Progress in the biological sciences relies on the availability of appropriate tools and strategies; in the areas of understanding and constructing multicellular systems, better methods of gene expression control are needed. Here, we work towards two approaches to controlling gene expression for applications related to multicellular coordination and pattern formation. First, we characterize the use of 2A self-cleaving peptides for polycistronic expression in Dictyostelium discoideum, a powerful model system for investigating the onset of coordinated behavior in a cell population. We also make novel observations about various factors that affect gene expression levels, which would inform future decisions on the use of 2A peptides in this system. Second, we examine the use of antisense RNA in mammalian cells for gene repression from an input that simultaneously activates other genes. We show that in transient overexpression systems, antisense RNA produced in trans can be a more effective repressor than convergent promoters and other well-established RNA-based methods for gene repression, and we explore ways in which this system could be improved for stable gene circuits. Overall, our results advance our understanding of available tools for gene expression control and the conditions under which they are appropriate to use.

Orientador: Celso de Almeida, Rodrigo Pereira Ramos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-08T10:36:25Z (GMT). No. of bitstreams: 1 Britto_PauloMarceloPerezRodriguesde_M.pdf: 4780408 bytes, checksum: c3b152e041128b5d94429d8b87c0068c (MD5) Previous issue date: 2006 / Resumo: O desempenho de sistemas de comunicações sem fio que utilizam técnicas de espalhamento espectral e múltiplo acesso por divisão de código (CDMA) é limitado pela interferência. Nesta dissertação, analisamos o desempenho de um algoritmo de seleção de seqüências de espalhamento de comprimento fixo, que busca a minimização dos efeitos de interferência em sistemas CDMA operando em canais com desvanecimento seletivo em freqüência e possuindo células adjacentes interferentes. Analisamos o desempenho deste sistema no qual o espalhamento espectral é feito usando duas seqüências: seqüências do usuário e seqüências identificadoras de células. Dentre as seqüências analisadas, podemos citar as Walsh, a Gold, as de comprimento máximo e também um tipo de seqüência de espalhamento baseado nas seqüências de comprimento máximo / Abstract: The performance of wireless communication systems using spread spectrum techniques and code division multiple access (CDMA) is interference-limited. In this dissertation, we evaluate the performance of a selection algorithm for fixed-length spreading sequences, aimed on minimizing the interference effects in CDMA systems operating in frequency selective fading channels and having adjacent interfering cells. We check the performance of this system where the spread spectrum operation is performed using two sequences: user sequences and cell identification sequences. Among the sequences considered, we use Walsh, Gold, maximum length and a type of spreading sequence based on the maximum length class / Mestrado / Telecomunicações e Telemática / Mestre em Engenharia Elétrica

Mechanics is known to play a fundamental role in many cellular and developmental processes. Beyond active forces and material properties, osmotic pressure is believed to control essential cell and tissue characteristics. However, it remains very challenging to perform in situ and in vivo measurements of osmotic pressure. Here we introduce double emulsion droplet sensors that enable local measurements of osmotic pressure intra- and extra-cellularly within 3D multicellular systems, including living tissues. After generating and calibrating the sensors, we measure the osmotic pressure in blastomeres of early zebrafish embryos as well as in the interstitial fluid between the cells of the blastula by monitoring the size of droplets previously inserted in the embryo. Our results show a balance between intracellular and interstitial osmotic pressures, with values of approximately 0.7 MPa, but a large pressure imbalance between the inside and outside of the embryo. The ability to measure osmotic pressure in 3D multicellular systems, including developing embryos and organoids, will help improve our understanding of its role in fundamental biological processes.

Background: Replacement of dysfunctional β-cells in the islets of Langerhans by transdifferentiation of pancreatic acinar cells has been proposed as a regenerative therapy for diabetes. Adult acinar cells spontaneously revert to a multipotent state upon tissue dissociation in vitro and can be stimulated to redifferentiate into β-cells. Despite accumulating evidence that contact-mediated signals are involved, the mechanisms regulating acinar-to-islet cell transdifferentiation remain poorly understood. Results: In this study, we propose that the crosstalk between two contact-mediated signaling mechanisms, lateral inhibition and lateral stabilization, controls cell fate stability and transdifferentiation of pancreatic cells. Analysis of a mathematical model combining gene regulation with contact-mediated signaling reveals the multistability of acinar and islet cell fates. Inhibition of one or both modes of signaling results in transdifferentiation from the acinar to the islet cell fate, either by dedifferentiation to a multipotent state or by direct lineage switching. Conclusions: This study provides a theoretical framework to understand the role of contact-mediated signaling in pancreatic cell fate control that may help to improve acinar-to-islet cell transdifferentiation strategies for β-cell neogenesis.

Background: Replacement of dysfunctional β-cells in the islets of Langerhans by transdifferentiation of pancreatic acinar cells has been proposed as a regenerative therapy for diabetes. Adult acinar cells spontaneously revert to a multipotent state upon tissue dissociation in vitro and can be stimulated to redifferentiate into β-cells. Despite accumulating evidence that contact-mediated signals are involved, the mechanisms regulating acinar-to-islet cell transdifferentiation remain poorly understood. Results: In this study, we propose that the crosstalk between two contact-mediated signaling mechanisms, lateral inhibition and lateral stabilization, controls cell fate stability and transdifferentiation of pancreatic cells. Analysis of a mathematical model combining gene regulation with contact-mediated signaling reveals the multistability of acinar and islet cell fates. Inhibition of one or both modes of signaling results in transdifferentiation from the acinar to the islet cell fate, either by dedifferentiation to a multipotent state or by direct lineage switching. Conclusions: This study provides a theoretical framework to understand the role of contact-mediated signaling in pancreatic cell fate control that may help to improve acinar-to-islet cell transdifferentiation strategies for β-cell neogenesis.