Cervical cancer is almost exclusively caused by the HPV virus, whit HPV 16 and 18 involved in the majority of cases. The HPV virus can be divided into high risk and low risk types, where the high risk types are most associated with cancer. HPV is spread by sexual skin to skin contact, many people get infected without getting cervical cancer. HPV is also involved in the development of several other types of cancers such as oral and other genital cancers. The HPV virus infects epithelium stem cells and disrupts basic functions of the cells. A high expression of the late genes early in an infection may result in that an HPV 16 infection dies out. The late gene expression was analysed by using a CAT ELISA method, in the cell lines used one of the late genes had been replaced by a CAT reporter gene. Several small molecules where investigated, to study the regulation of the late gene expression. The results of the study was that a regulation of the late gene expression could be seen when pBELMCAT was treated with TPA, TA and RA where TPA gave the highest increase in the late gene expression. TA/RA combined with TPA increased the expression even more. As a conclusion it seems possible for small molecules to be used in treatments for cervical cancer that is caused by HPV 16, to upregulate the late gene expression and maybe be able to eliminate the infection before serious damage and disease can develop.

Although conventional two-dimensional (2D) cell culture is convenient for routine work, researchers are turning to three-dimensional (3D) cell culture for more accurate, physiologically representative information on the way their cells behave and respond to stimuli. Cells can now be routinely cultured in the many commercially available 3D formats. In this study, we developed non-woven scaffolds for 3D cell culture and enhanced cell function. By making use of methods that measure the behaviour of liver cells in the 3D system we were able to demonstrate, compared to standard 2D systems, significantly higher expression of key liver enzymes involved in drug metabolism and albumin production (specifically cytochrome P450). Cell proliferation on the various scaffolds was comparable to that of a commercially available hydrogel 3D cell culture system, AlgimatrixTM. When culturing cells in 3D, the means by which cells are harvested or extracted from the 3D scaffold for downstream applications is more challenging than in 2D. For this reason, many of the 3D scaffolds currently manufactured are either bio-degradable or require the use of salts to dissolve the scaffold which may negatively impact on the cells they contain. By grafting the non-woven scaffolds with the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), we demonstrated that cells growing on the scaffolds are able to be released from the scaffold in a 3D conformation, non-enzymatically, through temperature changes. Selected thermoresponsive non-woven fabrics were also tested in an automated cell culture device for cell proliferation and thermally induced harvesting. One of the applications of a 3D cell culturing system would be in exploration of the many diseases plaguing mankind, in particular malaria which is still responsible for severe disease and mortality, especially in Africa. Most available antimalarials are designed to target the pathogenic blood stages in humans and to address the constant threat of drug resistance. However, to meet the objective of malaria eradication, medicines that block parasite transmission also need to be developed. Molecules that efficiently target the parasite stages in the liver would prevent pathogenesis, symptoms and transmission. Equipped with the knowledge that the infectious sporozoites traverse several hepatocytes prior to cell infection, it may be physiologically limiting to culture the exo-erythrocytic stage in vitro in a 2D cell culture system where the hepatocytes are in an unnatural flat conformation, distinctly different to their in vivo counterparts. Moreover, monolayer cell cultures lose their tissue-related functions rapidly, greatly impairing the predictive power of such assays. Thus, the second aim of this thesis was to establish if hepatocytes that have been cultured on 3D non-woven scaffolds improve in vitro sporozoite invasion compared to conventional 2D systems. Sporozoite invasion was detected in the conventional 2D monolayers using a TaqMan® assay but not in the hepatocytes growing in 3D. Future studies beyond the scope of this thesis will include modifications to the 3D scaffold to attempt achieving superior sporozoite invasion in this model system. / Thesis (PhD)--University of Pretoria, 2013. / Biochemistry / unrestricted

