Die Schwingungsspektroskopie, besonders die Raman-Spektroskopie, stellt ein wichtiges Werkzeug für Untersuchungen von Biomineralien dar. Raman-Spektroskopie wurde zur Untersuchung der organischen und anorganischen Bestandteile von Schwammskeletten eingesetzt. Die Raman-Spektroskopie trug auch zur Charakterisierung von biomimetischen Silikat-Präzipitaten bei. Durch ortsaufgelöste Raman-Spektroskopie konnte erstmalig die Verteilung von organischem Material in den extrahierten Silikatzellwänden von Kieselalgen nachgewiesen werden. Die ortsaufgelöste Raman-Spektroskopie wurde ebenfalls zur Untersuchung des SERS-Effekts an Zellwänden von Kieselalgen an die Silber-Nanopartikel gekoppelt waren eingesetzt.

The silica-based cell walls of diatoms are outstanding examples of nature’s capability to synthesize complex porous structures with genetically controlled patterns from the nanometer scale to the range of hundreds of micrometers. Formation of the cell wall building blocks (valves and girdle bands) occurs in membrane-bound compartments, termed silica deposition vesicles (SDVs), which are unique organelles in silica-forming protists. Isolation of the SDVs has not yet been achieved, which has severely hampered the efforts to understand the mechanisms of biological silica morphogenesis. The present thesis aimed to address this shortcoming. The foundation was the development of an improved cell cycle synchronization and a fluorescence labeling method for the model diatom Thalassiosira pseudonana that enabled rapid identification of valve SDVs in a cell lysate. Correlative fluorescence and electron microscopy allowed visualizing the development of valve silica with unprecedented spatio-temporal resolution. Elemental analysis and demineralization of immature valves provided the first direct chemical evidence that silica morphogenesis is an interplay of inorganic and organic molecules inside the valve SDVs. Cryo TEM imaging of valve SDVs indicated the formation of organic patterns that precede silica depostion. From these observations, an organic biomolecule dependent, liquid-liquid phase separation based model for pore formation in the diatom T. pseudonana was proposed. The second part of this thesis was focused on the enrichment of valve SDVs from T. pseudonana and the subsequent proteomics based identification of more than 40 potential valve SDV proteins. Among these, three diatom-specific proteins contained conserved protein protein interaction domains (ankyrin-repeats) and were surprisingly predicted to be located in the cytoplasm. The fluorescent tagging of the three proteins (termed dANK1-3) confirmed their association with the valve SDVs. When the respective dank genes were knocked out by CRISPR/Cas9, the valves displayed permanent anomalies in the quantity and the pattern of ~22 nm sized pores. Double knockout mutants lacking both dank1 and dank3 were almost completely devoid of pores. The analysis of valve morphogenesis in the single and double knockout mutants revealed phenotypic changes that were consistent with the liquid-liquid phase separation based model for pore pattern formation in diatom biosilica. The work of this thesis has provided for the first time direct access to valve SDVs, which has opened entirely new possibilities for studying the composition, properties, and working mechanism of an organelle that forms a complex shaped mineral.

Estruturas da glândula pineal humana foram estudadas empregando os métodos de microscopia de luz, microscopia eletrônica de varredura, microscopia de varredura por espectrometria de raio-x e difração de raio-x. As peças para microscopia de luz foram fixadas em solução de formalina a 10% durante 48h e incluídas em parafina. Para a microscopia eletrônica de varredura, as peças foram fixadas em solução de Karnovsky modificada, sendo que parte das peças foram fraturadas em nitrogênio líquido para o exame das características internas do corpo píneal. Os resultados evidenciaram que o corpo píneal apresenta formações calcáreas distribuídas no interior do tecido conjuntivo. As formações calcáreas possuem tamanhos e formas diferentes. As estruturas calcáreas apresentam uma cápsula constituída pelo tecido conjuntivo. Na porção interna, a estrutura calcárea é constituída por uma série de !ameias concêntricas, com porosidade de aspecto amorfo. Evidenciou-se pela análise de difração de raio-x, a estrutura cristalina formada pelos átomos de vários elementos que compõem a formação calc área . / The structure of human pineal gland was studied by optic microscopy, scanning eletromicroscopy, spectrometer x-ray scanning eletron microscopy with energy dispersion (EDS). The tissues to analysis by optic microscopy were fixed in formalin at 1O % during 48 h and put in parffin. For S.E.M., the tissues were fixed in modified Kamovsky solution, and a part of the material was fracturated in liquid nitrogen to analysis of internal characteristic of the pineal body. The results showed that the pineal body has calcareon concretion distributed in the connective tissue. In the internai position, the calcareon structure was made of concentrics lamelae with amorf aspect. The x-ray difractor showed the cristalin structure of the atom arrangement of the elements that compose the calcarean concretions.

