Ce travail de thèse décrit la préparation et l'étude des propriétés d'auto-assemblage de nouveaux amphiphiles fonctionnalisés par des sucres. Des glycosides propargyliques du lactose et de la N-acétyl-glucosamine ont été conjugués par chimie click (cycloaddition de Huisgen catalysée par des sels de cuivre) à des dérivés de poly(ethyleneglycol) dont une des extrémités a au préalable été modifiée par une fonction azide et l'autre par un bloc hydrophobe de type polyphénylène ou bien aliphatique. Après une caractérisation par résonance magnétique nucléaire et spectrométrie de masse, les propriétés d'auto-assemblage de ces amphiphiles ont été étudiées par diffusion dynamique de la lumière (DLS), diffraction des rayons-X aux petits angles (SAXS) et microscopie électronique. Il a été montré qu'en phase aqueuse, les systèmes amphiphiles dérivés du PEG 900 s'auto-assemblent pour former de micelles de taille extrêmement régulière dont le diamètre moyen est de l'ordre de 10 nm. La présence et la biodisponibilité des sucres à la surface de ces nanoparticules ont également pu être démontrées par diffusion dynamique de la lumière avec les lectines PNA et WGA. Les interactions spécifiques observées entre les lectines et micelles associées aux propriétés d'encapsulation de ce type de nanoparticules permettent d'imaginer de futures applications pour la délivrance de médicaments ou encore l'imagerie médicale. / This thesis reports the preparation and characterization of new rod-coil amphiphiles functionalized with oligo- and polysaccharides through Huisgen 1,3-dipolar cycloaddition reactions between species functionalized by an azide group on one side and an terminal alkyne on the other catalyzed by copper. The amphiphiles were synthesized and characterized based on different hydrophobic parts conjugated with the polymer poly(ethylene oxide) PEO with a hydrophilic spacer arm and the oligo- and polyssaccharides 2-propargyl-2-acetamido-2-deoxy-β-D-glucopyranose (GlcNAc) and propargyl β-D-galactopyranosyl-(14)-β-D-glucopyranose (Lac). The amphiphiles synthesized were characterized in terms of their chemical structure and composition through nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, mass spectroscopy (MALDI-TOF-MS and ESI-MS) and high resolution mass spectroscopy (HRMS). After the dissolution in water, the amphiphiles self-associate in highly regular micelles with an average diameter of 2RH ~ 10 nm. Dynamic light scattering (DLS), transmission electron microscopy (TEM) and small angle x-ray scattering (SAXS) were used in order to investigate the structure and dynamics of these saccharide amphiphiles. The presence of carbohydrate epitopes on the surface of the micelles and their bioavailability for the segmentation of lectin were also demonstrated by DLS. Specific interactions of GlcNAc and Lac residues with lectins from wheat germ agglutinin (WGA) and peanut agglutinin (PNA), respectively, reveal the potential applications of such amphiphilic derivatives of carbohydrates as vectorizing systems, both simple and specific to a drug delivery site.

Ce travail de thèse décrit la préparation et l'étude des propriétés d'auto-assemblage de nouveaux amphiphiles fonctionnalisés par des sucres. Des glycosides propargyliques du lactose et de la N-acétyl-glucosamine ont été conjugués par chimie click (cycloaddition de Huisgen catalysée par des sels de cuivre) à des dérivés de poly(ethyleneglycol) dont une des extrémités a au préalable été modifiée par une fonction azide et l'autre par un bloc hydrophobe de type polyphénylène ou bien aliphatique. Après une caractérisation par résonance magnétique nucléaire et spectrométrie de masse, les propriétés d'auto-assemblage de ces amphiphiles ont été étudiées par diffusion dynamique de la lumière (DLS), diffraction des rayons-X aux petits angles (SAXS) et microscopie électronique. Il a été montré qu'en phase aqueuse, les systèmes amphiphiles dérivés du PEG 900 s'auto-assemblent pour former de micelles de taille extrêmement régulière dont le diamètre moyen est de l'ordre de 10 nm. La présence et la biodisponibilité des sucres à la surface de ces nanoparticules ont également pu être démontrées par diffusion dynamique de la lumière avec les lectines PNA et WGA. Les interactions spécifiques observées entre les lectines et micelles associées aux propriétés d'encapsulation de ce type de nanoparticules permettent d'imaginer de futures applications pour la délivrance de médicaments ou encore l'imagerie médicale.

