Prognostic modelling of breast cancer patients: a benchmark of predictive models with external validation

Disserta����o apresentada para obten����o do Grau de Doutor em Engenharia Electrot��cnica e de Computadores ��� Sistemas Digitais e Percepcionais pela Universidade Nova de Lisboa, Faculdade de Ci��ncias e Tecnologia

Biological activity of well defined hydantoin derivatives on efflux pump systems of bacteria and cancer cells

A multi-resist��ncia a antibi��ticos e medicamentos usados em quimioterapia �� um dos grandes problemas com os quais as institui����es de sa��de se debatem hoje em dia. A ac����o provocada por bombas de efluxo �� uma das suas causas. Estas bombas t��m uma import��ncia fundamental, uma vez que, ao expelirem todo o tipo de t��xicos para o exterior das c��lulas, tamb��m expelem medicamentos, fazendo com que estes n��o tenham o efeito desejado dentro delas. As bombas de efluxo s��o transportadores que se encontram nas membranas de todo o tipo de c��lulas. Existem dois grandes tipos de bombas de efluxo: as prim��rias e as secund��rias. As primeiras conferem multi-resist��ncia principalmente em c��lulas eucariotas, como as c��lulas do cancro em humanos, tendo como fun����o a media����o da repulsa de subst��ncias t��xicas por interm��dio da hidr��lise de ATP. A primeira a ser descoberta e mais estudada destas bombas foi a ABCB1 que �� o gene que codifica a glicoprote��na-P (P de permeabilidade). Enquanto as secund��rias, que s��o a maior fonte de multi-resist��ncia em bact��rias, promovem a extrus��o de subst��ncias t��xicas atrav��s da for��a motriz de prot��es. Neste tipo de bombas s��o conhecidas quatro fam��lias principais, das quais uma das mais importantes �� a superfam��lia RND, uma vez que inclui a bomba AcrAB-TolC, que �� muito importante no metabolismo xenobi��tico de bact��rias Gramnegativas, nomeadamente a E.coli. Com o objectivo de reverter a multi-resist��ncia, tanto em c��lulas eucariotas como procariotas, t��m-se desenvolvido estrat��gias de combate que envolvem a descoberta de subst��ncias que inibam as bombas de efluxo. Assim sendo, ao longo dos tempos t��m sido descobertas variadas subst��ncias que cumprem este objectivo. �� o caso, por exemplo, dos derivados de fluoroquinolonas usados como inibidores de bombas de efluxo em bact��rias ou do Tamoxifen, utilizado na terapia de pacientes com cancro da mama. Um dos grupos de subst��ncias estudados para o desenvolvimento de poss��veis compostos que actuem como reversores de multi-resist��ncia s��o os compostos derivados de hidanto��nas. Estes, s��o conhecidos por possu��rem uma grande variedade de propriedades bioqu��micas e farmacol��gicas, sendo portanto usados para tratarem algumas doen��as em humanos, como a epilepsia. Nestes, est��o englobados compostos com actividade anti-convuls��o que constitui a sua grande mais-valia e, dependente da substitui����o no anel que os constitui, uma grande variedade de outras propriedades farmacol��gicas como a anti-fungica, a anti-arritmica, a anti-viral, a anti-diab��tica ou por exemplo a antagoniza����o de determinados receptores, como os da serotonina. Apesar de pouco usados em estudos experimentais para desenvolver subst��ncias anti-carcinog��nicas, existem alguns estudos com este efeito. Objectivos: O presente projecto envolve o estudo de bombas de efluxo prim��rias e secund��rias, em c��lulas eucariotas e procariotas, respectivamente. Em bact��rias, foram usados quatro modelos experimentais: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, E. coli AG 100 e Salmonella Enteritidis NCTC 13349. Em c��lulas de cancro foram usadas, c��lulas T de linfoma de rato parentais e c��lulas T de linfoma de rato transfectadas com o gene humano MDR-1. O