Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

What do broadband consumers want? Design and execution of a questionnaire on demand for bundled services on the Portuguese telecomunications market

The report addresses the question of what are the preferences of broadband consumers on the Portuguese telecommunication market. A triple play bundle is being investigated. The discrete choice analysis, adopted in the study, base on 110 responses, mainly from NOVA students. The data for the analysis was collected via manually designed on-line survey. The results show that the price attribute is relatively the most important one while the television attribute is being overlooked in the decision making process. Main effects examined in the research are robust. In addition, "extras" components are being tested in terms of users' preferences.

Caracterização da carga da doença arterial periférica dos membros inferiores : contributos para um melhor conhecimento do peso desta doença no internamento hospitalar

RESUMO - A doença arterial periférica (DAP) apresenta uma carga de doença significativa, afetando cerca de 3 a 10% da população em geral e 15 a 20% dos indivíduos com idade superior a 70 anos. A sua prevalência em Portugal foi estimada em cerca de 5,9% no continente; 6,6% na Região Autónoma dos Açores (RAA) e 3,8% na Região Autónoma da Madeira (RAM). Para além da importante carga de doença, quer em termos epidemiológicos, quer económicos, a DAP confere aos seus portadores um risco cardiovascular agravado, sendo que os mesmos apresentam cerca do triplo do risco de mortalidade e de eventos cardiovasculares quando comparados com indivíduos sem DAP. Tratou-se de um estudo observacional, transversal e descritivo tendo como base duas populações de estudo. A primeira é referente aos hospitais do Serviço Nacional de Saúde (SNS) com a valência de cirurgia vascular e a segunda à população portuguesa com episódios de internamento por diagnóstico de DAP dos membros inferiores (MI) nos anos de 2013 e 2014 na totalidade dos hospitais do SNS. Através da análise dos resultados do questionário procedeu-se à descrição de algumas das características dos serviços e unidades de cirurgia vascular de sete hospitais do SNS; através da análise da base de dados dos GDH para os anos de 2013 e 2014 procedeu-se à caracterização do peso do internamento por DAP dos membros inferiores a nível nacional no mesmo período. A DAP tem uma carga significativa e atendendo aos seus fatores de risco e história natural da doença, apresenta uma tendência crescente durante os próximos anos, representando por isso um enorme desafio para os sistemas de saúde. São, no entanto, necessários estudos mais aprofundados sobre o tema que permitam conhecer melhor o peso desta patologia e, de forma global, melhorar o planeamento, tendo por base a caracterização quer do lado da procura (dados epidemiológicos e peso no internamento), quer da oferta (capacidade instalada).

