Development of multifunctional lipid nanocapsules for the co-delivery of paclitaxel and CpG-ODN in the treatment of glioblastoma

International audience

A Novel integration approach : perturbation studies in vitro using PDK1 inhibitors in glioblastoma multiforme

Os objetivos a atingir em 2020 no que respeita ao processo de investigação e desenvolvimento de medicamentos estão claramente focados na redução em termos temporais na investigação pré-clínica e clínica e na diminuição da taxa de atrito entre as novas moléculas. De forma a atingir estes objetivos, um novo conceito tem sido desenvolvido e aplicado a este complexo e moroso processo, este é a Farmacologia Quantitativa e de Sistemas. Além disso, esta abordagem inovadora pode ser crucial para o tratamento de determinados tipos de tumores cerebrais letais – Glioblastoma Multiforme (GBM) – que permanecem um desafio terapêutico, e por tanto, uma doença com um destino fatal para os doentes. Por estas razões, esta dissertação de mestrado apresenta uma especial relevância, tendo por objetivos avaliar o potencial impacto e importância biológica da variação de parâmetros farmacológicos, para além da potência, no contexto da resposta celular ao fármaco, pela avaliação da perturbação induzida em células do GBM por inibidores do PDK1 e pela realização de uma caracterização multiparamêtrica dose-resposta destas novas moléculas. A presente dissertação assume em Portugal a vanguarda na área da Farmacologia Quantitativa e de Sistemas aplicada ao processo de investigação e desenvolvimento de medicamentos. Em última estância, esta dissertação poderá contribuir para uma melhor previsão dos fármacos durante este processo, significando assim possíveis vantagens para os utentes, indústrias farmacêuticas, institutos de investigação, governo e institutos superiores.

Silenciamento do canal de potássio Eag1 potencializa efeitos da temozolomida em células U-87 MG de glioblastoma multiforme

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.

Business-IT Alignment in PSS Value Networks: A Capability-Based Framework

Part 8: Business Strategies Alignment

Hierarchical spatial segregation of two Mediterranean vole species: the role of patch‑network structure and matrix composition

According to ecological theory, the coexistence of competitors in patchy environments may be facilitated by hierarchical spatial segregation along axes of environmental variation, but empirical evidence is limited. Cabrera and water voles show a metapopulation-like structure in Mediterranean farmland, where they are known to segregate along space, habitat, and time axes within habitat patches. Here, we assess whether segregation also occurs among and within landscapes, and how this is influenced by patch-network and matrix composition. We surveyed 75 landscapes, each covering 78 ha, where we mapped all habitat patches potentially suitable for Cabrera and water voles, and the area effectively occupied by each species (extent of occupancy). The relatively large water vole tended to be the sole occupant of landscapes with high habitat amount but relatively low patch density (i.e., with a few large patches), and with a predominantly agricultural matrix, whereas landscapes with high patch density (i.e.,many small patches) and low agricultural cover, tended to be occupied exclusively by the small Cabrera vole. The two species tended to co-occur in landscapes with intermediate patch-network and matrix characteristics, though their extents of occurrence were negatively correlated after controlling for environmental effects. In combination with our previous studies on the Cabrera-water vole system, these findings illustrated empirically the occurrence of hierarchical spatial segregation, ranging from withinpatches to among-landscapes. Overall, our study suggests that recognizing the hierarchical nature of spatial segregation patterns and their major environmental drivers should enhance our understanding of species coexistence in patchy environments.

Isogeometric analysis of large-deformation thin shells using RHT-splines for multiple-patch coupling

We present an isogeometric thin shell formulation for multi-patches based on rational splines over hierarchical T-meshes (RHT-splines). Nitsche’s method is employed to efficiently couple the patches. The RHT-splines have the advantages of allowing a computationally feasible local refine- ment, are free from linear independence, possess high order continuity and satisfy the partition of unity and non-negativity, properties. In addition, C 1 continuity of the RHT-splines obviates to use of rotational degrees of freedom. The good performance of the present method is demonstrated by a number of numerical examples.

