Programa de exercício e promoção da actividade física após cirurgia por cancro da mama

RESUMO: Objectivo: O exercício tem sido identificado como um factor de promoção da qualidade de vida em mulheres submetidas a cirurgia por cancro de mama. Mas os níveis de actividade física tendem a reduzir após o diagnóstico de cancro de mama e a manterem-se baixos após fim dos tratamentos. O objectivo deste estudo é verificar se um programa de exercício físico supervisionado, associado a estratégias motivacionais em mulheres submetidas a cirurgia por cancro da mama e que mantêm a intervenção usual praticada, é mais efectivo no aumento dos níveis de actividade física, na redução do índice de massa corporal e na melhoria da qualidade de vida, do que o tratamento usualmente praticado. Metodologia: Trata-se de um estudo piloto experimental, aleatorizado e controlado. Cumpriram os critérios de inclusão no estudo 37 utentes submetidas a cirurgia por cancro de mama no Hospital Fernando Fonseca, Amadora, e submetidos à intervenção usual da fisioterapia. Foram aleatoriamente distribuídas pelos grupos experimental (n=19) e de controlo (n=18). O contacto telefónico foi feito por um elemento externo, cego em relação à distribuição dos sujeitos. Alguns elementos desistiram ou não puderam participar no estudo. O número de sujeitos final foi de 11 para o grupo experimental e de 10 para o grupo de controlo, idênticos na média de idades. Todos os sujeitos mantiveram a intervenção usual da fisioterapia. Os sujeitos do grupo experimental foram ainda submetidos ao programa de exercício e promoção da actividade física. Todo o grupo foi avaliado no início do programa, no final do mesmo (12 semanas) e após 6 meses de follow-up, nos seguintes indicadores: nível de actividade física, pelo International Physical Activity Questionnaire (IPAQ), índice de massa corporal e qualidade de vida, pelos questionários: European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) e Supplementary Questionnaire Breast Cancer Module (QLQ-BR23). Resultados: O programa implementado demonstrou-se efectivo no aumento dos níveis de actividade física (p=0,017) e na qualidade de vida associada ao estado funcional (p=0,016) e ao estado de saúde global auto-percepcionado (p=0,030) no final do programa. Foi ainda possível identificar resultados positivos noutras variáveis estudadas, como por exemplo a fadiga auto-reportada, mas que não se demonstraram estatisticamente significativos, facto que pode atribuir-se à reduzida dimensão da amostra. Conclusões: Apesar de algumas limitações, este estudo é de grande a importância para a comunidade científica preocupada com crescente problemática do cancro de mama. Mais uma vez se demonstra que o exercício físico é uma estratégia importante para a melhoria do estado de saúde de pacientes confrontados com doença. Esta comprovação é de grande interesse para os profissionais de saúde e particularmente para os fisioterapeutas dedicados à promoção da saúde nesta população.---------------------ABSTRACT: Purpose: Exercise has been identified as a quality of life promoting factor, amongst women after breast cancer surgery. But the physical activity levels reduce significantly after a diagnosis of breast cancer and remain low after treatment is completed. The aim of this study is to determine whether a supervised group exercise program allied to motivating strategies in women after breast cancer surgery in conjunction with standard treatment, is more effective in improving physical activity levels, reducing body mass index and promoting quality of life, than standard treatment on its own. Methods: This is a pilot randomized controlled trial. Thirty-seven women, submitted to breast cancer surgery in the Hospital Fernando Fonseca, Amadora, and submitted to standard physiotherapy intervention, completed the inclusion criteria. They were randomly allocated into intervention group (n=19) and control group (n=18). The telephone contact was done by an external element, blind to the subjects’ allocation. Some elements declined or could not participate. The final intervention group was n=11 and the control n=10, similar in age. All subjects maintained the standard physiotherapy intervention. The experimental group was submitted to the exercise and health promotion program. The complete group was evaluated in the beginning of the program, at the final (after 12 weeks) and after 6 months follow-up, using as outcomes: physical activity index with the international Physical Activity Questionnaire (IPAQ), the body mass index and quality of life using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and Supplementary Questionnaire Breast Cancer Module (QLQBR23). Results: The studied program demonstrated to be effective in improving the physical activity index (p=0,017) and quality of live related to physical functioning (p=0,016) and to global health status (p=0,030) at the end of the program. It was possible to find positive results in some other outcomes, such as fatigue, although the differences were not statistically significant. We believe that these results can be attributed to the small sample size. Conclusions: Although we can identify some methodological constrains, this is a very important study for scientific community working on the breast cancer subject. Once more, exercise was identified as an important strategy to improve global heath status in breast cancer patients. This represents an important contribution to the health professionals and mostly for physiotherapists working on health promotion subject.

