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Establishing a quantitative understanding of tumor heterogeneity, a major feature arising from the evolutionary processes taking place within the tumor microenvironment, is an important challenge for cancer biologists. Recently established experimental techniques enabled summarizing the variety of tumor cell phenotypes in proliferative or migratory. In the former, cells mostly proliferate and rarely migrate, while the opposite happens with cells having the latter phenotype, a “go-and-grow” description of heterogeneity. In this manuscript we present a discrete time Markov chain to simulate the spatial evolution of a tumor which heterogeneity is described by cells having those two phenotypes. The cell density curves have two qualitatively distinct temporal regimes, as they recover the Gompertz curve widely used for tumor growth description, and a bi-phasic growth which temporal shape resembles the tumor growth dynamics under influence of immunoediting. We also show how our representation of heterogeneity gives rise to variable spatial patterning even when the tumors have similar size and dynamics.Author summaryWe present a spatial stochastic model to represent the growth of a tumor as a structure having cells of two phenotypes: one whose cells have division as their predominant transition, and another where cells are mostly migrating. The migratory phenotype results from a transformation of the proliferative. Our proposition is based on the assumption that a tumor grows initially within a limited region while its cells are capable of acquire nutrients. During that phase, the cancer cells start changing their phenotype because of stress in their microenvironment and exhaustion of nutrients that lead them to become more migratory and capable of generating metastasis. Our model enables us to recover the usual dynamics observed in tumor growth such as a logistic-like curve, called Gompertz model, widely observed, or the bi-phasic growth observed characterized by equilibrium phase interspersed between two growth regimes. Our approach also enable us to understand the internal spatial and temporal structure of the two sub-populations and can be useful to investigate the phenomena underpinning heterogeneous tumor growth, a feature that helps on the design of treatment strategies based on mitigating heterogeneity related drug resistance.

Breast cancer arising in very young patients may be biologically distinct; however, these tumors have been less well studied. We characterized a group of very young patients (≤ 35 years) for BRCA germline mutation and for somatic mutations in luminal (HER2 negative) breast cancer. Thirteen of 79 unselected very young patients were BRCA1/2 germline mutation carriers. Of the non-BRCA tumors, eight with luminal subtype (HER2 negative) were submitted for whole exome sequencing and integrated with 29 luminal samples from the COSMIC database or previous literature for analysis. We identified C to T single nucleotide variants (SNVs) as the most common base-change. A median of six candidate driver genes was mutated by SNVs in each sample and the most frequently mutated genes were PIK3CA, GATA3, TP53 and MAP2K4. Potential cancer drivers affected in the present non-BRCA tumors include GRHL2, PIK3AP1, CACNA1E, SEMA6D, SMURF2, RSBN1 and MTHFD2. Sixteen out of 37 luminal tumors (43%) harbored SNVs in DNA repair genes, such as ATR, BAP1, ERCC6, FANCD2, FANCL, MLH1, MUTYH, PALB2, POLD1, POLE, RAD9A, RAD51 and TP53, and 54% presented pathogenic mutations (frameshift or nonsense) in at least one gene involved in gene transcription. The differential biology of luminal early-age onset breast cancer needs a deeper genomic investigation.

Sources of cell lines used at this study. (DOCX 19 kb)

Sources of cell lines used at this study. (DOCX 19 kb)

Supplemental methods. (PDF 316 kb)

Gating strategies to analyze lymphoid and myeloid subpopulations – To analyze T lymphocytes a triple staining strategy was used with CD5, CD4 and CD8 antibodies. First, all CD5+ cells were gated, and then, CD4 and CD8 expression was analyzed in this R1 population, characterizing CD5+CD4+ and CD5+CD8+ T lymphocytes (A). To quantify peritoneal B cells, a double staining strategy was used with B220 and CD11b antibodies, where B220+CD11b− cells were considered B2 lymphocytes and B220+CD11b+ cells were considered B1 lymphocytes (B). And, finally, to characterize myeloid cells, a triple staining strategy was used with CD11b, Gr-1 and Ly-6G antibodies. All CD11bhigh Gr-1high cells are neutrophils, as observed by Ly-6G expression, and cells which expressed low levels of Gr-1 (CD11bhigh Gr-1low cells) were considered myeloid progenitors (C). (PDF 53 kb)

