Noble metal nanoparticles - Au and Ag - for biodetection

Dissertation submitted for obtainment of the Master’s Degree in Biotechnology, by the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Novos circuitos regulatórios de controlo espacio-temporal do desenvolvimento em bacillus subtilis

RESUMO: A esporulação em Bacillus subtilis é controlada por uma cascata de factores sigma da polimerase do RNA. F e E controlam os estágios precoces do desenvolvimento no pré-esporo e na célula mãe, respectivamente. Numa fase intermédia da diferenciação, quando a célula mãe acaba por envolver o pré-esporo, F é substituído por G e E é substituído por K. Vários mecanismos asseguram que a actividade dos diferentes factores sigma seja confinada a uma janela temporal precisa na célula adequada. Neste estudo, investigámos a função de um factor anti-G, designado por CsfB. Mostramos que para além da sua função de inibição da actividade do factor G em células pré-divisionais, CsfB é também necessário na célula mãe num estágio tardio do desenvolvimento. Mostramos que a expressão de csfB é activada na célula mãe a partir de um promotor dependente de K. Contudo, demonstramos que CsfB interage directamente com E e não com K, e que CsfB é suficiente para inibir a actividade transcricional dependente de E em células vegetativas de B. subtilis. Propomos que CsfB contribui para reduzir o período dependente de E, na linha de expressão genética da célula mãe, desse modo reduzindo a sobreposição entre os regulões E e K e aumentado a fidelidade do processo de desenvolvimento. Uma segunda proteína, YabK, partilha semelhança estrutural com CsfB. YabK é produzida no pré-esporo sob o comando de F, e é necessária para a esporulação. YabK contribui para a transição F/G no programa genético do pré-esporo, porque uma mutação que torna F sensível a CsfB ultrapassa parcialmente a função de YabK na esporulação. No entanto, YabK e CsfB funcionam por mecanismos diferentes, uma vez que YabK não liga directamente a F.---------ABSTRACT: Gene expression during spore development in Bacillus subtilis is governed by a cascade of RNA polymerase sigma factors. F and E control the early stages of development in the forespore and in the mother cell, respectively. At an intermediate stage in the differentiation process, when the larger mother cell finishes engulfment of the smaller forespore, F is replaced by G and E is replaced by K. Several mechanisms ensure the proper timing of activation of the cell type-specific sigma factors. Here, we have investigated the funtion of an anti-sigma G factor, called CsfB. We show here that in addition to its role in inhibiting G in pre-divisional cells, CsfB is also required in the mother cell at a late stage in development. We show that the expression of csfB is activated in the mother cell from a K-specific promoter. However, we demonstrate that CsfB binds directly to E but not to K in a yeast two-hybrid assay, and that CsfB is sufficient to inhibit E-dependent transcriptional activity in vegetative cells of B. subtilis. We posit that CsfB contributes to shutting off the early, E-controlled period in the mother cell line of gene expression, thus reducing the overlap between deployment of the E and K regulons and increasing the fidelity of the developmental process. A second protein, YabK, shares structural similarity with CsfB. YabK is produced in the forespore under F control, and is required for efficient sporulation. YabK contributes to the transition from the F- to the G-dependent period of gene expression, because a mutation that renders F sensitive to CsfB partially bypasses the need for YabK. Yet, YabK and CsfB must function in the control of sigma factor activity by different mechanisms because YabK does not bind directly to F.

G-quadruplex ligands for cancer therapy

DNA may fold into a diversity of structures and topologies such as duplexes and triplexes. Some specific guanine-rich DNA sequences may even fold into a higher order structures denominated guanine G-quadruplexes (G4). These G-quadruplex forming sequences have shown biological interest since were found in telomeres and in promoter region of oncogenes. Thus, these G4 forming sequences have been explored as therapeutic targets for cancer therapy, since G4 formation was demonstrated to inhibit RNA-polymerase and telomerase activity. However, the G4 structures are transient and are only formed under specific conditions. Hence the main objective of this work is to develop new G4-specific ligands which may potentially find applications in the therapeutic area. Several potential G4-binding ligands were synthesized and characterized. The synthesis of these compounds consisted on a procedure based on van Leusen chemistry and a cross-coupling reaction through C-H activation, affording phenanthroline compounds (Phen-1, 50%; Phen-2, 20%), phenyl (Iso-1, 61%; Iso-2, 21%; Ter-1, 85%; Ter-2, 35%), and quinolyl (Quin-1, 85%; Quin-2, 45%) compounds. Screening assays for selecting the potential G4 compounds were performed by FRET-melting, G4-FID, CD-melting and DSF. Qualitative biophysical studies were performed by fluorescence and CD spectroscopy. Two high-specific G-quadruplex ligands, Phen-1 and Phen-2, were found to effectively bind telomeric and c-myc G4 structures. Phen-1 was found to stabilize parallel telomeric 22AG and c-myc sequence by 4.1 and 4.3 ˚C, respectively. Phen-2 also displayed high affinity towards 22AG (

Towards modification of Medicago truncatula epigenome: genome editing with engineered nucleases

Periodic drought is the primary limitation of plant growth and crop yield. The rise of water demand caused by the increase in world population and climate change, leads to one of the biggest challenges of modern agriculture: to increase food and feed production. De novo DNA methylation is a process regulated by small interfering RNA (siRNAs), which play a role in plant response and adaptation to abiotic stress. In the particular case of water deficit, growing evidences suggest a link between the siRNA pathways and drought response in the model legume Medicago truncatula. As a first step to understand the role of DNA methylation under water stress, we have set up several bioinformatics and molecular methodologies allowing the design of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems and the assembly of TALENs (transcription activator-like effector nucleases), to target both dicer-like 3 (MtDCL3) and RNA-Dependent RNA polymerase (MtRDR2), enzymes of the RNA-directed DNA methylation pathway. TALENs efficiency was evaluated prior to plant transformation by a yeast-based assay using two different strategies to test TALENs activity: Polyacrylamide gel electrophoresis (PAGE) and Single strand conformation polymorphisms (SSCP). In this assay, yeast cells triple transformation emerged as good and rapid alternative to laborious yeast mating strategies. PAGE analysis might be a valuable tool to test TALENs efficacy in vivo if we could increase TALENs activity. SSCP-based approach proved to be ineffective due to the generation of several false positives. TALENs and CRISPR/Cas9 system constructed and designed in this work will in the future certainly enable the successful disruption of DCL3 and RDR2 genes and shed the light on the relationship between plant stress resistance and epigenetic regulation mediated by siRNAs in M.truncatula.

Light controlled synthesis of nucleic acids

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica - especialidade Biotecnologia, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia