A utilização de novas rotas de síntese de materiais nanoestruturados tem levado a obtenção de materiais apresentando formas anisotrópicas, que podem dar origem a novas propriedades e aplicações. Nesse contexto, este trabalho tem por objetivo a caracterização estrutural e magnética de Óxidos Magnéticos Diluídos nanoestruturados a base de ZnO dopados com Co (ZnO:Co) preparados a partir de moagem mecânica. Recentemente, grande atenção se tem dado na utilização do ZnO como OMDs para aplicação em Spintrônica. O objetivo é obtermos nanocristais de óxidos magnéticos sem a formação de fases secundárias e que apresentem ordenamento ferromagnético com temperatura de Curie acima da temperatura ambiente. A incorporação do Co na estrutura de ZnO foi avaliada através de analises microestruturais e químicas utilizando as técnicas de Difração de Raios X, Microscopia Eletrônica de Varredura, Espectroscopia Dispersiva de Energia, Espectroscopia Raman, X-Ray Absorption Near Edge Structure. A caracterização magnética se fez por magnetometria SQUID. Conjugando os resultados dessas diferentes técnicas, confirmou-se a substituição do Zn pelo Co na estrutura do ZnO, sem alteração da mesma. Observamos que o processo de moagem mecânica empregado possui um limite de eficiência que nos permitiu atingir diâmetros medianos dos grãos da ordem de 100 nm. Observamos também que o processo de moagem mecânica introduz defeitos estruturais no sistema ZnO:Co em correlação com a eficiência de moagem. A magnetometria SQUID revelou a coexistência de uma fase paramagnética e uma fase ferromagnética. As análises da fase ferromagnética demonstram claramente sua origem intrínseca e a sua associação direta à presença de defeitos estruturais introduzidos pela moagem mecânica / The synthesis of nanostructured material trough new routes has conducted to materials with anisotropic shapes that would lead to new properties and applications. In this context, the aim of this work is to characterize structural and magnetically nanostructured Dilute Magnetic Oxides based on the ZnO matrix and doped with Co prepared by the mechanical milling process. The goal is to obtain nanocrystals of magnetic oxides without the observation of undesired secondary phases that would present a ferromagnetic order with Curie temperature above the room temperature. In order to evaluate the incorporation of Co in the ZnO host structure, it was performed a careful microstructural and chemical characterization using the following techniques: X-Ray Diffractometry, Scanning Electron Microscope, Energy Dispersive X-Ray Spectrometry, Raman Spectroscopy and X-Ray Absorption Near Edge Structure. The magnetic characterization was performed by SQUID magnetometry. Conjugating the results of different techniques, confirmed the replacement of Zn by Co in the structure of ZnO without significant distortion. We observed that the mechanical milling has an efficiency limit, which led us to reach median diameters for the milled grains of the order of 100 nm. We also observed that the mechanical milling process introduces structural defects in the ZnO:Co system in correlation with the mechanical milling efficiency. The SQUID magnetometry reveled the coexistence of paramagnetic and ferromagnetic phases. The analyses of the ferromagnetic phase showed clearly its intrinsic origin associated directly to the presence of the structural defects introduced by the mechanical milling. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Foram sintetizadas amostras de ZnO puro e ZnO dopadas com Mn (ZnO:Mn) na forma de pós com tamanho de partícula/grão controlado pelo método dos precursores poliméricos. Os parâmetros do método foram manipulados visando obter amostras com diferentes tamanhos de partículas (entre 60 nm a 800 nm). Tanto o ZnO quanto o ZnO dopado com metais de transição (MT), que é um Óxido Semicondutor Magnético Diluído (OSMD), possuem grande potencial em aplicações na spintrônica. A dependência das propriedades magnéticas com o tamanho de partícula é um aspecto relacionado com a compreensão do comportamento ferromagnético deste tipo de material e ele é investigado neste trabalho. As amostras dopadas com Mn foram produzidas nas concentrações de 0,5, 1% e 3% de Mn (% em mol). A identificação das fases cristalinas das amostras e uma estimativa preliminar do tamanho de grão foram realizadas por difração de raios X por pó (DRXP). Medidas de microscopia eletrônica de varredura (MEV) complementaram o estudo da morfologia e tamanho das partículas. Estudo estrutural complementar por espalhamento Raman foi feito nas amostras. A análise de Fotoluminescência prevê informações a respeito da formação de defeitos estruturais e medidas de propriedades magnéticas foram feitas através da técnica de magnetometria SQUID. / We have prepared samples of ZnO pure and Mn-doped Zno (ZnO:Mn) in the form of powders with particle size/grain controlled by the polymeric precursor method. The method parameters were manipulated to obtain samples having different particle sizes (from 60 nm to 800 nm). Both ZnO pure as doped ZnO with transition metal (TM), which is a diluted magnetic oxides (DMOs) has great potential for applications in spintronics. The dependence of the magnetic properties with the particle size is an important aspect to understand the behavior of the ferromagnetic type of material and it is clarified in this work. The samples with Co were prepared with molar concentrations of 0,5, 1% and 3% of Mn (% in mol). The crystal structures of the samples were characterized using X-ray diffraction (XRD) and RAMAN scattering spectroscopy. The microstructure and composition distributions were characterized by scattering electron microscopy (SEM). The photoluminescence analysis clarifies the issues regarding the formation of structural defects and magnetic properties measurements were performed using a superconducting quantum interference device (SQUID) magnetometer. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Orientador: Clélia Akiko Hiruma Lima / Coorientador: Lúcia Regina Machado da Rocha / Banca: Cláudia Helena Pellizzon / Banca: Luis Vitor Silva Sacramento / Resumo: Cissus sicyoides (Linneu) pertencente à família Vitaceae é uma planta nativa do Caribe, América do Sul e especialmente no Brasil, onde é conhecida como ―Insulina vegetal‖. O chá das folhas desta espécie é utilizada popularmente como anti-inflamatório, anti-hipertensivo, antitérmico, antidiabético, antireumático, contra infecções respiratórias, dislipidemias, distúrbios gastrointestinais e indigestão, para anemia, acidente vascular cerebral, tremores e como ativador da circulação sanguínea. A análise fitoquímica das partes aéreas de C. sicyoides mostra a presença de flavonóides glicosilados, e várias atividades biológicas já foram descritas para esta espécie, dentre elas, atividade gastroprotetora do extrato metanólico de suas folhas. O objetivo deste trabalho foi investigar a atividade antiulcerogênica do extrato hidroalcoólico das folhas de C. sicyoides (EHCS), elucidar os mecanismos envolvidos no efeito antiulcerogênico, em processos inflamatórios e em modelos experimentais de diarreia em roedores. A administração oral do EHCS nas doses 250 e 500 mg/Kg protegeu a mucosa gástrica da ação dos agentes lesivos etanol e anti-inflamatório não esteroidal em ratos Wistar machos. O EHCS na menor dose efetiva (250 mg/Kg) não alterou os parâmetros bioquímicos do suco gástrico em modelo de ligadura do piloro e não preveniu a úlcera duodenal induzida por cisteamina hidroclorídrica. Este extrato aumentou a quantidade de muco aderido à parede gástrica, restabeleceu o fluxo sanguíneo atuando como regulador na microcirculação e a ação gastroprotetora do extrato é dependente de óxido nítrico por atuação da enzima óxido nítrico sintase induzível (iNOS), ao mesmo tempo que independe de grupamentos sulfidrilas e receptores vanilóides. Este extrato possui propriedade antioxidante ao promover a proteção das lesões gástricas induzidas por isquemia-reperfusão, reduzindo ... / Abstract: Cissus sicyoides (Linneu) belongs to the Vitaceae family is a plant native to the Caribbean, South America and especially in Brazil, where it is known as "vegetable insulin". The tea from leaves