Clinical decisions in breast cancer, a heterogenous disease

RESUMO: Introdução: Tratamento do carcinoma da mama Este trabalho inicia-se com a história do tratamento do carcinoma da mama, desde os primeiros documentos que descrevem doentes com carcinoma da mama até 1950. Desde 1950 até 2000 o diagnóstico, risco e as modalidades terapêuticas usadas no tratamento das doentes são mais detalhadas com ênfase nas terapêuticas locais, regionais e sistémicas. Parte 1:Quem tratar com terapêutica sistémica adjuvante Capítulo 1: A classificação TNM não está morta no carcinoma da mama Tem sido dito que a classificação TNM não é adequada para usar como ferramenta de prognóstico e decisão terapêutica no carcinoma da mama, especialmente em doentes com carcinoma detectado através de rastreio, que tem geralmente menores dimensões. A razão desta classificação não ser adequada prendese com o facto de não estarem incluidos parâmetros biológicos na classificação TNM atual. Pusemos a hipótese de que numa população com alta percentagem de carcinoma da mama não detectado em exames de rastreio, com uma mediana de idade baixa e com alta percentagem de estadios II e III, o estadiamento clássico, pela classificação TNM, é mais descriminatório que as características biológicas na determinação do prognóstico. Para isto analisámos uma população de doentes com carcinoma da mama tratados consecutivamente na mesma instituição, durante 10 anos. Caracterizámos os fatores de prognóstico do estadiamento clássico incluídos na classificação TNM e as variantes biológicas, presentemente não incluídas na classificação TNM. Quantificámos a capacidade de cada um dos factores de prognóstico para para prever a sobrevivência. A população é de 1699 doentes com carcinoma da mama que foram tratádos com terapêutica sistémica adjuvante. Individualmente, cada um dos fatores de prognostico, clássicos ou biológicos, diferem significativamente entre doentes que sobrevivem e que não sobrevivem. Explicitamente, como previsto, doentes com tumores maiores, envolvimento dos gânglios axilares, estadios TNM mais avançados, que não expressam recetor de esrogéneo, com amplificação do gene Her2, triplos negativos ou de menor diferenciação têm menor sobrevida. Na análise multivariada, só os fatores de prognostico da classificação TNM, o grau histológico e a amplificação do gene Her2, esta última com menos significância estatistica são preditores independentes de sobrevivência. Capítulo 2: Em busca de novos factores de prognostico: Poder preditivo e mecanismo das alterações de centrossomas em carcinoma da mama Compilámos inúmeros grupos de experiências de genómica feitas em tumores primários de doentes com carcinoma da mama para as quais existe informação prognóstica. Estas experiências são feitas com o objectivo de descobrir novos factores de prognóstico. Reanalisámos os dados, repetindo a mesma pergunta: Quais são os genes com expressão diferencial estatisticamente significativa entre doentes que recaíram e doentes que não recaíram. Identificámos 65 genes nestas condições e o MKI67, o gene que codifica a proteina Ki67, estava nesse grupo. Identificámos vários genes que se sabe estarem envolvidos no processo de agregação de centrossomas. O gene que considerámos mais promissor foi a kinesina KiFC1, que já tinha sido identificada como regulador da agregação de centrossomas. Anomalias cetrossomais numéricas e estruturais têm sido observadas em neoplasias. Há dados correlacionando anolmalias centrossomais estruturais e e numéricas com o grau de malignidade e os eventos precoces da carcinogénese. Mas estas anomalias centrossomais têm um peso para a célula que deve adapatar-se ou entrará em apoptose. Os nossos resultados sugerem que existe um mecanismo adaptativo, a agregação de centrossomas, com impacto prognóstico negativo. O nosso objetivo foi quantificar o valor prognóstico das anomalias centrossomais no carcinoma da mama. Para isto usámos material de doentes dos quais sabemos a história natural. Avaliámos os genes de agregação de centrossomas, KIFC1 e TACC3, nas amostras tumorais arquivadas em parafina: primeiro com PCR (polymerase chain reaction) quantitativa e depois com imunohistoquímica (IHQ). Apenas a proteína KIFC1 foi discriminatória em IHQ, não se tendo conseguido otimizar o anticorpo da TACC3. Os níveis proteicos de KIFC1 correlacionam-se com mau prognóstico. Nas doentes que recaíram observámos, no tumor primário, maior abundância desta proteína com localização nuclear. Em seguida, demonstrámos que a agregação de centrossomas é um fenómeno que ocorre in vivo. Identificámos centrossomas agregados em amostras de tumores primários de doentes que recaíram. Tecnicamente usámos microscopia de fluorescência e IHQ contra proteínas centrossomais que avaliámos nos tumores primários arquivados em blocos de parafina. Observámos agregação de centrossomas num pequeno número de doentes que recaíram, não validámos, ainda, este fenótipo celular em larga escala. Parte 2: Como tratar com terapêutica sistémica os vários subtipos de carcinoma da mama Capítulo 3: Quantas doenças estão englobadas na definição carcinoma da mama triplo negativo? (revisão) O carcinoma da mama triplo negativo é um tumor que não expressa três proteínas: recetor de estrogénio, recetor de progesterona e o recetor do fator de crescimento epidermico tipo 2 (Her2). As doentes com estes tumores não são ainda tratadas com terapêutica dirigida, possivelmente porque esta definição negativa não tem ajudado. Sabemos apenas as alterações genéticas que estes tumores não têm, não as que eles têm. Talvez por esta razão, estes tumores são o subtipo mais agressivo de carcinoma da mama. No entanto, na prática clínica observamos que estas doentes não têm sempre mau prognóstico, além de que dados de histopatologia e epidemiologia sugerem que esta definição negativa não está a capturar um único subtipo de carcinoma da mama, mas vários. Avaliámos criticamente esta evidência, clínica, histopatológica, epidemiológica e molecular. Há evidência de heterogeneidade, mas não é claro quantos subtipos estão englobados nesta definição de carcinoma da mama triplo negativo. A resposta a esta pergunta, e a identificação do fundamento molecular desta heterogeneidade vai ajudar a melhor definir o prognóstico e eventualmente a definir novos alvos terapêuticos nesta população difícil. Capítulo 4: Terapêuica sistémica em carcinoma da mama triplo negativo (revisão) A quimioterapia é a única terapêutica sistémica disponível para as doentes com carcinoma da mama triplo negativo, ao contrário dos outros dois subtipo de carcinoma da mama que têm com a terapêutica antiestrogénica e anti Her2, importantes benefícios. Apesar de terem surgido várias opções terapêuticas para estes doentes nennhuma terapêutica dirigida foi validada pelos ensaios clínicos conduzidos, possivelmente porque a biologia deste carcinoma ainda não foi elucidada. Muitos ensaios demonstram que os tumores triplos negativos beneficiam com quimioterapia e que as mais altas taxas de resposta patológica completa à terapêutica neoadjuvante são observadas precisamente nestes tumors. A resposta patológica completa correlaciona-se com a sobrevivência. Estamos a estudar regimes adjuvantes específicos para doentes com estes tumors, mas, neste momento, regimes de terceira geração com taxanos e antraciclinas são os mais promissores. O papel de subgrupos de fármacos específicos, como os sais de platina, mantémse mal definido. Quanto às antraciclinas e taxanos, estes grupos não mostraram beneficio específico em carcinoma da mama triplo negativo quando comparado com os outros subtipos. Os próprios carcinomas da mama triplos negativos são heterogéneos e carcinomas da mama basais triplos negativos com elevada taxa de proliferação e carcinomas da mama triplos negativos surgidos em doentes com mutação germinal BRCA1 poderão ser mais sensíveis a sais de platino e menos sensíveis a taxanos. Como a definição molecular ainda não foi explicada a busca de terapêutica dirigida vai continuar. Capítulo 5: Ensaio randomizado de fase II do anticorpo monoclonal contra o recetor do fator de crescimento epidérmico tipo 1 combinado com cisplatino versus cisplatino em monoterapia em doentes com carcinoma da mama triplo negativo metastizado O recetor do fator de crescimento epidérmico tipo 1 está sobre expresso nos tumores das doentes com carcinoma da mama triplo negativo metastizado, um subtipo agressivo de carcinoma da mama. Este ensaio investigou a combinação de cetuximab e cisplatino versus cisplatino isolado em doentes deste tipo. Doentes em primeira ou segunda linha de terapêutica para doença metastizada foram randomizadas, num sistema de 2 para 1, para receber até 6 ciclos da combinação de cisplatino e cetuximab ou cisplatino isolado. Às doentes randomizadas para o braço de monoterapia podiamos, após progressão, acrescentar cetuximab ou tratá-las com cetuximab isolado. O objetivo primário foi a taxa de resposta global. Os objetivos secundários foram a sobrevivência livre de doença, a sobrevivência global e o perfil de segurança dos fármacos. A população em análise foram 115 doentes tratadas com a combinação e 58 doentes tratadas com cisplatino em monoterapia, 31 destas em quem se documentou progressão passaram a