Remodeling of Yeast Genome Expression in Response to Environmental Changes

We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity. The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition. These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves ∼10% of yeast genes. The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change. These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response.

Transcriptional analysis of PRRSV-infected porcine dendritic cell response to Streptococcus suis infection reveals up-regulation of inflammatory-related genes expression

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.

Transcriptional analysis of PRRSV-infected porcine dendritic cell response to Streptococcus suis infection reveals up-regulation of inflammatory-related genes expression

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.

Transition and acoustic response of recirculation structures in an unconfined co-axial isothermal swirling flow

This paper reports the first observations of transition from a pre-vortex breakdown (Pre-VB) flowreversal to a fully developed central toroidal recirculation zone in a non-reacting, double-concentric swirling jet configuration and its response to longitudinal acoustic excitation. This transition proceeds with the formation of two intermediate, critical flow regimes. First, a partially penetrated vortex breakdown bubble (VBB) is formed that indicates the first occurrence of an enclosed structure as the centre jet penetration is suppressed by the growing outer roll-up eddy; resulting in an opposed flow stagnation region. Second, a metastable transition structure is formed that marks the collapse of inner mixing vortices. In this study, the time-averaged topological changes in the coherent recirculation structures are discussed based on the non-dimensional modified Rossby number (Ro(m)) which appears to describe the spreading of the zone of swirl influence in different flow regimes. Further, the time-mean global acoustic response of pre-VB and VBB is measured as a function of pulsing frequency using the relative aerodynamic blockage factor (i.e., maximum radial width of the inner recirculation zone). It is observed that all flow modes except VBB are structurally unstable as they exhibit severe transverse radial shrinkage (similar to 20%) at the burner Helmholtz resonant modes (100-110 Hz). In contrast, all flow regimes show positional instability as seen by the large-scale, asymmetric spatial shifting of the vortex core centres. Finally, the mixing transfer function M (f) and magnitude squared coherence lambda(2)(f) analysis is presented to determine the natural couplingmodes of the system dynamic parameters (u', p'), i.e., local acoustic response. It is seen that the pre-VB flow mode exhibits a narrow-band, low pass filter behavior with a linear response window of 100-105 Hz. However, in the VBB structure, presence of critical regions such as the opposed flow stagnation region alters the linearity range with the structure showing a response even at higher pulsing frequencies (100-300 Hz). (C) 2013 AIP Publishing LLC.

Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

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.

The proportionality of global warming to cumulative carbon emissions

The global temperature response to increasing atmospheric CO2 is often quantified by metrics such as equilibrium climate sensitivity and transient climate response1. These approaches, however, do not account for carbon cycle feedbacks and therefore do not fully represent the net response of the Earth system to anthropogenic CO2 emissions. Climate–carbon modelling experiments have shown that: (1) the warming per unit CO2 emitted does not depend on the background CO2 concentration2; (2) the total allowable emissions for climate stabilization do not depend on the timing of those emissions3, 4, 5; and (3) the temperature response to a pulse of CO2 is approximately constant on timescales of decades to centuries3, 6, 7, 8. Here we generalize these results and show that the carbon–climate response (CCR), defined as the ratio of temperature change to cumulative carbon emissions, is approximately independent of both the atmospheric CO2 concentration and its rate of change on these timescales. From observational constraints, we estimate CCR to be in the range 1.0–2.1 °C per trillion tonnes of carbon (Tt C) emitted (5th to 95th percentiles), consistent with twenty-first-century CCR values simulated by climate–carbon models. Uncertainty in land-use CO2 emissions and aerosol forcing, however, means that higher observationally constrained values cannot be excluded. The CCR, when evaluated from climate–carbon models under idealized conditions, represents a simple yet robust metric for comparing models, which aggregates both climate feedbacks and carbon cycle feedbacks. CCR is also likely to be a useful concept for climate change mitigation and policy; by combining the uncertainties associated with climate sensitivity, carbon sinks and climate–carbon feedbacks into a single quantity, the CCR allows CO2-induced global mean temperature change to be inferred directly from cumulative carbon emissions.