Evidence for a role of sphingosine-1 phosphate in cardiovascular remodelling in Fabry disease.

AIMS: A hallmark of Fabry disease is the concomitant development of left-ventricular hypertrophy and arterial intima-media thickening, the pathogenesis of which is thought to be related to the presence of a plasmatic circulating growth-promoting factor. We therefore characterized the plasma of patients with Fabry disease in order to identify this factor. METHODS AND RESULTS: Using a classical biochemical strategy, we isolated and identified sphingosine-1 phosphate (S1P) as a proliferative factor present in the plasma of patients with Fabry disease. Plasma S1P levels were significantly higher in 17 patients with Fabry disease compared with 17 healthy controls (225 +/- 40 vs. 164 +/- 17 ng/mL; P = 0.005). There was a positive correlation between plasma S1P levels and both common carotid artery intima-media thickness and left-ventricular mass index (r(2) = 0.47; P = 0.006 and r(2) = 0.53; P = 0.0007, respectively). In an experimental model, mice treated with S1P developed cardiovascular remodelling similar to that observed in patients with Fabry disease. CONCLUSION: Sphingosine-1 phosphate participates in cardiovascular remodelling in Fabry disease. Our findings have implications for the treatment of cardiovascular involvement in Fabry disease.

Cardiac and vascular hypertrophy in Fabry disease: evidence for a new mechanism independent of blood pressure and glycosphingolipid deposition.

OBJECTIVE: Fabry disease is an X-linked disorder resulting from alpha-galactosidase A deficiency. The cardiovascular findings include left ventricular hypertrophy (LVH) and increased intima-media thickness of the common carotid artery (CCA IMT). The current study examined the possible correlation between these parameters. To corroborate these clinical findings in vitro, plasma from Fabry patients was tested for possible proliferative effect on rat vascular smooth muscle cells (vascular smooth muscle cell [VSMC]) and mouse neonatal cardiomyocytes. METHODS AND RESULTS: Thirty male and 38 female patients were enrolled. LVH was found in 60% of men and 39% of women. Increased CCA IMT was equally present in males and females. There was a strong positive correlation between LV mass and CCA IMT (r2=0.27; P<0.0001). VSMC and neonatal cardiomyocyte proliferative response in vitro correlated with CCA IMT (r2=0.39; P<0.0004) and LV mass index (r2=0.19; P=0.028), respectively. CONCLUSIONS: LVH and CCA IMT occur concomitantly in Fabry suggesting common pathogenesis. The underlying cause may be a circulating growth-promoting factor whose presence has been confirmed in vitro.

Intracranial Nimodipine Implant: Feasibility and Implications for the Treatment of Subarachnoid Hemorrhage – A Pre-Clinical Study

Intracranial aneurysmal subarachnoid hemorrhage (aSAH) is a life-threatening condition requiring immediate neurocritical care. A ruptured aneurysm must be isolated from arterial circulation to prevent rebleeding. Open surgical clipping of the neck of the aneurysm or intra-arterial filling of the aneurysm sack with platinum coils are major treatment strategies in an acute phase. About 40% of the patients suffering from aSAH die within a year of the bleeding despite the intensive treatment. After aSAH, the patient may develop a serious complication called vasospasm. Major risk for the vasospasm takes place at days 5–14 after the primary bleeding. In vasospasm, cerebral arteries contract uncontrollably causing brain ischemia that may lead to death. Nimodipine (NDP) is used to treat of vasospasm and it is administrated intravenously or orally every four hours for 21 days. NDP treatment has been scientifically proven to improve patients’ clinical outcome. The therapeutic effect of L-type calcium channel blocker NDP is due to the ability to dilate cerebral arteries. In addition to vasodilatation, recent research has shown the pleiotropic effect of NDP such as inhibition of neuronal apoptosis and inhibition of microthrombi formation. Indeed, NDP inhibits cortical spreading ischemia. Knowledge of the pathophysiology of the vasospasm has evolved in recent years to a complex entity of early brain injury, secondary injuries and cortical spreading ischemia, instead of being pure intracranial vessel spasm. High NDP levels are beneficial since they protect neurons and inhibit the cortical spreading ischemia. One of the drawbacks of the intravenous or oral administration of NPD is systemic hypotension, which is harmful particularly when the brain is injured. Maximizing the beneficial effects and avoiding systemic hypotension of NDP, we developed a sustained release biodegradable NDP implant that was surgically positioned in the basal cistern of animal models (dog and pig). Higher concentrations were achieved locally and lower concentrations systemically. Using this treatment approach in humans, it may be possible to reduce incidence of harmful hypotension and potentiate beneficial effects of NDP on neurons. Intracellular calcium regulation has a pivotal role in brain plasticity. NDP blocks L-type calcium channels in neurons, substantially decreasing intracellular calcium levels. Thus, we were interested in how NDP affects brain plasticity and tested the hypothesis in a mouse model. We found that NDP activates Brain-derived neurotrophic factor (BDNF) receptor TrkB and its downstream signaling in a reminiscent of antidepressant drugs. In contrast to antidepressant drugs, NDP activates Akt, a major survival-promoting factor. Our group’s previous findings demonstrate that long-term antidepressant treatment reactivates developmental-type of plasticity mechanisms in the adult brain, which allows the remodeling of neuronal networks if combined with appropriate rehabilitation. It seems that NDP has antidepressant-like properties and it is able to induce neuronal plasticity. In general, drug induced neuronal plasticity has a huge potential in neurorehabilitation and more studies are warranted.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.