principal objectivo deste estudo foi a pesquisa de novos moduladores de bombas de efluxo presentes em bact��rias e c��lulas do cancro, tentando assim contribuir para o desenvolvimento de novos agentes farmacol��gicos que consigam reverter a multi-resist��ncia a medicamentos. Assim sendo foram testados trinta compostos derivados de hidanto��nas: SZ-2, SZ-7, LL-9, BS-1, JH-63, MN-3, TD-7k, GG-5k, P3, P7, P10, P11, RW-15b, AD-26, RW-13, AD-29, KF-2, PDPH-3, Mor-1, KK-XV, Thioam-1, JHF-1, JHC-2, JHP-1, Fur-2, GL-1, GL-7, GL-14, GL-16, GL-18. Como forma de atingir estes objectivos, a actividade biol��gica dos trinta compostos derivados de hidanto��nas foi avaliada nas quatro estirpes de bact��rias da seguinte forma: foram determinadas as concentra����es m��nimas inibit��rias dos trinta compostos como forma de definir as concentra����es em que os compostos seriam utilizados. Os compostos foram posteriormente testadas com um m��todo fluorom��trico de acumula����o de brometo de et��deo, que �� um substrato comum em bombas de efluxo bacterianas, desenvolvido por Viveiros et al. A actividade biol��gica dos compostos derivados de hidanto��nas nas c��lulas de cancro foi demonstrada por diferentes m��todos. O efeito anti-proliferativo e citot��xico dos trinta compostos foi avaliado nas c��lulas T de linfoma de rato transfectadas com o gene humano MDR-1 pelo m��todo de thiazolyl de tetraz��lio (MTT). Como o brometo de et��deo tamb��m �� expelido pelos transportadores ABC, estes compostos foram posteriormente testados com um m��todo fluorom��trico de acumula����o de brometo de et��deo desenvolvido por Spengler et al nos dois diferentes tipos de c��lulas eucariotas. Resultados: A maioria dos compostos derivados de hidanto��nas foi eficaz na modula����o de bombas de efluxo, nas duas estirpes de bact��rias Gram-negativas e nos dois diferentes tipos de c��lulas T de linfoma. Em contraste com estes resultados, nas duas estirpes de c��lulas Gram-positivas, a maioria dos compostos tiveram pouco efeito na inibi����o de bombas de efluxo ou at�� nenhum, em muitos dos casos. De uma maneira geral os melhores compostos nas diferentes estirpes de bact��rias foram: Thioam-1, SZ-2, P3, Rw-15b, AD-26, AD-29, GL-18, GL-7, KF-2, SZ-7, MN-3, GL-16 e GL- 14. Foram portanto estes os compostos que provocaram maior acumula����o de brometo de et��deo, inibindo assim com maior efic��cia as bombas de efluxo. No presente estudo, a maioria dos compostos conseguiu inibir a resist��ncia provocada pela bomba de efluxo ABCB1, tanto nas c��lulas parentais bem como nas c��lulas que sobre-expressam esta bomba, causando a acumula����o de brometo de et��deo dentro das c��lulas. As c��lulas que sobreexpressam a bomba ABCB1 foram posteriormente testadas com citometria de fluxo que �� a t��cnica padr��o para pesquisa de inibidores de bombas de efluxo. Os compostos que foram mais efectivos na inibi����o da bomba ABCB1, causando assim maior acumula����o de brometo de et��deo nas c��lulas que sobre-expressam esta bomba foram: PDPH-3, GL-7, KK-XV, AD-29, Thioam-1, SZ-7, KF-2, MN-3, RW-13, LL-9, P3, AD-26, JH-63 e RW- 15b. Este facto n��o corroborou totalmente os resultados da citometria de fluxo uma vez que os moduladores que provocaram maior inibi����o da bomba ABCB1 foram o MN-3, JH-63 e o BS-1, sendo que o ��ltimo n��o foi seleccionado como um bom composto usando o m��todo fluorom��trico de acumula����o de brometo de et��deo. Conclus��o: Os compostos derivados de hidanto��nas testados tiveram maior efeito nas estirpes de bact��rias Gram-negativas do que nas Gram-positivas. Relativamente ��s c��lulas eucariotas, as estruturas mais activas apresentam substituintes arom��ticos bem como alguns fragmentos aminicos terci��rios.