ser tratadas com um regime que incluía cetuximab, isolado ou em combinação. A taxa de resposta global foi de 20% no braço da combinaçao e de 10% no braço da monoterapia (odds ratio, 2.13). A sobrevivência livre de doença foi de 3.7 meses no braço da combinação e de 1.5 meses no braço em monoterapia (hazard ratio, 0.67). A sobrevivência global foi de 12.9 meses no braço da combinação versus 9.4 meses no braço de cisplatino. Conclui-se que, apesar de não ter sido alcançado o objectivo primário, acrescentar cetuximab, duplica a resposta e prolonga tanto a sobrevivência livre de doença como a sobrevivência global. Capítulo 6: Bloquear a angiogénese para tratar o carcinoma da mama (revisão) A angiogénese é uma característica que define a neoplasia, porque tumores com mais de 1mm precisam de formar novos vasos para poderem crescer. Desde que se descobriram as moléculas que orquestram esta transformação, que se têm procurado desenvolver e testar fármacos que interfiram com este processo. No carcinoma da mama o bevacizumab foi o primeiro fármaco aprovado pela FDA em primeira linha para tratar doença metastática. Depois foram estudados um grupo de inibidores de tirosina cinase associados aos recetores transmembranares envolvidos na angiogénese como o VEGFR, PDGFR, KIT, RET, BRAF e Flt3: sunitinib, sorafenib, pazopanib e axitinib Neste capítulo, analisaram-se e resumiram-se os dados dos ensaios clínicos das drogas anti-angiogénicas no tratamaneto do carcinoma da mama. Os ensaios de fase III do bevacizumab em carcinoma da mama mostraram uma redução na progressão de doença de 22 a 52% e aumento da sobrevivência livre de doença de 1.2 a 5.5 meses mas nunca foi demonstrado prolongamento de sobrevivência. Os ensaios de fase III em carcinoma da mama adjuvante com bevacizumab são dois e foram ambos negativos. O ensaio de fase III com o inibidor da tirosina cinase, sunitinib foi negativo, enquanto que os ensaios de fase II com os inibidores da tirosina cinase sorafenib e pazopanib melhoraram alguns indicadores de resposta e sobrevivência. A endostatina foi testada no contexto neoadjuvante com antraciclinas e melhorou a taxa de resposta, mas, mais ensaios são necessários para estabelecer este fármaco. A maioria dos ensaios clínicos dos agentes antiangiogénicos em carcinoma da mama reportaram aumento da taxa de resposta e de sobrevivência livre de doença mas nunca aumento da sobrevivência global quando comparado com quimioterapia isolada o que levou ao cepticismo a que assistimos atualmente em relação ao bloqueio da angiogénese. Ensaios clínicos selecionados em doentes específicas com objetivos translacionais relacionados com material biológico colhido, preferefencialmente em diferentes intervalos da terapêutica, serão cruciais para o bloqueio da angiogénese sobreviver como estratégia terapêutica em carcinoma da mama. Capítulo 7: A resposta à hipoxia medeia a resistência primária ao sunitinib em carcinoma da mama localmente avançado O sunitinib é um fármaco antiangiogénico que nunca foi avaliado isolado em doentes com carcinoma da mama não tratadas. O nosso objetivo foi caracaterizar a atividade do sunitinib isolado e em combinação com o docetaxel em carcinoma da mama não tratado, localmente avançado ou operável, mas de dimensão superior a 2 cm, para compreender os mecanismos de resposta. Doze doentes foram tratadas com duas semanas iniciais de sunitinib seguido de quatro ciclos de combinação de sunitinib e docetaxel. A resposta, a reistência e a toxicidade foram avaliadas de acordo com parametros clínicos, ressonância magnética nuclear, tomografia de emissão de positrões, histopatologia e perfis de expressão genómica. Detetámos resistência primária ao sunitinib na janela inicial de duas semanas, evidenciada em quatro doentes que não responderam. À data da cirurgia, cinco doentes tinham tumor viável na mama e axila, quatro tinahm tumor viável na mama e três foram retiradas do ensaio. Não houve respostas patológicas completas. A comparação dos perfis de expressão genómica entre os respondedores e os não respondedores, aos quinze dias iniciais, permitiu-nos identificar sobre expressão de VEGF e outras vias angiogénicas nos não respondedores. Especificamente, em tumores resistentes ao sunitinib isolado detectámos uma resposta transcricional à hipoxia caracterizada por sobre expressão de vários dos genes alvo do HIF1α. Neste ensaio de sunitinib isolado em doentes não tratadas com carcinoma da mama localmente avançado, encontrámos evidência molecular de resistência primária ao sunitinib possivelmente mediada por sobre expressão de genes que respondem à hipoxia. Parte 3: Quando parar a terapêutica sistémica às doentes com carcinoma da mama Capítulo 8: Agressividade terapêutica ns últimos três meses de vida num estudo retrospetivo dum centro único Incluímos todos os adultos que morreram com tumores sólidos na instituição em 2003 e foram tratados com quimioterapia para tratar neoplaias metastizadas. Colhemos dados detalhados relacionados com quimioterapia e toxicidade nos últimos três meses de vida a partir do processo clínico. Trezentas e dezanove doentes foram incluídos, a mediana de idade foi 61 anos. A mediana de sobrevivência de doença metastática foi de 11 meses. 66% (211) dos doentes foram tratados com QT nos últimos 3 meses de vida, 37% foram tratados com QT no úlimo mês de vida e 21% nas últimas duas semanas. Nos doentes que foram tratados com QT nos últimos três meses de vida, 50% começaram um novo regime terapêutico neste período e 14% começaram um novo regime no último mês. Identificámos como determinantes de tratamento com QT no fim de vida a idade jovem, o carcinoma da mama, do ovário e do pâncreas. Concluímos que administrámos QT no fim de vida frequentemente e iniciámos novos regimes terapêuticos no último mês de vida em 14% dos casos. Precisamos de aprofundar este trabalho para compreender se esta atitude agressiva resulta em melhor paliação de sintomas e qualidade de vida no fim de vida dos doentes com neoplasias disseminadas. Capítulo 9: O tratamento do carcinoma da mama no fim de vida está a mudar? Quisémos caracterizar a modificação da tendência no uso de QT e de estratégias paliativas no fim de vida das doentes com carcinoma da mama em diferentes instituições e em intervalos de tempo diferentes. Para isto selecionámos doentes que morreram de carcinoma da mama durante 6 anos, entre 2007 e 2012, num hospital geral e comparámos com as doentes que morreram de carcinoma da mama em 2003 num centro oncológico. Avaliámos um total de 232 doentes. O grupo mais recente tem 114 doentes e o grupo anterior tem 118 doentes. Usámos estatística descritiva para caracterizar QT no fim de vida e o uso de estratégias paliativas. Ambas as coortes são comparáveis em termos das características do carcinoma da mama. Observámos aumento do uso de estatégias paliativas: consulta da dor, consulta de cuidados paliativos e radioterapia paliativa no cuidado das doentes com carcinoma da mama metastizado. Evidenciámos aumento do número de mortes em serviços de cuidados paliativos. No entanto, a QT paliativa continua a ser prolongada até aos últimos meses de vida, embora tenhamos mostrado uma diminuição desta prática. Outros indicadores de agressividade como a admissão hospitalar também mostraram diminuição. Confirmámos a nossa hipótese de que há maior integração da medicina paliativa multidisciplinar e menos agressividade na terapêutica sistémica das doentes com carcinoma da mama nos últimos meses de vida. Chapter 10: Porque é que os nossos doentes são tratados com quimioterapia até ao fim da vida? (editorial) Este capítulo começa por dar o exmeplo duma jovem de 22 anos que viveu três meses após começar QT paliatva. Este caso epitomiza a futilidade terapêutica e é usado como ponto de partida para explorar as razões pelas quais administramos QT no fim de vida aos doentes quando é inútil, tóxica, logisticamente complexa e cara. Será que estamos a prescrever QT até tarde demais? Os oncologistas fazem previsões excessivamente otimistas e têm uma atitude pró terapêutica excessiva e são criticados por outros intervenientes nas instituições de saúde por isto. Crescentemente doentes, familiares, associações de doentes, definidores de políticas de saúde, jornalistas e a sociedade em geral afloram este tema mas tornam-se inconsistentes quando se trata dum doente próximo em que se modifica o discurso para que se façam terapêuticas sitémicas agressivas. Há uma crescente cultura de preservação da qualidade de vida, paliação, abordagem sintomática, referenciação a unidades de cuidados paliativos e outros temas do fim de vida dos doentes oncológicos terminais. Infelizmente, este tema tem ganhado momentum não porque os oncologistas estejam a refletir criticamente sobre a sua prática, mas porque os custos dos cuidados de saúde são crescentes e incomportáveis. Seja qual fôr o motivo, as razões que levam os oncologistas a administrar QT no fim de vida devem ser criticamente elucidadas. Mas há poucos dados para nos guiar nesta fase delicada da vida dos doentes e os que existem são por vezes irreconciliáveis, é uma revisão destes dados que foi feita neste capítulo. Conclusão: A abordagem do carcinoma da mama no futuro? Na conclusão, tenta-se olhar para o futuro e