluation de la sélectivité de marqueurs candidats dans des échantillons urinaires de patients atteints de cancer de la vessie. Cinq des vingt marqueurs initiaux ont été sélectionnés pour leur performance diagnostique, définissant le Panel 1 : VEGF, MMP9, IL8, PTGS2 et EN2. Une seconde étude a été réalisée afin d'évaluer le potentiel de marqueurs et de paramètres cliniques pour le diagnostic de la récidive des tumeurs urothéliales. Les échantillons urinaires évalués provenaient donc de patients présentant une récidive du cancer de la vessie et de patients ne présentant pas de récidive. Le Panel 2 a ainsi été défini, basé sur le modèle de régression multiple le plus performant. Il comprend les paramètres cliniques et moléculaires suivants : nombre de récidives antérieures, nombre de thérapies par BCG, stade de la tumeur au moment du diagnostic, CDH1, IL8, ErbB2, IL6, EN2 et VEGF.La seconde partie concerne le développement d'un test multiparamétrique pour la quantification des marqueurs sélectionnés. Il s'agit d'une plateforme automatisée, à haut-débit et sous un format de plaque de microtitration 96-puits. La méthode de quantification choisie est un immunoessai de type sandwich sous la forme de puce à protéines. Le développement de la plateforme a débuté avec le Panel 1 dont trois des cinq marqueurs (VEGF, MMP9 et IL8) ont été intégrés avec succès. Suite à la seconde étude de sélection de marqueurs, le développement de l'immunoessai multiparamétrique a été orienté vers le Panel 2. À l'exception du marqueur EN2, nécessitant une configuration d'immunoessai différente, tous les marqueurs du Panel 2 ont pu être intégrés à la plateforme
The work reported in this thesis focuses on the development of a multiplex analytical tool for the quantification of selected bladder cancer urinary biomarkers.The aim of the first part of this work is the selection of urinary biomarkers for the diagnosis and recurrence of urothelial tumors. A first study evaluated the selectivity of candidate markers in urine samples of bladder cancer patients. Five of the twenty initial markers were selected for their diagnostic performance. They define Panel 1: VEGF, MMP9, IL8, PTGS2 and EN2. A second study was then conducted to assess the potential of urinary markers and clinical parameters for the diagnosis of bladder cancer recurrence. Two types of urine samples were thus evaluated: samples from recurrent bladder cancer patients and samples from bladder cancer patients without recurrence. Panel 2 was then defined based on the best performing multivariate regression model. It includes the following clinical and molecular parameters: number of past recurrences, number of BCG therapies, tumor stage at diagnosis, CDH1, IL8, ErbB2, IL6, EN2 and VEGF.The second part involves the development of a multiplex test for the quantification of the selected markers. It is a high-throughput automated platform in a 96-well microtiter plate format. It was designed as a multiplex sandwich immunoassay based on a protein microarray. The platform development began with Panel 1 for which three of the five markers (VEGF, MMP9 and IL8) were successful integrated into a multiplex immunoassay. The end of the second marker selection study marked the development transition from Panel 1 to Panel 2. With the exception of EN2, requiring a different immunoassay configuration, all the Panel 2 markers were integrated into the platform

This thesis addresses the three principal questions concerning the development of CT urography for investigating haematuria and each question is the subject of a separate chapter. The questions are: What is the reasoning behind using CT urography? What is the optimum diagnostic strategy using CT urography? What are the problems with using CT urography and how may solutions be provided? Haematuria can signify serious disease such as urinary tract stones, renal cell cancer, upper tract urothelial cancer (UTUC) and bladder cancer (BCa). CT urography is defined as contrast enhanced CT examination of kidneys, ureters and bladder. The technique used here includes unenhanced, nephrographic and excretory-phases for optimized diagnosis of stones, renal masses and urothelial cancer respectively. The reasoning behind using excretory-phase CT urography for investigating haematuria is based on results showing its high diagnostic accuracy for UTUC and BCa. Patients with haematuria are classified as low risk or high risk for UTUC and BCa, by a risk score, determined by the presence/absence of risk factors: age > 50 years, visible or nonvisible haematuria, history of smoking and occupational exposure. The optimum diagnostic strategy for patients at high risk for urothelial cancer, uses CT urography as a replacement test for ultrasonography and intravenous urography and as a triage test for flexible and rigid cystoscopy, resulting in earlier diagnosis and potentially improving prognosis. For patients at low risk, ultrasonography, unenhanced and nephrographic-phase CT urography are proposed as initial imaging tests. Problems with using CT urography include false positive results for UTUC, which are eliminated by retrograde ureteropyelography-guided biopsy, an innovative technique, for histopathological confirmation of diagnosis. Recommendations for the NHS and possible future developments are discussed. CT urography, including excretory-phase imaging, is recommended as the initial diagnostic imaging test before cystoscopy for patients with haematuria at high risk for urothelial cancer.