The involvement of CDH1 in cancer angiogenesis

Disserta����o para obten����o do Grau de Mestre em Biotecnologia

Cancer theranostics: multifunctional gold nanoparticles for diagnostics and therapy

Doctorate in Biology, Specialty in Biotechnology

Evaluation of chemotherapeutic potential of natural extracts using 3D models of colon cancer

Dissertation to obtain master degree in Biotechnology

Development of a "green process" for the isolation of natural functional extracts with anti-cancer activity - Application of high-pressure technology

Disserta����o para obten����o do Grau de Mestre em Engenharia Qu��mica e Bioqu��mica

Cues for cancer stem cells origin

Disserta����o para obten����o do Grau de Mestre em Gen��tica Molecular e Biomedicina

Transimpedance amplifier for early detection of breast cancer

Breast cancer is the most common type of cancer worldwide. The effectiveness of its treatment depends on early stage detection, as well as on the accuracy of its diagnosis. Recently, diagnosis techniques have been submitted to relevant breakthroughs with the upcoming of Magnetic Resonance Imaging, Ultrasound Sonograms and Positron Emission Tomography (PET) scans, among others. The work presented here is focused on studying the application of a PET system to a Positron Emission Mammography (PEM) system. A PET/PEM system works under the principle that a scintillating crystal will detect a gamma-ray pulse, originated at the cancerous cells, converting it into a correspondent visible light pulse. The latter must then be converted into an electrical current pulse by means of a Photo- -Sensitive Device (PSD). After the PSD there must be a Transimpedance Amplifier (TIA) in order to convert the current pulse into a suitable output voltage, in a time period lower than 40 ns. In this Thesis, the PSD considered is a Silicon Photo-Multiplier (SiPM). The usage of this recently developed type of PSD is impracticable with the conventional TIA topologies, as it will be proven. Therefore, the usage of the Regulated Common-Gate (RCG) topology will be studied in the design of the amplifier. There will be also presented two RCG variations, comprising a noise response improvement and differential operation of the circuit. The mentioned topology will also be tested in a Radio-Frequency front-end, showing the versatility of the RCG. A study comprising a low-voltage self-biasing feedback TIA will also be shown. The proposed circuits will be simulated with standard CMOS technology (UMC 130 nm), using a 1.2 V power supply. A power consumption of 0.34 mW with a signal-to-noise ratio of 43 dB was achieved.

Assessment of Notch1 ligands production - key protein targets in breast cancer

Cell-to-cell communication is required for many biological processes in development and adult life. One of the most common systems utilized by a wide range of eukaryotes is the Notch signalling pathway. Four Notch receptors and five ligands have been identified in mammals that interact via their extracellular domains leading to transcription activation. Studies have shown that the Notch ligands expression is undetectable in normal breast tissues, but moderate to high expression has been detected in breast cancer. Thus, any of the Notch1 ligands can be studied as possible therapeutic targets for breast cancer. To study Notch pathway proteins there is the need to obtain stable protein solutions. E. coli is the host of excellence for recombinant proteins for the ease of use, fast growth and high cell densities. However, the expression of mammalian proteins in such systems may overwhelm the bacterial cellular machinery, which does not possess the ability for post-translational modifications, or dedicated compartments for protein synthesis. Mammalian cells are therefore preferred, despite their technical and financial increased demands. We aim to determine the best expression and purification conditions for the different ligand protein constructs, to develop specific function-blocking antibodies using the Phage Display technology. Moreover, we propose to crystallize the Notch1 ligands alone and in complex with the phage display selected antibodies, unveiling molecular details. hJag2DE3 and hDll1DE6 proteins were purified from refolded inclusion bodies or mammalian cell culture supernatants, respectively, and purity was confirmed by SDS-PAGE (>95%). Protein produced in mammalian cells showed to be more stable, apparently with the physiological disulfide pattern, contrary to what was observed in the refolded protein. Several nano-scale crystallization experiments were set up in 96-well plates, but no positive result was obtained. We will continue to pursue for the best expression for the Notch ligand constructs in both expression systems.