Schistosoma mansoni synthesizes glycoconjugates which interact with galectin-3, eliciting an intense humoral immune response. Moreover, it was demonstrated that galectin-3 regulates B cell differentiation into plasma cells. Splenomegaly is a hallmark event characterized by polyclonal B cell activation and enhancement of antibody production. Here, we investigated whether galectin-3 interferes with spleen organization and B cell compartment during chronic schistosomiasis, using wild type (WT) and galectin-3-/- mice. In chronically-infected galectin-3-/- mice the histological architecture of the spleen, including white and red pulps, was disturbed with heterogeneous lymphoid follicles, an increased number of plasma cells (CD19-B220-/lowCD138+) and a reduced number of macrophages (CD19-B220-Mac-1+CD138-) and B lymphocytes (CD19+B220+/highCD138-), compared with the WT infected mice. In the absence of galectin-3 there was an increase of annexin-V+PI- cells and a major presence of apoptotic cells in spleen compared with WT infected mice. In spleen of WT infected mice galectin-3 was largely expressed in lymphoid follicles and extrafollicular sites. Thus, we propose that galectin-3 plays a role in splenic architecture, controlling distinct events such as apoptosis, macrophage activity, B cell differentiation and plasmacytogenesis in the course of S. mansoni infection

Orientadores: Carmen Silvia Passos Lima, Gustavo Jacob Lourenço Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: As associações de alterações genéticas herdadas, como polimorfismos gênicos de base única (SNPs) com o risco de melanoma cutâneo (MC), com suas manifestações clinicopatológicas e prognóstico de portadores do tumor não foram estabelecidas e, desta forma, estes foram os objetivos do presente estudo. O DNA genômico de sangue periférico de 103 pacientes com MC e de 103 controles foi analisado por meio de lâminas com microarranjos de DNA (SNPs). As frequências de 12.994 SNPs foram distintas entre pacientes e controles; 6.814 SNPs (52,4%) estiveram localizados em regiões regulatórias da transcrição do DNA (regiões 5¿ e 3¿ não traduzidas e intergênicas), 26 (0,2%) em regiões codificantes de aminoácidos, 6.042 (46,5%) em íntrons e 112 (0,9%) em regiões não identificadas. Dez SNPs localizados em regiões regulatórias da expressão de genes, relacionados com a melanogênese, WNT8B c.-101T>C (rs rs3793772), KIT c.67+12766T>C (rs2017472), GNAI1 g.80130766C>T (rs2079162), ADCY3 c.675+9196T>G (rs11900505), PLCB1 g.8908966C>A (rs4295085), PRKCA c.205+407G>A (rs12601850), PRKCA c.288+33994A>G (rs1806448), CALM2 g.47384601A>G (rs11884600), CREB1 c.303+373G>A (rs10932201) e MITF c.938-325A>G (rs7623610), foram considerados de interesse para o estudo e foram utilizados para a validação dos resultados obtidos na GELE por meio da reação em cadeia da polimerase em tempo real com ensaios TaqMan® (Applied Biosystems®). Todos os SNPs isolados e em combinações específicas de dois a dois alteraram o risco de ocorrência de MC. Os SNPs ADCY3 c.675+9196T>G e MITF c.938-325A>G merecem destaque entre os demais. Os genótipos isolados ADCY3 TT e MITF AA e o genótipo combinado TT+GG + AA+AG dos SNPs estiveram associados a riscos 4,86, 3,13 e 19,17 vezes maiores de ocorrência de MC do que os demais genótipos. Aos 60 meses de observação, os pacientes com o genótipo MITF AA e com o genótipo combinado ADCY3 + MITF TT+AA apresentaram menor SLP (44,5% versus 68,2%, P= 0,007; 22,2% versus 61,3%, P= 0,006) e menor SG (67,7% versus 81,7%, P= 0,009; 44,4% versus 72,5%, P= 0,006) do que os pacientes com os outros genótipos dos genes (Estimativas de Kaplan-Meier). Pacientes com os repectivos genótipos estiveram sob riscos 2,40 e 3,53 vezes maiores de apresentarem progressão da doença e 2,85 e 3,84 vezes maiores de apresentarem evolução para o óbito (análise univariada de Cox). Nossos resultados indicam, pela primeira vez, que SNPs em genes de melanogênese podem contribuir para identificar indivíduos com alto risco de MC e de pacientes com prognóstico desfavorável, que mereçam maior atenção na prevenção, diagnóstico precoce ou terapêutica do tumor Abstract: Combinations of inherited genetic alterations, such as single nucleotide polymorphisms (SNPs) in association with cutaneous melanoma (CM) risk, clinical manifestation and prognosis in patients have not been clarified. Therefore, this was the aim of the present study. Genomic DNA was extracted of the peripheral blood samples of 103 CM patients and 103 controls. Each sample was genotyped using DNA high-resolution microarrays (Affymetrix® SNP 6.0). We observed 12.994 SNPs differentially between patients and controls groups. Among them, 6.814 SNPs (52.4%) were located in regulatory regions of DNA transcription (in 3¿ and 5¿ untranslated), 26 (0.2%) in coding sequence of amino acids regions, 6.042 (46.5%) in introns and 112 (0.9%) unidentified regions. Ten SNPs, located in regulatory regions of gene associated with melanogenesis pathway, WNT8B c.