Oligo array is a high throughput technology and is widely used in many scopes of biology and medical researches for quantitative and highly parallel measurements of gene expression. When one faulty step occurs during the synthesis process, it affects all probes using the faulty step. In this thesis, a two-phase genetic algorithm (GA) is proposed to design optimal quality control of oligo array for detecting any single faulty step. The first phase performs the wide search to obtain the approximate solutions and the second phase performs the local search on the approximate solutions to achieve the optimal solution. Besides, the proposed algorithm could hold many non-duplicate individuals and parallelly search multiple regions simultaneously. The superior searching capability of the two-phase GA helps us to find out the 275 nonequireplicate cases that settled by the hill-climbing algorithm. Furthermore, the proposed algorithm also discovers five more open issues.

Implantes de titânio têm sido extensivamente utilizados na Ortopedia e na Odontologia, principalmente como substitutos de elementos dentários ausentes. O titânio é um implante metálico de escolha devido à sua alta biocompatibilidade e resistência à corrosão, e também porque não provoca reações imunológicas, ao mesmo tempo em que promove a osseointegração. A biocompatibilidade do implante depende da resposta celular em contato com a sua superfície; sendo assim, mudanças nesta superfície podem provocar impactos benéficos na osseointegração através de alterações nas interações com as células presentes no local do implante, como, por exemplo, células osteoblásticas. O objetivo do presente trabalho foi caracterizar a resposta celular de células osteoblásticas humanas provenientes da crista óssea alveolar em contato com diferentes superfícies de titânio: controle (polido), nanotextura, nano+submicrotextura e microtextura rugosa. Os ensaios bioquímicos realizados foram: proliferação e viabilidade celular, quantidade de proteína total e atividade de fosfatase alcalina, além da detecção e quantificação de nódulos mineralizados, com a utilização do teste estatístico não paramétrico de Kruskal-Wallis e de Mann-Whitney com p≤0,05. Também foi realizada a avaliação da modulação gênica nas células em contato com as diferentes superfícies de titânio por meio do método de oligo microarray, utilizando lâminas Agilent formato 4x44 K e análise de microRNAs utilizando lâminas Agilent 8x15 K. A fim de identificar alterações na expressão gênica foi utilizado o programa GeneSpring GX. A expressão gênica foi validada pela reação de PCR quantitativa em tempo real (qRT-PCR). Observamos um pico na proliferação celular aos 10 dias de cultura e um aumento gradual da viabilidade celular ao longo do tempo. Entre as superfícies tratadas observou-se maior quantidade de proteína total na nanotextura em relação à nano+submicrotextura aos 10 dias de cultura (p≤0,05) e um aumento progressivo da atividade de fosfatase alcalina, com maior atividade na nanotextura em relação à nano+submicrotextura aos 14 dias de cultura (p≤0,05). Não houve diferença qualitativa na formação dos nódulos mineralizados, apesar de a microtextura rugosa apresentar maior quantidade de cálcio que a nanotextura (p≤0,05). Os resultados encontrados evidenciaram expressão diferenciada de 716 mRNAs (fold change≥2,0 e p≤0,05) e 32 microRNAs (fold change≥1,5 e p≤0,01) com funções associadas ao processo de osteogênese, principalmente mineralização, adesão celular, apoptose, proliferação e diferenciação celular. Os resultados sugerem que, diante do protocolo utilizado neste trabalho, o tratamento químico realizado na superfície do titânio provoca variações no metabolismo de células osteoblásticas tanto em nível celular como de expressão gênica / Titanium implants have been extensively used in orthopedics and dentistry, mainly as a replacement for missing teeth. Titanium is the metal implant of choice due to its high biocompatibility and corrosion resistance, as well as absence of immune response, while promoting osseointegration. The biocompatibility of the material depends on cellular response in contact with the surface; therefore, changes on this surface can have beneficial impacts on osseointegration through