In recent years, the degradation and subsequent loss of tissues is an issue that has affected people worldwide. Although there are treatments addressing the degradation of tissues, such treatments involve complicated and expensive procedures, where full tissue regeneration is not achieved. For these reasons, in recent years, tissue engineering has developed cutting-edge biomaterials capable of inducing effective tissue regeneration both under cellular or acellular conditions. Peptide hydrogels are versatile biomaterials composed of the basic components of life amino acids, which act as building blocks to form hierarchical structures, which subsequently go on to form well-defined scaffolds. Biomaterials have been widely used for the culture of mammalian cells, tissue engineering, regenerative medicine, drug delivery, etc. This is thanks to their capability of providing a three-dimensional architecture to cells, which mimics the natural architecture of the extracellular matrix (ECM). Peptide- based hydrogels can be easily functionalised with active biological cues, which can direct the cellular response. It has been shown that the ionic-complementary FEFEFKFK hydrogel, succeeded to support the culture of mammalian cells such as bovine chondrocytes. In this work, we used the same FEFEFKFK hydrogel to investigate the capability of this hydrogel to support the three-dimensional culture of both human osteoblasts (hOBs), and human mesenchymal stem cells (hMSCs) for bone regeneration applications. To achieve this goal, hOBs were cultured within both FEFEFKFK (non-functionalised) and RGD-FEFEFKFK (functionalised) gels. Then the suitability of the FEFEFKFK gels to induce cellular proliferation, synthesis of bone ECM and mineralisation was explored. In addition, taking advantage of the inherent plasticity of hMSCs, we also investigated the capability of the FEFEFKFK gel to foster the osteogenic differentiation of hMSCs, and subsequently to induce bone mineralisation in 3-D under osteogenic stimulation. Based on the results obtained in this work, the FEFEFKFK gel arises as a promising biomaterial for both bone and dental tissue regeneration applications.

Molecular self-assembly provides a simple and efficient route of constructing well-defined nanostructures which may serve as extra cellular matrix (ECM) mimics. This work focuses on two specific octapeptides: FEFEFKFK and FEFKFEFK (F: phenylalanine, E: glutamic acid, K: lysine) with alternating charge distribution. The peptides were shown to self-assemble in solution and form β-sheet rich nanofibres which, above a critical gelation concentration (CGC), entangle to form self-supporting hydrogels. The fibre morphology of the hydrogels was analysed using TEM and Cryo-SEM illustrating the dense fibrillar network of nanometer size fibres. Oscillatory rheology results showed that the hydrogels possesses viscoelastic properties. By varying peptide concentration and type hydrogel stiffness, viscosity, water content, fibre density and other mechanical properties were tailored to control cell interactions and subsequent tissue growth. Bovine chondrocytes were used to assess the biocompatibility of these novel scaffolds over 21 days under 2D and 3D cell culture conditions, particularly looking into cell morphology, proliferation and matrix deposition. 2D culture resulted in cell viability and collagen type I deposition. In 3D culture, the mechanically stable gel was shown to support viability, retention of cell morphology and collagen type II deposition. Subsequently, the scaffold may serve as a template for cartilage repair. In addition, this research also focused on developing novel injectable scaffold design with in situ gelation properties to encapsulate chondrocytes for cell culture applications.

Non-invasive and controlled release of bioactive compounds is an important goal in the development of drug delivery systems and novel biomaterials for tissue engineering. This project aims to exert spatio-temporal control over the release of bioactive compounds from phospholipid vesicle carriers by crosslinking them with superparamagnetic iron oxide nanoparticles to form a magnetic release nanostructure. The magnetic properties of the nanoparticles allow release to be triggered by an alternating magnetic field (AMF), which induces localised heating and “melts” the vesicle membranes. The aggregates can also be manipulated in space by a static magnetic field to create patterned materials. Incorporation of these aggregates into hydrogels has created novel responsive biomaterials. Controlled release of ascorbic acid-2-phosphate has been used to induce collagen production by chondrocytes, demonstrating an AMF triggered cellular response in vitro. The existing system has been redesigned after detailed characterisation and assessment of the performance of each component. Magnetic release has been extensively assessed using fluorescence techniques to quantify release, and optimised through the development of new silica-derived nanoparticle coatings and aggregate formulations informed by quantitative characterisation of nanoparticle functionalisation. The replacement of calcium alginate hydrogels as a 3D cell culture matrix with hyaluronic acid- based hydrogels was found to eliminate gel-induced leakage of vesicle contents and also improves the compatibility of the system with a greater range of cell types. Recently the effective encapsulation and AMF-triggered release of proteins including enzymes has been demonstrated and released enzymes have been demonstrated to retain their activity. Released trypsin was shown to retain proteolytic activity while hyaluronidase released into hyaluronan-derived hydrogels has been demonstrated to influence the rheological properties of the gel. A galactose-terminated lipid has been synthesised that enables specific targeting of the asialoglycoprotein (ASGPR) cell surface receptor receptor found in human hepatocytes, demonstrating the potential for customisation of the MNPV system to particular requirements.

Orientadores: Maria Ligia Rodrigues Macedo, Tomomasa Yano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T00:07:13Z (GMT). No. of bitstreams: 1 Ventura_ClaudioAngelo_D.pdf: 5407567 bytes, checksum: 8408eb98403ba9f693c16020c7c85169 (MD5) Previous issue date: 2006 / Resumo: Lectinas constituem uma classe de proteínas ou glicoproteínas que são capazes de ligar-se, reversivelmente e seletivamente, a carboidratos sem causar transformação química. Estudos têm mostrado que a ligação de lectinas a carboidratos de superficie celular de células normais e malignas leva a vários efeitos biológicos tais como proliferação celular e apoptose. Neste trabalho, nós investigamos os efeitos citotóxicos induzidos por TEL (uma lectina, isolada de sementes de Talisia esculenta, a qual preferencialmente liga-se a resíduos de manose) sobre linhagens de células tumorais e não-tumorais. Células Vero (rim de macaco verde afiicano) e Rela (carcinoma cervical humano) tratadas com TEL exibiram perda da integridade da membrana plasmática, retração celular, condensação da cromatina, fragmentação nuclear e formação de corpos apoptóticos. Além disso, células Vero e Rela tratadas com TEL também revelaram uma drástica desorganização do citoesqueleto de actina. A Fragmentação intemuc1eossomal do DNA foi detectada pelo método de TUNEL e eletroforese em gel de agarose. Os efeitos citotóxicos induzidos por TEL foram progressivos e mostraram que as alterações morfológicas e os danos ao DNA ocorreram após a perda da integridade da membrana. Adicionalmente, TEL inibiu significantemente a proliferação das células Rela de maneira dependente da concentração. Nós também mostramos através de microscopia de tluorescência que TEL é intemalizada nas células Vero dentro de pequenas vesículas que depois se acumulam da região perinuclear; nas células Rela, a intemalização não foi observada. Foi estabelecido que a atividade das caspases-3 e -9 aumentaram nas células Vero e Rela de uma maneira dependente do tempo. Em contraste com as células Vero, a atividade da caspase-3 precedeu a ativação da caspase9 nas células Rela. Os efeitos citotóxicos e a intemalização de TEL foram bloqueados pela mano se, sugerindo que a ligação de TEL ao carboidrato específico na superficie celular é um pré-requisito para esses processos. Os resultados mostraram que a morte celular induzida por TEL nas células Vero e Rela seguiu uma série de eventos que culminou em um modo apoptótico de morte celular. Finalmente, uma vez que as células Rela foram altamente sensíveis a citotoxicidade induzida pela lectina, TEL merece futuras investigações porque suas propriedades podem ser uma ferramenta útil na terapia contra câncer cervical humano / Abstract: Lectins constitute a class of proteins or glycoproteins which are capable of binding carbohydrates selectively and reversibly without causing their chemical transformation. Studies have shown that lectin binding to cell surface carbohydrates of normal and malignants cells triggers various biological effects such as cellular proliferation and apoptosis. In this work, we investigate the cytotoxic effects induced by TEL (a lectin, isolated from Talisia esculenta seeds, which binds preferentially to mannose residues), on tumoral and non-tumoral cell lines. TEL- treated Vero (African green monkey kidney) and Hela (human cervical carcinoma) cells exhibited loss of plasma membrane integrity, cellular retraction, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies. Furthermore, TEL-treated Vero and Hela cells also revealed a drastic disorganization of the actin cytoeskeleton. Internucleossomal DNA fragmentation was detected by TUNEL method. and agarose gel electrophoresis. TEL-induced cytotoxic effects were progressive and showed that the morphological alterations and DNA damage occurred after the loss of membrane integrity. Additionally, TEL significantly inhibited the proliferation of Hela cells in a concentration- dependent manner. We also show through of fluorescence microspy