The impact of phosphoinositide metabolic enzymes in glioblastoma: a tale of phosphoinositide-specific phospholipase C PLCβ1 and 5-phosphatase SKIP

Background: Glioblastoma multiforme (GBM) is one of the deadliest and most aggressive form of primary brain tumor. Unfortunately, current GBM treatment therapies are not effective in treating GBM patients. They usually experience very poor prognosis with a median survival of approximately 12 months. Only 3-5% survive up to 3 years or more. A large-scale gene profile study revealed that several genes involved in essential cellular processes are altered in GBM, thus, explaining why existing therapies are not effective. The survival of GBM patients depends on understanding the molecular and key signaling events associated with these altered physiological processes in GBM. Phosphoinositides (PI) form just a tiny fraction of the total lipid content in humans, however they are implicated in almost all essential biological processes, such as acting as second messengers in spatio-temporal regulation of cell signaling, cytoskeletal reorganization, cell adhesion, migration, apoptosis, vesicular trafficking, differentiation, cell cycle and post-translational modifications. Interestingly, these essential processes are altered in GBM. More importantly, incoming reports have associated PI metabolism, which is mediated by several PI phosphatases such as SKIP, lipases such as PLCβ1, and other kinases, to regulate GBM associated cellular processes. Even as PLCβ1 and SKIP are involved in regulating aberrant cellular processes in several other cancers, very few studies, of which majority are in-silico-based, have focused on the impact of PLCβ1 and SKIP in GBM. Hence, it is important to employ clinical, in vitro, and in vivo GBM models to define the actual impact of PLCβ1 and SKIP in GBM. AIM: Since studies of PLCβ1 and SKIP in GBM are limited, this study aimed at determining the pathological impact of PI metabolic enzymes, PLCB1 and SKIP, in GBM patient samples, GBM cell line models, and xenograft models for SKIP. Results: For the first time, this study confirmed through qPCR that PLCβ1 gene expression is lower in human GBM patient samples. Moreover, PLCβ1 gene expression inversely correlates with pathological grades of glioma; it decreases as glioma grades increases or worsens. Silencing PLCβ1 in U87MG GBM cells produces a dual impact in GBM by participating in both pro-tumoral and anti-tumoral roles. PLCβ1 knockdown cells were observed to have more migratory abilities, increased cell to extracellular matrix (ECM) adhesion, transition from epithelial phenotype to mesenchymal phenotype through the upregulation of EMT transcription factors Twist1 and Slug, and mesenchymal marker, vimentin. On the other hand, p-Akt and p-mTOR protein expression were downregulated in PLCβ1 knockdown cells. Thus, the oncogenic pathway PI3K/Akt/mTOR pathway is inhibited during PLCβ1 knockdown. Consistently, cell viability in PLCβ1 knockdown cells were significantly decreased compared to controls. As for SKIP, this study demonstrated that about 48% of SKIP colocalizes with nuclear PtdIns(4,5)P2 to nuclear speckles and that SKIP knockdown alters nuclear PtdIns(4,5)P2 in a cell-type dependent manner. In addition, SKIP silencing increased tumor volume and weight in xenografts than controls by reducing apoptosis and increasing viability. All in all, these data confirm that PLCβ1 and SKIP are involved in GBM pathology and a complete understanding of their roles in GBM may be beneficial.