Microwave imaging of the axilla to aid breast cancer diagnosis

Breast cancer is the most common type of cancer among women all over the world. An important issue that is not commonly addressed in breast cancer imaging literature is the importance of imaging the underarm region���where up to 80% of breast cancer cells can metastasise to. The first axillary lymph nodes to receive drainage from the primary tumour in the breast are called Sentinel Node. If cancer cells are found in the Sentinel Node, there is an increased risk of metastatic breast cancer which makes this evaluation crucial to decide what follow-up exams and therapy to follow. However, non-invasive detection of cancer cells in the lymph nodes is often inconclusive, leading to the surgical removal of too many nodes which causes adverse side-effects for patients. Microwave Imaging is one of the most promising non-invasive imaging modalities for breast cancer early screening and monitoring. This novel study tests the feasibility of imaging the axilla region by means of the simulation of an Ultra-Wideband Microwave Imaging system. Simulations of such system are completed in several 2D underarm models that mimic the axilla. Initial imaging results are obtained by means of processing the simulated backscattered signals by eliminating artefacts caused by the skin and beamforming the processed signals in order to time-align all the signals recorded at each antenna. In this dissertation several image formation algorithms are implemented and compared by visual inspection of the resulting images and through a range of performance metrics, such as Signal-to-Clutter Ratio and FullWidth Half Maximum calculations. The results in this study showed that Microwave Imaging is a promising technique that might allow to identify the presence and location of metastasised cancer cells in axillary lymph nodes, enabling the non-invasive evaluation of breast cancer staging.

Phage display as a tool for development of novel therapeutics for breast cancer

Phage display technology is a powerful platform for the generation of highly specific human monoclonal antibodies (Abs) with potential use in clinical applications. Moreover, this technique has also proven to be a reliable approach in identifying and validating new cancer-related targets. For scientific or medical applications, different types of Ab libraries can be constructed. The use of Fab Immune libraries allows the production of high quality and affinity antigen-specific Abs. In this work, two immune human phage display IgG Fab libraries were generated from the Ab repertoire of 16 breast cancer patients, in order to obtain a tool for the development of new therapeutic Abs for breast cancer, a condition that has great impact worldwide. The generated libraries are estimated to contain more than 108 independent clones and a diversity over 90%. Libraries validation was pursued by selection against BSA, a foreign and highly immunogenic protein, and HER2, a well established cancer target. Preliminary results suggested that phage pools with affinity for these antigens were selected and enriched. Individual clones were isolated, however, it was not possible to obtain enough data to further characterize them. Selection against the DLL1 protein was also performed, once it is a known ligand of the Notch pathway, whose deregulation is associated to breast cancer, making it an interesting target for the generation of function-blocking Abs. Selection resulted in the isolation of a clone with low affinity and Fab expression levels. The validation process was not completed and further effort will have to be put in this task in the future. Although immune libraries concept implies limited applicability, the library reported here has a wide range of use possibilities, since it was not restrained to a single antigen but instead thought to be used against any breast cancer associated target, thus being a valuable tool.

Immunohistochemistry detection of putative miR-200c and miR-203 targets in breast cancer patients

Part of this thesis will be published in the following: Gomes, B.C., Santos, B. 2015. Methods for studying microRNAs expression and their targets in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. In Methods in Molecular Biology: Cancer Drug Resistance (Rueff, J. & Rodrigues, A.S. eds), Springer Protocols.

Impact of using different radiation therapy techniques in breat cancer: contralateral breast dose

