O câncer (neoplasia) é uma das principais causas de mortalidade no mundo. Apesar dos grandes avanços no diagnóstico e nas formas de tratamento, ele ainda representa um enorme desafio para os pesquisadores de muitas áreas. Recentemente houve uma importante descoberta que poderá fornecer um paradigma completamente diferente no entendimento de como o câncer se inicia e cresce, com eventualmente profundas consequências nas formas de tratamento. Essa descoberta diz respeito a presença de células tronco adultas em tumores e seu possível papel no surgimento e crescimento destes. Propomos nesse trabalho um modelo matemático que considera a presença de células com propriedades de (a) auto renovação , (b) diferenciação e (c) mobilidade , características das células tronco. O modelo proposto é um autômato celular probabilístico com atualização assíncrona em uma rede. Cada elemento da rede pode estar vazio ou conter uma célula tumoral. Há dois tipos de células: as células rotuladas como do tipo 2 que são aquelas associadas com as células tronco tumorais e aquelas rotuladas como do tipo 1 que são as células diferenciadas, somente com capacidade de reprodução. A taxa de reprodução de cada célula é definida como uma função de sua vizinhança e tipo. Diferentes taxas de reprodução foram usadas nas simulações e as células do tipo 2 podem diferenciar-se ou mover-se. Os resultados das simulações mostram como a motilidade das células 2 e as taxas de reprodução de ambos os tipos de células influenciam os padrões morfológicos do tumor. Também investigamos uma possível transição de fase que pode estar relacionada a metástase. Essa transição de fase representa algo de grande interesse biológico, uma vez que a metástase é o mecanismo mais importante que leva o organismo a óbito. Compararemos nossos resultados com dados experimentais dos colaboradores Nascimento TL et al [1] da UNIFESP- Escola Paulista de Medicina. / Cancer (neoplasia) is one of the most dangerous diseases and one of the main cause of mortality around the world. Despite the great advances in diagnosis and treatment, it still represents a huge challenge to researchers of many areas. Recently there was a strinking discovery that may give rise to a complete different paradigm in the understanding of how cancer starts and grows, with eventually profound consequences in the forms of treatment. It is related to the ending of adult stem cells in tumors, and its possible role in the birth and growth of them. We propose in this work a mathematical model that takes into account the presence of cells with the properties of (a) self renewal, (b) differentiation and (c) mobility, characteristics of stem cells. The model developed is a probabilistic cellular automaton with asynchronous update set in a grid. Each element of the grid may be empty or contain a tumor cell. There are two types of cells: the cells labeled as type 2 are those associated with cancer stem cells and those labeled as type 1 are differentiated cells only capable of reproducing. The reproduction rate of each cell is defined as a function of its neighborhood and its type. Different rates of reproduction have been used in the simulations, and type 2 cells may differentiate and some motility. Our simulation results show how the motility of cells 2 and the reproduction rates of both types of cells influence the morphological patterns of tumor. We have also investigated a possible phase transition that may be related to metastasis. This phase transition represents something of great biological interest because metastasis is the most important mechanism that leads to death. We will compare our results with experimental data from collaborators Nascimento TL et al [1] in UNIFESP-Escola Paulista de Medicina.

Células de Schwann (CS) cultivadas a partir de nervo ciático de ratos foram enxertadas, associadas ou não ao fator de crescimento derivado de células gliais (GDNF), em ratos adultos submetidos à transecção total da medula espinhal (T11). Análises do comportamento motor do 1º ao 3º mês pós-operatório e do crescimento de fibras nervosas imunorreativas no epicentro do enxerto ao término deste período mostraram: melhora temporal dos parâmetros motores superior nos grupos que receberam CS, que foi potencializada na presença do GDNF; aumento na quantidade de axônios nos grupos que receberam CS, efeito potencializado na presença do GDNF. Em outro experimento, as reatividades astrocitária, microglial e do fator neurotrófico bFGF astrocitário foram avaliados na medula espinhal parcialmente transectada de ratos, 1 semana e 3 meses após a cirurgia. Ativação astrocitária e microglial e maior intensidade de bFGF nas substâncias branca e cinzenta foram encontradas em toda a extensão da medula, fatos relacionados ao processo local de cicatrização e fenômenos tróficos e plásticos à distância. / Schwann cells (SC) obtained from rat sciatic nerve were grafted, in the presence or not of glial derived neurotrophic factor (GDNF), in adult rats submitted to a complete spinal cord transection at T11. Behavioral analysis from the 1st to the 3rd post operative month and of the immunoreactive fiber outgrowth in the graft epicenter at the end of this period showed: temporal improvements on motor parameters that were superior in the groups that received SC, being even higher in the presence of GDNF; increased amount of axonal fibers in the groups treated with SC, being even higher in the presence of GDNF. In another experiment, astrocytic and microglial activation and astrocytic neurotrophic factor bFGF expression were analyzed after a partial spinal cord transection, 1 week and 3 months after surgery. Astrocitic and microglial activation as well as a higher intensity of astrocitic bFGF in the white and gray matters were found in the entire spinal cord, what is related to the local scar formation and distant trophic and plastic phenomena.