prever como será a tomada a cargo dum doente com carcioma da mama amanhã. Faz-se uma avaliação das várias àreas desde prevenção, rastreio, suscetibilidade genética e comportamental e terapêutica. Na terapêutica separa-se a terapêutica locoregional, sistémica adjuvante e da doença metastizada. Nos três últimos parágrafos a história duma mulher com um carcinoma localmente avançado que sobre expressa o recetor Her2, serve como ilustração de como devemos estar preparados para incorporar evolução, heterogeneidade e dinamismo no cuidado de doentes com carcinoma da mama. -------------------------------------------------------------------------------------------------- ABSTRACT: Introduction: Breast cancer care in the past This work starts with an overview of the treatment of breast cancer (BC). From the first reports of patients ill with BC until 1950. From 1950 until 2000, there is a more detailed account on how BC patients were treated with emphasis on the different modalities, local, regional and systemic treatments and their evolution. Part 1: Who to treat with adjuvant systemic therapy? Chapter 1: TNM is not dead in breast cancer It has been said that the current TNM staging system might not be suitable for predicting breast cancer (BC) outcomes and for making therapeutic decisions, especially for patients with screen detected BC which is smaller. The reason for this is also due to the non inclusion of tumor biology parameters in the current TNM system. We hypothesize that in a population where there is still a large abundance of non screen detected BC, with a low median age of incidence and abundance of high TNM staged lesions, biology is still second to classical staging in predicting prognosis. We analyzed a population of consecutive BC patients from a single institution during ten years. We characterized current established prognostic factors, classical staging variables included in the current TNM staging system and biological variables, currently not included in the TNM system. We quantified the capacity of individual prognostic factors to predict survival. We analyzed a population of 1699 consecutive BC patients. We found that individually both the TNM system prognostic factors and the biological prognostic factors are differing among BC survivors and dead patients in a statistically significant distribution. Explicitly, patients with larger tumors, positive nodes, higher stage lesions, ER negative, HER2 positive, TN or lower differentiation tumors show decreased survival. In the multivariate analysis we can conclude that in a population such as ours classical TNM staging variables, irrespective of tumor biological features, are still the most powerful outcome predictors. Chapter 2: Defining breast cancer prognosis: The predictive power and mechanism of centrosome alterations in breast cancer We performed a systematic analysis of the literature and compiled an extensive data set of gene expression data originated in primary tumours of BC patients with prognostic information. We analysed this data seeking for genes consistently up or down regulated in poor prognosis BC, i.e. that relapsed after initial treatment. In the course this bioinformatics analysis our lab identified 65 genes statistically significant across multiple datasets that can discriminate between relapsed and non-relapsed BC patients. Among the identified genes, we have detected genes such as MKI67, a marker of mitotic activity which is routinely used in the clinic. Unexpectedly, we also discovered several genes found to be involved in centrosome clustering, The most prominent of these is the kinesin KIFC1, also called HSET, and previously identified as regulator of centrosome clustering. Centrosome abnormalities (numerical, structural) have been observed in cancer. Indeed, compelling data has shown that cells from many cancers have multiple and abnormal centrosomes, that are either correlated with tumour malignancy or considered an early tumorigenesis event. However, extra centrosomes come at a cost and cells must be able to handle such abnormalities or otherwise die. Thus our results suggested a new mechanism of breast cancer progression with negative prognostic value. We aimed at quantifying the predictive power of centrosome clustering in BC clinical setting and at detecting this process in BC patient material. We validated the centrosome clustering genes KIFC1 and TACC3 in formalin fixed paraffin embedded (FFPE) BC patient material, using quantitative real-time PCR (RT-qPCR) technology. Our results indicate that the tested KIFC1 has a clear IHC signal (1) and that the protein expression patterns and levels correlate with prognosis, with relapsing patients having increased expression and nuclear localisation of this kinesin (2). Next we were able to show that centrosome clustering does occur in vivo. We identified centrosome amplification and clustering in breast cancer samples, and we established a fluorescence microscopy-based IHC approach by staining FFPE samples with centrosomal markers. Using this approach we have observed centrosome amplification and clustering in a small set of poor prognosis samples. By expanding the number of samples in which we have characterised the number of centrosomes, we were able to confirm our preliminary observation that centrosomes are clustered in relapsed BC. Part 2: How to treat breast cancer subtypes? Chapter 3: How many diseases is triple negative breast cancer? (review) Triple negative breast cancer is a subtype of breast cancer that does not express the estrogen receptor, the progesterone receptor and the epidermal growth factor receptor type 2 (Her2). These tumors are not yet treated with targeted therapies probably because no positive markers have been described to reliably classify them - they are described for what they are not. Perhaps for this reason, they are among the most aggressive of breast carcinomas, albeit with very heterogenous clinical behavior. The clinical observation that these patients do not carry a uniformly dismal prognosis, coupled with data coming from pathology and epidemiology, suggests that this negative definition is not capturing a single clinical entity, but several. We critically evaluate this evidence in this paper, reviewing clinical and epidemiological data, as well as molecular data. There is evidence for heterogeneity, but it is not clear how many diseases are grouped into triple negative breast cancer. Answering this question, and identifying the molecular basis of heterogeneity will help define prognosis and, eventually, the identification of new targeted therapies. Chapter 4: Systemic treatment for triple negative breast cancer (review) Chemotherapy remains the backbone of treatment for triple negative breast cancer (TNBC). Despite the appearance of new targeted and biologic agents there has been no targeted therapy validated for TNBC, possibly because the biology of TNBC has not been conclusively elucidated. Many studies have shown that TNBC derive significant benefit of chemotherapy in the neoadjuvant, adjuvant and metastatic treatment, possibly more benefit than other BC subtypes. Neoadjuvant chemotherapy studies have repeatedly shown higher response rates in TNBC than non-TNBC. Pathologic complete response has been shown to predict improved long term outcomes in BC. Although specific adjuvant regimens for TNBC are under study, third generation chemotherapy regimens utilizing dose dense or metronomic polychemotherapy are among the most effective tools presently available. The role of specific chemotherapy agents, namely platinum salts, in the treatment of TNBC remains undefined. Taxanes and anthracyclines are active in TNBC and remain important agents, but have not shown specific benefit over non-TNBC. TNBC is itself a heterogeneous group in which subgroups like basal like BC defined by higher proliferation and including those TNBC arising in BRCA1 mutation carriers may be more sensitive to platinum agents and relatively less sensitive to taxanes. The molecular characterization of TNBC is lacking and therefore the search for targeted therapy is still ongoing. Chapter 5: Randomized phase II study of the anti-epidermal growth factor receptor monoclonal antibody cetuximab with cisplatin versus cisplatin alone in patients with metastatic triple-negative breast cancer Epidermal growth factor receptor is overexpressed in metastatic triple-negative breast cancers, an aggressive subtype of breast cancer. Our randomized phase II study investigated cisplatin with or without cetuximab in this setting. Patients who had received no more than one previous chemotherapy regimen were randomly assigned on a 2:1 schedule to receive no more than six cycles of cisplatin plus cetuximab or cisplatin alone. Patients receiving cisplatin alone could switch to cisplatin plus cetuximab or cetuximab alone on disease progression. The primary end point was overall response rate (ORR). Secondary end points studied included progressionfree survival (PFS), overall survival (OS), and safety profiles. The full analysis set comprised 115 patients receiving cisplatin plus cetuximab and 58 receiving cisplatin alone; 31 patients whose disease progressed on cisplatin alone switched to