of this species is popularly used as anti-inflammatory, anti-hypertensive, antipyretic, antidiabetic, antirheumatic, against respiratory infections, dyslipidemia, gastrointestinal disorders and indigestion, for anemia, cerebral tremors and as an activator of circulation blood. The phytochemical analysis of aerial parts of C. sicyoides showed predominantly the presence of glycosylated flavonoids and various biological activities have been described for this species, among them, gastroprotective activity of the methanol extract of its leaves. With these considerations, the aim of this study was to investigate the antiulcer activity of hydroalcoholic extract of C. sicyoides (HECS), as well as to elucidate the mechanisms involved in antiulcerogenic effect, inflammatory processes and in experimental models of diarrhea in rodents. The HECS in doses 250 and 500 mg/Kg protected the gastric mucosa from the damaging agents action, ethanol and anti-inflammatory non-steroidal when administered orally to male Wistar rats. The EHCS at the lowest effective dose (250 mg/Kg) didn't alter biochemical parameters of gastric juice in pylorus ligature model and did not prevent the duodenal ulcer induced by cysteamine hidroclorídrica. This extract increased the amount of mucus adhered to the gastric wall, restored blood flow in the microcirculation acting as a regulator and gastroprotective activity of the extract is dependent on nitric oxide activity of the enzyme inducible nitric oxide synthase (iNOS), while independent groups of sulfhydryl and vanilloid receptors. This extract has antioxidant properties to promote the protection of gastric lesions induced by ischemia-reperfusion injury by reducing lipid peroxidation (LPO) and ... / Mestre

The biopharmaceutical market has been on the rise for the past two decades and is expected to continue to excel, currently presenting a growing rate of more than double than conventional pharma. Traditionally this growth has been hindered by multiple formulation issues such as poor bioavailability and poor stability. Consequently, the drive to optimise the stability of protein drug candidates via formulation impels the need for development of novel excipients. Novel glycopolymer excipients were reported to confer improved protein stability in selected cases. Nonetheless,their structure-function relationship and wider applicability remain largely unknown. Here we report the synthesis of glycopolymers with different molecular architectures based on mannose, galactose, arabinose, N-acetyl glucosamine, lactose and trehalose, and nvestigate their utility as excipients for the solution formulation of a monoclonal antibody (mAb). In this thesis work the physical stability of selected antibodies was measured as the unfolding transition temperature (Tm) and aggregation onset temperature (Tagg), as a function of glycopolymer properties, such as the nature of sugar repeating unit, macromolecular architecture and concentration. Results show that, in contrast to the stabilising effect of the corresponding mono- and di-saccharide constituents, both linear and 4-arm star glycopolymers generally destabilised the antibody, decreasing both Tm and Tagg. Accelerated stability studies of a concentrated mAb solution followed the same trend, where an increasing glycopolymer:mAb molar ratio generally decreased the percentage monomer(i.e. increased soluble aggregates). Importantly, trehalose-based glycopolymers further generated visible aggregates that could not be predicted from Tm or Tagg data. The data demonstrate a complex interplay of sugar chemistry and solution concentration of synthetic glycopolymers on their modulation of protein conformational stability and aggregation propensity. The mechanisms involved in protein:glycopolymer interaction, both in solution and dry state were further investigated, thus unravelling the behaviour reported in terms of protein stabilisation. Finally, the glycopolymers were studied as drug delivery systems, acting as solubility enhancers for hydrophobic species in aqueous solutions, through the use of extrinsic fluorescent dyes.