changes in interactions with cells at the implant site, such as osteoblastic cells. The objective of this study was to characterize the cellular response of osteoblastic cells from human alveolar crest in contact with different titanium surfaces: control (polished), nanotextured, nano+submicrotextured and rough microtexture. The performed biochemical assays included cell proliferation and viability, total protein content and alkaline phosphatase activity, in addition to the detection and quantification of mineralized nodules, using the non-parametric statistical tests of Kruskal-Wallis and Mann-Whitney (p≤0,05). Osteoblastic gene modulation was evaluated by means of an oligo microarray method using Agilent format 4 x 44 K slides and Agilent 8x15 K slides for microRNAs analysis. In order to identify changes in gene expression, it was used the GeneSpring GX program. Gene expression was validated by quantitative PCR real time (qRT-PCR). It was observed a peak in cell culture proliferation at 10 days and a gradual increase in cell viability over the periods. Among the treated surfaces, we observed an increase in the amount of total protein in nanotextured when compared to nano+submicrotextured at 10 days of culture (p≤0,05), and a progressive increase of alkaline phosphatase activity, with higher activity in nanotextured compared to nano+submicrotextured at 14 days of culture (p≤0,05). There was no qualitative difference among the groups with regards to mineralized nodules, although the rough microtexture group showed higher amounts of calcium than the nanotextured group (p≤0,05). Our results showed a differential expression of 716 mRNAs (fold change≥2,0 and p≤0,05) and 32 microRNAs (fold change≥1,5 and p≤0,01), with functions associated to the osteogenesis process, mainly mineralization, cell adhesion, apoptosis, cell proliferation and differentiation. The results suggest that, with the protocols used in this investigation, the treatments performed in the titanium surface induce changes in the metabolism of osteoblastic cells, both at the cellular as well as at the gene expression levels

Implantes de titânio têm sido extensivamente utilizados na Ortopedia e na Odontologia, principalmente como substitutos de elementos dentários ausentes. O titânio é um implante metálico de escolha devido à sua alta biocompatibilidade e resistência à corrosão, e também porque não provoca reações imunológicas, ao mesmo tempo em que promove a osseointegração. A biocompatibilidade do implante depende da resposta celular em contato com a sua superfície; sendo assim, mudanças nesta superfície podem provocar impactos benéficos na osseointegração através de alterações nas interações com as células presentes no local do implante, como, por exemplo, células osteoblásticas. O objetivo do presente trabalho foi caracterizar a resposta celular de células osteoblásticas humanas provenientes da crista óssea alveolar em contato com diferentes superfícies de titânio: controle (polido), nanotextura, nano+submicrotextura e microtextura rugosa. Os ensaios bioquímicos realizados foram: proliferação e viabilidade celular, quantidade de proteína total e atividade de fosfatase alcalina, além da detecção e quantificação de nódulos mineralizados, com a utilização do teste estatístico não paramétrico de Kruskal-Wallis e de Mann-Whitney com p≤0,05. Também foi realizada a avaliação da modulação gênica nas células em contato com as diferentes superfícies de titânio por meio do método de oligo microarray, utilizando lâminas Agilent formato 4x44 K e análise de microRNAs utilizando lâminas Agilent 8x15 K. A fim de identificar alterações na expressão gênica foi utilizado o programa GeneSpring GX. A expressão gênica foi validada pela reação de PCR quantitativa em tempo real (qRT-PCR). Observamos um pico na proliferação celular aos 10 dias de cultura e um aumento gradual da viabilidade celular ao longo do tempo. Entre as superfícies tratadas observou-se maior quantidade de proteína total na nanotextura em relação à nano+submicrotextura aos 10 dias de cultura (p≤0,05) e um aumento progressivo da atividade de fosfatase alcalina, com maior atividade na nanotextura em relação à nano+submicrotextura aos 14 dias de cultura (p≤0,05). Não houve diferença qualitativa