cetuximab-containing therapy. The ORR was 20% with cisplatin plus cetuximab and 10% with cisplatin alone (odds ratio, 2.13). Cisplatin plus cetuximab resulted in longer PFS compared with cisplatin alone (median, 3.7 v 1.5 months; hazard ratio, 0.67. Corresponding median OS was 12.9 versus 9.4 months. While the primary study end point was not met, adding cetuximab to cisplatin doubled the ORR and appeared to prolong PFS and OS, warranting further investigation in mTNBC. Chapter 6: Blocking angiogenesis to treat breast cancer (review) Angiogenesis is a hallmark of cancer because tumors larger than 1mm need new vessels to sustain their growth. Since the discovery of the molecular players of this process and some inhibitors, that angiogenesis became a promising therapeutic target. Bevacizumab was the first molecular-targeted antiangiogenic therapy approved by the FDA and is used as first-line therapy in metastatic breast cancer. A second class of approved inhibitors (sunitinib, sorafenib, pazopanib and axitinib) include oral small-molecule tyrosine kinase inhibitors that target vascular endothelial growth factor receptors, platelet-derived growth factor receptors, and other kinases including KIT, Ret, BRAF and Flt-3, but none of these have gained approval to treat breast cancer. This review analyzes and summarizes data from clinical trials of anti-angiogenic agents in the treatment of BC. Phase III trials of bevacizumab in advanced BC have demonstrated a reduction in disease progression (22–52%), increased response rates and improvements in progression-free survival of 1.2 to 5.5 months, but no improvements in OS. Bevacizumab phase III trials in early BC have both been negative. Bevacizumab combined with chemotherapy is associated with more adverse events. Phase III trials of the tyrosine kinase inhibitor sunitinib were negative, while randomized phase II trials of sorafenib and pazopanib have improved some outcomes. Endostatin has been tested in neoadjuvant clinical trials in combination with anthracyclinebased chemotherapy in treatment-naive patients and has increased the clinical response rate, but more trials are needed to establish this drug. Most trials of anti-angiogenic agents in BC have reported improved RR and PFS but no increase in OS compared to chemotherapy alone, leading to skepticism towards blocking angiogenesis. Selected trials in selected BC populations with translational endpoints related to harvested tumor tissue and other biological material samples, preferentially at several timepoints, will be crucial if antiangiogenesis is to survive as a strategy to treat BC. Chapter 7: Does hypoxic response mediate primary resistance to sunitinib in untreated locally advanced breast cancer? The antiangiogenic drug sunitinib has never been evaluated as single agent in untreated BC patients. We aimed to characterize the activity of sunitinib, alone and with docetaxel, in untreated locally advanced or operable BC, and, to uncover the mechanisms of response. Twelve patients were treated with an upfront window of sunitinib followed by four cycles of sunitinib plus docetaxel. Response, resistance and toxicity were evaluated according to standard clinical parameters, magnetic resonance imaging, positron emission tomography, pathology characterization and gene expression profiling. We detected primary resistance to sunitinib upfront window in untreated BC, as evidenced by four non-responding patients. At surgery, five patients had viable disease in the breast and axilla, four had viable tumor cells in the breast alone and three were taken off study due to unacceptable toxicity and thus not evaluated. Early functional imaging was useful in predicting response. There were no pathologic complete responses (pCR). Comparison of gene expression profiling tumor data between early responders and non-responders allowed us to identify upregulation of VEGF and angiogenic pathways in non responders. Specifically, in tumors resistant to the single-agent sunitinib we detected a transcriptional response to hypoxia characterized by over-expression of several HIF1α target genes. In this report of single-agent sunitinib treatment of untreated localized BC patients, we found molecular evidence of primary resistance to sunitinib likely mediated by up-regulation of hypoxia responsive genes. Part 3: When to stop systemic treatment of breast cancer patients? Chapter 8: The aggressiveness of cancer care in the last three months of life: a retrospective single centre analysis. All adult patients with solid tumors who died in our hospital in 2003 and received chemotherapy for advanced cancer, were included. Detailed data concerning chemotherapy and toxicity, in the last three months of life, were collected from patientsʼ clinical charts. A total of 319 patients were included. Median age was 61 years. Median time from diagnosis of metastatic disease to death was 11 months. The proportion of patients who received chemotherapy in the last three months of life was 66% (n=211), in the last month 37% and in the last two weeks 21%. Among patients who received chemotherapy in the last three months of life, 50% started a new chemotherapy regimen in this period and 14% in the last month. There was an increased probability of receiving chemotherapy in the last three months of life in younger patients and in patients with breast, ovarian and pancreatic carcinomas. There was a large proportion of patients who received chemotherapy in the last three months of life, including initiation of a new regimen within the last 30 days. Thus, further study is needed to evaluate if such aggressive attitude results in better palliation of symptoms at the end of life. Chapter 9: Is breast cancer treatment in the end of life changing? We aimed to characterize the shifting trends in use of anti-cancer chemotherapy and palliative care approaches in the end of life of BC patients in different institutions and times. For this, we selected women that died of BC during six years, from 2007 to 2012, and were treated in a central acute care general hospital and compared it with the BC patients that died in 2003 and were treated in a large cancer center. We analyzed a total of 232 patients: the more recent group has 114 women and the older cohort has 118. We used descriptive statistics to characterize CT in the EoL and use of palliative care resources. Both populations were similar in terms of BC characteristics. We observed more palliative care resources, pain clinic, palliative care teams and palliative radiotherapy, involved in the care of MBC patients and a shift towards more deaths at hospices. Systemic anti cancer treatments continue to be prolonged until very late in patients’ lives, notwithstanding, we could show a decrease in the use of such treatments. Other indicators of aggressiveness, namely hospital admissions, also show a decrease. We confirmed our hypothesis that there is more integration of multidisciplinary palliative care and less aggressiveness in the treatment of metastatic cancer patients, specifically, use of palliative anti-cancer treatment and hospital admissions. Nonetheless, we use systemic therapy until too late with underutilization of palliative medicine. Chapter 10: Why do our patients get chemotherapy until the end of life? (editorial) The editorial starts with a clinical case of a 21 year old patient that lives three months after starting palliative chemotherapy for the first time, a case that illustrates therapeutic futility at the end of life. Why are we not ceasing chemotherapy when it is useless, toxic, logistically complex and expensive? Are we prescribing chemotherapy until too late in solid tumor patientsʼ lives? Medical oncologists have overly optimistic predictions and, excessive, treatment-prone attitude and they are criticized by other health care providers for this. Increasingly, patients, their families, advocacy groups, policy makers, journalists and society at large dwell on this topic, which is a perplexing conundrum, because sometimes they are the ones demanding not to stop aggressive systemic anticancer treatments, when it comes to their loved ones. There is a growing culture of awareness toward preserving quality of life, palliative care, symptom-directed care, hospice referral and end of life issues regarding terminal cancer patients. Sadly, this issue is gaining momentum, not because oncologists are questioning their practice but because health care costs are soaring. Whatever the motive, the reasons for administering chemotherapy at the end of life should be known. There are few and conflicting scientific data to guide treatments in this delicate setting and we review this evidence in this paper. Conclusion: What is the future of breast cancer care? This work ends with a view into the future of BC care. Looking into the different areas from prevention, screening, hereditary BC, local, regional and systemic treatments of adjuvant and metastatic patients. The last three paragraphs are a final comment where the story of a patient with Her2 positive locally advanced breast cancer is used as paradigm of evolution, heterogeneity and dynamism in the management of BC.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia

Evidence of Wγγ production in pp collisions at s√=8 TeV and limits on anomalous quartic gauge couplings with the ATLAS detector

This Letter reports evidence of triple gauge boson production pp→W(ℓν)γγ+X, which is accessible for the first time with the 8 TeV LHC data set. The fiducial cross section for this process is measured in a data sample corresponding to an integrated luminosity of 20.3 fb−1, collected by the ATLAS detector in 2012. Events are selected using the W boson decay to eν or μν as well as requiring two isolated photons. The measured cross section is used to set limits on anomalous quartic gauge couplings in the high diphoton mass region.

Nonlinear behavior of gelatin networks reveals a hierarchical structure

We investigate the strain hardening behavior of various gelatin networks-namely physical gelatin gel, chemically cross-linked gelatin gel, and a hybrid gel made of a combination of the former two-under large shear deformations using the pre-stress, strain ramp, and large amplitude oscillations shear protocols. Further, the internal structures of physical gelatin gels and chemically cross-linked gelatin gels were characterized by small angle neutron scattering (SANS) to enable their internal structures to be correlated with their nonlinear rheology. The Kratky plots of SANS data demonstrate the presence of small cross-linked aggregates within the chemically cross-linked network whereas, in the physical gelatin gels, a relatively homogeneous structure is observed. Through model fitting to the scattering data, we were able to obtain structural parameters, such as the correlation length (ξ), the cross-sectional polymer chain radius (Rc) and the fractal dimension (df) of the gel networks. The fractal dimension df obtained from the SANS data of the physical and chemically cross-linked gels is 1.31 and 1.53, respectively. These values are in excellent agreement with the ones obtained from a generalized nonlinear elastic theory that has been used to fit the stress-strain curves. The chemical cross-linking that generates coils and aggregates hinders the free stretching of the triple helix bundles in the physical gels.

Bilateral carotid endarterectomy combined with myocardial revascularization during the same surgical act

The best surgical approach for the treatment of patients with severe cerebral artery disease and simultaneous serious coronary artery disease still remains controversial. In this report we present a case of a 72-year-old female patient admitted to the hospital with unstable angina. Triple coronary artery obstructive disease and severe bilateral carotid artery stenosis were diagnosed. A combined, simultaneous surgical procedure was performed. After total circulatory by-pass with a membrane oxygenator, the patient's body temperature was lowered to 32°C. During the cool-down period, three proximal anastomoses of segments of autologous saphenous veins were performed in the ascending aorta. Immediately afterwards, bilateral carotid endarterectomy was performed, followed by three distal anastomoses to coronary arteries. The patient showed a satisfactory post-operative outcome. It was concluded that the combination of moderate hypothermia, hemodilution with appropriate hemodynamic control, as used in this patient, was an effective method of cerebral protection. The simultaneous approach of carotid endarterectomy and coronary artery by-pass surgery should be seen as a safe option for the treatment of this type of patient.

Synthesis and biological evaluation of mono and bis-naphthalimide derivatives against SH-SY5Y, human brain cancer cells

Dissertação de mestrado em Medicinal Chemistry

Aneurysm of the left main coronary artery

Aneurysm of the left main coronary artery is a rare angiographic finding, with few cases described in the international literature. We report the case of a 42-year-old male with a previous history of acute myocardial infarction, whose coronariography indicated triple vessel coronary disease and an aneurysm of the left main coronary artery. A review of the etiology, clinical aspects, and surgical management of coronary arterial aneurysm is presented.