that TEL is internalized in Vero cells into small visicles that further coalesce in the perinuclear region; in Hela cells, TEL internalization was not observed. It was established that caspase-3 and -9 activity increased in Vero and Hela cells after TEL treatment in a time- dependent manner. In contrast to Vero cells, caspase- 3 activity preceded the activation of caspase-9 in Hela cells. The cytotoxic effects and the internalization of TEL was blocked by manose, suggesting that the binding of TELto specific carbohydrate on the cell-surface is a prerequisite for these processes. The results showed that TEL- induced cell death in Vero and HeLa cells followed by down stream events leading to apoptotic mode of cell death. Finally, since Hela cells were highly sensitive to lectin-induced cytotoxicity, TEL merits further investigation due to its properties can be a useful tool in therapy against human cervical cancer / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientadores: Rubens Maciel Filho, Jorge Vicente Lopes da Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-18T15:30:30Z (GMT). No. of bitstreams: 1 Senedese_AnaLiviaChemeli_M.pdf: 22505646 bytes, checksum: 1c68dba091d7e2e2fc02cd7387c9852e (MD5) Previous issue date: 2011 / Resumo: Em decorrência da grande demanda de transplantes de órgãos e tecidos no Brasil, ha estímulos para criação de terapias alternativas como o desenvolvimento de substitutos biológicos temporários, isto e, scaffolds, através da Bioengenharia e Engenharia Tecidual, descartando a necessidade de doadores. Neste trabalho, foi utilizado o polímero policaprolactona (PCL) para estruturar scaffolds 3D por meio da plataforma experimental de manufatura aditiva Fab@CTI, a qual apresenta um cabeçote de extrusão intercambiável construído para entrada de material em forma de filamento. Uma nova proposta de orientação de raster foi criada para o design dos scaffolds. Foram estruturados scaffolds com raster regular e randômico com poros de 0.25, 0.5 e 1 mm. Analises de Difração de raios-x (DRX), Calorimetria exploratória diferencial (DSC) e Espectroscopia de absorção no infravermelho com transformada em Fourier (FTIR) foram realizadas para verificar possíveis mudanças nas propriedades térmicas do material durante o processamento. Microscopia eletrônica de varredura (MEV) foi feita para checar a morfologia dos scaffolds e medir o diâmetro dos filamentos extrudados na Fab@CTI alem do espaçamento entre eles. Também foram realizadas analises de citotoxicidade e viabilidade celular com células tronco mesenquimais de tecido adiposo. Ainda, utilizando o software modeFRONTIER, foi feita uma simulação de valores de modulo de compressão do PCL possíveis de serem obtidos. Os resultados de DRX e FTIR não mostraram degradação do PCL e o DSC revelou algumas alterações no ponto de fusão e diminuição da cristalinidade. Os scaffolds não se mostraram tóxicos e os modelos com poros de 0.25 mm e raster regular e randômico foram os que apresentaram viabilidade celular / Abstract: Due to the demand for organs and tissues transplants in Brazil, is indeed the creation of new therapies as the development of temporary biological substitutes, ie, scaffolds, through Bioengineering and Tissue Engineering, discarding the require for donors. In this work, we used the polymer polycaprolactone (PCL) to structure 3D scaffolds by the additive manufacturing experimental platform, Fab@CTI, which presents an interchangeable extrusion head build to filaments materials input. A new proposal for random raster was presented to design the scaffolds. Were structured scaffolds with regular and random raster and pores of 0.25, 0.5 and 1 mm. Analysis of X-Ray Diffraction (XRD), Differential Scanning Calorimetry (DSC) and Fourier Transform Spectroscopy (FTIR) were conducted to check for possible changes in thermal properties of the material during the processing. Scanning Electron Microscopy (SEM) was done to check the morphology of scaffolds and measure the diameter