Scenario and modelling uncertainty in global mean temperature change derived from emission-driven global climate models

We compare future changes in global mean temperature in response to different future scenarios which, for the first time, arise from emission-driven rather than concentration-driven perturbed parameter ensemble of a global climate model (GCM). These new GCM simulations sample uncertainties in atmospheric feedbacks, land carbon cycle, ocean physics and aerosol sulphur cycle processes. We find broader ranges of projected temperature responses arising when considering emission rather than concentration-driven simulations (with 10–90th percentile ranges of 1.7 K for the aggressive mitigation scenario, up to 3.9 K for the high-end, business as usual scenario). A small minority of simulations resulting from combinations of strong atmospheric feedbacks and carbon cycle responses show temperature increases in excess of 9 K (RCP8.5) and even under aggressive mitigation (RCP2.6) temperatures in excess of 4 K. While the simulations point to much larger temperature ranges for emission-driven experiments, they do not change existing expectations (based on previous concentration-driven experiments) on the timescales over which different sources of uncertainty are important. The new simulations sample a range of future atmospheric concentrations for each emission scenario. Both in the case of SRES A1B and the Representative Concentration Pathways (RCPs), the concentration scenarios used to drive GCM ensembles, lies towards the lower end of our simulated distribution. This design decision (a legacy of previous assessments) is likely to lead concentration-driven experiments to under-sample strong feedback responses in future projections. Our ensemble of emission-driven simulations span the global temperature response of the CMIP5 emission-driven simulations, except at the low end. Combinations of low climate sensitivity and low carbon cycle feedbacks lead to a number of CMIP5 responses to lie below our ensemble range. The ensemble simulates a number of high-end responses which lie above the CMIP5 carbon cycle range. These high-end simulations can be linked to sampling a number of stronger carbon cycle feedbacks and to sampling climate sensitivities above 4.5 K. This latter aspect highlights the priority in identifying real-world climate-sensitivity constraints which, if achieved, would lead to reductions on the upper bound of projected global mean temperature change. The ensembles of simulations presented here provides a framework to explore relationships between present-day observables and future changes, while the large spread of future-projected changes highlights the ongoing need for such work.

Climate and CO2 controls on global vegetation distribution at the last glacial maximum: analysis based on palaeovegetation data, biome modelling and palaeoclimate simulations.

The global vegetation response to climate and atmospheric CO2 changes between the last glacial maximum and recent times is examined using an equilibrium vegetation model (BIOME4), driven by output from 17 climate simulations from the Palaeoclimate Modelling Intercomparison Project. Features common to all of the simulations include expansion of treeless vegetation in high northern latitudes; southward displacement and fragmentation of boreal and temperate forests; and expansion of drought-tolerant biomes in the tropics. These features are broadly consistent with pollen-based reconstructions of vegetation distribution at the last glacial maximum. Glacial vegetation in high latitudes reflects cold and dry conditions due to the low CO2 concentration and the presence of large continental ice sheets. The extent of drought-tolerant vegetation in tropical and subtropical latitudes reflects a generally drier low-latitude climate. Comparisons of the observations with BIOME4 simulations, with and without consideration of the direct physiological effect of CO2 concentration on C3 photosynthesis, suggest an important additional role of low CO2 concentration in restricting the extent of forests, especially in the tropics. Global forest cover was overestimated by all models when climate change alone was used to drive BIOME4, and estimated more accurately when physiological effects of CO2 concentration were included. This result suggests that both CO2 effects and climate effects were important in determining glacial-interglacial changes in vegetation. More realistic simulations of glacial vegetation and climate will need to take into account the feedback effects of these structural and physiological changes on the climate.

Heterologous microarray experiments used to identify the early gene response to heat stress in a coral reef fish

Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. In this study, we tested the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Results from a comparative genomic hybridization experiment and direct sequence comparisons indicate that for most genes there is considerable sequence similarity between the two species, suggesting that the D. rerio array is useful for genomic studies of P. moluccensis. We employed this heterologous microarray approach to characterize the early transcriptional response to heat stress in P. moluccensis. A total of 111 gene loci, many of which are involved in protein processing, transcription, and cell growth, showed significant changes in transcript abundance following exposure to elevated temperatures. Changes in transcript abundance were validated for a selection of candidate genes using quantitative real-time polymerase chain reaction. This study demonstrates that heterologous microarrays can be successfully employed to study species for which specific microarrays have not yet been developed, and so have the potential to greatly enhance the utility of microarray technology to the field of environmental and functional genomics.

