Response to Frequency Shifted Artificial Echoes in the Bat Rhinolophus ferrumequinum

In 5 roosting bats the resting frequency, that is the mean frequency of the cf-portion of consecutive sounds, is kept constant with a standard deviation which varies between 30 120 Hz in different bats and at different days. In 15 bats the emitted sounds were electronically shifted in frequency and played back as artificial echoes. Upward frequency shifts were responded by a decrease of the emission frequency. This frequency compensation occurred at frequency shifts of up to 4400 Hz in all bats and up to 6000 ttz in a few bats. The frequency decrease in different bats over the whole compensation range was 50-300 tIz smaller than the frequency shifts in the echoes. The echoes, therefore, returned at a frequency, called the reference frequency, which was by this compensation offset higher than the resting frequency. The standard deviations of the emission frequency in compensating bats were only slightly larger than in roosting bats and the same in the whole compensation range. All bats started to compensate frequency shifts when they were slightly larger than the compensation offset. Downward frequency shifts were not responded by a change of the emission frequency, but the accuracy with which the emission frequency was kept decreased somewhat. From these results it is concluded that the Doppler shift compensation system of the Horseshoe bats compares the echo frequency with the reference frequency and compensates deviations of upward frequency shifts

O anel de inteiros quadráticos

Trabalho de Conclusão de Curso - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas, Curso de Matemátic

Construction of Enzymes with Synthetic Allosteric Regulation to Control Metabolic Pathways of Escherichia coli

In metabolic engineering strains are created that overproduce a certain product. For that, the production pathway is often released from any transcriptional, translational and post-translational regulation, resulting in a high abundance of enzymes in the production pathway and enzyme variants with feedback-resistance. However, complete dysregulation has several disadvantages: the pathway cannot respond to internal and external perturbations and metabolism of the host is overloaded, resulting in a lowered cellular fitness and reduced growth rates. To circumvent this problem, it is desirable to implement new layers of regulation in the metabolic network and in particular in the overproduction pathway. So far, such regulation has usually been implemented by controlling enzyme abundance. Two-phase processes for instance are dividing a bioprocess in two phases, a growth phase in which a sufficient amount of biomass is accumulated, and a production phase. In this second phase, the expression of enzymes needed for overproduction is induced, often in combination with the introduction of metabolic bottlenecks in competing pathways. However, the regulation of enzyme abundances does not allow fast response at the second or minute time-scale. Especially in large-scale bioreactors fast response is important, because of fluctuating availabilities of nutrients and oxygen caused by insufficient mixing which leads to the formation of microenvironments and dead-zones. Cells in which the overproduction pathway is either dysregulated or regulated only by implemented control of enzyme abundance are not able to adjust their metabolic networks according to fast changing microenvironments, leading to stressed and therefore unproductive strains which might negatively affect the stability and durability of bioprocesses. This highlights the need for faster acting dynamic control of metabolic pathways, for example through enzymes with synthetic allosteric regulation. However, the creation and usage of such enzymes is very challenging. A major goal of this work was to create such enzymes with synthetic allosteric regulation and to test their ability to control fluxes through their pathway. Synthetic allosteric enzymes are ‘metabolic valves’ that implement bottlenecks in the reaction they are catalyzing and we sought to characterize functioning of these valves in vivo. Therefore, our first goal was to examine functioning of these valves, resulting metabolic bottlenecks and their impact on the general fitness (Chapter 3). For that, we analyzed growth and metabolic profiles of wildtype isolate and laboratory strains and could show that in laboratory strains a previously reported bottleneck caused by low pyrE gene expression causes insufficient fluxes through the pyrimidine biosynthesis pathway and subsequently lowered growth rates. In addition to that, we used CRISPR interference to introduce artificial bottlenecks in 30 reactions in different parts of the metabolic network. In 16 of the resulting 30 strains we were able to detect elevated substrate or lowered product concentration, indicating a metabolic bottleneck. However, only 6 of these 16 strains also had a reduced growth rate, underlining that the impact of metabolic bottlenecks on the growth rate is generally dependent on the reaction and strength of the bottleneck. In the second part, we then evaluated two methods to create synthetic allosteric enzymes, both of which are based on the concept of directed evolution: Split Proteins (Protein Fragment Complementation, Chapter 4) and Domain Insertion (Chapter 5). With the Split Protein approach we were able to couple two fragments of a split dihydrofolate reductase (DHFR) to the conditionally interacting proteins FRAP and FKBP12, resulting in a rapamycin-dependent metabolic enzyme that can be used to control the folate biosynthesis pathway and consequently the growth rate. With the Domain Insertion approach, we created enzyme-regulatory domain chimera consisting of 2-Isopropylmalate synthase (LeuA) and murine DHFR as enzymes and the maltose binding protein MBP as regulatory domain. We isolated functional proteins, but could so far not identify a variant that is sensitive to the effector. We optimized the protocol to an extent that libraries of thousands of strain variants expressing potentially switching enzymes can be generated and the screening for strains of interest became the limiting factor. In a third part of this work we therefore evaluated the fluorescent single cell growth rate reporter TIMER for its utilization in E. coli and especially to enrich slow growing cells out of large genetic variant strain libraries in a high-throughput manner using fluorescence-activated cell sorting (Chapter 6). The herewith developed enrichment method is planned to be applied in the future to strain libraries created with the Domain Insertion library approach

Plaza de Las Glorias Catalanas: la periferia desde el tiovivo

Este trabajo presenta una visión sobre la Plaza de las Glorias Catalanas dividida en dos partes. En la primera se muestran intencionadamente algunos aspectos que han condicionado este lugar y le han dado el carácter de periferia que se subraya en la presente tesina. En la segunda parte se analiza el desarrollo del lugar en los últimos veinte años, mostrándolo tal como es hoy

Using a virtual cortical module implementing a neural field model to modulate brain rhythms in Parkinson’s disease

We propose a new method for selective modulation of cortical rhythms based on neural field theory, in which the activity of a cortical area is extensively monitored using a two-dimensional microelectrode array. The example of Parkinson’s disease illustrates the proposed method, in which a neural field model is assumed to accurately describe experimentally recorded activity. In addition, we propose a new closed-loop stimulation signal that is both space- and time- dependent. This method is especially designed to specifically modulate a targeted brain rhythm, without interfering with other rhythms. A new class of neuroprosthetic devices is also proposed, in which the multielectrode array is seen as an artificial neural network interacting with biological tissue. Such a bio-inspired approach may provide a solution to optimize interactions between the stimulation device and the cortex aiming to attenuate or augment specific cortical rhythms. The next step will be to validate this new approach experimentally in patients with Parkinson’s disease