of filaments extruded at Fab@CTI beyond the distance between them. Analyses of cytotoxicity and cell viability with mesenchymal stem cells from adipose tissue were also performed. In addition, using the software modeFRONTIER, a simulation of compressive modulus of PCL was done with values possible to obtain. The result of the XRD and FTIR showed no degradation of PCL and DSC revealed some changes in melting point and decrease of crystallinity. The scaffolds were not toxic and models of 0.25 mm pores and both regular and random raster showed cell viability / Mestrado / Desenvolvimento de Processos Químicos / Mestre em Engenharia Química

Orientador: Gabriel Forato Anhê / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T19:10:52Z (GMT). No. of bitstreams: 1 Barbosa_AnaPauladeLima_D.pdf: 1915868 bytes, checksum: d146b07dfcd99b2676c519233cddf2f2 (MD5) Previous issue date: 2014 / Resumo: O Diabetes Mellitus tipo II (DMT2) é uma doença caracterizada pela diminuição da sensibilidade à insulina e consequente prejuízo na homeostasia glicêmica. Estudos recentes mostraram que a melatonina, um hormônio produzido pela glândula pineal, melhora a tolerância à glicose e a resistência à insulina, diminui a expressão de proteínas ligadas à gliconeogênese hepática (G6Pase e PEPCK) e reduz a apoptose de células beta pancreáticas in vivo. Entretanto, ainda não estava claro se a melatonina conseguia, de fato, reverter in situ as ações lipotóxicas dos ácidos graxos saturados em células beta pancreáticas e na musculatura esquelética, e se podia diminuir a expressão dos genes reguladores da gliconeogênese hepática. Para responder a esse questionamento, utilizamos culturas celulares de insulinoma (MIN6), de músculo esquelético (miotubos de L6) e de hepatoma (HepG2). Os resultados mostraram que a melatonina reverte a resistência à insulina gerada por palmitato em miotubos de L6, mas somente em passagens baixas, não diminui a apoptose em MIN6 e não altera a expressão do G6pc, o gene que codifica a G6Pase, em HepG2 / Abstract: Type II Diabetes Mellitus (T2DM) is characterized by decrease of insulin sensitivity, resulting impairment glucose homeostasis. Recent studies have shown that melatonin, a hormone produced by the pineal gland, improves glucose tolerance and insulin resistance, reduce the expression of hepatic gluconeogenesis proteins (G6Pase and PEPCK) and diminish pancreatic beta cell apoptosis in vivo. However, it was unclear if melatonin could reverse the lipotoxic actions of saturated fatty acid in pancreatic beta cells and skeletal muscle, and if melatonin could decrease the expression of regulatory genes of hepatic gluconeogenesis in situ. To answer this question, we used cell cultures of insulinoma (MIN6), skeletal muscle (L6 myotubes) and hepatoma (HepG2). The results showed that melatonin reverses palmitate insulin resistance in L6 myotubes, but only at low passages, melatonin does not decrease apoptosis in MIN6 and does not alter the expression of G6pc, the encoding gene of G6Pase, in culture cell of HepG2 / Doutorado / Farmacologia / Doutora em Farmacologia

O estudo de agentes que interferem no funcionamento das proteínas relacionadas com o ciclo celular é importante para a compreensão dos processos de transformação e de morte celular. Alterações de ploidia, embora presentes na maioria dos tumores humanos, não têm ainda seu papel conhecido no processo de oncogênese. A alteração do número cromossômico é conseqüência primária de erros que envolvem o fuso mitótico e o cinetócoro. Dessa maneira, drogas que agem sobre os microtúbulos são consideradas aneugênicas potenciais. O presente trabalho enfocou o estudo do mecanismo pelo qual drogas que atuam sobre microtúbulos levam ao aparecimento de células multinucleadas e o significado biológico destas. Os resultados mostraram que a vincristina induziu bloqueio em mitose das células BBnt e MDCK, com conseqüente entrada em interfase no estado multinucleado. As células multinucleadas não apresentaram sinais