L’organisme humain présente de nombreuses constantes de régénération tissulaires et c’est cette caractéristique essentielle qui maintient l’équilibre physiologique. Toutefois, l’existence de lésions importantes provoquée par un déséquilibre interne ou externe peut empêcher l’organisme de s’auto-régénerer. Dans ce cas, l’application des biomatériaux développés pour des applications biomédicales peuvent améliorer le processus de guérison. Pour les applications en tissus durs, les biomatériaux doivent posséder des propriétés similaires aux matrices naturelles tant sur le plan biologique que physico-mécanique. Dans les applications en bioingénierie osseuse, les composites à base de collagène (Col) et d’hydroxiapatite (HA) sont devenus tellement performant qu’ils peuvent être classifiés comme des matériaux biomimétiques. Cette thèse propose la production d’une matrice 3-D poreuse à base d’HA et de Col (50:50wt%). Ce composite réticulé par le glutaraldéhyde a été caractérisé par des différentes techniques et servira de support pour la culture cellulaire. Des cellules estromales ostéoprogénitrices ont été cultivées dans un environnement statique et dynamique (deux vitesse de flux) et leurs capacités de colonisation ainsi que leurs comportements d’adhésion, de prolifération, de différentiation seront observés. A travers les résultats de diffraction de rayons X et de spectroscopie infrarouge, il est possible d’affirmer la présence dans la matrice collagène d’une phase minérale peu cristalline constituée par de l’hydroxiapatite carbonatée du type-B déficiente en calcium. La viabilité cellulaire a été fortement influencée par les systèmes de culture au cours des 21 jours. Les résultats du système dynamique en haute vitesse montrent une excellente capacité du composite à supporter les processus cellulaires. Les cellules sont capables d’adhérer, de proliférer et de coloniser la matrice tridimensionnelle. / The progress in Tissue Engineering area allows the development of biomaterials that mimic the properties of natural tissues. For biomedical applications in mineralized tissues, composites based on hydroxyapatite (HA) and collagen (Col) have presented good results when implanted in vivo. The aim of this work was to produce a 3-D matrix and to observe the cell behaviour when stromal cells are cultured in contact with HA-Col scaffold under static and dynamic conditions. For in vitro biological evaluation, osteoprogenitor human cells (Stro+1A cells) were grown and their colonization capacity and adhesion, proliferation and differentiation behaviour were quantified. Two perfusion flow rates (0,03ml/min and 0,3ml/min) were proposed for dynamic culture. The HA-Col composite was prepared by reorganization of Col fibrils simultaneously with HA crystal nucleation and precipitation from calcium and phosphate rich solutions. Afterwards, the composites were crosslinked and sterilized by gamma radiation. Stro+1A cells were inoculated (5x105 cells/sample) into the scaffolds and cultured over 21 days in a humid incubator at 37°C and 5% CO2. Infrared spectroscopy and X-ray diffraction results suggested a calcium-deficient hydroxyapatite as mineral phase. About cell culture, the cell number increased under higher flow rate dynamic culture. By scanning electron microscopy and histological sections, we observed cells adhered and spread inside colonized scaffolds.

This work resulted in the development of multiple distinct and novel methods of cheaply producing large numbers of biologically derived, complex, 3-dimensional microstructures in a multitude of possible compositions. The biologically derived structures employed in this work were diatoms, a type of single celled algae, which grow complex silica shells in species-specific shapes. Due to the wide diversity of naturally occurring diatom shapes (on the order of 105), and the flexibility in tailoring chemical compositions using the methods developed here, real potential exists for cheaply mass-producing industrially relevant quantities of controlled shape and size 3-d particles for the first time. The central theme of this research is the use of diatoms as a transient scaffold onto which a coating is applied. After curing the coating, and in some cases firing the coating to form ceramic, the diatom can be selectively etched away leaving a free standing replica of the original structure with the salient features of the pre-form intact, but now composed of a completely different material. Using this concept, specific methods were developed to suit various precursors. Dip coating techniques were used to create epoxy diatoms, and silicon carbide diatoms. The Sol-Gel method was used to synthesize zirconia diatoms in both the tetragonal and monoclinic phases. A multi step method was developed in which previously synthesized epoxy diatoms were used as a template for deposition of a silicon carbide precursor and then heat treated to produce a silicon carbide/carbon multi-component ceramic. A hydrothermal reaction was also developed to convert Titania diatoms to barium titanate by reaction with barium hydroxide. Finally, the device potential of diatom-derived structures was conclusively demonstrated by constructing a gas sensor from a single Titania diatom. Under suitable conditions, the sensor was found to have the fastest response and recovery time of any sensor of this type reported in the literature. Furthermore, this work has laid the groundwork for the synthesis of many other tailored compositions of diatoms, and provided several compositions for device creation.