RESUMO: O cancro de mama e o mais frequente diagnoticado a indiv duos do sexo feminino. O conhecimento cientifico e a tecnologia tem permitido a cria ����o de muitas e diferentes estrat egias para tratar esta patologia. A Radioterapia (RT) est a entre as diretrizes atuais para a maioria dos tratamentos de cancro de mama. No entanto, a radia ����o e como uma arma de dois canos: apesar de tratar, pode ser indutora de neoplasias secund arias. A mama contralateral (CLB) e um org��o susceptivel de absorver doses com o tratamento da outra mama, potenciando o risco de desenvolver um tumor secund ario. Nos departamentos de radioterapia tem sido implementadas novas tecnicas relacionadas com a radia ����o, com complexas estrat egias de administra ����o da dose e resultados promissores. No entanto, algumas quest��es precisam de ser devidamente colocadas, tais como: E seguro avan��ar para tecnicas complexas para obter melhores indices de conformidade nos volumes alvo, em radioterapia de mama? O que acontece aos volumes alvo e aos tecidos saudaveis adjacentes? Qu��o exata e a administra����o de dose? Quais s��o as limita����es e vantagens das t��cnicas e algoritmos atualmente usados? A resposta a estas quest��es e conseguida recorrendo a m etodos de Monte Carlo para modelar com precis��o os diferentes componentes do equipamento produtor de radia ����o(alvos, ltros, colimadores, etc), a m de obter uma descri c��o apropriada dos campos de radia c��o usados, bem como uma representa ����o geometrica detalhada e a composi����o dos materiais que constituem os org��os e os tecidos envolvidos. Este trabalho visa investigar o impacto de tratar cancro de mama esquerda usando diferentes tecnicas de radioterapia f-IMRT (intensidade modulada por planeamento direto), IMRT por planeamento inverso (IMRT2, usando 2 feixes; IMRT5, com 5 feixes) e DCART (arco conformacional dinamico) e os seus impactos em irradia ����o da mama e na irradia ����o indesejada dos tecidos saud aveis adjacentes. Dois algoritmos do sistema de planeamento iPlan da BrainLAB foram usados: Pencil Beam Convolution (PBC) e Monte Carlo comercial iMC. Foi ainda usado um modelo de Monte Carlo criado para o acelerador usado (Trilogy da VARIAN Medical Systems), no c odigo EGSnrc MC, para determinar as doses depositadas na mama contralateral. Para atingir este objetivo foi necess ario modelar o novo colimador multi-laminas High- De nition que nunca antes havia sido simulado. O modelo desenvolvido est a agora dispon�� vel no pacote do c odigo EGSnrc MC do National Research Council Canada (NRC). O acelerador simulado foi validado com medidas realizadas em agua e posteriormente com c alculos realizados no sistema de planeamento (TPS).As distribui ����es de dose no volume alvo (PTV) e a dose nos org��os de risco (OAR) foram comparadas atrav es da an alise de histogramas de dose-volume; an alise estati stica complementar foi realizadas usando o software IBM SPSS v20. Para o algoritmo PBC, todas as tecnicas proporcionaram uma cobertura adequada do PTV. No entanto, foram encontradas diferen cas estatisticamente significativas entre as t ecnicas, no PTV, nos OAR e ainda no padr��o da distribui ����o de dose pelos tecidos s��os. IMRT5 e DCART contribuem para maior dispers��o de doses baixas pelos tecidos normais, mama direita, pulm��o direito, cora c��o e at e pelo pulm��o esquerdo, quando comparados com as tecnicas tangenciais (f-IMRT e IMRT2). No entanto, os planos de IMRT5 melhoram a distribui����o de dose no PTV apresentando melhor conformidade e homogeneidade no volume alvo e percentagens de dose mais baixas nos org��os do mesmo lado. A t ecnica de DCART n��o apresenta vantagens comparativamente com as restantes t ecnicas investigadas. Foram tamb em identi cadas diferen cas entre os algoritmos de c alculos: em geral, o PBC estimou doses mais elevadas para o PTV, pulm��o esquerdo e cora ����o, do que os algoritmos de MC. Os algoritmos de MC, entre si, apresentaram resultados semelhantes (com dferen cas at e 2%). Considera-se que o PBC n��o e preciso na determina ����o de dose em meios homog eneos e na regi��o de build-up. Nesse sentido, atualmente na cl nica, a equipa da F sica realiza medi ����es para adquirir dados para outro algoritmo de c alculo. Apesar de melhor homogeneidade e conformidade no PTV considera-se que h a um aumento de risco de cancro na mama contralateral quando se utilizam t ecnicas n��o-tangenciais. Os resultados globais dos estudos apresentados confirmam o excelente poder de previs��o com precis��o na determina����o e c alculo das distribui ����es de dose nos org��os e tecidos das tecnicas de simula����o de Monte Carlo usados.