Dissertations / Theses on the topic 'Clock control'

Have you thought of why you get tired or why you get hungry? Something in your body keeps track of time. It is almost like you have a clock that tells you all those things.

And indeed, in the suparachiasmatic region of our hypothalamus reside cells which each act like an oscillator, and together form a coherent circadian rhythm to help our body keep track of time. In fact, such circadian clocks are not limited to mammals but can be found in many organisms including single-cell, reptiles and birds. The study of such rhythms constitutes a field of biology, chronobiology, and forms the background for my research and this thesis.

Pioneers of chronobiology, Pittendrigh and Aschoff, studied biological clocks from an input-output view, across a range of organisms by observing and analyzing their overt activity in response to stimulus such as light. Their study was made without recourse to knowledge of the biological underpinnings of the circadian pacemaker. The advent of the new biology has now made it possible to "break open the box" and identify biological feedback systems comprised of gene transcription and protein translation as the core mechanism of a biological clock.

My research has focused on a simple transcription-translation clock model which nevertheless possesses many of the features of a circadian pacemaker including its entrainability by light. This model consists of two nonlinear coupled and delayed differential equations. Light pulses can reset the phase of this clock, whereas constant light of different intensity can speed it up or slow it down. This latter property is a signature property of circadian clocks and is referred to in chronobiology as "Aschoff's rule". The discussion in this thesis focus on develop a connection and also a understanding of how constant light effect this clock model.

Circadian rhythms in biological processes occur in a wide range of organisms and are generated by endogenous oscillators. In Neurospora crassa, the FRQ-oscillator (comprised of FRQ, WC-1 and WC-2) is essential for rhythms in asexual sporulation and gene expression. How this oscillator signals to the cell to control rhythmicity is unknown. Furthermore, under certain growth conditions, rhythms are observed in FRQ-null strains, indicating the presence of one or more FRQ-less oscillators (FLOs). Interestingly, while circadian rhythms are observed in the related Aspergillus spp., they lack the frq gene, leading to the hypothesis that a FLO is responsible for rhythms in Aspergillus. Thus, Aspergillus provides a useful organism to investigate the components of the FLO. To investigate how an oscillator controls circadian output, we characterized the role of N. crassa NRC-2. The nrc-2 gene is under control of the clock and encodes a putative serine-threonine protein kinase. In a NRC-2-null strain cultured in low glucose conditions, FRQ-oscillator-dependent outputs are arrhythmic, but are rhythmic in high glucose. Our data suggests a model whereby NRC-2 relays metabolic information to the FRQ-oscillator to control rhythmic output. To understand the role of FLO(s) in the N. crassa circadian system, we examined regulation of the ccg-16 gene. We show that ccg-16 transcript rhythmicity is FRQ-independent, but WC-1-dependent. Furthermore, in contrast to current models for the FRQ-oscillator, we observed that rhythms in WC-1 protein accumulation persist in the absence of FRQ. These data support a new model involving two oscillators that are coupled through the WC-1 protein and that regulate different outputs. One approach to identify components of the FLO involved characterizing circadian rhythms in Aspergillus spp, which lacks FRQ. We find that A. flavus and A. nidulans, display circadian rhythms in sporulation and gene expression, respectively. Together, these findings provide a foundation for the identification of FLO components in both Aspergillus and N. crassa, that will ultimately lead to an understanding of how a multi-oscillator system can generate and coordinate circadian rhythmicity.