Evaluation of the theranostic use of miRNAs in correlation to the radiopharmaceutical 64CuCl2 in glioblastoma

B:Glioblastoma multiforme(GBM) is one of the most prevalent and aggressive malignant primary brain tumors in adult patients. 64CuCl2 is an innovative radiopharmaceutical investigated as theranostic agent in GBM patients. The therapeutic scheme is still under evaluation, therefore the research focused on the possibility of radioresistance development. The actors responsible for modulating radioresistance could be miRNAs, thus their potential use was investigated both in radioresistant cell lines and in GBM patients plasma samples. M:Radioresistant cell lines were generated by exposing U87MG, U373MG lines to increasing doses of radiation for 32 weeks. Cell membrane permeability alterations and DNA damage were assessed to characterize the lines. Moreover, 64Cu cell incorporation and subcellular distribution were investigated measuring gamma-radiation emission. miRNA expression was evaluated: in parental and radioresistant cell lines, both in cell pellet and media exosomes; in plasma samples of GBM patients using TaqMan Array MicroRNA Cards. R:Radioresistant lines exhibited reduction in membrane permeability and in DNA DSBs indicating the capability to skip the drug killing effect. Cell uptake assays showed internalization of 64Cu both in the sensitive and radioresistant lines. Radioresistant lines showed a different miRNA expression profile compared to the parental lines. 5 miRNAs were selected as possible biomarkers of response to treatment (miR-339-3p, miR-133b, miR-103a-3p, miR-32-5p, miR-335-5p) and 6 miRNAs as possible predictive biomarkers of response to treatment (let-7e-5p, miR-15a-5p, miR-29c-3p, miR-495, miR-146b-5p, miR-199a-5p). miR-32-5p was selected as possible molecule to be used to restore 64CuCl2 responsiveness in the radioresistant cell lines. C: This is the first study describing the development and characterization of 64CuCl2 radioresistant cell lines useful to implement the approach for dosimetric analysis to avoid radioresistance uprising. miRNAs could bring to a better understanding of 64CuCl2 treatment, becoming a useful tool both in detection of treatment response and both as molecule that could restore responsiveness to 64CuCl2 treatment.

The role of Phospholipases as novel potential modulators of aggressiveness in Glioblastoma

Glioblastoma is the most malignant brain tumor in adults. The standard care of treatment is tumor resection, radiotherapy, and chemotherapy. Despite these invasive therapeutic approaches, glioblastoma prognosis remains unchanged. Therefore, a better understanding of the molecular mechanisms driving tumor transformation is needed to uncover novel therapeutic strategies. Several studies have shown the significance of lipid signaling and phospholipases (PLCs) in the regulation of different mechanisms in the central nervous system as well as in glioblastoma pathogenesis. This work suggests a potential role of PLCβ1 in the maintenance of a less aggressive phenotype of the tumor. Indeed, it was demonstrated that PLCβ1 gene was relatively less expressed in glioblastoma patients compared to their healthy/low-grade counterparts. Moreover, PLCβ1 silencing, in both immortalized and primary cell lines, led to increased cell migration, invasion, proliferation, cell survival and induced the upregulation of mesenchymal markers and metalloproteinases. Moreover, PLCγ1, another abundant PLC isoform in the brain, has been identified as a key element for the aggressiveness of glioblastoma. Data collected on patients’ biopsies and engineered cell models, suggested a strong correlation between PLCγ1 expression level and the acquisition of a more aggressive tumor phenotype. Finally, this trend was further probed using patient-derived glioblastoma stem cells (GSCs), which are a specific tumor population that drives aggressiveness, resistance, and recurrence in glioblastoma. GSCs analysis on the transcriptomic profiles confirmed that PLCγ1 downregulation modulated positively the activation of pathways that negatively regulate cell motility and migration and led to a decreased expression of genes involved in cancer development and progression. Taken together, these data highlight the importance of further investigating phospholipases as potential prognostic biomarkers and targets in the development of new therapeutic strategies for glioblastoma.