-101T>C (rs rs3793772), KIT c.67+12766T>C (rs rs2017472), GNAI1 g.80130766C>T (rs2079162), ADCY3 c.675+9196T>G (rs11900505), PLCB1 g.8908966C>A (rs4295085), PRKCA c.205+407G>A (rs12601850), PRKCA c.288+33994A>G (rs1806448), CALM2 g.47384601A>G (rs11884600), CREB1 c.303+373G>A (rs10932201) e MITF c.938-325A>G (rs7623610), were considered of interest for the study and validated by polymerase chain reaction in real time using TaqMan assays (Applied Biosystems®). All SNPs isolated and combined alter the risk for CM. SNPs ADCY3 c.675+9196T>G and MITF c.938-325A>G deserves highlight. The isolated genotypes ADCY3 TT and MITF AA and TT+GG + AA+AG combined were associated with 4,86, 3,13 and 19,17-fold increased risk for CM. During 60 months of observation, patients with MITF AA genotypes and ADCY3 + MITF TT+AA combined genotypes were associated with poor progression free survival (44,5% versus 68,2%, P= 0,007; 22,2% versus 61,3%, P= 0,006) and lower overall survival (67,7% versus 81,7%, P= 0,009; 44,4% versus 72,5%, P= 0,006) than the patients with others genotypes (Kaplan-Meier estimates). Carriers of the same genotypes had 2,40 and 3,53 more chances of disease progression and 2,85 and 3,84 more chances to evolve to death, respectively (Cox univariate). Our results suggest, for the first time, that SNPs related in melanogenesis pathway may contributed for identify people with high risk for CM and patients with poor prognosis, that need to receive more attention on preventing, early diagnosis or treatment Doutorado Fisiopatologia Médica Doutora em Ciências FAPESP 2012/16617-4, 2012/15880-3 CNPQ 479529/2012-4 FAEPEX 191/14

Orientadores: Patrícia Maria Bergamo Favaro, Sara Teresinha Olalla Saad Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: As síndromes mielodisplásicas (SMD) são um grupo heterogêneo de doenças caracterizadas por hematopoese ineficaz e risco de progressão para leucemia mieloide aguda (LMA). SMD de baixo de risco é caracterizada por um aumento de apoptose na medula óssea e alterações clínicas com perfil autoimune, enquanto que na SMD de alto risco há uma evasão imune, baixa apoptose e danos secundários ao DNA, contribuindo para a progressão para LMA. Essas evidências, junto com os dados de terapia imunossupressora em pacientes com SMD, sugerem o papel do sistema imune na progressão desta doença. Entretanto, o papel do sistema imune não é claro, e estudos que abordem o perfil das células T são importantes para o melhor entendimento da patogênese da SMD. Formin-like 1 (FMNL1) pertence à família de proteínas formina, indispensáveis para muitos processos fundamentais actina-dependentes. FMNL1 é restritamente expressa em células derivadas de linhagem hematopoética e superexpressa em células neoplásicas hematopoéticas malignas. Recentemente, foi descrito que FMNL1 está envolvida no processo de citotoxicidade de células CD8+. Desse modo, estudar a expressão de FMNL1 tanto nos linfócitos como nas células da MO dos pacientes com SMD, poderia contribuir para o melhor entendimento do papel dessa nova proteína neste modelo de neoplasia hematológica. No presente estudo, foi observada uma diminuição significativa na contagem absoluta de linfócitos periféricos no grupo SMD, após ajuste para idade, quando comparada com o grupo de doadores saudáveis (controle). Entretanto, houve um aumento da frequência de células CD3+, resultante do aumento significativo das subpopulações de células CD3+CD4+ no grupo de alto risco e CD3+CD8+ no grupo de baixo risco, de acordo com as classificações FAB e WHO. A razão CD4:CD8 encontrou-se aumentada no grupo de alto risco comparado com o de baixo risco. Dependência transfusional foi correlacionada positivamente com a porcentagem de CD3+CD4+, enquanto que a idade dos pacientes correlacionou-se de forma negativa com a porcentagem de CD3+ e CD3+CD8+. Os níveis de expressão de FOXP3, nas células CD3+ de sangue periférico, foram significativamente menores no grupo de baixo risco quando comparado com o grupo controle, e esse padrão se repetiu para a expressão de IL10. A quantificação dos transcritos de IL10 correlacionou-se negativamente com a porcentagem de células CD3+CD8+. Em conclusão, evidenciamos que pacientes com SMD apresentaram um menor número de linfócitos, porém com a frequências das células T CD3+, CD3+CD4+ e CD3+CD8+ aumentadas. Os pacientes de baixo risco apresentaram uma diminuição da expressão de FOXP3 e de IL10, quadro característico de um microambiente apoptótico e inflamatório. Já no grupo de alto risco, a expressão de FOXP3 e de IL10 aumenta em relação ao grupo de baixo risco. É interessante ressaltar que nos pacientes com SMD houve uma correlação entre o aumento da expressão de IL10 e a diminuição das células T CD3+CD8+, sugerindo a contribuição das Tregs na progressão da doença através da produção de IL10. A análise da expressão de FMNL1 em células CD3+ de sangue periférico não denotou diferenças significativas entre os pacientes com SMD e o grupo controle. Entretanto observou-se uma correlação positiva entre a expressão de FMNL1 e o número de células CD3+CD4+ e ambos com a dependência transfusional. Quanto à expressão de FMNL1 em amostras de MO, houve uma expressão significativamente menor nos pacientes com SMD quando comparado com as células de doadores normais, além de uma correlação negativa entre FMNL1 e número de