L'objectif de cette thèse est de faciliter le diagnostic du cancer de la vessie. Durant une cystoscopie, un endoscope est introduit dans la vessie pour explorer la paroi interne de l'organe qui est visualisée sur un écran. Cependant, le faible champ de vue de l'instrument complique le diagnostic et le suivi des lésions. Cette thèse présente des algorithmes pour la création de cartes bi- et tridimensionnelles à large champ de vue à partir de vidéo-séquences cystoscopiques. En utilisant les avancées récentes dans le domaine de la minimisation d'énergies discrètes, nous proposons des fonctions coût indépendantes des transformations géométriques requises pour recaler de façon robuste et précise des paires d'images avec un faible recouvrement spatial. Ces transformations sont requises pour construire des cartes lorsque des trajectoires d'images se croisent ou se superposent. Nos algorithmes détectent automatiquement de telles trajectoires et réalisent une correction globale de la position des images dans la carte. Finalement, un algorithme de minimisation d'énergie compense les faibles discontinuités de textures restantes et atténue les fortes variations d'illuminations de la scène. Ainsi, les cartes texturées sont uniquement construites avec les meilleures informations (couleurs et textures) pouvant être extraites des données redondantes des vidéo-séquences. Les algorithmes sont évalués quantitativement et qualitativement avec des fantômes réalistes et des données cliniques. Ces tests mettent en lumière la robustesse et la précision de nos algorithmes. La cohérence visuelle des cartes obtenues dépasse celles des méthodes de cartographie de la vessie de la littérature.

L'objectif de cette thèse est de faciliter le diagnostic du cancer de la vessie. Durant une cystoscopie, un endoscope est introduit dans la vessie pour explorer la paroi interne de l'organe qui est visualisée sur un écran. Cependant, le faible champ de vue de l'instrument complique le diagnostic et le suivi des lésions. Cette thèse présente des algorithmes pour la création de cartes bi- et tridimensionnelles à large champ de vue à partir de vidéo-séquences cystoscopiques. En utilisant les avancées récentes dans le domaine de la minimisation d'énergies discrètes, nous proposons des fonctions coût indépendantes des transformations géométriques requises pour recaler de façon robuste et précise des paires d'images avec un faible recouvrement spatial. Ces transformations sont requises pour construire des cartes lorsque des trajectoires d'images se croisent ou se superposent. Nos algorithmes détectent automatiquement de telles trajectoires et réalisent une correction globale de la position des images dans la carte. Finalement, un algorithme de minimisation d'énergie compense les faibles discontinuités de textures restantes et atténue les fortes variations d'illuminations de la scène. Ainsi, les cartes texturées sont uniquement construites avec les meilleures informations (couleurs et textures) pouvant être extraites des données redondantes des vidéo-séquences. Les algorithmes sont évalués quantitativement et qualitativement avec des fantômes réalistes et des données cliniques. Ces tests mettent en lumière la robustesse et la précision de nos algorithmes. La cohérence visuelle des cartes obtenues dépassent celles des méthodes de cartographie de la vessie de la littérature
The aim of this thesis is to facilitate bladder cancer diagnosis. The reference clinical examination is cystoscopy, where an endoscope, inserted into the bladder, allows to visually explore the organ's internal walls on a monitor. The main restriction is the small field of view (FOV) of the instrument, which complicates lesion diagnosis, follow-up and treatment traceability.In this thesis, we propose robust and accurate algorithms to create two- and three-dimensional large FOV maps from cystoscopic video-sequences. Based on recent advances in the field of discrete energy minimization, we propose transformation-invariant cost functions, which allow to robustly register image pairs, related by large viewpoint changes, with sub-pixel accuracy. The transformations linking such image pairs, which current state-of-the-art bladder image registration techniques are unable to robustly estimate, are required to construct maps with several overlapping image trajectories. We detect such overlapping trajectories automatically and perform non-linear global map correction. Finally, the proposed energy minimization based map compositing algorithm compensates small texture misalignments and attenuates strong exposure differences. The obtained textured maps are composed by a maximum of information/quality available from the redundant data of the video-sequence. We evaluate the proposed methods both quantitatively and qualitatively on realistic phantom and clinical data sets. The results demonstrate the robustness of the algorithms, and the obtained maps outperform state-of-the-art approaches in registration accuracy and global map coherence