Synthese und präklinische Evaluation von neuen antioxidativen Substanzen zur Neuroprotektion

Oxidativer Stress ist seit über 25 Jahren als ein Charakteristikum vieler pathologischer Prozesse bekannt. Helmut Sies beschrieb bereits in den 1980er Jahren oxidativen Stress als Störung in der prooxidativ – antioxidativen Balance zugunsten der prooxidativen Seite, wodurch es potentiell zu Schäden in verschiedenen Geweben kommt. Oxidativer Stress tritt sowohl bei neurodegenerativen Erkrankungen wie Morbus Alzheimer, Morbus Parkinson und zerebraler Ischämie, bei peripheren Erkrankungen wie Arteriosklerose, als auch beim Alterungsprozess per se auf und wird als Ursache oder zumindest als ein krankheitsfördernder Faktor diskutiert. Die in in vitro-Experimenten als vielversprechend antioxidativ getesteten Substanzen (meist phenolhaltig) ergaben in mehreren klinischen Studien keinen signifikanten Vorteil. Um die Ursachen dieser Ergebnisse näher zu analysieren, wurde in der vorliegenden Arbeit auf Basis des cytoprotektiven Phenothiazins, einem aromatischen trizyklischen Amin, der Einfluss von verschiedenen Substituenten im Hinblick auf Lipophilie, Radikalstabilisierung und Löslichkeit des Moleküls chemisch vorhergesagt. Anhand dieser in silicio Struktur-Wirkungs-Beziehung wurden anschließend neue Modellsubstanzen synthetisiert, welche sich systematisch in den drei zuvor genannten Parametern unterschieden. Dies wurde durch Substitution von unterschiedlich langen Fettsäureketten, von löslichkeitsbeeinflussenden funktionellen Gruppen, oder durch Anellierung zusätzlicher aromatischer Ringe erreicht. In den folgenden Versuchen zu antioxidativer Kapazität, zellulärem Überleben, Lipidperoxidation und Proteinoxidation zeigte sich, dass mit gesteigerter Stabilität der korrespondierenden Radikale und mit wachsender Lipophilie die antioxidativ cytoprotektive Aktivität der neuen Derivate bis zu einer gewissen Grenze (logP ≈ 7) signifikant zunahm; über diesen Wert hinaus sank die Effektivität wieder ab. Benzanellierte Phenothiazine entwickelten mit EC50-Werten von ungefähr 8-10 nM die höchste mittlere effektive Wirkkonzentration in oxidativ geschädigten, klonalen hippocampalen Neuronen (HT-22 Zellen). Dies entspricht einer etwa 20-fachen Verbesserung gegenüber α-Tocopherol, welches bisher als bestes natürliches lipophiles Antioxidans angesehen wurde. Im Vergleich zu Phenothiazin erreichen die neuen Antioxidantien immerhin eine höhere Effektivität um den Faktor 4. Folglich sind es sowohl Aspekte der Löslichkeit und der Distribution, welche die Potenz der gegenwärtigen Antioxidantien limitieren als auch Aspekte der Radikalstabilisierung, die Einfluss auf die primäre Wirksamkeit nehmen. Dieses Wissen sollte beim zukünftigen Design neuer, antioxidativ potenter Moleküle im Hinblick auf ihren langfristigen Einsatz bei neurodegenerativen Erkrankungen von Nutzen sein.