citopenias. Usando modelos de linhagens celulares hematopoéticas para a diferenciação, observou-se um aumento significativo na expressão gênica e protéica de FMNL1 durante a diferenciação megacariocítica. Esses resultados sugerem a participação de FMNL1 na ativação de linfócitos CD4+ no sangue periférico e na diferenciação hematopoética na medula óssea Abstract: Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by ineffective hematopoiesis and risk of progression towards acute myeloid leukemia. Low-risk MDS is characterized by increased apoptosis in the bone marrow (BM), with a clinical autoimmune profile, whereas in high-risk MDS an immune evasion, low apoptosis and secondary DNA damage occurs, contributing to the progression of AML. This evidence, together with the data of immunosuppressive therapy in patients with MDS, suggests a role of the immune system in the progression of this disease. However, this role of the immune system is remains unclear, and studies that address the profile of T cells are important for a better understanding of the pathogenesis of MDS. Formin-like 1 (FMNL1) belongs to the family of proteins formina indispensable for many fundamental processes in actin-dependent. FMNL1 is strictly expressed in hematopoietic lineage derived cells, and overexpressed in malignant hematopoietic neoplastic cells. FMNL1 has recently been reported to be involved in the cytotoxicity of CD8+ cells. Thus, studies on the expression of FMNL1, both in lymphocytes and BM cells of MDS patients, could contribute to a better understanding of the role of this protein in this new model of hematologic malignancy. In the present study, we observed a significant decrease in absolute peripheral lymphocyte counts in the MDS group, after adjusting for age, compared with the healthy donor group (control). However, there was an increased frequency of CD3+, resulting in a significant increase of the CD3+CD4+ subpopulation in high risk and CD3+CD8+ in MDS low risk, according to FAB and WHO classifications. CD4:CD8 ratio was increased in the high risk when compared to the low risk group. Transfusion dependence was positively correlated with the percentage of CD3+CD4+, whereas the age of patients correlated negatively with the percentage of CD3+ and CD3+CD8+. The expression levels of FOXP3, in peripheral blood CD3+ cells, was significantly lower in the low risk group compared to controls and this pattern was repeated for the expression of IL10. Interestingly, IL10 transcripts correlated negatively with the percentage of CD3+CD8+. In conclusion, we found that patients with MDS had a lower lymphocyte number, however presented an increased frequency of CD3+ and CD3+CD8+ T cells. Our low risk patients showed a decreased expression of FOXP3 and IL10, characteristic of apoptotic and inflammatory microenvironment. In the high risk group, the expression of FOXP3 and IL10 was normal. Interestingly, there was a correlation between increasing expression of IL10 and reduction of CD3+CD8+ T cells in patients, suggesting the contribution of Treg in disease progression due to IL10 production. Analysis of FMNL1expression in CD3+ cells of peripheral blood showed no significant differences between patients with MDS and the control group. However, there was a positive correlation between FMNL1 expression and the number of CD3+CD4+, and both were transfusion dependence. FMNL1 expression in BM samples was significantly lower in MDS patients when compared with cells from normal donors, and there was a negative correlation between FMNL1 and number of cytopenias. Using models of hematopoietic cell lineages for differentiation, we observed an increase in gene and protein expression of FMNL1 during megakaryocytic and granulocytic differentiation. These results suggest the participation of FMNL1 in the activation of CD4+ lymphocytes in peripheral blood and bone marrow hematopoietic differentiation Mestrado Fisiopatologia Médica Mestre em Ciências

Orientador: Carmen Silvia Passos Lima Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Melanócitos anormais podem ser eliminados do organismo por atividade de linfócitos T. O gene PDCD1 codifica o receptor PD1 em linfócitos T, relacionado à regulação do sistema imune, e é polimórfico em humanos. Assim, é possível que indivíduos saudáveis herdem habilidades distintas para eliminar melanócitos anormais e desenvolver melanoma cutâneo (MC). Os objetivos do estudo foram os de verificar se os polimorfismos de nucleotídeo único (SNPs) PD1.1 c.