碩士
國立中山大學
醫學科技研究所
107
We have developed self-linkable magnetic graphene oxide-bPEI-prussian blue (MPP) as a peroxidase mimicking nanozyme with high oxidizability to 3,3’,5,5’-tetramethylbenzidine (TMB), which generates significant absorption intensity for the colorimetric immunosensing of apolipoprotein A1 (ApoA1) in early bladder cancer (BC) diagnosis and prognosis monitoring. The ultrasensitive immunosensor was constructed using an ApoA1 antibody (ApoA1Ab)-functionalized chip (biochipApoA1) and self-linkable magnetic graphene oxide-bPEI-prussian blue (MPP). After incubating the sample and capturing ApoA1 proteins captured on the biochipApoA1, the MPP functionalized with ApoA1Ab and mouse IgG (MPP-1), rabbit anti-mouse IgG antibody (MPP-2), and goat anti-rabbit IgG antibody (MPP-3) were added together. We envisioned that each captured ApoA1 protein would allow the retention of a large amount of MPP through a self-linking process to amplify the colorimetric signal of TMB in the presence of H2O2. The linear detection range could be obviously widened in the presence of self-linkable MPP—from 0.05 ng/mL to 100 ng/mL—compared with the group without signal amplification (from 1 ng/mL to 100 ng/mL). Our immunosensor analysis of ApoA1 in the urine of BC patients and healthy individuals was highly correlated with enzyme-linked immunosorbent assay measurements; moreover, the ApoA1 concentrations of patients with high-grade BC were significantly higher than those of patients with low-grade BC. After standard clinical treatment, a significant drop of ApoA1 concentration occurred in urine that was lower than the cut-off concentration, suggesting potential clinical applications of the new self-linkable MPP-generating colorimetric immunosensor in early BC diagnosis and prognosis monitoring.

碩士
長庚大學
生物醫學研究所
99
Bladder cancer (BCa) is the 4th most common malignancy in men, and the number of diagnosed cases is increasing. However, five years after surgery in patients with advanced BCa survival rate is only fifty percent within, or even lower. Therefore, it is important to find a convenient and accurate method to detect bladder cancer. MicroRNAs (microRNAs) are small (~22nt) regulatory RNAs frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. In this study, we aim to identify microRNAs in urine as potential biomarkers for BCa diagnosis. We first established a method to prepare total RNA from small volume of urine samples and demonstrated that microRNAs indeed present in human urine in a remarkably stable form. To identify urine microRNAs differentially expressed in normal and BCa samples, we analyzed the expression levels of 270 microRNAs in urine samples from 8 BCa patients and 8 normal controls. We identified a subset of 34 microRNAs whose expression levels were significantly altered in normal and BCa urine. To validate the utility of these urine microRNAs for BCa detection, we conducted a large scale validation using urine samples from 37 BCa and 43 age- and gender-matched controls and validated that seven microRNAs which are up-modulated, were significantly ( p&lt;0.05 and 1.5 fold-change ) altered in BCa urine. To determine the best microRNA or microRNA panel for BCa detection, we conducted ROC analyses to evaluate the predicting power of individual urine microRNAs, either alone or in combination. Our data demonstrated that combination of two urine microRNAs significantly improved the specificity and sensitivity in BCa detection. Moreover, we compared differential expressed microRNAs in tissue and urine to find out whether these microRNAs have biology meaning or not. On the other hand, lysine-specific demethylase1 (LSD1) overexpressed in BCa tissues, which may contributes genes activated or silenced, even microRNAs. Therefore, we aim to 1. Analyze the expression levels of 270 microRNAs in tissue samples from 4 BCa tissues and 4 adjacent normal tissues. 2. Analyze the expression levels of 270 microRNAs in two BCa cell lines which were knocked down LSD1. Combine both we find out there are two microRNAs may be regulated by LSD1, which may result in gene dysregulation and cause bladder cancer.

Chan Wing Yan Michael.