de morte celular por apoptose, entretanto, quando em prófase apresentaram vários centrossomos, que poderiam originar divisões celulares com fusos multipolares. Estes resultados indicam que essas linhagens celulares possuem pontos de checagem mitótico funcionais e células multinucleadas são viáveis e capazes de prosseguir no ciclo celular. A presença de mitoses com fusos multipolares é indício de que as células multinucleadas passam por divisões anormais, que progrediriam para apoptose resultando na eliminação desta população. / The study of agents that interfere in the functionality of proteins related to cell cycle is important for the understanding of the transformation and cell death processes. Although ploidy alterations are presented in the majority of human tumors, their role in oncogenic process is not understood yet. The alteration on chromosomal number is the primary consequence of errors involving the mitotic spindle and kinetocore. Thus, drugs acting on the microtubules are considered as potentially aneugenic agents. The present work aimed to study the mechanism of multinucleated cells induction by action of antimicrotubule drug and biological meaning of these cells. The results showed that vincristine induced mitotic arrest of both BBnt and MDCK cells, with consequent entrance into interphasic-multinucleated status. Multinucleated cells did not present features of cell death by apoptosis; they were still viable and able to go further in cell-cycle progression. The presence of many structures suggested microtubule enucleation, centrosomes-like were detected on treated cells and could be responsible for the multi-spindle assembling that leads the multinucleated cells to abnormal divisions. Later on, when the multinucleated cells accumulated more abnormalities they were eliminated from the cell population by apoptosis.

S. pneumoniae é um importante patógeno humano que afeta sobretudo crianças. Vacinas são a principal estratégia de combate e o polissacarídeo (PS) da cápsula é o antígeno. Dentre os sorotipos do patógeno, o tipo 1 é prevalente em crianças e o PS1 componente obrigatório das vacinas. Neste trabalho objetivou-se: estabelecer um método de dosagem do PS1, selecionar a melhor cepa produtora de PS1, avaliar 3 peptonas, investigar 4 componentes do meio de cultura em planejamento experimental (PE) em reator e propor um novo meio e estratégia de cultivo. A cepa ST595/01 foi selecionada e o Phytone (Phy) foi a peptona com maior produção de PS1, 298mg/L, medido por m-hidroxidifenil com sulfamato. O PE 24-1 com extrato de levedura (EL), Phy, Asn e Gln mostrou os maiores efeitos positivos do Phy para produção de biomassa (Cx) e PS1. EL foi positivo para Cx, Asn não apresentou efeitos e Gln menor efeito positivo para PS1. Assim, testou-se o novo meio com Phy 15g/L e EL 2g/L sob duas estratégias: a batelada alimentada superou a batelada simples em 2x para Cx e 2,5x para PS1. / S. pneumoniae is an important human pathogen that affects mainly children. Vaccines are the main strategy to fight the disease and capsular polysaccharide (PS) is the antigen. Among pneumococcal serotypes, type 1 is prevalent in children. The aims of this work were: establish a method to measure PS1, screen strains for the best PS1 producer, evaluate 3 peptones, investigate the effects of 4 medium components by a design of experiment (DoE) and propose a new medium/strategy for cultivation. The strain ST595/01 was selected. Phytone (Phy) was the peptone with the highest PS1 production (298mg/L, measured by m-hydroxydiphenyl + sulfamate method). DoE 24-1 was carried out to assess the effects of yeast extract (YE), Phy, Asn and Gln. Phy showed the major positive effects for biomass (Cx) and PS1. YE demonstrated effects for Cx. Gln showed minor effect on PS1 production and Asn did not present effects. A new medium based on 15g/L Phy and 2g/L YE was evaluated by 2 strategies: fed-batch showed Cx production 2-fold higher and PS1 2.5-fold higher than simple batch.