-606G>A, PD1 c.627+252C>T, PD1.5 c.804C>T e PD1.9 c.644C>T do gene PDCD1 influenciam no risco de MC, nas manifestações clínicas e biológicas do tumor, na expressão do gene PDCD1 e na sobrevida dos pacientes com MC. Foram avaliados 250 pacientes com MC e 250 controles (doadores de sangue). Os genótipos dos SNPs foram identificados em DNA em leucócitos do sangue periférico, por meio da reação em cadeia da polimerase (PCR) em tempo real. A expressão do gene PDCD1 foi avaliada por PCR quantitativo. As diferenças entre grupos foram avaliadas por meio do teste de Fisher ou qui-quadrado. Os riscos de ocorrência de MC a que os indivíduos foram submetidos, foram obtidas por meio das razões das chances (ORs). As comparações da expressão gênica de indivíduos com os genótipos distintos de cada SNPs foram realizadas por meio do teste de Mann-Whitney. A sobrevida livre de recidiva (SLR) e a global (SG) dos pacientes foram avaliadas pelo método de Kaplan-Meier/teste log-rank e por análises univariada e multivariada de Cox. Indivíduos com o genótipo PD1 CC isolado e associado ao genótipo PD1.5 CC estiveram sob riscos 2,20 e 2,51 vezes maiores de desenvolvimento do MC do que os demais, respectivamente. Indivíduos com o fototipo I ou II e com o genótipo CC do SNP PD1 e CC + CC dos SNPs PD1 e PD1.5 estiveram sob riscos 5,89 vezes e 6,71 vezes maiores de MC do que os demais. O genótipo PD1.5 TT foi associado com maior expressão do gene PDCD1 do que os genótipos CT ou CC (1,28 versus 2,49 UA; p= 0.03). Os pacientes com o genótipo AA do PD1.1 apresentaram menor SLR do que os com os genótipos GA ou GG. Acreditamos que os nossos resultados possam contribuir para identificar indivíduos com alto risco para a ocorrência do MC e de portadores do tumor com prognóstico desfavorável, que mereçam receber atenção especial na prevenção, diagnóstico precoce e terapêutica diferenciada Abstract: Abnormal melanocytes can be eliminated from the body by T lymphocytes. The PDCD1 gene encoding the receptor PD1 on T cells is related to the immune system regulation, and it is polymorphic in humans. Thus, it is possible for healthy individuals to inherit different abilities for eliminating abnormal melanocytes and developing cutaneous melanoma (CM). This study aimed to assess whether the single nucleotide polymorphisms (SNPs) PD1.1 c.-606G>A, PD1 c.627+252C>T, PD1.5 c.804C>T and PD1.9 c.644C>T of PDCD1 gene influence the risk of CM, clinical and biological manifestations of the tumor, expression of PDCD1 gene, and survival of patients with CM. We evaluated 250 patients with CM and 250 controls (blood donors). Genotypes of SNPs were identified in DNA from peripheral blood leukocytes, by real-time polymerase chain reaction (PCR). The PDCD1 gene expression was assessed by quantitative PCR. The statistical significance of differences between groups was calculated using the Fisher's exact or chi-square test. The occurrence of CM risks in patients and controls, was accessed by the odds ratios (ORs). Comparisons of gene expression in individuals with different genotypes for each SNP were performed using the Mann-Whitney test. The recurrence-free survival (RFS) and overall (OS) of patients were evaluated by the Kaplan-Meier method/log-rank test, univariate and multivariate analyses of Cox. Individuals with CC genotype PD1 isolated and associated with PD1.5 CC genotype were under 2.20 and 2.51 times greater risk of developing CM than others, respectively. Indivíduas with I or II fototype and the PD1 CC genotype and CC + CC of PD1 e PD1.5 SNPs were under 5.89 times and 6.71 times greater risks of developing CM than others. PD1.5 TT genotype was associated with increased expression of PDCD1 gene than CT or CC genotype (1.28 versus 2.49 UA; p= 0.03). Patients with PD1.1 AA genotype had lower RFS than those with GA or GG genotypes. We believe that our results may help to identify individuals at high risk of developing CM and tumor patients with poor prognosis, deserving special attention in prevention, early diagnosis and differentiated treatment Mestrado Fisiopatologia Médica Mestra em Ciências FAPESP 2014/10042-5

Author(s): Malone, Christopher D; Olson, Emilia S; Mattrey, Robert F; Jiang, Tao; Tsien, Roger Y; Nguyen, Quyen T | Abstract: PurposeThe ability to detect small malignant lesions with magnetic resonance imaging (MRI) is limited by inadequate accumulations of Gd with standard chelate agents. To date, no T1-targeted agents have proven superiority to Gd chelates in their ability to detect small tumors at clinically relevant field strengths. Activatable cell-penetrating peptides and their Gd-loaded dendrimeric form (ACPPD-Gd) have been shown to selectively accumulate in tumors. In this study we compared the performance of ACPPD-Gd vs. untargeted Gd chelates to detect small tumors in rodent models using a clinical 3T-MR system.Materials and methodsThis study was approved by the Institutional-Animal Care-and-Use Committee. 