---------ABSTRACT:Breast cancer is the most frequent in women. Scienti c knowledge and technology have created many and di erent strategies to treat this pathology. Radiotherapy (RT) is in the actual standard guidelines for most of breast cancer treatments. However, radiation is a two-sword weapon: although it may heal cancer, it may also induce secondary cancer. The contralateral breast (CLB) is a susceptible organ to absorb doses with the treatment of the other breast, being at signi cant risk to develop a secondary tumor. New radiation related techniques, with more complex delivery strategies and promising results are being implemented and used in radiotherapy departments. However some questions have to be properly addressed, such as: Is it safe to move to complex techniques to achieve better conformation in the target volumes, in breast radiotherapy? What happens to the target volumes and surrounding healthy tissues? How accurate is dose delivery? What are the shortcomings and limitations of currently used treatment planning systems (TPS)? The answers to these questions largely rely in the use of Monte Carlo (MC) simulations using state-of-the-art computer programs to accurately model the di erent components of the equipment (target, lters, collimators, etc.) and obtain an adequate description of the radiation elds used, as well as the detailed geometric representation and material composition of organs and tissues. This work aims at investigating the impact of treating left breast cancer using di erent radiation therapy (RT) techniques f-IMRT (forwardly-planned intensity-modulated), inversely-planned IMRT (IMRT2, using 2 beams; IMRT5, using 5 beams) and dynamic conformal arc (DCART) RT and their e ects on the whole-breast irradiation and in the undesirable irradiation of the surrounding healthy tissues. Two algorithms of iPlan BrainLAB TPS were used: Pencil Beam Convolution (PBC)and commercial Monte Carlo (iMC). Furthermore, an accurate Monte Carlo (MC) model of the linear accelerator used (a Trilogy R VARIANR) was done with the EGSnrc MC code, to accurately determine the doses that reach the CLB. For this purpose it was necessary to model the new High De nition multileaf collimator that had never before been simulated. The model developed was then included on the EGSnrc MC package of National Research Council Canada (NRC). The linac was benchmarked with water measurements and later on validated against the TPS calculations. The dose distributions in the planning target volume (PTV) and the dose to the organs at risk (OAR) were compared analyzing dose-volume histograms; further statistical analysis was performed using IBM SPSS v20 software. For PBC, all the techniques provided adequate coverage of the PTV. However, statistically significant dose di erences were observed between the techniques, in the PTV, OAR and also in the pattern of dose distribution spreading into normal tissues. IMRT5 and DCART spread low doses into greater volumes of normal tissue, right breast, right lung, heart and even the left lung than tangential techniques (f-IMRT and IMRT2). However,IMRT5 plans improved distributions for the PTV, exhibiting better conformity and homogeneity in target and reduced high dose percentages in ipsilateral OAR. DCART did not present advantages over any of the techniques investigated. Di erences were also found comparing the calculation algorithms: PBC estimated higher doses for the PTV, ipsilateral lung and heart than the MC algorithms predicted. The MC algorithms presented similar results (within 2% di erences). The PBC algorithm was considered not accurate in determining the dose in heterogeneous media and in build-up regions. Therefore, a major e ort is being done at the clinic to acquire data to move from PBC to another calculation algorithm. Despite better PTV homogeneity and conformity there is an increased risk of CLB cancer development, when using non-tangential techniques. The overall results of the studies performed con rm the outstanding predictive power and accuracy in the assessment and calculation of dose distributions in organs and tissues rendered possible by the utilization and implementation of MC simulation techniques in RT TPS.