na formação dos nódulos mineralizados, apesar de a microtextura rugosa apresentar maior quantidade de cálcio que a nanotextura (p≤0,05). Os resultados encontrados evidenciaram expressão diferenciada de 716 mRNAs (fold change≥2,0 e p≤0,05) e 32 microRNAs (fold change≥1,5 e p≤0,01) com funções associadas ao processo de osteogênese, principalmente mineralização, adesão celular, apoptose, proliferação e diferenciação celular. Os resultados sugerem que, diante do protocolo utilizado neste trabalho, o tratamento químico realizado na superfície do titânio provoca variações no metabolismo de células osteoblásticas tanto em nível celular como de expressão gênica / Titanium implants have been extensively used in orthopedics and dentistry, mainly as a replacement for missing teeth. Titanium is the metal implant of choice due to its high biocompatibility and corrosion resistance, as well as absence of immune response, while promoting osseointegration. The biocompatibility of the material depends on cellular response in contact with the surface; therefore, changes on this surface can have beneficial impacts on osseointegration through changes in interactions with cells at the implant site, such as osteoblastic cells. The objective of this study was to characterize the cellular response of osteoblastic cells from human alveolar crest in contact with different titanium surfaces: control (polished), nanotextured, nano+submicrotextured and rough microtexture. The performed biochemical assays included cell proliferation and viability, total protein content and alkaline phosphatase activity, in addition to the detection and quantification of mineralized nodules, using the non-parametric statistical tests of Kruskal-Wallis and Mann-Whitney (p≤0,05). Osteoblastic gene modulation was evaluated by means of an oligo microarray method using Agilent format 4 x 44 K slides and Agilent 8x15 K slides for microRNAs analysis. In order to identify changes in gene expression, it was used the GeneSpring GX program. Gene expression was validated by quantitative PCR real time (qRT-PCR). It was observed a peak in cell culture proliferation at 10 days and a gradual increase in cell viability over the periods. Among the treated surfaces, we observed an increase in the amount of total protein in nanotextured when compared to nano+submicrotextured at 10 days of culture (p≤0,05), and a progressive increase of alkaline phosphatase activity, with higher activity in nanotextured compared to nano+submicrotextured at 14 days of culture (p≤0,05). There was no qualitative difference among the groups with regards to mineralized nodules, although the rough microtexture group showed higher amounts of calcium than the nanotextured group (p≤0,05). Our results showed a differential expression of 716 mRNAs (fold change≥2,0 and p≤0,05) and 32 microRNAs (fold change≥1,5 and p≤0,01), with functions associated to the osteogenesis process, mainly mineralization, cell adhesion, apoptosis, cell proliferation and differentiation. The results suggest that, with the protocols used in this investigation, the treatments performed in the titanium surface induce changes in the metabolism of osteoblastic cells, both at the cellular as well as at the gene expression levels

Data management and analysis represent a major challenge for microarray studies. In this study, Affymetrix software was used to analyze an HIV-infection data. The microarray analysis shows remarkably different results when using different parameters provided by the software. This highlights the fact that a standardized analysis tool, incorporating biological information about the genes is needed in order to better interpret the microarray study. To address the data management problem, in-house programs, including scripts and a database, were designed. The in-house programs were also used to overcome problems and inconveniences discovered during the data analysis, including management of the gene lists. The database provides rapid connection to many online public databases, as well as the integration of the original microarray data, relevant publications and other useful information. The in-house programs allow investigators to process and analyze the full Affymetrix microarray data in a speedy manner.

