The Cell Biology of Genomes: Bringing the Double Helix to Life

The recent ability to routinely probe genome function at a global scale has revolutionized our view of genomes. One of the most important realizations from these approaches is that the functional output of genomes is affected by the nuclear environment in which they exist. Integration of sequence information with molecular and cellular features of the genome promises a fuller understanding of genome function

Physiological importance of RNA and protein mobility in the cell nucleus

Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways

Analysis of Hox10 specific peptide motifs in their patterning functions of the axial skeleton

Tese de mestrado, Biologia (Biologia Evolutiva e do Desenvolvimento), 2009, Universidade de Lisboa, Faculdade de CiênciasHox genes play a fundamental role in anterior-posterior patterning and are remarkably conserved throughout evolution (Slack et al., 1993). Their products are transcription factors that regulate a specific set of genes with essential functions in development. Although different Hox genes show a notable functional specificity in vivo, they demonstrate a surprisingly low DNA-binding specificity in vitro. Sequence analysis can provide a way to understand how Hox genes achieve their biological specificity (Prince, 2002). Genetic experiments revealed that Hox genes are involved in global patterning processes in the axial skeleton to produce the axial formulae. Hox group 10 genes, in particular, have been shown to repress thoracic rib formation, since their overexpression in the presomitic mesoderm causes a ribless phenotype and their global inactivation resulted in extra ribs (Wellik et al., 2003, Carapuço et al., 2005). Two peptide domains were identified in Hox10 proteins which are conserved among all the Hox 10 members and are absent from all other Hox proteins. One of these is an octapeptide located just N-terminal to the homeodomain. The purpose of this work is to understand the role of this octapeptide in Hox10 protein function. This is being approached by the genesis and functional analysis of transgenic mice expressing mutant Hoxa10 proteins that contain specific deletions or amino acid changes in this domain. In previous transgenic assays, the overexpression of Hoxb9 gene in the presomitic mesoderm did not produce an abnormal axial skeleton phenotype. For this reason, this gene was used to generate chimeric contructs with the Hoxa10 gene. The results obtained show that the removal of the octapeptide is sufficient to block the rib-repressing activity of Hoxa10 when expressed in the presomitic mesoderm. In addition, introduction of this peptide motif, as well as the whole Hoxa10 sequence N-terminal to it, into the Hoxb9 protein produced a partial ribless phenotype. These results indicate that the octapeptide is necessary for the rib-repressing activity of Hoxa10 but it does not seem to be sufficient for this function, at least individuallyResumo alargado em português disponível no document

The concept of self-organization in cellular architecture

In vivo microscopy has recently revealed the dynamic nature of many cellular organelles. The dynamic properties of several cellular structures are consistent with a role for self-organization in their formation, maintenance, and function; therefore, self-organization might be a general principle in cellular organization