Circadian rhythms are visible as daily oscillations in biochemical, physiological, or behavioral processes. These rhythms are produced by an endogenous clock that maintains synchrony with the external environment through responses to external stimuli such as light or temperature. The clock, in turn, coordinates internal processes in a time-dependent fashion. Genetic and molecular analysis of the filamentous fungus Neurospora crassa has demonstrated that the products of the frequency (frq) and white-collar (wc-1 and wc-2) genes interact to form an interlocked feedback loop that lies at the heart of the clock in this fungus. This feedback loop, termed the FRQ/WC oscillator, produces a ~24h oscillation in frq mRNA, FRQ protein, and WC-1 protein. In turn, the FRQ/WC oscillator regulates rhythmic behavior and gene expression. The goal of this dissertation is to understand how rhythmic outputs are regulated by the FRQ/WC oscillator in Neurospora. To this end, we have taken a microarray approach to first determine the extent of clock-controlled gene expression in Neurospora. Here, we show that circadian regulation of gene expression is widespread; 145 genes, representing 20% of the genes we analyzed, are clock-controlled. We show that clockregulation is complex; clock-controlled genes peak at all phases of the circadian cycle. Furthermore, we demonstrate the clock regulates diverse biological processes, such as intermediary metabolism, translation, sexual development and asexual development. WC-1 is required for all light- and clock-regulated gene expression in Neurospora. We have shown that overexpression of WC-1 is sufficient to activate clock-controlled gene expression, but is not sufficient to induce all light-regulated genes in Neurospora. This result indicates that cycling of WC-1 is sufficient to regulate rhythmic expression of a subset of clockcontrolled genes. Conversely, a post-translational mechanism underlies WC-1 mediated light signal transduction in Neurospora. Finally, we have demonstrated the Neurospora circadian system is comprised of mutually coupled oscillators that interact to regulate output gene expression in the fungus.

Periodic oscillations underlie many intracellular functions, such as circadian time keeping, cell cycle control and locomotor pattern generation in nerve cells. These intracellular oscillations are generated in intricate biochemical reaction networks involving genes, proteins and other biochemical components. In most cases, robust oscillations are of pivotal importance for the organism, i.e., the oscillations must be maintained in the presence of internal and external perturbations.

Model based analysis of robustness in intracellular oscillators has attracted considerable attention in recent years. The analysis has almost exclusively been based on either complete removal of network components, e.g., single genes, or perturbation of model parameters. In this thesis, a control theoretic approach to analyze structural robustness of intracellular oscillators is proposed. The method is based on adding dynamic perturbations to the network interactions. Determination of the smallest perturbation translating the underlying steady-state into a Hopf bifurcation point is used to quantify the robustness. The method can be used to determine critical substructures within the overall network and to identify specific network fragilities. Also, an approach to nonlinear model reduction based on the robustness analysis is proposed.

The proposed robustness analysis method is applied to elucidate mechanisms underlying robust oscillations in circadian clocks. Circadian clocks, molecular oscillators generating 24 hour rhythms in many organisms, are known to display a striking robustness towards internal and external perturbations. The underlying networks involve a large number of genes that are transcribed into mRNA which produce proteins subsequently regulating the activity of other genes, together forming an intricate network with a large number of embedded feedback loops. An often recurring hypothesis is that the interlocked feedback loop structure of circadian clocks serves the purpose of robustness. From analysis of several recently published models of circadian clocks, it is found in this thesis that the robustness of circadian clocks primarily results from a high gain in a single gene regulatory feedback loop generating the oscillations. This gain can be elevated by additional feedback loops, involving either gene regulation or post-translational feedback, but a similar robustness can be achieved by simply increasing the amplification within the master feedback loop.