Glioblastoma multiforme (GBM) is the most lethal primary central nervous system tumor, with median survival after diagnosis of less than 12 months because dissemination into the brain parenchyma limits the long-term effectiveness of surgical resection, and because GBM cells are resistant to radiation and chemotherapy. This sad dismal prognosis for patients with GBM emphasizes the need for greater understand of the fundamental biology of the disease. Invasion is one of the major causes of treatment failure and death from glioma, because disseminated tumor cells provide the seeds for tumor recurrence. Inflammation is increasingly recognized as an important component of invasion. In the brain, inflammation can occur by activation of microglia, the resident macrophages of the brain, or by tumor-associated blood macrophages. Therefore, we hypothesize that activity of the innate immune system in the brain can influence tumor progression by secreting cytokines such as Tumor Necrosis Factor alpha (TNF-α). In this study, we show that patient-derived glioma spheres undergo morphological changes in response to TNF‑α that are associated with changes in migration behavior in vitro. These morphological changes include appearance of tumor islands in site different from where the primary tumor cells were seeded. We further showed that TNF‑α treated cells significantly increased expression of cell adhesion molecules such as CD44 and VCAM-1. Furthermore, we demonstrate increased cell density also caused increased in expression of cell adhesion molecules. The extent to which these are recapitulated in vivo will be investigated.

O câncer (neoplasia) é uma das principais causas de mortalidade no mundo. Apesar dos grandes avanços no diagnóstico e nas formas de tratamento, ele ainda representa um enorme desafio para os pesquisadores de muitas áreas. Recentemente houve uma importante descoberta que poderá fornecer um paradigma completamente diferente no entendimento de como o câncer se inicia e cresce, com eventualmente profundas consequências nas formas de tratamento. Essa descoberta diz respeito a presença de células tronco adultas em tumores e seu possível papel no surgimento e crescimento destes. Propomos nesse trabalho um modelo matemático que considera a presença de células com propriedades de (a) auto renovação , (b) diferenciação e (c) mobilidade , características das células tronco. O modelo proposto é um autômato celular probabilístico com atualização assíncrona em uma rede. Cada elemento da rede pode estar vazio ou conter uma célula tumoral. Há dois tipos de células: as células rotuladas como do tipo 2 que são aquelas associadas com as células tronco tumorais e aquelas rotuladas como do tipo 1 que são as células diferenciadas, somente com capacidade de reprodução. A taxa de reprodução de cada célula é definida como uma função de sua vizinhança e tipo. Diferentes taxas de reprodução foram usadas nas simulações e as células do tipo 2 podem diferenciar-se ou mover-se. Os resultados das simulações mostram como a motilidade das células 2 e as taxas de reprodução de ambos os tipos de células influenciam os padrões morfológicos do tumor. Também investigamos uma possível transição de fase que pode estar relacionada a metástase. Essa transição de fase representa algo de grande interesse biológico, uma vez que a metástase é o mecanismo mais importante que leva o organismo a óbito. Compararemos nossos resultados com dados experimentais dos colaboradores Nascimento TL et al [1] da UNIFESP- Escola Paulista de Medicina. / Cancer (neoplasia) is one of the most dangerous diseases and one of the main cause of mortality around the world. Despite the great advances in diagnosis and treatment, it still represents a huge challenge to researchers of many areas. Recently there was a strinking discovery that may give rise to a complete different paradigm in the understanding of how cancer starts and grows, with eventually profound consequences in the forms of treatment. It is related to the ending of adult stem cells in tumors, and its possible role in the birth and growth of them. We propose in this work a mathematical model that takes into account the presence of cells with the properties of (a) self renewal, (b) differentiation and (c) mobility, characteristics of stem cells. The model developed is a probabilistic cellular automaton with asynchronous update set in a grid. Each element of the grid may be empty or contain a tumor cell. There are two types of cells: the cells labeled as type 2 are those associated with cancer stem cells and those labeled as type 1 are differentiated cells only capable of reproducing. The reproduction rate of each cell is defined as a function of its neighborhood and its type. Different rates of reproduction have been used in the simulations, and type 2 cells may differentiate and some motility. Our simulation results show how the motility of cells 2 and the reproduction rates of both types of cells influence the morphological patterns of tumor. We have also investigated a possible phase transition that may be related to metastasis. This phase transition represents something of great biological interest because metastasis is the most important mechanism that leads to death. We will compare our results with experimental data from collaborators Nascimento TL et al [1] in UNIFESP-Escola Paulista de Medicina.