2 of 4 inguinal breast fat pads of 16 albino-C57BL/6 mice were inoculated with tumor Py8119 cells and the other 2 with saline at random. MRI at 3T was performed at 4, 9, and 14 days after inoculation on 8 mice 24-hours after injection of 0.036mmol Gd/kg (ACPPD-Gd), and before and 2-3 minutes after 0.1 mmol/kg gadobutrol on the other 8 mice. T1-weighted (T1w) tumor signal normalized to muscle, was compared among the non-contrast, gadobutrol, and ACPPD-Gd groups using ANOVA. Experienced and trainee readers blinded to experimental conditions assessed for the presence of tumor in each of the 4 breast regions. Receiver operator characteristic (ROC) curves and area-under-curve (AUC) values were constructed and analyzed.ResultsTumors ≥1mm3 were iso-intense to muscle without contrast on T1w sequences. They enhanced diffusely and homogeneously by 57±20% (pl0.001) 24 hours after ACPPD-Gd and by 25±13% (pl0.001) immediately after gadobutrol. The nearly 2-fold difference was similar for small tumors (1-5mm3) (45±19% vs. 19±18%, p = 0.03). ACPPD-Gd tended to improve tumor detection by an experienced reader (AUC 0.98 vs 0.91) and significantly more for a trainee (0.93 vs. 0.82, p = 0.02) compared to gadobutrol. This improvement was more pronounced when obvious tumors (g5mm3) were removed from the ROC analysis for both the experienced observer (0.96 vs. 0.86) and more so for the trainee (0.86 vs. 0.69, p = 0.04).ConclusionACPPD-Gd enhances MMP-expressing tumors of any size at 3T 24 hours after administration, improving their detection by blinded observers when compared to non-contrast and contrast groups given commercial Gd-chelates and imaged during the equilibrium phase.

Orientador: Sara Teresinha Olalla Saad Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Os eventos de migração e adesão das células progenitoras hematopoéticas são cruciais para a sua manutenção nos nichos da medula óssea e são regulados por diversos fatores, dentre eles as RhoGTPases. ARHGAP21 é uma proteína RhoGAP (RhoGTPase activating protein, que regulam negativamente as RhoGTPases), e atua na migração e adesão celulares. O papel da Arhgap21 na hematopoese ainda é desconhecido. O objetivo geral do presente estudo foi estudar a função da Arhgap21 na migração e adesão das células progenitoras hematopoéticas. Neste trabalho, mostramos que ainda não foi possível a obtenção do camundongo nocaute para Arhgap21, pois parece que morrem antes do nascimento. O estudo foi realizado com os camundongos Arhgap21+/-, cuja expressão gênica e protéica de Arhgap21 está reduzida. As células progenitoras hematopoéticas Arhgap21+/- apresentaram menor quimiotaxia induzida por CXCL-12 e redução da adesão à fibronectina, o que provavelmente levou a um homing ineficiente para a medula óssea e para o baço. Observou-se também que o microambiente da medula óssea e do baço com redução de Arhgap21 leva à ineficiência do homing das células progenitoras hematopoéticas normais. Não foi observada alteração na expressão gênica e na freqüência de células expressando CXCR-4, bem como não houve diferença na secreção de CXCL-12. As células da medula óssea Arhgap21+/- apresentaram maior expressão de Cdc42 e parecem possuir maior atividade desta RhoGTPase. Pela primeira vez se descreve o papel da Arhgap21 na hematopoese normal e ela parece ser um importante regulador da migração, adesão e homing das células progenitoras hematopoéticas, provavelmente pela regulação negativa de Cdc42, através da sua função GAP Abstract: Migration and adhesion of hematopoietic progenitor cells are crucial events for their maintenance on bone marrow niches. The role and mechanisms underlying hematopoietic progenitors migration and adhesion has been extensively studied and RhoGTPases have been implicated in these events. Regulators and effectors of RhoGTPases functions also have been studied in biology of hematopoietic progenitor cells. ARHGAP21 is a RhoGAP member, which function as negative regulators of RhoGTPases, and plays role in cell migration and adhesion. The aim of this study was to investigate the role of Arhgap21 in migration and adhesion of hematopoietic progenitor cells. It was not possible to generate a knockout mouse until now, because they die before birth, as results indicate. Heterozygous mice (Arhgap21+/-)v have reduction in Arhgap21 gene and protein expression and we used it as model. Hematopoietic Progenitor cells from Arhgap21+/- mice showed reduction in CXCL-12- induced chemotaxis as well as in fibronectin adhesion, which probably leaded to inefficient homing to wild-type bone marrow and spleen observed. In addition, homing of wild-type hematopoietic progenitor cells to Arhgap21+/- bone marrow and spleen was also reduced. It was not observed differences in CXCR-4 gene expression and frequency of progenitor hematopoietic cells expressing this receptor as well as in CXCL-12 secretion in Arhgap21+/- bone marrow or serum. It is the first time that the role of Arhgap21 in hematopoiesis is described and it seems to be an important regulator of hematopoietic progenitor cell migration, adhesion and homing, probably due to Cdc42 negative regulation by its RhoGAP function Doutorado Biologia Estrutural, Celular, Molecular e do Desenvolvimento Doutora em Fisiopatologia Médica