Micronouvelles. Des nouvelles en trois lignes à la twittérature

Qu'est-ce qu'une micronouvelle ou microfiction ou microrécit? Qu'est-ce qui est en jeu dans ces différentes désignations? Quelles sont les coordonnées de l’émergence et de l’essor des formes minuscules ? Quelles questions théoriques soulèvent ces tout petits récits et comment sont-elles traitées dans le discours critique? Pour répondre à ces questions, cet essai situe les micronouvelles dans la dynamique complexe du paysage littéraire et culturel contemporain, en proposant une triple approche génologique, narratologique et transfictionnelle, qui met en lumière les connexions des micronouvelles avec des faits divers, des légendes urbaines, des bandes dessinées et des contes de fées.

Ventricular resynchronization through biventricular cardiac pacing for the treatment of refractory heart failure in dilated cardiomyopathy

OBJECTIVE: The biventricular pacing (BVP) approach has good results in the treatment of congestive heart failure (CHF) in patients (pts) with disorders of intraventricular conduction. METHODS: We have applied BVP to 28 pts, with left ventricular pacing using minitoracotomy in 3 pts and the transvenous aproach via coronary sinus in 25 pts. The mean duration of the QRS complexes was 187 ms, in the presence of the left branch block in 22 pts, and right branch block + divisional hemiblock in 6 pts. All pts had been considerated candidates to cardiac transplantation, and were under optimized drug therapy. Sixteen pts were in Functional Class (NYHA) IV, and 12 in class III. The ejection fraction varied from 22 to 46% (average = 34%). The pacing mode employed was biventricular triple-chamber in 22 pts, and bi-ventricular dual-chamber in 6 pts (one with ICD). RESULTS: The pts were followed up for a period that ranged from 10 days to 14 months (mean 5 months). All pts presented clinical improvement after implant, chaging the NYHA Functional Class at the end of follow-up to Class I (9pts), Class II (10 pts) and Class III (6 pts). The initial mean ejection fraction have-raised to 37%. Two pts died suddenly. One patient died due to a pulmonary fungal infection. CONCLUSION: Ventricular resynchronization through BVP, improved significantly the Functional Class and, therefore, the quality of life. Assessments of myocardial function acutely performed do not reflect the clinical improvement observed.

Tenencia responsable de mascotas y su impacto en la salud pública.

Desde el año 2007 y ante la demanda de municipios cordobeses, se comenzó a trabajar, mediante el enfoque de la Responsabilidad Social Universitaria (RSU) de la UCC, en el concepto de tenencia responsable de mascotas, como herramienta indispensable para abordar de manera integral la problemática asociada con animales vagabundos, en el amplio marco de la salud pública. El objetivo del presente proyecto es caracterizar la problemática particular de las distintas comunidades, para la posterior implementación de acciones concretas y dirigidas hacia las mismas, mediante la articulación con programas de RSU de la UCC afines. De ese modo se analizará la problemática desde una triple perspectiva analizando los principales actores involucrados, ellos son: la sociedad en su conjunto (Estado, organizaciones intermedias y la propia comunidad), los animales y el ambiente como variable propia de cada comunidad. De tal análisis se busca crear un sistema de diagnóstico situacional en tenencia responsable de mascotas, asignándole así una puntuación y clasificandolas en comunidades mayor o menormente responsables. De manera particular se podrá analizar el conocimiento general de la población en la temática, la distribución local de enfermedades zoonóticas (transmisibles al ser humano) o no zoonóticas en animales, las características demográficas de las mascotas en la comunidad, detectado así puntos críticos de acción para futuras acciones a realizar, por parte de la comunidad en su conjunto, sean vecinos, ONGs y/o organismos gubernamentales. En todas las intervenciones, se busca la interacción con la sociedad en todas las instancias posibles, haciéndola partícipe y dueña de sus propios cambios, fomentando así la sustentabilidad a largo plazo de las acciones realizadas.

Formação das castas no gênero Melípona (Illiger, 1806)

The present work is destinated to prove that the castes : workers and queens, in Melipona bees are due to genetic factors and not to differences in food. 2) Material used: Hives of Melipona quadri-fasciata anthidioides (Lep. 1836), M. schenki schenki (Gribodo, 1893), M. fasciata rufiventris (Lep. 1836), M. quadri-fasciata vicina (Lep. 1836), M. marginata marginata (Lep. 1836), Apis mellifera (L. 1758). 3) It should be pointed out that in Melipona bees there are no royal cells for the queens, but all the cells are of the same size independently of being destinated for workers, queens or drones. The numerous queens which are born are killed soon after emerging from their cells. 4) Changes of feeding in quality and in quantity caused no variation of castes. The only variable factor is the size, which becomes bigger when the bee is well nourished. 5) The offsprings of 5 hives were examined : 3 of M. quadri-fasciata anthidioides (n.o 1, n.o 2 and n.o 3), 1 of M. quadri-fasciata vicina (n.o 4) and 1 of M. marginata marginata (n.o 5). Combs of about 40 cells were taken into laboratory and the type of bee registered immediately after emerging. The results of the counts were: BOX COMB WORKER QUEEN PERCENTAGE Σ X2 to 12,5% Nº 1 1th 69 8 10,4% 0, 3139 " 1 2nd 144 18 11,1% 0, 2856 " 2 1th 52 8 13,3% 0, 0384 " 3 1th 45 10 18,2% 1, 6736 " 4 1th 56 4 6,7% 1, 8686 " 4 2nd 29 4 12,1% 0,00432 Σ X2 to 25% " 5 1th 34 14 29,2% 0,44444 "5 2nd 83 27 24,5% 0, 0121 In the 4 first boxes there is a percentage of 11,63% queens and in the last there is a percentage of 25,95%. 6) These percentages are very near two genetical ratios: 12,5% or 7:1, and 25% or 3:1, which correspond to a trifactorial and a bifactorial back-cross. Carrying out a X² test no significant deviations were found ( X² to 12,5% and to 25% and table 1 to 4). 7) We suppose that the formula for the queen in the first case (11,65%) is: AaBbCc. Since the Melipona bees are arrhenotokous hymenopteres, the drones are haploid and may have any one of the following eight formulas, corresponding to the gonic segregation of the queem : ABC, ABc, Abc, Abc, AbC, aBC, aBc, abC, abc. Anyone combination of these males with the queen will give a segregation of 7 workers to 1 queen, since there is always only one triple heterozygote among the eight possible segregates (table 5). 8) In order to explain the second case, it is suffient to assume that in this species there are only two pairs of factors, the queen being the double heterozygote : AaBb, while the drones may have any one of the following constitutions: AB, Ab, aB and ab. Workers are again all diploids which are homozygous for one or both factors, for instance: AABB, AABb, AaBB, aaBb, AAbb, etc. (table 6). 9) It is suggested that the genus Melipona is an intermediary type between the solitary bees, where all females are fertile independently of their feeding, and the genera Apis and Trigona, where without special feeding all females are born sterile, while only specially fed females develop into fertile queens. 10) No speculations are put forward with regards to the evolutionary mechanism which may have been responsible for the development of the genetical determination of castes in Melipona, since it seems advisable point to extend the studies to other insects with complicated caste systems.