Orientador: Taís Maria Bauab / Banca: Izabela Dutra Alvim / Banca: Carlos Henrique Gomes Martins / Resumo: Os fitoterápicos têm apresentado resultados surpreendentes no tratamento de doenças crônicas, principalmente do trato digestório (úlceras gástricas e duodenais, colite ulcerativa e doença de Crohn), diabetes e câncer. A Política Nacional de Plantas Medicinais e Fitoterápicos - PNPMF (Ministério da Saúde - MS) propõe o estudo de diversas espécies vegetais, dentre estas, plantas do gênero Astronium (Anacardiaceae), que reúne espécies como Astronium fraxinifolium, Astronium graveolens e Astronium urundeuva, com propriedades anti-inflamatória, antiulcerogênica, cicatrizante e antimicrobiana. Nesse contexto, a inclusão da tecnologia no aprimoramento de plantas medicinais deve ser estimulada já que a estratégia de incorporação de extratos vegetais em sistemas nanoestruturados tem otimizado suas propriedades. A atividade antimicrobiana dos extratos incorporados ou não em sistemas nanoestruturados foi avaliada pela técnica de microdiluição frente aos micro-organismos Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Salmonella setubal (ATCC 19196), Helicobacter pylori (ATCC 43504) e Candida albicans (ATCC 18804). Os extratos vegetais foram testados nas concentrações de 1000 a 7,81 μg/mL e como controle foram utilizados a amoxicilina (100 μg/mL) para H. pylori; a ampicilina (50 μg/mL) para as demais bactérias, e a anfotericina B (32 μg/mL) e fluconazol (256 μg/mL) para C. albicans. Após incubação a 37 ºC/72 horas sob condição de microaerofilia para H. pylori; 37 ºC/24 horas para as demais bactérias e 37 ºC/48 horas para a levedura, foram realizadas as leituras espectrofotométrica e visual, com os reveladores resazurina 0,01% (bactérias), e cloreto de 2,3,5-trifeniltetrazólio 2% - TTC (levedura) para a determinação da concentração inibitória mínima (CIM). A incorporação foi feita em um sistema nanoestruturado constituído por 10%... / Abstract: Herbal medicines have shown amazing results for the treatment of chronic diseases, especially the ones of the digestive tract (gastric and duodenal ulcers, ulcerative colitis and Crohn's disease), diabetes and cancer. The National Policy of Medicinal Plants and Herbal Medicines (Ministry of Health) proposes the study of several plant species, including plants containing the genus Astronium (Anacardiaceae) that includes species such as A. fraxinifolium, A. graveolens and A. urundeuva, which comprise anti-inflammatory, anti-ulcerogenic, healing and antimicrobial properties. In this context, the inclusion of technology in the improvement of medicinal plants should be encouraged since the strategy to incorporate plant extracts in nanostructured systems has optimized their properties. The antimicrobial activity of the extracts, which were incorporated or not into nanostructured systems, was evaluated by microdilution technique for the microorganisms Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Salmonella setubal (ATCC 19196), Helicobacter pylori (ATCC 43504) and Candida albicans (ATCC 18804). The plant extracts were tested at concentrations from 1000 to 7.81 μg/mL, and the following medications were used to check in order to see if they will work with each type of microorganisms: amoxicillin (100 μg/mL) for H. pylori; ampicillin (50 μg/mL) for the other bacteria, amphotericin B (32 μg/mL) and fluconazole (256 μg/mL) for C. albicans. After the incubation at 37 ºC/72 hours under microaerophilic condition for H. pylori; 37 ºC/24 hours for the other bacteria and 37 °C/48 hours for the yeast, spectrophotometric and visual readings were performed, with the developers 0.01% resazurin (bacteria) and 2% 2,3,5-triphenyltetrazolium chloride in order to determine the minimum inhibitory concentration (MIC). The incorporation was prepared into a nanostructured system com... / Mestre

The mupirocin biosynthetic pathway belongs to the trans-AT group in which acyltransferase (AT) activity is provided by a separate polypeptide (MmpC) rather than in cis as found in the typical type I polyketide synthases. AT docking domains have been documented in trans-AT PKS clusters for ten years yet little functional evidence is available. The cluster shows many interesting features that must be understood to create novel products. Specificity studies demonstrated that AT2 performs the typical AT function of loading malonyl-CoA to ACPs throughout the cluster. Mutagenesis studies demonstrated the importance of AT active site residues for protein structural integrity, acquisition and transfer of malonate and propose an alternate role for AT1 as a proofreading enzyme responsible for hydrolysing truncated intermediates from the pathway. Consequently an edit, reload, reduce model for MmpC is proposed. Mutagenesis of docking domains led to a halt in mupirocin production and suggested that docking domains are required for structural integrity of the Mmps or for guiding the ACPs into the correct position for interactions with their respective partners. Studies involving a mutated ACP3 protein confirmed the importance of Trp55, as demonstrated by structural changes and the inability of the protein to accept malonate from AT2.