Thomsen-Friedenreich antigens in bladder cancer: evaluation of their prognostic value

RESUMO: Introdu����o. O cancro de bexiga �� uma patologia comum que representa o 6�� e o 5�� cancro mais incidente em Portugal e na It��lia, respetivamente. Em mais de metade dos casos ocorre reincid��ncia durante o primeiro ano, requerendo acompanhamento cl��nico ao longo da vida. A instila����o intravesical de Bacillus Calmette-Gu��rin (BCG) (uma estirpe atenuada do Mycobacterium bovis) representa uma imunoterapia eficaz no combate ao cancro de bexiga, no entanto, muitos aspetos da intera����o de BCG com as c��lulas tumorais bem como com as c��lulas do sistema imunit��rio permanecem por desvendar. As c��lulas tumorais de bexiga expressam frequentemente as formas sialiladas dos antig��nios de Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). Contudo ainda se desconhece o significado da sua express��o na malignidade tumoral e se afeta a efic��cia da terap��utica BCG. Objetivo do estudo. Investigar o papel dos antig��nios sT e sTn no fen��tipo maligno de c��lulas de cancro de bexiga bem como na resposta mediada pelo sistema imunit��rio �� terapia com BCG. Metodologia. Para tal, foram utilizadas as linhas celulares de cancro da bexiga HT1376 e MCR, geneticamente modificadas por transdu����o com vetores codificantes para as sialiltransferases ST3GAL1 ou ST6GALNAC1, de forma a expressar homogeneamente os antig��nios sT ou sTn respetivamente. Estes modelos celulares foram estudados ap��s confronto com BCG. O n��vel de BCG internalizado foi avaliado por citometria de fluxo. O perfil global de express��o gen��tica dos modelos celulares antes e ap��s incuba����o com BCG foi analisado pela tecnologia de microarray. O perfil de citocinas secretadas pelos modelos celulares ap��s incuba����o com BCG, bem como de macr��fagos estimulados pelo secretoma de c��lulas de cancro de bexiga que por sua vez foram estimuladas previamente por BCG, foi estudado pelo sistema multiplex de ���imuno-esferas���. Resultados. A an��lise do transcritoma dos modelos celulares revelou que grupos de genes envolvidos em fun����es espec��ficas foram modulados em paralelo nos dois modelos celulares, ap��s transdu����o, independentemente da sialiltransferase expressa. Ou seja, em c��lulas que expressavam a sialiltransferase ST3GAL1 ou ST6GALNAC1, os genes envolvidos na regula����o da segrega����o cromoss��mica e na repara����o do DNA foram consistentemente regulados negativamente. Genes descritos na literatura como marcadores para o cancro de bexiga foram tamb��m modulados. A incuba����o com BCG resultou numa tend��ncia ao aumento da express��o de genes relevantes na preserva����o e estabilidade gen��mica e menor malignidade, no entanto, apenas em c��lulas que expressavam sT ou sTn. Entre as dez citocinas testadas, apenas a IL-6 e IL-8 foram expressas pelas linhas celulares de cancro da bexiga, com indu����o destas ap��s estimula����o com BCG, e principalmente em c��lulas que expressavam ST3GAL1 ou ST6GALNAC1. Em macr��fagos, citocinas inflamat��rias, tais como IL-1��, IL-6 e TNF��, e a citocina anti-inflamat��ria IL-10, foram induzidas apenas pelo secretoma de c��lulas de cancro da bexiga confrontadas com BCG, com maior relev��ncia quando estas expressavam ST3GAL1 ou ST6GALNAC1, prevendo a estimula����o de macr��fagos semelhantes aos de tipo M1 e uma melhor resposta �� terapia com BCG. Conclus��es. O efeito geral da express��o destas sialiltransferases e dos produtos enzim��ticos sT ou sTn nas c��lulas de cancro de bexiga conduz a um fen��tipo de maior malignidade. Contudo, a maior avidez de estas na produ����o de citocinas inflamat��rias ap��s confronto com BCG, bem como a maior capacidade de estimula����o de macr��fagos, predir�� uma resposta �� terapia com BCG mais eficaz em tumores que expressem os antig��nios de TF sialilados. Tais conclus��es s��o totalmente concordantes com os nossos mais recentes dados cl��nicos obtidos em colabora����o, que mostram que em doentes com cancro de bexiga que expressam sTn respondem melhor a terapia BCG. ----------ABSTRACT: Background. Bladder cancer is a common malignancy representing the 6th and the 5th most incident cancer in Portugal and in Italy, respectively. More than half of the cases relapse within one year, requiring though a lifelong follow-up. Intravesical instillation of Bacillus Calmette-Gu��rin (BCG) (an