"August 2002."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (p. 168-200).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

O cancro da bexiga (CB) é o sexto tipo de cancro mais prevalente no mundo, com um aumento constante na sua incidência e prevalência, e é acompanhado por uma alta morbimortalidade. O CB é uma doença complexa com várias vias moleculares e patológicas, reflectindo comportamentos diferentes, dependendo do estadiamento clínico do tumor e do tipo molecular. O diagnóstico e a monitorização do CB são realizados principalmente por testes invasivos, nomeadamente cistoscopias periódicas; esse procedimento, embora seja um método confiável, é altamente desconfortável para o paciente e não é isento de comorbidades. Actualmente, não há indicação formal para o uso de biomarcadores moleculares na prática clínica, apesar de existirem vários testes disponíveis. Há uma necessidade imperativa de um teste clínico não invasivo para detecção precoce, monitorização da doença e resposta ao tratamento no CB. Nesta revisão, o nosso objectivo é avaliar e comparar diferentes testes baseados em biomarcadores moleculares e avaliar seu papel potencial como novas moléculas para o diagnóstico, acompanhamento e monitorização da resposta ao tratamento do CB.
Bladder cancer (BC) ranks as the sixth most prevalent cancer in the world, with a steady rise in its incidence and prevalence, and is accompanied by a high morbidity and mortality. BC is a complex disease with several molecular and pathological pathways, thus reflecting different behaviors depending on the clinical staging of the tumor and molecular type. Diagnosis and monitoring of BC is mainly performed by invasive tests, namely periodic cystoscopies; this procedure, although a reliable method, is highly uncomfortable for the patient and it is not exempt of comorbidities. Currently, there is no formal indication for the use of molecular biomarkers in clinical practice, even though there are several tests available. There is an imperative need for a clinical non-invasive testing for early detection, disease monitoring, and treatment response in BC. In this review, we aim to assess and compare different tests based on molecular biomarkers and evaluate their potential role as new molecules for bladder cancer diagnosis, follow-up, and treatment response monitoring.

O cancro da bexiga (CB) é o sexto tipo de cancro mais prevalente no mundo, com um aumento constante na sua incidência e prevalência, e é acompanhado por uma alta morbimortalidade. O CB é uma doença complexa com várias vias moleculares e patológicas, reflectindo comportamentos diferentes, dependendo do estadiamento clínico do tumor e do tipo molecular. O diagnóstico e a monitorização do CB são realizados principalmente por testes invasivos, nomeadamente cistoscopias periódicas; esse procedimento, embora seja um método confiável, é altamente desconfortável para o paciente e não é isento de comorbidades. Actualmente, não há indicação formal para o uso de biomarcadores moleculares na prática clínica, apesar de existirem vários testes disponíveis. Há uma necessidade imperativa de um teste clínico não invasivo para detecção precoce, monitorização da doença e resposta ao tratamento no CB. Nesta revisão, o nosso objectivo é avaliar e comparar diferentes testes baseados em biomarcadores moleculares e avaliar seu papel potencial como novas moléculas para o diagnóstico, acompanhamento e monitorização da resposta ao tratamento do CB.
Bladder cancer (BC) ranks as the sixth most prevalent cancer in the world, with a steady rise in its incidence and prevalence, and is accompanied by a high morbidity and mortality. BC is a complex disease with several molecular and pathological pathways, thus reflecting different behaviors depending on the clinical staging of the tumor and molecular type. Diagnosis and monitoring of BC is mainly performed by invasive tests, namely periodic cystoscopies; this procedure, although a reliable method, is highly uncomfortable for the patient and it is not exempt of comorbidities. Currently, there is no formal indication for the use of molecular biomarkers in clinical practice, even though there are several tests available. There is an imperative need for a clinical non-invasive testing for early detection, disease monitoring, and treatment response in BC. In this review, we aim to assess and compare different tests based on molecular biomarkers and evaluate their potential role as new molecules for bladder cancer diagnosis, follow-up, and treatment response monitoring.

碩士
國立中正大學
光機電整合工程研究所
100
The multispectral image reproduction for diagnosis of bladder cancer with different stages is proposed. The study can be divided into two parts: the investigation of multispectral system, which simulate the spectra of bladder cell, and the research of score plot, which categorize bladder cell with different stages. A system combined with multi-spectral imaging (MSI), color reproduction technique and score plot is applied to achieve the study. The results show that using the first principle component and the second principle component are more satisfying than the first principle component and the third principle component in analyzing of socre plot. According to this, we can categorize bladder cell line (E7), bladder cell line (TSHG-8301), bladder cell line (J82) and bladder cell line (TCC-SUP) into three groups using ellipse function, which can divide these bladder cell line into normal, stage2,3 and stage 4. In the future, we hope we can make medical diagnosis more efficiency and help patients for early treatment applying our technique.