“Clinical and biological role of adjuvant dendritic cells vaccination in newly glioblastoma patients”

Background. Glioblastoma (GBM) is the most common primary tumor of central nervous system and it has a poor prognosis. Standard first line treatment, which includes surgery followed by adjuvant radio-chemotherapy,produces only modest benefits to survival. The interest for immunotherapy in this field derives from the development of new drugs and effective therapies as immune-check points inhibitors, adoptive T-cell approaches or dendritic cell (DC) based vaccines or a combinations of these. GBM is described as a typical “immune-deserted” cancer exhibiting a number of systemic and environmental immunosuppressive factors. Considering the role of microenvironment, and above all the lower tumor load and depletion of immunosuppressive cells in GBM, our hypothesis is that DC vaccine may induce an immune response. Main aims and study design. The main aim of this project is to study the role of immune system in GBM, including identification of potential prognostic and predictive markers of outcome and response to dendritic cell vaccine. Firstly, we performed a retrospective analysis on blood samples. Then, we analyzed the immuno-component in tissues samples of enrolled patients; and compared that with blood results. Then, the last part of the project is based on a prospective clinical trial on patients enrolled in DC-based vaccination produced at IRST Cell Factory and actually used for patients with melanoma and other tumors. The enrollment is still ongoing. Expected results. The project will i) develop an immune-panel of prognostic and predictive markers to help clinicians to improve the therapeutic strategy for GBM patients; ii) provide preliminary results on the effectiveness of immunotherapy on GBM patients.

Synthesis of Na-PSS geopolymers and analysis of the H2O/Na2O molar ratio influence over their mechanical properties

Geopolymers are solid aluminosilicate material made by mixing an activating solution and a solid precursor. This work studied the mechanisms of synthesis of metakaolin-based geopolymers and the influence of water content, described by the molar ratio H2O/Na2O, on the final product. The samples were tested using a Uniaxial Compressive Test (UCT) to define their compressive resistance. Two geopolymers series were synthetized and let them rest for 7- days and 28-days, each of them composed by six different sets. 7-day rest series showed that water addition had no relevant effect over its resistance while the 28-day rest series almost doubled the compressive resistance, although those with the highest H2O/Na2O molar ratio showed instead a drastic reduction. Two other series were synthesized by adding silt aggregate, a waste material obtained in the production of aggregate for concrete, corresponding to 10wt% and 20wt%of the metakaolin used. After 28 days of aging, these samples were tested via UCT to measure the variation of the compressive resistance after the silt addition. The aggregate has disruptive effects over the compressive resistance, but the 20wt% samples achieved a higher compressive resistance. Samples with highest and lowest compressive resistance have been chosen to carry out an XRD analysis. In all the samples it has been recognized the presence of Anatase (TiO2), a titanium oxide found in the metakaolin and Thermonatrite, a hydrated sodium carbonate [Na2CO3 • (H2O)]. Scanning Electron Microscopy was carried out on the samples with the highest compressive resistance and showed that the samples with lower water content developed a homogeneous geopolymeric texture, while those with higher water content showed instead a spongy-like texture and a higher air or pore solution bubbles presence. Silt/geopolymer composites showed a fracture system developing across the interstitial transition zone between the geopolymer matrix and the aggregate particle.

Thermodynamic Study Of The Micellization Of Zwitterionic Surfactants And Their Interaction With Polymers In Water By Isothermal Titration Calorimetry.

The micellization of a homologous series of zwitterionic surfactants, a group of sulfobetaines, was studied using isothermal titration calorimetry (ITC) in the temperature range from 15 to 65 °C. The increase in both temperature and the alkyl chain length leads to more negative values of ΔGmic(0) , favoring the micellization. The entropic term (ΔSmic(0)) is predominant at lower temperatures, and above ca. 55-65 °C, the enthalpic term (ΔHmic(0)) becomes prevalent, figuring a jointly driven process as the temperature increases. The interaction of these sulfobetaines with different polymers was also studied by ITC. Among the polymers studied, only two induced the formation of micellar aggregates at lower surfactant concentration: poly(acrylic acid), PAA, probably due to the formation of hydrogen bonds between the carboxylic group of the polymer and the sulfonate group of the surfactant, and poly(sodium 4-styrenesulfonate), PSS, probably due to the incorporation of the hydrophobic styrene group into the micelles. The prevalence of the hydrophobic and not the electrostatic contributions to the interaction between sulfobetaine and PSS was confirmed by an increased interaction enthalpy in the presence of electrolytes (NaCl) and by the observation of a significant temperature dependence, the latter consistent with the proposed removal of hydrophobic groups from water.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Epicardial And Pericardial Fat In Type 2 Diabetes: Favourable Effects Of Biliopancreatic Diversion.