Células de Schwann (CS) cultivadas a partir de nervo ciático de ratos foram enxertadas, associadas ou não ao fator de crescimento derivado de células gliais (GDNF), em ratos adultos submetidos à transecção total da medula espinhal (T11). Análises do comportamento motor do 1º ao 3º mês pós-operatório e do crescimento de fibras nervosas imunorreativas no epicentro do enxerto ao término deste período mostraram: melhora temporal dos parâmetros motores superior nos grupos que receberam CS, que foi potencializada na presença do GDNF; aumento na quantidade de axônios nos grupos que receberam CS, efeito potencializado na presença do GDNF. Em outro experimento, as reatividades astrocitária, microglial e do fator neurotrófico bFGF astrocitário foram avaliados na medula espinhal parcialmente transectada de ratos, 1 semana e 3 meses após a cirurgia. Ativação astrocitária e microglial e maior intensidade de bFGF nas substâncias branca e cinzenta foram encontradas em toda a extensão da medula, fatos relacionados ao processo local de cicatrização e fenômenos tróficos e plásticos à distância. / Schwann cells (SC) obtained from rat sciatic nerve were grafted, in the presence or not of glial derived neurotrophic factor (GDNF), in adult rats submitted to a complete spinal cord transection at T11. Behavioral analysis from the 1st to the 3rd post operative month and of the immunoreactive fiber outgrowth in the graft epicenter at the end of this period showed: temporal improvements on motor parameters that were superior in the groups that received SC, being even higher in the presence of GDNF; increased amount of axonal fibers in the groups treated with SC, being even higher in the presence of GDNF. In another experiment, astrocytic and microglial activation and astrocytic neurotrophic factor bFGF expression were analyzed after a partial spinal cord transection, 1 week and 3 months after surgery. Astrocitic and microglial activation as well as a higher intensity of astrocitic bFGF in the white and gray matters were found in the entire spinal cord, what is related to the local scar formation and distant trophic and plastic phenomena.

Pubertal development is a time of growth and development in the brain, leading to high sensitivity during this period. Past research in our lab has shown that shipping female inbred and outbred mice during pubertal development alters their sensitivity to steroid hormones in adulthood, thus affecting sexual receptivity, cognition, depression-like behavior, and anxiety-like behavior. Here, we test the hypothesis that mice treated with lipopolysaccharide during pubertal development would have more active microglia, the brain's immune cells, after injection than mice treated with lipopolysaccharide in adulthood. No significant interactions were observed between treatment and age between any brain area measured, suggesting that pubertal development does not render the brain's immune system hypersensitive to environmental stressors.

