O estudo das propriedades das membranas celulares, assim como sua interação com íons e moléculas é de fundamental importância para o entendimento de processos biológicos complexos, como a formação do tecido ósseo. O osso natural é constituído por compostos orgânicos, especialmente proteínas como o colágeno, e por minerais, como a hidroxiapatita. O processo pelo qual ocorre a formação do componente inorgânico é chamado de biomineralização, um processo complexo mediado pela liberação de vesículas da matriz (VM), secretadas em locais específicos a partir de osteoblastos. Essas VM são ricas nas principais biomoléculas envolvidas nesse processo, como as proteínas anexina, em especial a anexina V (AnxA5), que exercem papel importante como, formação de canais de cálcio e ligação com o colágeno, criando assim um microambiente adequado para a formação inicial e propagação dos cristais de hidroxiapatita a partir de fibrilas de colágeno. No presente estudo foi possível incorporar colágeno e AnxA5 em sistemas modelo para estudos de biomineralização, utilizando-se monocamadas de Langmuir e os filmes Langmuir-Blodgett (LB). As monocamadas foram formadas com os principais grupos carregados de lipídios encontrados nas VM: fosfatidilcolina (PC) e fosfatidilserina (PS). As monocamadas formadas por lipídios, colágeno e AnxA5 foram depositadas sobre discos de titânio utilizando-se a técnica dos filmes LB. Os filmes formados foram expostos a uma solução que simula o pH e força iônica do fluido corpóreo, de maneira a mimetizar a precipitação de hidroxiapatita. As amostras foram caracterizadas por microscopia eletrônica de varredura (MEV), espectroscopia vibracional na região do infravermelho (FTIR) e difração de raios X (EDX). Além disso, estudou-se as propriedades de molhabilidade e energia livre de superfície das amostras de titânio modificadas. Colágeno e AnxA5 interagiram com os lipídios, como mostrado nas isotermas. Interação preferencial foi observada no sistema binário contendo DPPC e DPPS e na presença de íons cálcio, como ocorre nos sistemas naturais. A presença das proteínas e dos fosfolipídios nos filmes LB sobre as superfícies de titânio foram essenciais para formação de nanopartículas de hidroxiapatita biomimética. / The study of the properties of cell membranes, as well as their interaction with ions and molecules, is of fundamental importance for the understanding of complex biological processes, such as the formation of bone tissue. The natural bone consists of organic compounds, especially proteins like collagen, and minerals, such as hydroxyapatite. The process which the formation of the inorganic component takes place is called biomineralization, a complex process mediated by the release of matrix vesicles (MV), secreted at specific sites from osteoblasts. These MVs are rich in the main biomolecules involved in this process, such as annexin proteins, especially annexin V (AnxA5), which play an important role as calcium channels formation and binding with collagen, thus creating a suitable microenvironment for formation initial and propagation of the hydroxyapatite crystals from collagen fibrils. In the present study, it was possible to incorporate collagen and AnxA5 into model systems for biomineralization studies using Langmuir monolayers and Langmuir-Blodgett (LB) films. The monolayers were formed with the main lipid groups found in MVs: phosphatidylcholine (PC) and phosphatidylserine (PS). The monolayers formed by lipids, collagen and AnxA5 were deposited on titanium plates using the LB film technique. The films were exposed to a solution that simulates the pH and ionic strength of the body fluid in order to mimic the precipitation of hydroxyapatite. The samples were characterized by scanning electron microscopy (SEM), infrared vibration spectroscopy (FTIR) and X-ray diffraction (XRD). In addition, the wettability and surface free energy properties of the modified titanium samples were studied. Collagen and AnxA5 interacted with lipids, as shown in the isotherms. Preferential interaction was observed in the binary system containing DPPC and DPPS and in the presence of calcium ions, as occurs in natural systems. The presence of the proteins and the phospholipids in the LB films on the titanium surfaces were essential for the formation of biomimetic hydroxyapatite nanoparticles.