No description available.

Submitted by Daniele Amaral (daniee_ni@hotmail.com) on 2016-09-15T12:52:47Z No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:50:05Z (GMT) No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:50:10Z (GMT) No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) / Made available in DSpace on 2016-09-16T19:50:14Z (GMT). No. of bitstreams: 1 TeseALL.pdf: 6666981 bytes, checksum: 706c82c0c9656012bd6e5a239f13e404 (MD5) Previous issue date: 2015-08-12 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Enamel formation is a two-step complex process by which proteins are first secreted to forming an extracellular matrix, followed by massive protein degradation simultaneously with the completion of mineralization. Excessive exposure to fluoride can disrupt this process and drive to a condition known as dental fluorosis. Recently it has been reported that genetic factors may influence the responses of mineralized tissues to fluoride, a phenomenon observed in A/J and 129P3/J mice strain. The present study aimed to map the protein profile of mouse enamel. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by two-dimensional electrophoresis and liquid chromatography, coupled to mass spectrometry. A total of 120 proteins were identified, from which 113 are well characterized, both experimentally and functionally. Seven proteins were classified as uncharacterized proteins. After functional enrichment, 9 proteins were significantly related to the terms "odontogenesis and tissue biomineralization”. Surprisingly, the COL1A1 and COL1A2 protein were found on secretory stage enamel of both strains. Protein interaction analysis showed interaction between the ENAM and other types of collagen. Another interesting finding was the possibility of uncharacterized sequence Q8BIS2 be an extracellular matrix protein involved on degradation of matrix proteins. These findings suggest that collagen is present on dental enamel and it seems to have a role in amelogensis. Furthermore, the existence of an new enzyme could be the key to elucidation of the mechanisms involved in the enamel biomineralization and the genetic susceptibility to dental fluorosis. / O desenvolvimento do esmalte dentário é um processo complexo no qual as proteínas da matriz do esmalte interagem para conduzir a deposição e o crescimento dos cristais de hidroxiapatita. Fatores como a exposição excessiva ao fluoreto podem atrapalhar esse processo, resultando em uma patologia conhecida como fluorose dentária. Recentemente tem sido relatado que fatores genéticos podem influenciar as respostas dos tecidos mineralizados ao fluoreto, fenômeno observado nas linhagens de camundongos A/J, tida como susceptível e 129P3/J, tida como resistente. Neste sentido, este estudo objetivou mapear as proteínas do esmalte de incisivos de camundongos A/J e 129P3/J, a fim de que se possa melhor entender os mecanismos moleculares envolvidos na patogênese da fluorose dentária. Para isso, amostras de matriz de esmalte foram obtidas de camundongos das linhagens A/J e 129P3/J e analisadas por eletroforese bidimensional e cromatografia liquida, associadas a espectrometria de massas. Foi identificada um total de 120 proteínas, das quais 113 são proteínas bem caracterizadas, tanto experimentalmente quanto funcionalmente, e 7 proteínas que foram classificadas como proteínas não caracterizadas. Após enriquecimento funcional utilizando ferramentas de bioinformática, verificou-se que 9 proteínas estavam significativamente relacionadas aos termos “odontogênese e biomineralização de tecidos”. Dentre essas, as proteínas COL1A1 e COL1A2 chamaram atenção, uma vez que o colágeno não é associado à amelogênese. As proteínas estavam presentes no esmalte em fase de secreção de ambas as linhagens, e, além disso, a análise de interação in silico mostrou a existência de interação física entre a enamelina e outros tipos de colágeno. Outro achado interessante foi a possibilidade da sequência não caracterizada Q8BIS2 ser uma proteína extracelular da matriz envolvida na degradação das proteínas de matriz. Em conclusão, os resultados levam a crer que o colágeno está presente no esmalte e é provável que tenha participação na amelogênese. Além disso, a existência de uma enzima ainda não caracterizada pode ser a chave para elucidação dos mecanismos envolvidos na biomineralização bem como na susceptibilidade genética à fluorose dentária. / 2008/03489-2 e 2009/53852-9