Orientador: José Barreto Campello Carvalheira Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: Embora a obesidade seja reconhecida como importante causa de diabetes e doença cardiovascular, a associação entre obesidade e diferentes tipos de câncer tem recebido muito menos atenção. A associação entre obesidade e o desenvolvimento de câncer de cólon representa um dos principais avanços conceituais na patogênese do câncer de cólon da última década. Recentemente a atuação da inflamação subclínica da obesidade na carcinogênese ganhou destaque. Mecanisticamente acredita-se que a obesidade atue como promotor tumoral, e seus efeitos pró-tumorigênicos dependam principalmente da resposta inflamatória de baixo grau ocasionada pela obesidade que envolve a produção de citocinas inflamatórias e pró-tumorigênicas (TNF e IL-6). Uma das principais características da inflamação induzida por obesidade é a infiltração de macrófagos no tecido adiposo, produzindo citocinas inflamatórias e outros mediadores que interferem na sinalização insulínica. Inflamação e estresse de retículo que são conectadas em diversos níveis, são sistemas adaptativos de curto período de expressão necessárias para a função e sobrevivência do organismo, e ambas são prejudiciais quando ativadas cronicamente. Neste sentido, a ativação da PKR durante a inflamação e posterior ativação de JNK pela PKR, também interfere e prejudica a via de sinalização da insulina. A relação entre o câncer de cólon e obesidade pode ser devido a ação, em nível molecular, da inflamação subclínica de baixo grau e ao estresse celular causado por essa sinalização inflamatória. Sendo a PKR responsiva à sinalização inflamatória e também à via insulínica em outros tecidos, e relacionada à carcinogênese e à progressão em diversos tipos de câncer, a investigação de sua participação é relevante a medida que propicia o entendimento da fisiopatologia molecular de tumores de cólon. Assim, o objetivo principal do estudo foi avaliar o papel da PKR no desenvolvimento de tumores de cólon em camundongos submetido a dieta hiperlipídica. A ausência de PKR previne a formação de tumores. Além disso, aparentemente a ausência de PKR em células mielóides também confere proteção contra a resistência à insulina induzida por dieta hiperlipídica, reduzindo a inflamação induzida pela obesidade. Essas observações demostram que a PKR pode ser um ponto principal durante a carcinogênese associada à inflamação e pode representar um promissor alvo para a intervenção terapêutica Abstract: Although obesity is recognized as a major cause of diabetes and cardiovascular disease, the association between obesity and different types of cancer has received much less attention. The association between obesity and the development of colon cancer is one of the major conceptual advances in the pathogenesis of colon cancer in the last decade. Recently the role of subclinical inflammation in obesity and in carcinogenesis gained prominence. Mechanistically it is believed that obesity acts as a tumor promoter, and their pro-tumorigenic effects depend mainly on low-grade inflammatory response caused by obesity, involving the production of inflammatory and pro-tumorigenic cytokines (TNF and IL-6). A key feature of obesity-induced inflammation is the infiltration of macrophages in adipose tissue, producing inflammatory cytokines and other mediators that interfere with insulin signaling. Reticulum stress and inflammation are connected on many levels and work as short period adaptive systems required for the function and survival of the organism, and both are detrimental when chronically activated. In this regard, the activation of PKR during inflammation and subsequent activation of JNK by PKR also interferes and impairs insulin signaling pathway. Thus, PKR can form a metabolically active inflammatory complex which then becomes part of the of insulin pathway and of the pathogens response pathway and control of translation sensible to nutrients. The relationship between colon cancer and obesity may be due to action at the molecular level, subclinical low-grade inflammation and cellular stress caused by this inflammatory signaling. PKR is responsive to inflammatory signaling and also to the insulin pathway in other tissues, and related to carcinogenesis and progression in several types of cancer. Thus, investigation of it's participation is relevant as it provides the understanding of the molecular pathophysiology of colon tumors. Thus, the main objective of the study was to evaluate the role of PKR in the development of colon tumors in mice subjected to a high-fat diet. The absence of PKR prevents the formation of tumors. Moreover, apparently the absence of PKR in myeloid cells also confers protection against resistance to insulin induced by a high-fat diet, reducing inflammation induced by obesity. These observations demonstrate that PKR can be a primary point during carcinogenesis associated with inflammation and may represent a promising target for therapeutic intervention Doutorado Fisiopatologia Médica Doutor em Ciências