Diabetic cardiomyopathy involves both forms of cardiac cell cell death such as apoptosis and necrosis. However, this remains unknown whether hyperglycemia induced apoptosis in the cell culture system is inhibited by parasympathetic nerve derived exosomes. We isolated parasympathetic and sympathetic nerves and derived exosomes. We developed hyperglycemia induced apoptosis in H9c2 cells. H9c2 cells were divided into 4 groups: 1) Control, 2) H9c2+ Glucose 100 mmol, 3) H9c2+ Glucose +parasympathetic-exo, 4) H9c2+ Glucose+sympathetic-exo. We determined cell proliferation and viability with MTT assay kit and apoptosis with TUNEL staining and cell death detection ELISA kit. Data was further confirmed with pro-apoptotic proteins caspase-3 and BAX and anti-apoptotic protein Bcl2. High glucose exposed H9c2 cells significantly reduced cell viability which is improved by parasympathetic-exo but not by sympathetic-exo. Increased apoptosis in hyperglycemia in H9c2 cells confirmed with TUNEL staining and cell death ELISA was significantly (p

The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle.

There is a need to develop novel and effective therapies targeting end-stage peripheral arterial occlusive disease (PAOD). Inducing collateral arteriogenesis is a possible novel treatment that produces a natural bypass for blood flow, a promising solution to the long-term issues seen with current treatments. Regenerative therapies, and progenitor cells specifically, have demonstrated great promise in repair after injury and disease. While most progenitor or stem cells have poor efficacy in this context, muscle progenitor cells, or myoblasts, have encouraging results. Myoblasts enhance arteriogenesis and secrete cytokines or chemokines that recruit monocytes to injury sites. Implanting these adherent cells in a hydrogel construct near natural bypasses in peripheral vasculature increases the size of nearby collateral vessels, pointing to a potentially effective cellular therapy for PAOD. However, many aspects of this novel therapy are yet to be characterized. Given the impact of inflammation and anoikis on transplanted cell survival, one such aspect is the beginning stages of the localized immune response to the implantation of the polymer/cell construct and the impact myoblasts have on this response. This research aims to elucidate the inflammatory response occurring within and locally around poly N-isopropylacrylamide (PNIPAM) polymer constructs post-implantation, focusing on the population and classification of cells within and on the surface of the construct. We hypothesize that the implantation will trigger a local foreign body response (FBR) and recruit multiple immune cell types, primarily macrophage lineage, to the injury site. Cell presence on the construct will be analyzed via confocal microscopy and cell populations within the construct will be typed and quantified via flow cytometry. The goal of this thesis is to characterize the impact of myoblasts in a PNIPAM construct implanted in-vivo in a mouse model on the immune response and use this characterization to help interpret how the construct is modulating the immune response and how we can adjust this response more favorably.

<p>The innate host response to virus infection is largely dominated by the production of type I interferons (IFNs). Fibroblasts, considered nonprofessional immune cells, respond to virus infection after recognition of viral components such as double-stranded (ds)RNA. The constitutively expressed transcription factor IFN regulatory factor 3 (IRF3) is rapidly activated and type I IFNs are produced. In the absence of IRF3, it was found that IFNs are still produced. This thesis identifies IRF9 as the transcription factor responsible for IFN production in the absence of IRF3 based on its ability to bind the murine (m)IFNβ promoter determined via oligonucleotide pull-down assays.</p> <p>In the absence of both IRF3 and IRF9, primary fibroblasts are deficient for IFN signalling. Surprisingly, significant inhibition of virus replication following dsRNA treatment of cells deficient for IRF3 and IFN signalling was recently observed with the large DNA virus herpes simplex virus type 1 (HSV-1) being more susceptible to inhibition than the small RNA virus vesicular stomatitis virus (VSV). As nitric oxide is known for its nonspecific antiviral effects against DNA viruses, involvement of this molecule in the antiviral response to HSV-1 in the absence of IRF3 and type I IFN induction and signalling was investigated. Here it is shown that in the absence of IRF3 and IFN, nitric oxide constitutes a major component of the innate response against HSV-1 in response to dsRNA in primary fibroblasts. In these cells, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IRF1 regulate inducible nitric oxide synthase (iNOS) expression, subsequently producing nitric oxide. As most viruses encode strategies to render their environment IRF3 and/or IFN deficient, it appears that IRF9 and nitric oxide serve as secondary responses to protect the host against viral infection. These data emphasize the importance and requirement of the host to employ multiple strategies to overcome infection.</p> / Master of Science (MSc)