Cell-targeted regulation of gene expression through synthetic RNA devices

The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function.

We describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation.

We next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein.

The foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.

A study to determine those areas in which vessel simulation may be effectively applied to the accomplishment of Coast Guard vessel operational training /

Mode of access: Internet.

The role of prediction in training with a simulated orbital docking task

Cover title.

Preliminary design study of a driving simulator, for the Liberty Mutual Insurance Company, Boston, Massachusetts.

"Final report, Contract no. CC-102."

In-flight simulation, theory and application,

"To be presented to the Fluid Dynamics Panel of the Advisory Group for Aeronautical Research and Development of the North Atlantic Treaty Organization, 10 April through 17 April 1961."

Distributed interactive simulation of combat.

Mode of access: Internet.

Tradevman 1 & C. /

"Rate training manual."

Project cyclone: solution of a flutter problem. U.S. Navy Bureau of Aeronautics contract no as 54-545-C.

Mode of access: Internet.

Dynamic test program for weapons system trainers. Project 413(2) Final report.

"Contract N61339-318."

Operator's manual for trainer, unit-conduct of fire (U-COFT), M1A1-120mm tank (sheltered), 60 Hz (6920-01-158-2638), 50 Hz (6920-01-158-2639).

Includes index.

Operator's manual for trainer, unit-conduct of fire (U-COFT), M2/M3 fighting vehicles (sheltered), 60 Hz (6920-01-158-6756), 50 Hz (6920-01-158-6757).

Includes index.

Programming Molecular Devices using Nucleic Acid Hairpins

Nucleic Acid hairpins have been a subject of study for the last four decades. They are composed of single strand that is

hybridized to itself, and the central section forming an unhybridized loop. In nature, they stabilize single stranded RNA, serve as nucleation

sites for RNA folding, protein recognition signals, mRNA localization and regulation of mRNA degradation. On the other hand,

DNA hairpins in biological contexts have been studied with respect to forming cruciform structures that can regulate gene expression.

The use of DNA hairpins as fuel for synthetic molecular devices, including locomotion, was proposed and experimental demonstrated in 2003. They

were interesting because they bring to the table an on-demand energy/information supply mechanism.

The energy/information is hidden (from hybridization) in the hairpin’s loop, until required.

The energy/information is harnessed by opening the stem region, and exposing the single stranded loop section.

The loop region is now free for possible hybridization and help move the system into a thermodynamically favourable state.

The hidden energy and information coupled with

programmability provides another functionality, of selectively choosing what reactions to hide and

what reactions to allow to proceed, that helps develop a topological sequence of events.

Hairpins have been utilized as a source of fuel for many different DNA devices. In this thesis, we program four different

molecular devices using DNA hairpins, and experimentally validate them in the

laboratory. 1) The first device: A

novel enzyme-free autocatalytic self-replicating system composed entirely of DNA that operates isothermally. 2) The second

device: Time-Responsive Circuits using DNA have two properties: a) asynchronous: the final output is always correct

regardless of differences in the arrival time of different inputs.

b) renewable circuits which can be used multiple times without major degradation of the gate motifs

(so if the inputs change over time, the DNA-based circuit can re-compute the output correctly based on the new inputs).

3) The third device: Activatable tiles are a theoretical extension to the Tile assembly model that enhances

its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly.

4) The fourth device: Controlled Amplification of DNA catalytic system: a device such that the amplification

of the system does not run uncontrollably until the system runs out of fuel, but instead achieves a finite

amount of gain.

Nucleic acid circuits with the ability

to perform complex logic operations have many potential practical applications, for example the ability to achieve point of care diagnostics.

We discuss the designs of our DNA Hairpin molecular devices, the results we have obtained, and the challenges we have overcome

to make these truly functional.

Electromyographic activity of trunk muscles during exercises with flexible and non-flexible poles

Objective: Hand-held flexible poles which are brought into oscillation to cause alternating forces on trunk, are advocated as training devices that are supposed to solicit increased levels of stabilizing trunk muscle activity. The aim of this study was to verify this claim by comparing electromyographic (EMG) activity of trunk muscles during exercises performed with a flexible pole and a rigid pole.Methods: Twelve healthy females performed three different exercises with flexible and rigid poles. EMG activity of iliocostalis lumborum (IL), multifidus (MU), rectus abdominis (RA), external oblique (EO) and internal oblique (IO), and was continuously measured. The EMG signals were analyzed in time domain by calculation of the Root Mean Square (RMS) amplitudes over 250 ms windows. The mean RMS-values over time were normalized by the maximum RMS obtained for each muscle.Results: The IO showed a 72% greater EMG activity during the exercises performed with the flexible pole than with the rigid pole (p = 0.035). In exercises performed in standing, the IO was significantly more active than when sitting (p = 0.006).Conclusion: As intended, the cyclic forces induced by the oscillating pole did increase trunk muscle activation. However, the effect was limited and significant for the IO muscle only.

Do acolhimento ao acompanhamento compartilhado: a construção colaborativa de uma proposta para o estágio curricular no curso de pedagogia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)