O estudo das propriedades das membranas celulares, assim como sua interação com íons e moléculas é de fundamental importância para o entendimento de processos biológicos complexos, como a formação do tecido ósseo. O osso natural é constituído por compostos orgânicos, especialmente proteínas como o colágeno, e por minerais, como a hidroxiapatita. O processo pelo qual ocorre a formação do componente inorgânico é chamado de biomineralização, um processo complexo mediado pela liberação de vesículas da matriz (VM), secretadas em locais específicos a partir de osteoblastos. Essas VM são ricas nas principais biomoléculas envolvidas nesse processo, como as proteínas anexina, em especial a anexina V (AnxA5), que exercem papel importante como, formação de canais de cálcio e ligação com o colágeno, criando assim um microambiente adequado para a formação inicial e propagação dos cristais de hidroxiapatita a partir de fibrilas de colágeno. No presente estudo foi possível incorporar colágeno e AnxA5 em sistemas modelo para estudos de biomineralização, utilizando-se monocamadas de Langmuir e os filmes Langmuir-Blodgett (LB). As monocamadas foram formadas com os principais grupos carregados de lipídios encontrados nas VM: fosfatidilcolina (PC) e fosfatidilserina (PS). As monocamadas formadas por lipídios, colágeno e AnxA5 foram depositadas sobre discos de titânio utilizando-se a técnica dos filmes LB. Os filmes formados foram expostos a uma solução que simula o pH e força iônica do fluido corpóreo, de maneira a mimetizar a precipitação de hidroxiapatita. As amostras foram caracterizadas por microscopia eletrônica de varredura (MEV), espectroscopia vibracional na região do infravermelho (FTIR) e difração de raios X (EDX). Além disso, estudou-se as propriedades de molhabilidade e energia livre de superfície das amostras de titânio modificadas. Colágeno e AnxA5 interagiram com os lipídios, como mostrado nas isotermas. Interação preferencial foi observada no sistema binário contendo DPPC e DPPS e na presença de íons cálcio, como ocorre nos sistemas naturais. A presença das proteínas e dos fosfolipídios nos filmes LB sobre as superfícies de titânio foram essenciais para formação de nanopartículas de hidroxiapatita biomimética. / The study of the properties of cell membranes, as well as their interaction with ions and molecules, is of fundamental importance for the understanding of complex biological processes, such as the formation of bone tissue. The natural bone consists of organic compounds, especially proteins like collagen, and minerals, such as hydroxyapatite. The process which the formation of the inorganic component takes place is called biomineralization, a complex process mediated by the release of matrix vesicles (MV), secreted at specific sites from osteoblasts. These MVs are rich in the main biomolecules involved in this process, such as annexin proteins, especially annexin V (AnxA5), which play an important role as calcium channels formation and binding with collagen, thus creating a suitable microenvironment for formation initial and propagation of the hydroxyapatite crystals from collagen fibrils. In the present study, it was possible to incorporate collagen and AnxA5 into model systems for biomineralization studies using Langmuir monolayers and Langmuir-Blodgett (LB) films. The monolayers were formed with the main lipid groups found in MVs: phosphatidylcholine (PC) and phosphatidylserine (PS). The monolayers formed by lipids, collagen and AnxA5 were deposited on titanium plates using the LB film technique. The films were exposed to a solution that simulates the pH and ionic strength of the body fluid in order to mimic the precipitation of hydroxyapatite. The samples were characterized by scanning electron microscopy (SEM), infrared vibration spectroscopy (FTIR) and X-ray diffraction (XRD). In addition, the wettability and surface free energy properties of the modified titanium samples were studied. Collagen and AnxA5 interacted with lipids, as shown in the isotherms. Preferential interaction was observed in the binary system containing DPPC and DPPS and in the presence of calcium ions, as occurs in natural systems. The presence of the proteins and the phospholipids in the LB films on the titanium surfaces were essential for the formation of biomimetic hydroxyapatite nanoparticles.