Graphene oxide (GO) and reduced GO (r-GO) nanomaterials exhibit great potential for several biomedical applications. Of foremost importance is to determine any potential health hazards related in their exposure. In this research, we hypothesised that the different material properties evidenced by GO and r-GO would elicit different biological responses. The first objective of this work was to synthesize Go and r-GO and characterize their physiochemical properties. The second aim was to investigate whether the two-distinct surface chemistries of GO and r-GO influenced their biological effect. The potential toxicity of these nanomaterials was investigated using the normal lung fibroplast cell line MRC-5 and cancerous epithelial lung cell line A549. The cytotoxicity of graphene derivatives was concentration-, time- and cell-dependent and varied according to the material used. Thus, the surface chemistry of graphene plays a critical role in its biocompatibility. Non-cancerous cells had a higher sensitivity to GO cytotoxicity than cancer cells. R-GO was highly biocompatible to MRC-5 cells and for A549 cells had a minimal effect of cell viability. At 37C˚, GO and r-GO were moderately hemolyric at concentration of 125 µg/ml and highly haemolytic at concentration of 300 µg/ml. Exposure of cells to both graphene derivatives led to reactive oxygen species (RO5) generation without genotoxicity. GO, but not r-GO, led to autophagy in both cell lines, possibly inhibiting the PIP3-Akt/mTOR pathway. For both cell lines and at non-lethal concentrations, GO downregulated the expression of glycogen synthase kinase-3 (GSK-3ß). GO was also found to dysregulate both Wnt/b-catenin and Akt cell signalling pathways which are vital for cellular function. The finding relating to cell signalling provide an insight to the safety of GO which is important to its use in cancer therapy.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, leading to hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. A vaccine is currently not available, while the standard of care is effective in only 50% of treated patients. The first specific anti-HCV drugs have been recently approved, and new classes of targeted agents are under clinical trials/investigation. Nevertheless, improved treatment strategies are needed, in order to bypass the rapid emergence of resistance. All the viral non-structural proteins are a possible target for the identification of novel and selective antivirals. Among them, the NS3 helicase is still underexploited, with no known inhibitor under pre-clinical or clinical development. This enzyme plays a crucial role in the virus life cycle: it catalyses the separation of double-stranded RNA strands, which is necessary for genome amplification and translation. Due to its essential function, the NS3 helicase was chosen as a target for the identification of new, specific anti-HCV compounds. Different computer-aided techniques were employed to identify potential smallmolecule inhibitors of the enzyme. Two structure-based virtual screenings of commercially available compounds were performed on the main nucleic acid binding site. A series of candidate inhibitors was evaluated in the HCV replicon assay, yielding two primary hits with low μM activity. Secondly, the model of the one known inhibitor co-crystallised with the enzyme was used as a starting point for a shape-comparison screening of small molecule libraries. A new series of compounds was selected and evaluated for anti-HCV activity, and one of them was found to inhibit the viral replication at a low μM concentration. Several new derivatives of the initial hits were synthesised, belonging to four main structural families: bis-aromatic piperazine derivatives, symmetrical phenylendiamine compounds, differently substituted thieno-pyrimidines, and triphenyl-pyrrolone analogues. Inhibition of HCV replication in the replicon assay was evaluated for the new compounds prepared and several structures showed a range of activity from low-μM to nM.