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

The effect of v-{\it mos\/} expression on the regulation of the {\it fos\/} promoter in 490N3T cells

The v-mos oncogene acquired by Moloney murine sarcoma viruses by recombination with the c-mos proto-oncogene encodes a 37kD cytoplasmic serine/threonine protein kinase which can phosphorylate tubulin and vimentin, as well as the cyclin B component of the maturation promotion factor complex (MPF). Our earliest experiments asked whether the v-mos protein could activate the transcription of transin. Since the transcription of transin was known to be mediated by both fos-dependent and fos-independent pathways, it seemed possible that the induction of transin transcription by v-mos might be mediated by p55$\sp{\rm c-}\sp{fos}$. Surprisingly, when we examined the effect of v-mos on the fos promoter, we observed a significant inhibition of transcription in 49ON3T cells, a subclone of N1H3T3 mouse fibroblasts.^ In this thesis we show that in mouse 49ON3T cells, transcription from the fos promoter is up to 10-fold repressed in the presence of v-mos. Moreover, in this cell line several other transforming constructs (v-ras, v-src, neu) also cause repression of the fos promoter. Interestingly, nontransforming oncogenes (e.g. myc) do not repress fos transcription. The repressive effect was lost in v-mos mutants lacking in ATP-binding or kinase domain, arguing that the effect on fos transcription was mediated by v-mos transforming kinase activity. As mos is a cytoplasmic protein, it was assumed that transcriptional repression was mediated by conversion of a transcriptional regulator to a repressor by mos-induced phosphorylation. As a first approximation of the identity of this factor, we mapped the position of the mos effect on the fos promoter using reporter (CAT) constructs. We found that repression was mediated by regions $-$221 to $-$106 and $-$122 to $-$65 relative to the fos transcriptional start site, both of which regions regulate baseline fos transcription. There are direct repeats containing E2F transcriptional activator/repressor recognition motifs in these regions which bind similar nuclear proteins independently of v-mos presence or absence. Our data show that the contribution of the direct repeat to baseline fos transcription is mediated by these E2F sites with perhaps some contribution from the overlapping retinoblastoma control element (RCE). We have shown that there is a separate DNA protein interaction in the direct repeat which is more pronounced in the presence of v-mos. The recognition site for this protein, which we speculate mediates the mos-induced downregulation of fos transcription, overlaps but is distinct from the E2F and RCE binding sites. (Abstract shortened by UMI.) ^

4HPR-X radiation mechanisms of interactions in non-small cell lung cancer (NSCLC) cells in vitro

Lung cancer is the leading cause of cancer death. However, poor survival using conventional therapies fuel the search for more rational interventions. The objective of this study was to design and implement a 4HPR-radiation interaction model in NSCLC, employing a traditional clinical modality (radiation), a relatively new, therapeutically unexplored agent (4HPR) and rationally combining them based on molecular mechanistic findings pertaining to their interactions. To test the hypothesis that 4HPR sensitizes cells to radiation-induced cell death via G2+M accumulation, we designed a working model consisting of H522 adenocarcinoma cells (p53, K-ras mutated) derived from an NSCLC patient; 4HPR at concentrations up to 10 μM; and X radiation up to 6 Gy generated by a patient-dedicated Phillips RT-250 X ray unit at 250 KV, 15 mA, 1.85 Gy/min. We found that 4HPR produced time- and dose-dependent morphological changes, growth inhibition, and DNA damage-inducing enhancement of reactive oxygen species. A transient G2+M accumulation of cells maximal at 24 h of continuous 4HPR exposure was used for irradiation time scheduling. Our data demonstrated enhanced cell death (both apoptotic and necrotic) in irradiated cells pre-treated with 4HPR versus those with either stressor alone. 4HPR's effect of increased NSCLC cells' radioresponse was confirmed by clonogenic assay. To explore these practical findings from a molecular mechanistic perspective, we further investigated and showed that levels of cyclin B1 and p34cdc2 kinase—both components of the mitosis promoting factor (MPF) regulating the G2/M transition—did not change following 4HPR treatment. Likewise, cdc25C phosphatase was not altered. However, enhanced p34cdc2 phosphorylation on its Thr14Tyr15 residues—indicative of its inactivation and increased expression of MPF negative regulators chk1 and wee1 kinases—were supportive of explaining 4HPR-treated cells' accumulation. Hence, p34cdc2 phosphorylation, chk1, and wee1 warrant further evaluation as potential molecular targets for 4HPR-X radiation combination. In summary, we (1) demonstrated that 4HPR not only induces cell death by itself, but also increases NSCLC cells' subsequent radioresponse, indicative of potential clinical applicability, and (2) for the first time, shed light on deciphering 4HPR-X radiation molecular mechanisms of interaction, including the finding of 4HPR's role as a p34cdc2 inactivator via Thr14Tyr15 phosphorylation. ^

Plo1 Kinase Recruitment to the Spindle Pole Body and Its Role in Cell Division in Schizosaccharomyces pombe

Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis (Ohkura et al., 1995; Bahler et al., 1998). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 (Hudson et al., 1990). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression of plo1+ activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation.

A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1 beta maturation

Background: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1 beta and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE(2) and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1 beta and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)alpha and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1 beta and PGE(2) did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-alpha and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1 beta was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE(2) production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1 beta maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE(2) production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.