膀胱尿路上皮腫瘤發病率在泌尿道腫瘤中排第二位，它具有高複發性的特點。目前，有創性尿道膀胱鏡檢查是診斷的金標準。儘管先後有很多血液或尿液中的分子被先後用於診斷膀胱癌的診斷研究，但到目前為止尚未有任何一種方法可以取代膀胱鏡檢查。有證據表明在膀胱上皮腫瘤組織中有很多異常表達的microRNA，但是內在機制的有關研究相對缺乏。在本研究中，我們利用在尿液上清中異常表達的microRNA來評估它們在膀胱癌診斷中的價值。而且，我們揭示了其潛在的調控機理。通過microRNA基因芯片，我們結合并對比來自膀胱腫瘤病人和正常對照患者的9個尿液上清樣本，以及4對腫瘤組織及臨近正常黏膜上皮中microRNA的表達，初步篩選出10個異常的microRNA。然後我們使用定量RT-PCR的方法在另外獨立的18對腫瘤組織和正常黏膜中進一步驗證芯片結果。最後我們就6個被帥選出來的microRNA在71例的膀胱癌患者和正常對照組的尿液上清中進行檢測並評估其診斷效能。我們發現，miR-125b和miR-99a的表達在膀胱癌患者的尿液上清中明顯下調。另外，它們下調程度與腫瘤的病理分級相關。結合miR-125b和miR-99b兩者作為診斷膀胱癌的指標，靈敏度達86.7%，特異度達81.1%，同時有陽性預測值達91.8%。當作為腫瘤分級指標，miR-125b具有81.4%的敏感度，87.0%的特異度，陽性預測值達93.4%。膀胱腫瘤切除之後，和術前比較，兩個microRNA的表達水平再度上升。我們將miR-99轉染到三個膀胱腫瘤細胞株中（T24，UMUC3和J82）。我們發現miR-99a對UMUC3細胞具有輕微的抗增殖功能。同時，miR-99a在3個細胞株中顯示均顯示具有抗遷移和抗侵襲能力。為尋找miR-99a的目標mRNA，我們結合數據庫算法預測，在Western blot中驗證到miR-99a能顯著下調VLDLR蛋白。隨後我們將帶有VLDLR的3'UTR質粒轉染進入細胞中并證實VLDLR mRNA是miR-99a直接作用的目標。另外，當VLDLR siRNA被轉入3個細胞株之後，我們觀察到相似的抗遷移和抗侵襲的現象。最後我們發現N-cadherin是該通路中的下游抑制遷移和侵襲的分子。本項研究證實研究尿液上清中的microRNA是可行的。MiR-125b和miR-99a是膀胱腫瘤的診斷和分級的有效指標。此外，miR-99a能夠通過和VLDLR mRNA直接結合從而抑制膀胱腫瘤遷移和侵襲功能。
Urothelial carcinoma of the bladder (UCB) is the second most common malignancy in the urological system with high recurrence rate. Current gold standard examination for diagnosis is urethrocystoscopy, which is an invasive procedure. Although numerous molecular markers in blood or urine have been proposed as diagnostic biomarkers for bladder cancer, none of them could replace urethrocystoscopy in clinical practice. There are accumulating evidences suggesting microRNA dysregulation might be related to the pathogenesis of UCB. However, the exact functions of these microRNAs in UCB remain unknown. In this thesis, the role of selected microRNAs in urine supernatant was investigated in the diagnosis of UCB and also the carcinogenesis of UCB.
In brief, a high-throughput microarray was carried out on nine supernatants of urine from UCB and normal subjects, and also four pairs of tissue from UCB and normal mucosa. Ten microRNA candidates were then identified. Quantitative RT-PCR was used to validate these microRNAs on a set of 18 pairs of tumor tissue and normal mucosa. Eventually, six potential candidate microRNAs were selected and then validated as diagnostic tools on the samples of urine supernatants from 71 patients (50 of known UCB and 21 of normal subjects). The expression levels of these selected microRNAs were further evaluated in the urine supernatants of 20 patients after tumors resections. MiR-125b and miR-99a were the two most significantly down-regulated microRNAs in the urine supernatants of patients with UCB. Moreover, the degree of down-regulation was associated with the pathological grade of the tumor. A combined index of miR-125b and miR-99a in urine supernatant had a sensitivity of 86.7%, specificity of 81.1%, and a positive predicted value of 91.8% for diagnosing UCB. When used to discriminate high-grade from low-grade UCB, miR-125b alone had a sensitivity of 81.4%, specificity of 87.0% and PPV of 93.4%. After transurethral resections, the expression levels of both microRNAs were significantly increased compared to pre-operative levels.