attenuated strain of Mycobacterium bovis) represents an effective immunotherapy of bladder cancer, although many aspects of the interaction of BCG with cancer cells and host immune cells remain obscure. Bladder cancer cells often express the sialylated forms of the Thomsen-Friedenreich (TF), i.e., sialil-T (sT) e sialil-Tn (sTn). However, it���s still unknown the sense of such expression in tumour malignancy and in the BCG therapy efficacy. Aim of the study. To investigate the role of the sT and sTn antigens on the malignant phenotype of bladder cancer cells and the immune mediated response to BCG therapy. Experimental. We have utilized populations of the bladder cancer cell lines HT1376 and MCR, genetically modified by transduction with the sialyltransferases ST3GAL1 or ST6GALNAC1 to express homogeneously sT or sTn antigens. The level of BCG internalized was assessed by flow cytometry. The whole gene expression profile of BCG-challenged or unchallenged bladder cancer cell lines was studied by microarray technology. The profile of cytokines secreted by BCG-challenged bladder cancer cells and that of macrophages challenged by the secretome of BCG-challenged bladder cancer cells was studied by multiplex immune-beads assay. Results. Transcriptome analysis of the sialyltransferase-transduced cells revealed that groups of genes involved in specific functions were regulated in parallel in the two cell lines, regardless the sialyltransferase expressed. Namely, in sialyltransferase-expressing cells, genes involved in the proper chromosomal segregation and in the DNA repair were consistently down-regulated, while genes reported in literature as markers for bladder cancer were modulated. BCG-challenging induced a tendency to up-regulation of the genes preserving genomic stability and reducing malignancy, but only in cells expressing either sT or sTn. Among the ten cytokines tested, only IL-6 and IL-8 were expressed by bladder cancer cell lines and up-regulated by BCG-challenging, mainly in sialyltransferases-expressing cells. In macrophages, inflammatory cytokines, such as IL-1��, IL-6 and TNF��, and the antinflammatory IL-10 were induced only by the secretome of BCG-challenged bladder cancer cells, particularly when expressing either sialyltransferase, predicting the stimulation of M1-like macrophages and a better response to BCG therapy. Conclusions. The general effect of the expression of the two sialyltransferases and their products in the bladder cancer cells is toward a more malignant phenotype. However, the stronger ability of sialyltransferase expressing cells to produce inflammatory cytokines upon BCG-challenging and to stimulate macrophages predicts a more effective response to BCG in tumours expressing the sialylated TF antigens. This is fully consistent with our recent clinical data obtained in collaboration, showing that patients with bladder cancer expressing sTn respond better to BCG therapy.

Design of a CMOS amplifier for breast cancer detection

A transimpedance amplifier (TIA) is used, in radiation detectors like the positron emission tomography(PET), to transform the current pulse produced by a photo-sensitive device into an output voltage pulse with a desired amplitude and shape. The TIA must have the lowest noise possible to maximize the output. To achieve a low noise, a circuit topology is proposed where an auxiliary path is added to the feedback TIA input, In this auxiliary path a differential transconductance block is used to transform the node voltage in to a current, this current is then converted to a voltage pulse by a second feedback TIA complementary to the first one, with the same amplitude but 180�� out of phase with the first feedback TIA. With this circuit the input signal of the TIA appears differential at the output, this is used to try an reduced the circuit noise. The circuit is tested with two different devices, the Avalanche photodiodes (APD) and the Silicon photomultiplier (SIPMs). From the simulations we find that when using s SIPM with Rx=20k�� and Cx=50fF the signal to noise ratio is increased from 59 when using only one feedback TIA to 68.3 when we use an auxiliary path in conjunction with the feedback TIA. This values where achieved with a total power consumption of 4.82mv. While the signal to noise ratio in the case of the SIPM is increased with some penalty in power consumption.