O intuito do desenvolvimento de biomateriais potencialmente aplicáveis como implantes de substituição óssea é sintetizar superfícies que induzam respostas biológicas positivas quando em contato com o tecido hospedeiro. Para isso, faz-se necessário não somente estudos de composição química, mas também é importante buscar compreender propriedades físicas e estruturais dos sistemas que estes materiais irão substituir. O osso natural é constituído por compostos orgânicos, especialmente proteínas como o colágeno, reforçado com minerais, tais como hidroxiapatita. O processo pelo qual ocorre a formação do componente inorgânico é chamado biomineralização. Uma maneira de introduzir macromoléculas em sistemas modelo para estudos de biomineralização é a formação de monocamadas Langmuir, que são camadas monomoleculares altamente organizadas de moléculas anfifílicas na interface líquido-ar. Essas monocamadas podem ser transferidas para suportes sólidos formando filmes de Langmuir-Blodgett. Neste estudo, foi investigado a incorporação de colágeno tipo-I em monocamadas insolúveis contendo os fosfolipídeos 1,2-dipalmitoil-sn-glicero-3-fosfatidilcolina (DPPC) ou ácido octadecilfosfônico (OPA), bem como a transferência dessas monocamadas para suportes de titânio por meio da técnica de Langmuir-Blodgett. Estes filmes foram utilizados como matrizes para o crescimento de hidroxiapatita biomimética após a exposição dos discos de titânio modificados a uma solução que simula a concentração de íons e pH do plasma sanguíneo humano. As amostras foram caracterizadas por microscopia eletrônica de varredura (MEV), microscopia de força atômica (AFM), espectroscopia vibracional na região do infravermelho (FTIR) e difração de raios-X (DRX). A razão molar Ca/P foi determinada por espectroscopia de dispersão de energia de raios-X (EDX). Propriedades dos biomateriais como molhabilidade, rugosidade e energia livre de superfície também foram estudadas. A biocompatibilidade das amostras foi investigada in vitro utilizando-se ensaios de viabilidade de osteoblastos. O colágeno interage com os fosfolipídios na interface água/ar, como evidenciado pelas isotermas de pressão de superfície, obtidas na presença da proteína. Além disso, a estabilidade das monocamadas de Langmuir foi aumentada devido à presença de colágeno na subfase. A incorporação de colágeno e fosfolipídios em filmes Langmuir-Blodgett sobre as superfícies de titânio são fatores determinantes para a formação de filmes inorgânicos altamente organizado, contínuos e uniformes, compostos por nanopartículas de hidroxiapatita biomimética, semelhante à encontrada no osso natural. As modificações nas superfícies aumentaram a proliferação de osteoblasto no titânio indicando que este material não é tóxico a este tipo de célula. / The development potential of biomaterials for bone replacement is guided by the necessity to provide a surface that induces positive biological responses when the material is in contact with the host tissue. For this, it is necessary the study of the chemistry and surface composition and also to understand the physical properties and the structural organization of the natural system which is in contact with the biomaterial. Natural bone is composed by organic compounds, especially protein like collagen, reinforced with minerals, such as hydroxyapatite. The process by which the formation of the inorganic component takes place is named biomineralization. One way to introduce macromolecules in model systems for biomineralization studies is the formation of Langmuir monolayers, which are highly organized monomolecular layers of amphiphilic molecules adsorved at the liquid-air interface. These monolayers can be transferred to solid substrate to form the Langmuir-Blodgett films. In this study, it was investigated the incorporation of type-I collagen in insoluble Langmuir monolayers containing either 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) or octadecylphosphonic acid (OPA) and their transference to titanium supports using the Langmuir-Blodgett technique. These films were used as matrices to the growth of biomimetic hydroxyapatite by the exposure of the modified titanium discs to a solution that mimics the ions concentration and pH of the human blood plasma. The samples were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), vibrational spectroscopy in the infrared region (FTIR), and X-ray diffraction (XRD). The Ca/P molar ratio was determined by energy dispersive X-ray spectroscopy (EDS). The properties of the biomaterials as wettability, roughness, and surface free energy were also studied. The biocompatibility of the samples was studied in vitro using osteoblasts viability assays. The collagen interacts with the phospholipids at air/water interface as evidenced by the pressure surface-surface area isotherms obtained in the presence of the protein. Moreover, the stability of the Langmuir monolayers was increased when collagen is present at the subphase. The incorporation of collagen and phospholipids on the titanium surfaces is important to the formation of organized, continuous and uniform inorganic films composed by biomimetic hydroxyapatite nanoparticles, similar to that found in the natural bone. These films are not toxic to osteoblasts as evidenced by the stimulation of their proliferation on the titanium-modified samples.