Extracytoplasmic function (ECF) sigma factor sigma(F) is involved in Caulobacter crescentus response to heavy metal stress

Background: The alpha-proteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. It has been reported that C. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes. Results: In this work, we show that the ECF sigma factor sigma(F) is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. Microarray experiments indicate that sigma(F) controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. Surprisingly, sigma(F) itself is not strongly auto-regulated under metal stress conditions. Interestingly, sigma(F)-dependent genes are not induced in the presence of agents that generate reactive oxygen species. Promoter analyses revealed that a conserved sigma(F)-dependent sequence is located upstream of all genes of the sigma(F) regulon. In addition, we show that the second gene in the sigF operon acts as a negative regulator of sigma(F) function, and the encoded protein has been named NrsF (Negative regulator of sigma F). Substitution of two conserved cysteine residues (C131 and C181) in NrsF affects its ability to maintain the expression of sigma(F)-dependent genes at basal levels. Furthermore, we show that sigma(F) is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein NrsF. Conclusion: A possible mechanism for induction of the sigma(F)-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor NrsF, leading to the release of sigma(F) to bind RNA polymerase core and drive transcription of its regulon.

Extracytoplasmic function (ECF) sigma factor σF is involved in Caulobacter crescentus response to heavy metal stress

Background The α-proteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. It has been reported that C. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes. Results In this work, we show that the ECF sigma factor σF is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. Microarray experiments indicate that σF controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. Surprisingly, σF itself is not strongly auto-regulated under metal stress conditions. Interestingly, σF-dependent genes are not induced in the presence of agents that generate reactive oxygen species. Promoter analyses revealed that a conserved σF-dependent sequence is located upstream of all genes of the σF regulon. In addition, we show that the second gene in the sigF operon acts as a negative regulator of σF function, and the encoded protein has been named NrsF (Negative regulator of sigma F). Substitution of two conserved cysteine residues (C131 and C181) in NrsF affects its ability to maintain the expression of σF-dependent genes at basal levels. Furthermore, we show that σF is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein NrsF. Conclusion A possible mechanism for induction of the σF-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor NrsF, leading to the release of σF to bind RNA polymerase core and drive transcription of its regulon.

Mechanisms of cell senescence: p21 and DNA damage response in aging and longevity

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

The role of the NadR regulator during infection and its implication for the coverage of a new Meningococcus B vaccine

Background: Neisseria meningitides represents a major cause of meningitis and sepsis. The meningococcal regulator NadR was previously shown to repress the expression of the Neisserial Adhesin A (NadA) and play a major role in its phase-variation. NadA is a surface exposed protein involved in epithelial cell adhesion and colonization and a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B infection. The NadR mediated repression of NadA is attenuated by 4-HPA, a natural molecule released in human saliva. Results: In this thesis we investigated the global role of NadR during meningogoccal infection, identifying through microarray analysis the NadR regulon. Two distinct types of NadR targets were identified, differing in their promoter architectures and 4HPA responsive activities: type I are induced, while type II are co-repressed in response to the same 4HPA signal. We then investigate the mechanism of regulation of NadR by 4-HPA, generating NadR mutants and identifying classes or residues involved in either NadR DNA binding or 4HPA responsive activities. Finally, we studied the impact of NadR mediated repression of NadA on the vaccine coverage of 4CMenB. A selected MenB strains is not killed by sera from immunized infants when the strain is grown in vitro, however, in an in vivo passive protection model, the same sera protected infant rats from bacteremia. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h post-infection. Conclusions: Our results suggest that NadR coordinates a broad transcriptional response to signals present in the human host, enabling the meningococcus to adapt to the relevant host niche. During infectious disease the effect of the same signal on NadR changes between different targets. In particular NadA expression is induced in vivo, leading to efficient killing of meningococcus by anti-NadA antibodies elicited by the 4CMenB vaccine.