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

Regulation of gene transcription plays a major role in mediating cellular responses and physiological behavior in all known organisms. The finding that similar genes are often regulated in a similar manner (co-regulated or "co-expressed") has directed several "guilt-by-association" approaches in order to reverse-engineer the cellular transcriptional networks using gene expression data as a compass. This kind of studies has been considerably assisted in the recent years by the development of high-throughput transcript measurement platforms, specifically gene microarrays and next-generation sequencing. In this thesis, I describe several approaches for improving the extraction and interpretation of the information contained in microarray based gene expression data, through four steps: (1) microarray platform design, (2) microarray data normalization, (3) gene network reverse engineering based on expression data and (4) experimental validation of expression-based guilt-by-association inferences. In the first part test case is shown aimed at the generation of a microarray for Thellungiella salsuginea, a salt and drought resistant close relative to the model plant Arabidopsis thaliana; the transcripts of this organism are generated on the combination of publicly available ESTs and newly generated ad-hoc next-generation sequencing data. Since the design of a microarray platform requires the availability of highly reliable and non-redundant transcript models, these issues are addressed consecutively, proposing several different technical solutions. In the second part I describe how inter-array correlation artifacts are generated by the common microarray normalization methods RMA and GCRMA, together with the technical and mathematical characteristics underlying the problem. A solution is proposed in the form of a novel normalization method, called tRMA. The third part of the thesis deals with the field of expression-based gene network reverse engineering. It is shown how different centrality measures in reverse engineered gene networks can be used to distinguish specific classes of genes, in particular essential genes in Arabidopsis thaliana, and how the use of conditional correlation can add a layer of understanding over the information flow processes underlying transcript regulation. Furthermore, several network reverse engineering approaches are compared, with a particular focus on the LASSO, a linear regression derivative rarely applied before in global gene network reconstruction, despite its theoretical advantages in robustness and interpretability over more standard methods. The performance of LASSO is assessed through several in silico analyses dealing with the reliability of the inferred gene networks. In the final part, LASSO and other reverse engineering methods are used to experimentally identify novel genes involved in two independent scenarios: the seed coat mucilage pathway in Arabidopsis thaliana and the hypoxic tuber development in Solanum tuberosum. In both cases an interesting method complementarity is shown, which strongly suggests a general use of hybrid approaches for transcript expression-based inferences. In conclusion, this work has helped to improve our understanding of gene transcription regulation through a better interpretation of high-throughput expression data. Part of the network reverse engineering methods described in this thesis have been included in a tool (CorTo) for gene network reverse engineering and annotated visualization from custom transcription