No description available.

Bayreuth, Univ., Diss., 2009

No description available.

No description available.

No description available.

The Langmuir – Blodgett (LB) technique is about forming insoluble monolayer on the surface of aqueous solution and recently, it has emerged as one of the best method to study floating monolayer at the air – water interface. It has gained popularity after the use of monolayer with chemical complexes as well as biological species, and recently it has been used for the formation of biosensors. Langmuir monolayer arrays the amphiphilic molecules in a fashion where the hydrophobic part points towards the air and the hydrophilic group remains in contact with the aqueous subphase. Due to this property of Langmuir monolayer to orient the molecules at the air – water interface in a particular fashion, it can successfully serve as a template for two – dimensional reactions with restricted freedom. Hence, Langmuir monolayer has been extensively employed to study chemical and biological reactions at the air – water interface. To understand the behavior of Langmuir monolayer, surface pressure – molecular area (P – A) isotherms are studied as these P – A isotherms illustrate general conditions regarding the phase behavior of the two-dimensional Langmuir monolayer. Any change occurring due to the alignment of aliphatic molecules forming the monolayer is reflected by the change in P – A isotherms, which is known as phase transition. The phase transition is the most important element of the P – A isotherms with a characteristic signature of a plateau region in the isotherms. This phase transition point changes with the change of certain external parameters such as temperature, pH, and ionic strength, and as a result gives general information regarding the phase transition behavior. Therefore, with the little change of external parameters, the arrangement of the molecules in the monolayer also changes, which is reflected in the change in the nature of the isotherms. Thus, the system can, in principle, be used to define several physical parameters associated with it. On account of the property of Langmuir monolayer to orient the molecules at the air – water interface with restricted mobility and due to their condensed nature known as solid like phase, it closely mimics the situation inside a biological cell. Hence, we wanted to test whether an artificial nucleus can be generated at LB films. This can be achieved by immobilizing DNA or protein at the air – water interface and then by promoting their biological properties through macromolecular recognition. Here, immobilization of a macromolecule of biological relevance, its interaction with another component of a cell and extracting the thermodynamic parameters utilizing the LB technique will be of significance. This thesis embodies the immobilization of some biologically important proteins then follows their activity as well as DNA recognition properties at the air – water interface. A set of equations are derived here for the two dimensional Langmuir monolayer, which are used to calculate the thermodynamics of the system under study. Chapter 1 outlines the information about Langmuir monolayer and LB films. It sketches the historical background of the Langmuir monolayer and also elucidates the theory behind the same. This chapter cites the technical details of formation of Langmuir monolayer and LB films viś – a – viś other methods available for the fabrication of monomolecular films. It adequately discusses the functional LB films and their utilization for various different purposes. Finally, the role of metal ions in the LB films and in immobilizing biological macromolecules is discussed. Chapter 2 discusses the different techniques employed to perform the experiments described in this thesis. It includes the purification methods for the different proteins and DNA; the details of formation of Langmuir monolayer and fabrication of LB films. This chapter also describes the various techniques used for the characterization of the LB films, i.e Atomic Force Microscopy (AFM) and Fourier Transform Infrared (FTIR) spectroscopy. In Chapter 3, immobilization and imaging of protein molecules and protein DNA complexes on a LB substrate have been explored. Firstly, we describe the preparation of a Ni (II) – arachidate (NiA) monolayer and its characterization through P – A isotherm on a LB trough. Then, recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the NiA monolayer through a Ni (II) – histidine interaction. A single molecule of RNA polymerase (RNAP) could be seen through intermittent-contact AFM. Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specific DNA molecules from the subphase in an oriented fashion. In Chapter 4, the interaction between NiA and histidine tagged RNAP (HisRNAP), and RNAP and DNA were studied. LB films of Arachidic acid – NiA, NiA -HisRNAP and NiA – HisRNAP – DNA with different mole fractions were fabricated systematically. P -A isotherms were registered, and the excess Gibbs energy of mixing was calculated. The LB films were then deposited on solid supports for FTIR spectroscopic measurements. The FTIR spectra revealed the change in the amount of incorporated Ni (II) ions into the AA monolayer with the change in pH. The increase in mole fraction of RNAP and DNA in the NiA and NiA – RNAP monolayer, respectively, with their increasing concentration in the subphase are also noticed. The system developed here is robust and can be utilized to follow macromolecular interactions. In chapter 5, the Langmuir monolayer has been utilized to array a protein, Dps, specific for Fe (II) and non-specific for DNA. Dps from Mycobacterium smegmatis is known to have a cage like structure, exists in two oligomeric states, trimer and dodecamer, and can accommodate Fe (II) ions in its internal cavity. In addition, it converts Fe (II) to Fe (III), both in trimeric and dodecameric form, whereas the latter species is specific for non-specific DNA binding. We demonstrate here that, histidine tagged Dps in both oligomeric states can be immobilized on NiA LB films, where both ferroxidation and DNA binding ability remained unaffected in the ordered protein assembly. Interestingly, when Fe (II) – arachidate was used to generate a LB layer instead of NiA, Dps protein not only recognizes Fe (II) ion in the monolayer, it also converts it to Fe (III) ion in a time dependent fashion. However, once Fe (III) – Dps complex is formed and arrayed on LB monolayers, it remains very stable.

The work in this thesis can be split into three areas. In the first, inelastic electron tunnelling spectroscopy (IETS) is used to investigate resistive and bias polarity dependent effects on vibrational mode energy in metal - aluminium oxide - metal tunnel junctions. It is shown that even when four point-probe techniques are used the ratio of width to thickness of the electrodes used in IET junctions has an effect on both the breadth and position of spectral lines. A simple treatment based upon work done by Giaever allows for such effects to be corrected. Work done on top metal and polarity effects in undoped IET junctions investigated the effect on the position of the 450 meV mode on reversing the applied bias. The investigation revealed that any dependence on the nature of the top-metal electrode was outside the accuracy of the work. Calculation of q/e for the hydroxyl group on alumina within the tunnel junction has been extended to include other electrode materials. A new and exciting facility within the university provided the impetus for the second area of work. A class 100 clean room housing a Langmuir Blodgett (LB) trough offered the opportunity to produce metal - insulator - Langmuir Blodgett film - metal tunnel junctions. It was realised at the outset that IETS using LB films would be difficult, previous workers had tried using a home-made tank with only limited success. However, the added sophistication of the new tank did not improve matters as was hoped. Although the results were disappointing, only a few junctions had resistances low enough to be usable in the spectrometer, the investigation produced some of the very few IET specra using junctions doped with LB films. The results also revealed the important role that imperfections and pin-holes play in the tunnelling process. The last area used IETS to investigate two commercially important and interesting polymers, hydrogels and polymeric electrolytes. Hydrogel have many applications in the field of implants, prosthetic, and cosmetics and have been studied and developed for many years. Polymeric electrolytes have many commercial applications especially in the field of solid-state batteries and conducting polymers. The way in which hydrogels swell as they absorb water is important, as is the way they adsorb onto a surface and much work has been done to investigate these characteristics using bulk samples. The study done by this group is the first to investigate the swelling and adsorption behaviour of a monolayer of the hydrogel poly 2-hydroxylethyl methacrylate. The results from both investigations indicate that ester cleavage occurs in p-HEMA and that water incorporated within a hydrogel has a limited structure with the first layer being thinner than the second and subsequent layers.