In further studies on the role of microRNAs on the development of UCB, miR-99a was selected for further studies. The precursor of miR-99a was temporally transfected into 3 bladder cancer cell lines: T24, UMUC3 and J82. The proliferation ability was noticed to be suppressed mildly in UMUC3, but not the other. Meanwhile, migration and invasion abilities were inhibited by miR-99a in the all 3 cell lines. Potential targets of miR-99a were predicted from several prediction databases. Subsequently, in Western Blot study, the protein level of very low density lipoprotein receptor (VLDLR) was showed to be down-regulated by miR-99a. Thereafter, a plasmid constructed with 3’UTR of VLDLR was transfected into cytoplasm, which confirmed VLDLR mRNA was a direct target of miR-99a. All 3 cells lines showed the same effect on suppression of migration and invasion after knockdown of VLDLR. N-cadherin was identified as a down-stream molecule responsible for the migration and invasion suppression in this pathway.
This study confirmed microRNA expression in urine supernatants was a feasible approach for the assessment of biomarkers, and miR-125b and miR-99a showed promising results in the diagnosis and grading of UCB. Furthermore, we showed that miR-99a suppressed tumor migration and invasion by directly targeting VLDLR.
Detailed summary in vernacular field only.
Zhang, Dingzuan.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 107-131).
Abstract and appendix also in Chinese.
Abstract --- p.I
摘要 --- p.III
Acknowledgments --- p.V
Abbreviations --- p.VII
List of figures --- p.IX
List of Tables --- p.XI
Content --- p.XII
Chapter Chapter I: --- General Introduction
Chapter 1.1 --- Bladder cancer --- p.1
Chapter 1.1.1 --- The incidence of bladder cancer
Chapter 1.1.2 --- The burden of bladder cancer to the health care system
Chapter 1.1.3 --- Risk factors for bladder cancer
Chapter 1.1.4 --- Pathology grading system in bladder cancer
Chapter 1.1.5 --- Current diagnostic methods and treatment for bladder cancer
Chapter 1.2 --- Biomarkers for bladder cancer --- p.7
Chapter 1.2.1 --- The advantages of biomarkers in blood and urine for the diagnosis of bladder cancer
Chapter 1.2.2 --- Biomarkers in blood for bladder cancer
Chapter 1.2.3 --- Biomarkers in the urine for bladder cancer
Chapter 1.2.4 --- Current concerning problems with biomarkers
Chapter 1.3 --- MicroRNAs and bladder cancer --- p.11
Chapter 1.3.1 --- Post-trancriptional function of microRNAs
Chapter 1.3.2 --- The function of microRNAs in tumor
Chapter 1.3.3 --- Prospects of detecting microRNA in cell-free fluid in tumor
Chapter 1.4 --- MicroRNA target identification --- p.15
Chapter 1.4.1 --- Prediction of microRNA target
Chapter 1.4.2 --- Validation of microRNA target
Chapter 1.4.3 --- Validation of direct interaction between microRNA and target RNA
Chapter 1.4.4 --- Validation of direct binding of microRNA and mRNA in vivo
Chapter 1.5 --- Migration and invasion of bladder cancer --- p.19
Chapter 1.5.1 --- The biological process of migration in bladder cancer
Chapter 1.5.2 --- Epithelial to mesenchymal transition in bladder cancer
Chapter 1.6 --- Objectives of this study --- p.21
Chapter Chapter II --- MicroRNAs in urine supernatant: potential useful markers for bladder cancer screening
Chapter 2.1 --- Introduction --- p.23
Chapter 2.2 --- Materials and methods --- p.26
Chapter 2.2.1 --- Ethics Statement
Chapter 2.2.2 --- Patients and samples
Chapter 2.2.3 --- RNA extraction
Chapter 2.2.4 --- MicroRNA microarray
Chapter 2.2.5 --- Quantitative real-time polymerase chain reaction (RT-PCR)
Chapter 2.2.6 --- Statistical methods
Chapter 2.3 --- Results --- p.31
Chapter 2.3.1 --- MicroRNA screening by microRNA microarray
Chapter 2.3.2 --- Independent validation of the ten selected microRNAs by qRT-PCR on tissue
Chapter 2.3.3 --- Verification of the six validated microRNAs in urine supernatants as tumor markers
Chapter 2.3.4 --- MiR-125b and miR-99a in urine supernatants were useful for the diagnosis of bladder cancer
Chapter 2. --- 3.5 MiR-125b and miR-99a were two highly correlated microRNAs
Chapter 2.3.6 --- Expression levels of miR-125b and miR-99a increased after tumor resection
Chapter 2.4 --- Discussion --- p.47
Chapter Chapter III: --- MiR-99a suppresses migration and invasion in bladder cancer by targeting VLDLR
Chapter 3.1 --- Introduction --- p.53