datasets. / Die Regulation der Gentranskription spielt eine wichtige Rolle bei der Steuerung des physiologischen Verhaltens in allen Organismen. Dass ähnliche Gene oft in gleicher Weise reguliert werden (koreguliert oder koexpimiert), hat zu diversen „guilt-by-association“-Ansätzen zur Rekonstruktion von zellulären Transkriptionsnetzwerken geführt, die Genexpressionsdaten zur Orientierung nutzen. Studien dieser Art wurden in den letzten Jahren durch die Entwicklung von Hochdurchsatzmessungen von Transkriptmengen mittels Mikroarrays und ‚Next Generation‘ Sequenziertechniken stark gefördert. In der vorliegenden Arbeit werden verschiedene Ansätze zur Verbesserung der Extraktion und Interpretation von Mikroarray-basierten Genexpressionsdaten in vier Schritten beschrieben: (1) Mikroarray-Sonden-Design, (2) Mikroarray Datennormalisierung, (3) Rekonstruktion von Gennetzwerken unter Verwendung von Expressionsdaten und (4) experimentelle Überprüfung von expressionsbasierten „guilt-by-association“ Schlussfolgerungen. Im ersten Teil wird ein Beispiel zur Erstellung eines Mikroarrays für Thelungiella salsuginea gezeigt, einem salz- und trockenresistenten Verwandten von Arabidopsis thaliana. Zur Rekonstruktion der Transkripte wurden sowohl öffentliche ESTs (‚expressed sequence tags‘) als auch neu erzeugte ‚Next Generation‘ Sequenzierdaten genutzt. Da das Design von Mikroarrays speziesspezifische, nicht-redundante Transkriptmodelle erfordert, werden diese Aufgaben nacheinander abgearbeitet und verschiedene technische Lösungsmöglichkeiten aufgezeigt. Im zweiten Teil wird beschrieben, wie übliche Mikroarray-Normalisierungsverfahren wie RMA und GCRMA zu Korrelationsartefakten führen können. Technische sowie mathematische Hintergründe werden erläutert und zur Lösung des Problems wird mit tRMA eine neue Normalisierungsmethode vorgestellt. Der dritte Teil der Arbeit beschäftigt sich der expressionsbasierten Rekonstruktion von Gennetzwerken. Es wird demonstriert, wie dabei verschiedene „Zentralitäten“ bei zur Unterscheidung von spezifischen Genklassen, hier beispielhaft essentielle Gene von Arabidopsis thaliana, genutzt werden können und wie die Verwendung von konditioneller Korrelation tieferes Verständnis des der Transkriptionsregulation zugrundeliegenden Informationsflusses ermöglicht. Weiterhin werden Ansätze zur Netzwerkrekonstruktion verglichen. Besonderes Augenmerk liegt dabei auf der LASSO Technik, einer Art linearer Regression, die trotz ihren theoretischen Vorteilen in Robustheit und Interpretierbarkeit gegenüber Standardmethoden bisher selten zur Rekonstruktion von globalen Gennetzwerken genutzt wurde. Die Leistungsfähigkeit von LASSO wird durch in silico Analysen der Zuverlässigkeit der erstellten Gennetzwerke gemessen. Im letzten Teil der Arbeit wurden LASSO und andere Rekonstruktionsmethoden genutzt um experimentell neue Gene der folgenden zwei Szenarien zu identifizieren: im Samenschleim von Arabidopsis thaliana und während der Knollenentwicklung von Solanum tuberosum unter Sauerstoffmangel. In beiden Fällen wird eine interessante Methodenkomplementarität gezeigt, nach welcher eine Mischung mehrerer Ansätze zu empfehlen ist um Schlüsse aufgrund von Transkriptexpression zu ziehen. Zusammenfassend zielt diese Arbeit darauf ab, das Verständnis der Regulation von Gentranskriptionsnetzwerken durch bessere Interpretation von Hochdurchsatzexpressionsdaten zu verbessern. Ein Teil der in dieser Arbeit beschriebenen Methoden wurden im Programm CorTo zur Gennetzwerkrekonstruktion und annotierten Visualisierung von benutzerdefinierten Transkriptionsdaten verarbeitet.