Tierärztliche Hochsch., Diss., 2005--Hannover.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Vita. Includes: Comparison of the [alpha]-glucosides of Saccharomyces produced in response to five non-allelic maltose genes / Harlyn O. Halvorson, Sara Winderman and John Gorman. Reprinted from Biochimica et biophysica acta, vol. 67 (1963), p. 42-53 -- Relationship between protein and nucleic acid synthesis in Pseudomonas azotogensis grown in hexetidine / John Gorman and Harlyn Halvorson. Reprinted from Archives of biochemistry and biophysics, vol. 84, no. 2 (Oct. 1959), p. 462-470 -- The abnormal pattern of protein synthesis in Pseudomonas azotogensis in the presence of hexetidine / H.O. Halvorson and John Gorman. Reprinted from Experimental cell research, vol. 17, p. 522-524. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Polymers which are sensitive towards external physical, chemical and electrical stimuli are termed as ‘intelligent materials’ and are widely used in medical and engineering applications. Presently, polymers which can undergo a physical change when heat is applied at a certain temperature (cloud point) in water are well-studied for this property in areas of separation chemistry, gene and drug delivery and as surface modifiers. One example of such a polymer is the poly (N-isopropylacrylamide) PNIPAAM, where it is dissolved well in water below 32 oC, while by increasing the temperature further leads to its precipitation. In this work, an alternative polymer poly (2-(2-methoxy ethoxy)ethyl methacrylate-co- oligo(ethylene glycol) methacrylate) (P(MEO2MA-co-OEGMA)) is studied due to its biocompatibility and the ability to vary its cloud points in water. When a layer of temperature responsive polymer was attached to a single continuous porous piece of silica-based material known as a monolith, the thermoresponsive characteristic was transferred to the column surfaces. The hybrid material was demonstrated to act as a simple temperature ‘switch’ in the separation of a mixture of five steroids under water. Different analytes were observed to be separated under varying column temperatures. Furthermore, more complex biochemical compounds such as proteins were also tested for separation. The importance of this work is attributed to separation processes utilizing environmentally friendly conditions, since harsh chemical environments conventionally used to resolve biocompounds could cause their biological activities to be rendered inactive. / Polymere, welche empfindlich gegenüber externen physikalischen, chemischen und elektrischen Einflüssen sind, werden „intelligente Materialien“ genannt. Diese werden weitverbreitet in medizinischen und technischen Anwendungen eingesetzt. Auf diesem Gebiet ausführlich erforschte Materialien sind Polymere, welche durch Hitze bei einer bestimmten Temperatur (Trübungspunkt) eine physikalische Veränderung eingehen können, genannt thermoresponsive Polymere. Eingesetzt werden diese z.B. in chromatographischen Trennverfahren, in Gen- und Wirkstofftransport Vorgängen und zur Oberflächenmodifikation. Ein Beispiel für so ein Polymer ist das poly(N-isopropylacrylamide) PNIPAAM, welches unter 32 °C in Wasser gelöst vorliegt und mit Erhöhung der Temperatur als Niederschlag ausfällt. In dieser Arbeit wurde ein alternatives Polymer, das poly(2-(2-methoxyethoxy)ethylmethacrylate-co-oligo(ethyleneglycol) methacrylate) (P(MEO2MA-co-OEGMA)), untersucht, in Bezug auf Biokompatibilität und der Änderung des Trübungspunktes in Wasser. Wenn eine Schicht eines temperaturempfindlichen Polymers auf einen Monolithen (einteiliger, poröser und auf Silika-basierendes Material) aufgebracht wird, werden die thermoresponsiven Eigenschaften auf die Oberfläche dieses