Chapter 3.2 --- Materials and methods --- p.56
Chapter 3.2.1 --- Human tissue samples and bladder cancer cell lines
Chapter 3.2.2 --- RNA extraction and Polymerase Chain Reaction
Chapter 3.2.3 --- MicroRNA and plasmid transfection
Chapter 3.2.4 --- Western Immunoblotting
Chapter 3.2.5 --- Agarose gel electrophoresis
Chapter 3.2.6 --- Luciferase assay
Chapter 3.2.7 --- MTT proliferation assay
Chapter 3.2.8 --- Apoptosis assay
Chapter 3.2.9 --- Cell cycle analysis
Chapter 3.2.10 --- Cell migration Assay
Chapter 3.1.11 --- Cell invasion assay:
Chapter 3.2.12 --- Statistical methods:
Chapter 3.3 --- Results --- p.67
Chapter 3.3.1 --- MiR-99a was significantly down-regulated in bladder cancer
Chapter 3.3.2 --- Precursor microRNA was successfully transfected into bladder cancer cell lines
Chapter 3.3.3 --- MiR-99a had little effect on cell proliferation
Chapter 3.3.4 --- MiR-99a had little effect on cell apoptosis and cell cycle
Chapter 3.3.5 --- Over-expression of miR-99a suppressed cell migration in bladder cancer
Chapter 3.3.6 --- Over-expression of miR-99a also suppressed invasion ability in bladder cancer
Chapter 3.3.7 --- Target prediction for miR-99a using 8 target prediction databases
Chapter 3.3.8 --- Protein level of VLDLR was down-regulated by miR-99a in bladder cancer
Chapter 3.3.9 --- VLDLR was a direct target of miR-99a
Chapter 3.3.10 --- VLDLR mRNA was not down-regulated correspondingly by miR-99a
Chapter 3.3.11 --- MiR-99a suppressed down-stream protein of VLDLR in Reelin pathway
Chapter 3.3.12 --- Knockdown of VLDLR also suppressed cell migration and invasion
Chapter 3.3.13 --- N-cadherin was the down-stream protein responsible for the suppression of migration and invasion in miR-99a/VLDLR pathway
Chapter 3.4 --- Discussion --- p.93
Chapter Chapter IV: --- Conclusion and prospective --- p.101
Appendix --- p.105
Reference --- p.107

碩士
中山醫學大學
生化暨生物科技研究所
97
Background: Malignant tumors are at the top leading cause of death in Taiwan in the past decades. Angiogenesis is not only essential for tumor growth but is also implicated in invasion of the cancer cells into the circulation, and growth of dormant micro-metastases into frank metastatic lesions. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis as well as in solid tumors. It also has a role for VEGF in Malignant tumors; although has not been fully elucidated. This study will examine the VEGF secretary activity of malignant cells in the patients with prostate cancer or bladder cancer. The urine samples were obtained before, during and after treatment. The purposes of this study are to assay the VEGF value by ELISA method and its correlation with disease prognosis in various prostate cancer or bladder cancer; and to evaluate the VEGF concentrations between prostate cancer and bladder cancer. Method: The urine samples were collected from 8 prostate cancer and 10 bladder cancer patients in this study. It also was compared to 11 normal samples. An enzyme-linked immunosorbent assay (ELISA) was used to quantify VEGF concentrations. Urine samples will be tested using a commercial Elisa system for VEGF, (R&D systems). Identification of VEGF tumor markers for malignant disease using Enzyme-Linked Immunosorbent Assay technologies. The differential VEGF concentrations were statistically analyzed using bioinformatic softwares. Results: Our research samples were 61 specimens. The VEGF value was low in 11 cases of Negative control(2.60 pg/ml), no obvious auto-secretary activity of those cells. In 8 cases of prostatic carcinoma and 10 cases of bladder cancer, the VEGF urine level were 96.80 pg/ml(p=0.001) and 271.13 pg/ml(p=0.012) respectively( Mann-Whitney test). High VEGF level decreased significantly during treatment . However, the cases were too small to had exact predict value (only 8 and 10 cases). Patients were with high VEGF auto-secretary activity and also had bad prognosis. Conclusion: Although our study is a primary result, study cases are varied, but it still provide important information that VEGF has an important role in prostate cancer and bladder cancer. We will process further research of single and specific disease in the future to analyze the exact correlation of VEGF and malignant tumor diseases before, during and after treatment. The VEGF tumor-specific markers show great potential for the detection of the Malignant tumors.