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

O diabetes mellitus gestacional (DMG) é o distúrbio metabólico mais comum da gravidez. A definição padrão do DMG consiste no metabolismo anormal da glicose diagnosticado pela primeira vez durante a gestação. Mulheres que têm história de DMG geralmente apresentam diabetes pós-parto, resistência à insulina, síndrome metabólica, hipertensão e dislipidemia. A detecção precoce deste estado metabólico anormal é importante para eventual intervenção na tentativa de impedir ou mesmo retardar o aparecimento dos outros tipos de diabetes. Alguns estudos têm apontado, em mulheres com DMG, indução de genes envolvidos com resposta imune, particularmente aqueles associados com inflamação. A identificação de genes de inflamação induzidos em gestantes com DMG tem fornecido a base para elucidar a ligação entre vias inflamatórias e DMG. Para testar esta hipótese foi realizada a comparação do perfil transcricional de células mononucleares de sangue periférico (PBMCs) de pacientes com DMG e controles. As amostras de RNA total foram hibridadas utilizando oligo microarrays Agilent ® 4 x 44 K englobando o genoma funcional humano total. Os mRNAs diferencialmente expressos foram identificados aplicando-se a análise de Rank Products, e posteriormente submetidos ao agrupamento hierárquico de Pearson por meio do software Cluster. Utilizando o programa TreeView, foi realizada a construção dos dendrogramas com as representações espaciais dos mRNAs, classificados de acordo com suas funções moleculares e vias biológicas. A partir do banco de dados DAVID, foram identificados 130 processos biológicos significantes (P<0.05) incluindo os de resposta imune e defesa, resposta inflamatória, regulação de citocinas, apoptose, desenvolvimento de vasos sanguíneos e proliferação celular. Entre as vias de maior relevância destacamos a via de interação entre receptores de citocinas e a de sinalização do receptor NOD-like, além das vias de câncer, lúpus e asma. Adicionalmente, encontramos os transcritos dos genes IGFBP2, TCF3, OLR1, TCF7L2, previamente associados a alterações metabólicas, diferencialmente expressos nas gestantes com DMG. Também observamos que genes do complexo principal de histocompatibilidade (MHC), HLA-DRB6, HLA-DQA2, HLA-DQB2, HLA-DQB1, HLA-DOA, apresentaram mRNAs induzidos nas pacientes com DMG. A partir deste estudo, constatamos que vias relacionadas ao sistema imunológico e categorias funcionais associadas à inflamação participam da patogenia do DMG. Além disso, evidenciamos que transcritos de genes que pertencem ao MHC e aqueles envolvidos em processos metabólicos, estiveram diferencialmente expressos no DMG. Estes resultados confirmam nossa hipótese inicial e contribuem para o melhor entendimento das bases genéticas desta doença. / Gestational Diabetes Mellitus (GDM) is the most common metabolic disorder found during pregnancy. The standard definition of GDM is the abnormal glucose metabolism first diagnosed during pregnancy. Women who have a history of GDM usually present postpartum diabetes, insulin resistance, metabolic syndrome, hypertension and dyslipidemia. Early detection of this abnormal metabolic status may permit early intervention to prevent or even delay the development of other types of diabetes. The induction of genes involved in immune response in women with GDM has been reported, particularly those associated with inflammatory pathways, providing basis proposing that inflammation genes might be associated to GDM. To test this hypothesis, we compared the transcriptome profiling of peripheral blood mononuclear cells (PBMCs) of GDM patients and controls. The total RNA samples were hybridized to Agilent ® 4 x 44 K oligo microarrays covering the whole human functional genome. Differentially expressed mRNAs were obtained by Rank Product analysis and then submitted to hierarchical clustering using the Cluster software . Dendrograms and spatial representations of mRNAs were constructed through the TreeView software . These mRNAs were classified according to their molecular functions and biological pathways using the DAVID database. We observed 130 significant biological processes (P<0.05), including immune and defense response, inflammatory response, regulation of cytokines, apoptosis, blood vessels development and cell proliferation. Among the most relevant pathways, we highlighted the interaction between cytokine receptors, NOD-like receptor signaling and cancer, lupus and asthma pathways. Additionally, we found transcripts of the genes IGFBP2, TCF3, OLR1, TCF7L2, which were previously associated with metabolic abnormalities, differentially expressed in pregnant women with GDM. Some major histocompatibility complex (MHC) genes (HLA-DRB6, HLA-DQA2, HLA-DQB2, HLA-DQB1, HLA-DOA) also presented mRNAs induced in patients with GDM. In conclusion, we found that immunerelated pathways and functional categories associated with inflammation participate in the pathogenesis of DMG. Furthermore, we showed that transcripts of genes belonging to MHC and those involved in metabolic processes were differentially expressed in DMG. These results confirmed our initial hypothesis and contribute to a better understanding of the genetics basis of this disease.