Monolithen übertragen. Der Monolith dient hier als Säule in einer HPLC-Anlage. Es wurde gezeigt, dass das Hybrid-Material als einfacher „Temperaturschalter“ in der Trennung von fünf verschiedenen Steroiden in Wasser agieren kann. Untersucht wurde die Separation verschiedener Analyten mit dem Variieren der Säulentemperatur. Zusätzlich wurden mehr komplexe biochemische Stoffe, wie Proteine, getestet. Die Bedeutung dieser Arbeit ist zurückzuführen auf Separationsprozesse, welche umweltfreundlichen Bedingungen nutzen, da die rauen chemischen Bedingungen in konventionellen Separationsprozessen die biologische Inaktivität der Verbindungen zur Folge haben können. Der zweite Teil der Arbeit beschäftigte sich mit der Entwicklung eines alternativen Trägermaterials als Ersatz zu den Silika-basierende Trennungssäulen. Kohlenstoffmaterialien sind aufgrund ihrer ausgezeichneten mechanischen Härte und chemischen Stabilität eine vielversprechend Alternative. Die Synthese von Kohlenstoffkugeln als Trägermaterial kann als „grüner“ Prozess in meiner Arbeit angesehen werden, da milde Synthesebedingungen in purem Wasser verwendet wurden. Die Leistungsfähigkeit des Materials wurde mit einer Serie von Separationsreaktionen gezeigt.

Die Immobilisierung von Oligo(ethylenglykol)-derivaten an Oberflächen von Metallen ist ein viel versprechender Ansatz, um unspezifische Adsorptionen von Proteinen, Bakterien und Zellen zu minimieren bzw. zu verhindern. Im Mittelpunkt der Arbeit stand die Entwicklung, Darstellung, Charakterisierung sowie Applikation maßgeschneiderter, self-assembly-fähiger Moleküle, die gezielt auf TiO2- und nicht auf SiO2-Oberflächen anbinden. Die resultierenden Monoschichten (SAMs) wiesen eine Biokompatibilität sowie Biofunktionalität auf. Dazu wurden neue bisher noch nicht beschriebene Moleküle entwickelt, die auf einer Kombination von funktionalisierten Oligo(ethylenglykol)-Einheiten mit Monoalkylphosphorsäure- und Alkylphosphonsäurederivaten basieren. Diese Verbindungen konnten durch die Anwendung der Self-Assembly-Technik erfolgreich aus wässriger Lösung auf TiO2-Substrate adsorbiert werden. Die hergestellten, ultradünnen monomolekularen Schichten wurden mit verschiedenen analytischen Methoden, wie Spektroskopische Ellipsometrie, winkelabhängiger XPS und SPR-Spektroskopie charakterisiert. Durch eine gezielte Anbindung an TiO2-Oberflächen und einer stabilen Ausbildung von SAMs konnten sowohl die unspezifische Proteinadsorption zurückgedrängt bzw. verhindert, als auch eine spezifische Anbindung von ausgewählten Proteinen realisiert werden. / The surface immobilization of oligo (ethylene glycol) on metals is a promising approach to minimize or prevent non-specific adsorption of proteins, bacteria and cells. The aim of this work was the design, preparation, characterization and application of tailor-made, self-assembly molecules, which are able to adsorbed selectively on TiO2 surfaces but not on SiO2. The resulting self-assembled monolayers (SAMs) had a biocompatibility and bio functionality. For this purpose new molecules have been developed, which are not described in the literature. These compounds are derivatives of monoalkyl phosphoric acids or alkyl phosphonic acids and contain a terminal functional oligo (ethylene glycol) unit. The compounds were assembled on the TiO2-surface by self-assembly technique from aqueous solution. The adsorbed layers were characterized by different analytical tools, like angle resolved XPS, spectroscopic ellipsometry and SPR-spectroscopy. The selective adsorption of SAMs on TiO2-surfaces and the formation of stable SAMs make it possible to prevent or minimize non specific protein adsorption and also to bind selected proteins via specific surface reactions.