Orientador: Jose Barreto Campello Carvalheira Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: A proteína mTOR é um proteína reguladora chave de vários processos celulares, dentre eles proliferação, crescimento e sobrevivência celular. Fatores de crescimento, oxigênio, status energético e a presença de aminoácidos são fundamentais para que todos esses processos ocorram normalmente. Descobertas realizadas nas últimas décadas mostraram que a via da mTOR encontra-se ativada em vários processos celulares, incluindo formação tumoral e angiogênese. A leucina é um aminoácido de cadeia ramificada que tem o maior potencial em ativar a via da mTOR. Devido sua capacidade de promover a síntese proteica e ganho de massa muscular, seu uso é constantemente estimulado em pacientes com câncer. No entanto, seus efeitos no crescimento tumoral não está claro. Dessa forma, realizamos um estudo cujo objetivo principal foi investigar os efeitos da dieta suplementada com leucina na modulação do crescimento tumoral em diferentes linhagens de células tumorais que se diferenciem em relação à ativação constitutiva da via IRS1/Akt/mTOR. Estudos in vivo e in vitro realizados demonstraram que as células que se diferenciam em relação à ativação da via IRS1/AKT/mTOR respondem de maneira distinta à suplementação com leucina. Linhagens de células tumorais que possuem a via da mTOR constitutivamente ativada, PC-3 e MCF-7, quando suplementadas com doses elevadas de leucina in vitro reduziram a proliferação celular e causaram retenção das células na fase G1 do ciclo celular. Já o xenoenxerto tumoral da PC-3 reduziu sua proliferação e aumentou a morte celular quando os animais foram suplementados com leucina na dieta. Nós também observamos aumento da atividade da mTOR e da p70S6K em todas as linhagens celulares quando suplementadas com leucina. O aumento da atividade da proteína mTOR foi acompanhado de redução na fosforilação de AKTser473 nas células que possuíam a via da PI3K hiperativada (PC-3 e MCF-7). Esse fato pode estar ocorrendo devido a ativação das alças de contraregulação ocasionadas pela estimulação excessiva provocada pela suplementação com leucina, naquelas linhagens celulares que já possuem a via hiperativada. Fato este comprovado pelo aumento da fosforilação em serina 307 da proteína IRS1. Dessa forma, nossos resultados sugerem que a ativação da via da mTOR é central para determinar a sensibilidade de tumores à dieta suplementada com leucina, podendo modular o desenvolvimento tumoral naquelas células que já possuem a via IRS1/AKT/mTOR constitutivamente ativada. O mecanismo pelo qual a leucina pode retardar o desenvolvimento tumoral em células que possuem a via da mTOR hiperativada parece estar relacionado com o eixo de regulação negativa p70S6K-PI3K, com consequente redução da fosforilação de AKT e liberação das vias apoptóticas nos tecidos tumorais Abstract: mTOR is a key regulatory protein in various cellular processes including proliferation, cell growth and survival. Growth factors, oxygen, energy status and amino acids are all essential to these processes. New findings in the last few decades have shown that the mTOR pathway is activated in many cellular processes, including tumorigenesis and angiogenesis. The branched chain amino acid leucine has the greatest potential to activate the mTOR pathway. Due to its ability to promote protein synthesis and muscle mass gain, use of leucine is frequently utilized in patients with cancer. However, the effect of leucine on tumor growth is not clear. The aim of this study is therefore to investigate the effect of diet-supplemented leucine on the modulation of tumor growth in several tumor cell lines that differ in the constitutive activation status of the insulin receptor substrate 1 (IRS1)/AKT/mTOR pathway. Both in vitro and in vivo experiments demonstrated different cell proliferation responses when cells were exposed to high doses of leucine. Tumor cell lines PC-3 and MCF-7, which have a constitutively activated mTOR signaling, displayed reduced cell proliferation and G1 phase cell cycle arrest when supplemented with high doses of leucine in vitro. Likewise, leucine-supplemented PC-3 cell tumor xenografts displayed reduced proliferation and increased cell death. We also observed increased activity of mTOR and its downstream substrate p70S6K in all cell lines supplemented with leucine. Increased mTOR activity was accompanied by a reduction in AKT serine 473 (ser473) phosphorylation in cell lines with a hyperactivated PI3K pathway (PC-3 and MCF-7). This most likely occurred because leucine supplementation further increased mTOR and p70S6K activity, triggering the inhibitory p70S6K/IRS1 axis. In fact, we found increased IRS1 ser307 phosphorylation in hyperactivated cell lines (PC-3 and MCF-7) supplemented with high doses of leucine. Therefore, our results suggest that mTOR pathway activation is central to determining the sensitivity of tumors to leucine supplementation. Furthermore, this could affect the response to leucine-supplemented therapies of those tumors in which the PI3K pathway is constitutively activated. The mechanism for this appears to be related to the negative p70S6K/IRS1 regulation axis, with consequent reduction of AKT phosphorylation and the release of apoptotic pathways in tumor tissues Doutorado Fisiopatologia Médica Doutora em Ciências