Ectopic fat is often identified in obese subjects who are susceptible to the development of type 2 diabetes mellitus (T2DM). The ectopic fat favours the decrease in insulin sensitivity (IS) and adiponectin levels. We aimed to evaluate the effect of biliopancreatic diversion (BPD) on the accumulation of ectopic fat, adiponectin levels and IS in obese with T2DM. A nonrandomised controlled study was performed on sixty-eight women: 19 lean-control (23.0 ± 2.2 kg/m(2)) and 18 obese-control (35.0 ± 4.8 kg/m(2)) with normal glucose tolerance and 31 obese with T2DM (36.3 ± 3.7 kg/m(2)). Of the 31 diabetic women, 20 underwent BPD and were reassessed 1 month and 12 months after surgery. The subcutaneous adipose tissue, visceral adipose tissue, epicardial adipose tissue and pericardial adipose tissue were evaluated by ultrasonography. The IS was assessed by a hyperglycaemic clamp, applying the minimal model of glucose. One month after surgery, there was a reduction in visceral and subcutaneous adipose tissues, whereas epicardial and pericardial adipose tissues exhibited significant reduction at the 12-month assessment (p < 0.01). Adiponectin levels and IS were normalised 1 month after surgery, resembling lean-control values and elevated above the obese-control values (p < 0.01). After 12 months, the improvement in IS and adiponectin was maintained, and 17 of the 20 operated patients exhibited fasting glucose and glycated haemoglobin within the normal range. After BPD, positive physiological adaptations occurred in grade I and II obese patients with T2DM. These adaptations relate to the restoration of IS and decreased adiposopathy and explain the acute (1 month) and chronic (12 months) improvements in the glycaemic control.

Biópsia hepática por laparotomia paracostal em bovinos e búfalos

A técnica de biópsia hepática em ruminantes tem importante valor no diagnóstico clínico de doenças tóxicas e metabólicas, em especial nos desequilíbrios minerais. As técnicas mais comumente utilizadas restringem análises devido ao limitado volume de tecido obtido. No presente trabalho, avaliou-se o uso de uma técnica de biópsia hepática por laparotomia paracostal em bovinos e búfalos. Foram utilizados 10 bovinos e 10 búfalos hígidos. Os animais foram mantidos em estação, sedados com xilazina e infiltrados localmente com lidocaína e epinefrina. O acesso à cavidade abdominal foi realizado por meio de uma incisão dorso-ventral de 15cm no flanco direito, iniciada ventralmente (cerca de 4-5cm) ao processo transverso da 2a ou 3a vértebra lombar e situada caudalmente (cerca de 4cm) e paralelamente à 13a costela, obtendo-se visualização do fígado. Foi então realizado pinçamento do bordo caudal do órgão com pinça Doyen para remoção de fragmento hepático (2 a 4g). Procedeu-se o fechamento da cavidade abdominal como de rotina. Foram analisados os parâmetros bioquímicos e hematológicos antes do procedimento (tempo zero) e após 24 horas, 48 horas, 5 dias e 10 dias após a biópsia. Todas as variáveis bioquímicas estudadas retornaram aos valores basais 5 e 10 dias após o procedimento nos bovinos e búfalos, respectivamente. O tempo médio de cirurgia por animal foi de 25 minutos. A biópsia hepática por laparotomia paracostal demonstrou ser uma técnica eficaz e de baixo risco à saúde dos animais, permitindo a coleta de suficiente quantidade de tecido hepática para realização de múltiplas análises.