published_or_final_version / abstract / Mathematics / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Photoreactive groups have been used in photoaffinity labelling of chemical macromolecules via the generation of highly reactive species upon short wave light irradiation. One of the most efficient photoreactive functional groups is trifluoromethyl aryldiazirine (TFMAD). This compound was synthesised as part of the work discussed in this thesis, making use of microwave irradiation to shorten reaction times (Chapter I). An investigation of properties allowed the development of three different applications for conjugation to biomolecules. The first application consisted of the development of an approach for generation of small-molecule microarrays, where a 2,000 compound library was immobilised onto the glass surface through carbene insertion. The microarray was then used to screen for potential binders to beta-transducin repeat containing protein (b-TrCP1) allowing the reduction of possible candidates to less than 25 compounds (Chapter II). The second application was the synthesis of two probes to allow the selective delivery of active compounds inside specific organelles or cells. The diazirine moiety was used as a rapid way to covalently capture a number of cargos. The approach allowed a peptoid and an anticancer drug to be conjugated to the two probes and their cell penetrability properties and therapeutic effect were studied, respectively (Chapter III). Finally, the insertion properties of TFMAD were used to develop approaches to attach DNA onto microspheres and the efficiency of this delivery system was evaluated (Chapter IV).

Peptide nucleic acids (PNAs) were used as encoding tags to enable the analysis of peptide libraries by PNA/DNA hybridisation onto DNA microarrays. This allowed entire peptide libraries to be organised and sorted in a two dimensional format whereby all library members could be interrogated and analysed on a one-byone basis. In this thesis, PNA-encoded peptide libraries, generated by split-and-mix library synthesis, were screened for a variety of functions. Peptide sequences identified from the screening of a PNA-encoded library were analysed in detail as the first specific substrates for chymopapain. A new PNAencoded library consisting of D-amino acids was synthesised and screened with a number of proteases in attempts to identify novel/unusual substrates. PNA-encoded libraries were also used in the screening of peptide libraries for other activities. Thus substrates for catalyst-free Hüisgen cycloaddition were identified following the reaction between an alkyne modified peptide library and azidofluorescein, while cell-penetrating peptides were identified by hybridization of an internalized encoded library onto a DNA microarray.

The global aim of this thesis was to study the use of microarray technology for the screening and identification of biocompatible polymers, to understand physiological phenomena, and the design of biomaterials, implant surfaces and tissue-engineering scaffolds. This work was based upon the polymer microarray platform developed by the Bradley group. Polymer microarrays were successfully applied to find the best polymer supports for: (i) mouse fibroblast cells and used to evaluate cell biocompatibility and cell morphology. Fourteen polyurethanes demonstrated significant cellular adhesion. (ii) Analysis of the adhesion of human erythroleukaemic K562 suspension cells onto biomaterials with particular families of polyurethanes and polyacrylates identified. A DNA microarray study (to access the global gene expression profiles upon cellular binding) demonstrated that interactions between cells and some polyacrylates induced a number of transcriptomic changes. These results suggested that, during these interactions, a chain of cellular changes is triggered, most notably resulting in the downregulation of membrane receptors and ligands. (iii) Identification of polymers with potential applications in the field of stem cell biology. Polymers were identified that showed attachment, promotion and stabilisation of hepatocyte-like cells. A polyurethane support (PU-134) was pinpointed, which significantly improved both hepatocyte-like cell function and “lifespan”. A second project investigated biomaterials that promoted adhesion, growth and function of endothelial progenitor cells. A new polymer matrix was identified which contained the necessary signals to promote endothelial phenotype and function. This has potential application in the creation of blood vessels and the endothelialisation of artificial vessel prostheses and stent coatings for improving angioplasty therapy. (iv) The study of bacterial adhesion, focusing on the adhesion of food-borne pathogenic bacterium Salmonella enterica serovar typhimurium, strain SL1344, and the commensal bacterium Escherichia coli, strain W3110. Several polymers were found to support selective bacterial enrichment, as well as others that minimised bacterial adhesion.