abstract: Early detection and treatment of disease is paramount for improving human health and wellness. Micro-scale devices promote new opportunities for the rapid, cost-effective, and accurate identification of altered biological states indicative of disease early-onset; these devices function at a scale more sensitive to numerous biological processes. The application of Micro-Electro-Mechanical Systems (MEMS) in biomedical settings has recently emerged and flourished over course of the last two decades, requiring a deep understanding of material biocompatibility, biosensing sensitively/selectively, biological constraints for artificial tissue/organ replacement, and the regulations in place to ensure device safety. Capitalizing on the inherent physical differences between cancerous and healthy cells, our ultra-thin silicone membrane enables earlier identification of bladder cancer—with a 70% recurrence rate. Building on this breakthrough, we have devised an array to multiplex this sample-analysis in real-time as well as expanding beyond bladder cancer. The introduction of new materials—with novel properties—to augment current and create innovative medical implants requires the careful analysis of material impact on cellular toxicity, mutagenicity, reactivity, and stability. Finally, the achievement of replacing defective biological systems with implanted artificial equivalents that must function within the same biological constraints, have consistent reliability, and ultimately show the promise of improving human health as demonstrated by our hydrogel check valve. The ongoing proliferation, expanding prevalence, and persistent improvement in MEMS devices through greater sensitivity, specificity, and integration with biological processes will undoubtedly bolster medical science with novel MEMS-based diagnostics and therapeutics.
Dissertation/Thesis
Doctoral Dissertation Electrical Engineering 2018

Noble metal nanostructures display collective oscillation of the surface conduction electrons upon light irradiation as a form of localized surface plasmon resonance (LSPR) properties. Size, shape and the refractive index of surrounding environment are the key features that controls the LSPR properties. Surface passivating ligands have the ability to modify the charge density of nanostructures to allow resonant wavelength to match that of the incident light, a phenomenon called “plasmoelectric effect,”. According to the drude model Red and blue shifts of LSPR peak of nanostructures are observed in the event of reducing and increasing charge density, respectively. However, herein we report unusual LSPR properties of gold triangular nanoprisms (Au TNPs) upon functionalization with para-substituted thiophenols (X-Ph-SH, X = -NH₂, -OCH₃, -CH₃, -H, -Cl, -CF₃, and -NO₂). Accordingly, we hypothesized that an appropriate energy level alignment between the Au Fermi energy and the HOMO or LUMO of ligands allows delocalization of surface plasmon excitation at the hybrid inorganic-organic interface, and thus provides a thermodynamically driven plasmoelectric effect. We further validated our hypothesis by calculating the HOMO and LUMO levels and also work function changes of Au TNPs upon functionalization with para substituted thiol. We further utilized our unique finding to design ultrasensitive plasmonic substrate for biosensing of cancer microRNA in bladder cancer and owe to unpresidential sensitivity of the developed Au TNPs based LSPR sensor, for the first time we have been utilized to analysis the tumor suppressor microRNA for more accurate diagnosis of BC. Additionally, we have been advancing our sensing platform to mitigate the false positive and negative responses of the sensing platform using surface enhanced fluorescence technique. This noninvasive, highly sensitive, highly specific, also does not have false positives technique provide strong key to detect cancer at very early stage, hence increase the cancer survival rate. Moreover, the electromagnetic field enhancement of Surface-Enhanced Raman Scattering (SERS) and other related surface-enhanced spectroscopic processes resulted from the LSPR property. This dissertation describes the design and development of entirely new SERS nanosensors using flexible SERS substrate based on unique LSPR property of Au TNPs and developed sensors shows excellent SERS activity (enhancement factor = ~6.0 x 106) and limit of detection (as low as 56 parts-per-quadrillions) with high selectivity by chemometric analyses among three commonly used explosives (TNT, RDX, and PETN). Further we achieved the programable self-assembly of Au TNPs using molecular tailoring to form a 3D supper lattice array based on the substrate effect. Here we achieved the highest reported sensitivity for potent drug analysis, including opioids and synthetic cannabinoids from human plasma obtained from the emergency room. This exquisite sensitivity is mainly due to the two reasons, including molecular resonance of the adsorbate molecules and the plasmonic coupling among the nanoparticles. Altogether we are highly optimistic that our research will not only increase the patient survival rate through early detection of cancer but also help to battle the “war against drugs” that together is expected to enhance the quality of human life.