Sôbre u'a modificação do meio de Monteverde

It is well known that the culture media used in the presumptive diagnosis of suspiciuous colonies from plates inoculated with stools for isolation of enteric organisms do not always correctly indicate the major groups of enterobacteria. In an effort to obtain a medium affording more exact indications, several media (1-9) have been tested. Modifications of some of these media have also been tested with the result that a satisfactory modification of Monteverde's medium was finaly selected. This proved to be most satisfactory, affording, as a result of only one inoculation, a complete series of basic indications. The modification involves changes in the formula, in the method of preparation and in the manner of storage. The formulae are: A. Thymol blue indicator: NaOH 0.1/N .............. 34.4 ml; Thymol blue .............. 1.6 g; Water .................... 65.6 ml. B. Andrade's indicator. C. Urea and sugar solution: Urea ..................... 20 g; Lactose ................... 30 g; Sucrose ................... 30 g; Water .................... 100 ml. The mixture (C.) should be warmed slightly in order to dissolve the ingredients rapidly. Sterilise by filtration (Seitz). Keep stock in refrigeratior. The modification of Monteverde's medium is prepared in two parts. Semi-solid part - Peptone (Difco) 2.0 g; NaCl 0.5 g; Agar 0.5 g; Water 100.0 ml. Boil to dissolve the ingredients. Adjust pH with NaOH to 7.3-7.4. Boil again for precipitation. Filter through cotton. Ad indicators "A" 0.3 ml and "B" 1.0 ml. Sterilise in autoclave 115ºC, 15 minutes in amounts not higher than 200 ml. Just before using, add solution "C" asseptically in amounts of 10 ml to 200 ml of the melted semi-solid medium, maintained at 48-50ºC. Solid part - Peptone (Difco) 1.5 g; Trypticase (BBL) 0.5 g; Agar 2.0 g; Water 100,00 ml. Boil to dissolve the ingredients. Adjust pH with NaOH to 7.3-7.4. Boils again. Filter through cotton. Add indicators "A" 0.3 ml and "B" 1.0 ml; ferrous ammonium sulfate 0.02 g; sodiun thiosulfate 0.02 g. Sterilise in autoclave 115ºC, 15 minutes in amounts not higher than 200 ml. Just before using, add solution "C" asseptically in amounts of 10 ml to 200 ml of the melted solid medium, maintained at 48-50ºC. Final medium - The semi-solid part is dispensed first (tubes about 12 x 120 mm) in 2.5 ml amounts and left to harden at room temperature, in vertical position. The solid part is dispensed over the hardened semi-solid one in amounts from 2.0 ml to 2.5 ml and left to harden in slant position, affording a butt of 12 to 15 mm. The tubes of medium should be subjected to a sterility test in the incubator, overnight. Tubes showing spontaneous gas bubbles (air) should then be discarded. The medium should be stored in the incubator (37ºC), for not more than 2 to 4 days. Storage of the tubes in the ice-box produces the absorption of air which is released as bubbles when the tubes are incubated at 37ºC after inoculation. This fact confirmed the observation of ARCHAMBAULT & McCRADY (10) who worked with liquid media and the aplication of their observation was found to be essential to the proper working conditions of this double-layer medium. Inoculation - The inoculation is made by means of a long straight needle, as is usually done on the triple sugar, but the needel should penetrate only to about half of the height of the semi-solid column. Indol detection - After inoculation, a strip of sterelized filter papaer previously moistened with Ehrlich's reagent, is suspended above the surface of the medium, being held between the cotton plug and the tube. Indications given - In addition to providing a mass of organisms on the slant for serological invetigations, the medium gives the following indications: 1. Acid from lactose and/or sucrose (red, of yellowsh with strains which reduce the indicators). 2. Gas from lactose and/or sucrose (bubbles). 3. H[2]S production, observed on the solid part (black). 4. Motility observed on the semi-solid part (tubidity). 5. Urease production, observed on solid and semi-solid parts (blue). 6. Indol production, observed on the strip of filter paper (red or purplish). Indol production is not observed with indol positive strains which rapidly acidify the surface o the slant, and the use of oxalic acid has proved to give less sensitive reaction (11). Reading of results - In most cases overnight incubation is enough; sometimes the reactions appear within only a few hours of incubation, affording a definitive orientation of the diagnosis. With some cultures it is necessary to observe the medium during 48 hours of incubation. A description showing typical differential reaction follows: Salmonella: Color of the medium unchanged, with blackening of the solid part when H[2]S is positive. The slant tends to alkalinity (greenish of bluish). Gas always absent. Indol negative. Motility positive or negative. Shigella: Color of the medium unchanged at the beginning of incubation period, but acquiring a red color when the strain is late lactose/sucrose positive. Slant tending to alkalinity (greenish or purplish). Indol positive or negative. Motility, gas and H[2]S always negative. Proteus: Color of the medium generally changes entirely to blue or sometimes to green (urease positive delayed), with blackening of solid part when H[2]S is positive. Motility positive of negative. Indol positive. Gas positive or negative. The strains which attack rapidly sucrose may give a yellow-greenish color to the medium. Sometimes the intense blue color of the medium renders difficult the reading of the H[2]S production. Escherichiae and Klebsiellae: Color of the medium red or yellow (acid) with great and rapid production of gas. Motility positive or negative. Indol generally impossible to observe. Paracoli: Those lactose of sucrose positive give the same reaction as Esherichia. Those lactose or sucrose negatives give the same reactions as Salmonellae. Sometimes indol positive and H[2]S negative. Pseudomonas: Color of the medium unchanged. The slant tends to alkalinity. It is impossible to observe motility because there is no growth in the bottom. Alkaligenes: Color of the medium unchanged. The slant tends to alkalinity. The medium does not alter the antigenic properties of the strains and with the mass of organisms on the slant we can make the serologic diagnosis. It is admitted that this medium is somewhat more laborious to prepare than others used for similar purposes. Nevertheless it can give informations generally obtained by two or three other media. Its use represents much saving in time, labor and material, and we suggest it for routine laboratory work in which a quick presumptive preliminary grouping of enteric organisms is needed.