Estruturas da glândula pineal humana foram estudadas empregando os métodos de microscopia de luz, microscopia eletrônica de varredura, microscopia de varredura por espectrometria de raio-x e difração de raio-x. As peças para microscopia de luz foram fixadas em solução de formalina a 10% durante 48h e incluídas em parafina. Para a microscopia eletrônica de varredura, as peças foram fixadas em solução de Karnovsky modificada, sendo que parte das peças foram fraturadas em nitrogênio líquido para o exame das características internas do corpo píneal. Os resultados evidenciaram que o corpo píneal apresenta formações calcáreas distribuídas no interior do tecido conjuntivo. As formações calcáreas possuem tamanhos e formas diferentes. As estruturas calcáreas apresentam uma cápsula constituída pelo tecido conjuntivo. Na porção interna, a estrutura calcárea é constituída por uma série de !ameias concêntricas, com porosidade de aspecto amorfo. Evidenciou-se pela análise de difração de raio-x, a estrutura cristalina formada pelos átomos de vários elementos que compõem a formação calc área . / The structure of human pineal gland was studied by optic microscopy, scanning eletromicroscopy, spectrometer x-ray scanning eletron microscopy with energy dispersion (EDS). The tissues to analysis by optic microscopy were fixed in formalin at 1O % during 48 h and put in parffin. For S.E.M., the tissues were fixed in modified Kamovsky solution, and a part of the material was fracturated in liquid nitrogen to analysis of internal characteristic of the pineal body. The results showed that the pineal body has calcareon concretion distributed in the connective tissue. In the internai position, the calcareon structure was made of concentrics lamelae with amorf aspect. The x-ray difractor showed the cristalin structure of the atom arrangement of the elements that compose the calcarean concretions.

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren. / Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.

Les crinoïdes sont bien connus pour leurs fossiles, mais la biominéralisation de leurs stades larvaires n’est que peu documentée. La première partie du projet présente le développement des ossicules des trois stades larvaires du comatule Florometra serratissima : doliolaria, cystidienne et pentacrinoïde. Les ossicules du crinoïde démontraient de la plasticité phénotypique et de la désynchronisation dans leur développement. Les crinoïdes étant la classe basale des échinodermes modernes, ceci porte à croire que ces traits étaient aussi caractéristiques des échinodermes ancestraux et auraient joué un rôle dans la radiation hâtive et la grande disparité des échinodermes. Pour notre deuxième étude, comme les patrons de morphologie des crinoïdes et des autres échinodermes sont nombreux et sont régulés par des protéines spécifiques, nous avons vérifié la présence de quatre familles de protéines de la matrice de spicules (SMAP) connues chez les oursins dans les transcriptomes des autres échinodermes et d’autres deutérostomes. La famille des spicules matrix (SM) et l’anhydrase carbonique CARA7LA étaient absentes chez tout autre organisme que les oursins, les protéines spécifiques au mésenchyme (MSP130) étaient présentes en nombres différents chez tous les ambulacraires suggérant de multiples duplications et pertes, et les métalloprotéases étaient nombreuses chez chacun. Le développement des ossicules chez les échinodermes est un sujet qui a gagné en popularité au cours des dernières décennies, spécialement chez les oursins, et inclure les crinoïdes dans ce type d’étude permettra de nous renseigner sur l’origine et l’évolution des échinodermes modernes. / While the fossil record of crinoids is widespread and largely known, biomineralization of their larval stages is poorly documented. The first part of the project focuses on the ossicle development of the three larval stages of the feather star Florometra serratissima: doliolaria, cystidean and pentacrinoid. The ossicles of the crinoid showed phenotypic plasticity and asynchronous development. Crinoids form the basal class of living echinoderms; this prompts one to believe that these traits were also characteristic of the ancestral echinoderms and would have played a role in the early radiation and large disparity of the modern echinoderms. For the second study, as patterns of morphology of crinoids and of other echinoderms are numerous and are regulated by specific proteins, we verified the presence of four families of spicule matrix associated proteins (SMAPs) known among sea urchins in transcriptomes of the other echinoderms and deuterostomes. The family of spicule matrix (SMs) proteins and the carbonic anhydrase CARA7LA were absent in any other organism aside from sea urchins, mesenchyme specific proteins (MSP130s) were present in varying numbers in all ambulacrarians suggesting multiple duplications and losses and matrix metalloproteases were numerous in every organisms. The development of ossicles in echinoderms is a topic that has gained popularity in the last decades, especially in sea urchins, and including crinoids in this type of study will inform us about the origin and evolution of the modern echinoderms.