The successful clinical translation of non-viral gene delivery systems has yet to be achieved due to the biological and technical obstacles to preparing a safe, potent and cost-effective vector. Hyperbranched polymers have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification compared to other polymers, which makes them more feasible for scale up and manufacturing. In the first part of this thesis, we compare hyperbranched poly(amino acids) synthesised by co-polymerising histidine and lysine, with hyperbranched polylysine prepared using the well-known 'ultra-facile' thermal polycondensation route, to investigate the effects of histidine units on the structure and gene delivery applications of the resultant materials. The conditions of polymerisation were optimised to afford water-soluble hyperbranched polylysine-co-histidine of three different molar ratios with molecular masses varying from 13-30 kDa. Spectroscopic, rheological and thermal analysis indicated that the incorporation of histidine modulated the structure of hyperbranched polylysine to produce a more dendritic polymer with less flexible branches. Experiments to probe gene delivery to A549 and H1299 cells, surprisingly, indicated that the co-polymers containing histidine were not more effective in transfecting a luciferase gene than hyperbranched polylysines synthesised as established literature comparators. We attribute the variations in gene delivery efficacy to the changes induced in polymer architecture by the branching points at histidine residues, and obtain structure-function information relating histidine content with polymer Tg, pKa and ability to form stable polyplexes with plasmid DNA. These results are of significance to nanomedicine design as they indicate that addition of histidine as a co-monomer in the synthetic route to hyperbranched polymers changes not only the buffering capacity of the polymer but has significant effects on the overall structure, architecture and gene delivery efficacy. It has become known that many cationic polymers are cytotoxic and although a large number of polycations have now designed to address the toxicity problem, there is still a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a high throughput and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Therefore, in the second part of this thesis, metabolomics was applied to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethylenimine polyplexes, on the metabolic pathways of A459 and H1299 cell lines. The study revealed that the polyplexes downregulated metabolites associated with glycolysis and the TCA cycle, and induced oxidative stress in both cell lines. The fold changes of the metabolites indicated that the polyplexes of polyethylenimine and hyperbranched polylysine affected the metabolism much more than the polyplexes of hyperbranched polylysine-co-histidine. This was in line with transfection results, suggesting a correlation between the toxicity and transfection efficiency of these polyplexes. This part highlights the importance of metabolomics approaches not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanisms underlying their action, which could help to design more efficient vectors. In the third part of this thesis, we investigated the ability of the hyperbranched polymers to condense and deliver siRNA. The results indicated that the higher molecular mass polymers achieved better siRNA delivery and gene silencing than the lower molecular mass form of the polymers and the lysine-only polymer was more efficient than the histidinylated one. These results can be attributed to the low charge (molecular mass) and stiffness of siRNA molecules in comparison with plasmid DNA, which in combination with the impact of histidine incorporation on the structure of the hyperbranched polymers can also explain the lower efficiency of histidinylated polymers. Overall, this thesis is highlighted the impacts of structural factors on the gene delivery applications of hyperbranched polymers and the importance of these factors to inform the design of new polymeric vectors. Also, metabolomics approaches were introduced to this area, not only to evaluate the safety of gene vectors but also to understand the molecular basis by which these vectors act. The data together suggest that the hyperbranched polymers prepared during thermal polycondensation of amino acids have some efficacy in preliminary gene delivery applications, and that these might be improved with future studies to be a candidate for clinical purposes.

A amortização via reação de estado sólido foi estudada em filmes finos multicamadas de Fe-Zr na composição 50% átomos de Fe, para três diferentes modulações (A=75 A, 150 A e 240 Á), tratadas a 350°C em tempos entre 15min e 24h. A formação e crescimento de fases foi caracterizada através da difração de raios-X em baixo e alto ângulos, espectroscopia de retroespalhamento Rutherford, espectroscopia Mõssbauer de elétrons de conversão e magnetometria. Foi verificado que apenas uma fase amorfa é formada independente da modulação. Determinou-se que a cinética de crescimento apresenta diferentes regimes em função do tempo de tratamento térmico. Foi mostrado que medidas magnéticas são complementares àquelas obtidas por espectroscopia Mõssbauer, e constituem-se uma opção para a caracterização da cinética da reação, a qual pode ser quantificada adotando-se um modelo de crescimento planar das fases reagidas na interface dos pares de reação. Foi calculado um diagrama de energia livre de Gibbs para o sistema Fe-Zr, determinando o comportamento da fase amorfa, bem como o intervalo de amorfizabilidade do mesmo. / The solid state amorphization reaction was studied in multilayered thin films of Fe-Zr at the composition 50% at. Fe, for three different modulations (A= 75 A, 150 A and 240 Á), annealed at 350°C for times ranging from 15 min to 24h. The reaction and consequent phase's growth were characterized by X-ray diffraction in low and high angles, Rutherford backscattering spectroscopy, conversion electron Mossbauer spectroscopy and magnetometry. Only one amorphous phase was formed with none dependency on the modulation. lt was determined that the growth kinetic has different regimes related to the annealing time. Magnetic measurements are complementary to the Mossbauer results, and are an option to the growth kinetic characterization. A Gibbs free energy diagram was calculated for the Fe-Zr system, describing the amorphous phase behavior, and the glass forming range o f the system.