Plants are exposed to a variety of abiotic stresses, including salinity and drought. These environmental stresses cause major losses in crop yield. High salinity stress alone impairs crop production on at least 20% of irrigated land worldwide. Thus, the development of stress-tolerant crops is of major importance for food security. Many physiological responses to ensure acclimation to adverse environmental conditions require the synthesis and perception of the plant hormone abscisic acid (ABA). Recent studies have shown that the function of the circadian clock is altered under some abiotic stress conditions such as drought, and osmotic stress. The first part of this thesis investigates the role of the stress response hormone abscisic acid in changing the function of the clock under osmotic stress. It was found that multiple core clock genes are responsive to ABA application, with sharp transient induction of morning associated genes in particular. In comparison, osmotic stress caused a damping of the amplitude of gene expression. It was then shown that the disruptive effect of osmotic stress on circadian leaf movement rhythms required the biosynthesis of ABA. This is important as it demonstrates that ABA is a key factor in mediating osmotic stress responses to the clock. The second half of this thesis then focuses on how altered function of the clock might impact plant performance under drought or osmotic stress. It was found that the morning associated LATE ELONGATED HYPOCOTYL (LHY) transcription factor, which functions as a key component of the circadian clock, regulates many of the components of the ABA signalling pathway. Evidence was provided that, while overexpression of LHY results in reduced ABA levels, ABA responsive gene expression is significantly increased upon ABA treatment. Finally, through phenotypic analysis it was determined that increased LHY expression leads to increased performance in drought and osmotic stress conditions. This is important as it suggests that manipulation of circadian clock function may be useful as a novel approach in the future engineering of stress tolerant crop lines.

All eukaryotic organisms display rhythms which persist under constant environmental conditions with periods of approximately, but very seldom exactly, 24 hours. Such rhythms are "circadian" and are driven by an internal "biological clock." Circadian rhythms of locomotor activity, retinal sensitivity to light and ommatidial morphology have been characterized in the cockroach, Leucophaea maderae (L.). It is not known whether the same clock(s) controls both circadian rhythms of electroretinogram (ERG) amplitude and morphological changes of the compound eye. In order to determine whether the location of the clock that controls morphological changes is in the same location as the one that controls ERG, brain lesions were made proximal or distal to the region of the putative clock regulating the expression of a circadian rhythm in eye sensitivity to light in anesthetized cockroaches. These and sham operated control animals were held for approximately two weeks under LD 12:12 at 25±2°C conditions in environmental chambers. After this time period, conditions of continuous darkness were established in order to allow rhythms to free run. Eye tissue was removed on subjective midday two and subjective midnight two, fixed, embedded, sectioned and the sections were examined and photographed using a Zeiss transmission electron microscope. Observations were made to establish the presence or absence of a circadian rhythm of submicrovillar cysternae area (SMC), as well as rhabdom area and screening pigment granules organization (SPG). The results indicate that the clock, located in the lobular neuropil region of the optic lobe that controls the rhythm of morphological changes in the eye, is in the same proximity as that which controls the changes in ERG amplitude.

The circadian clock regulates a wide range of biological processes, allowing plants to be prepared for predictable daily diurnal changes in environmental cues such as light and temperature. Recent studies have suggested that the circadian clock may also control plant immunity. The exact nature of the interaction between the circadian clock and plant pathogens remains unknown. Our focus in this study is on the elucidation of the role of the biological clock in plant immunity against the necrotrophic pathogen to Botrytis cinerea. In order to do this we tested the level of susceptibility to B. cinerea in Arabidopsis thaliana wild type and transgenic plants: toc1, cca1/lhy, cca1/toc1, lhy/toc1, cca1/lhy/toc1, GLK1 OE, GLK2 OE, glk1, glk2, and glk1/glk2. We demonstrated that the time of infection plays a role in susceptibility to B. cinerea. Specifically, we found that plants are more susceptible to infection in the subjective morning. We also found that genetic mutations in core clock components or in GLK genes leads to changes in susceptibility to B. cinerea. Our data suggests that clock genes are not solely responsible for plant immune responses to B. cinerea but rather the ways in which the biological clock system regulates outcome pathways. Furthermore, when we entrain the biological clock by changing the photoperiod (day length) in normal earth conditions LD 24h and SD 24h, we observed that short day plants had higher susceptibility to B. cinerea than long day plants. In addition, when we entrain the biological clock in different photoperiods, the LD 30h photoperiod plants displayed similar responses as those in the SD 24h photoperiod. The data indicates that day length is not responsible for the control of plant immunity; it is the ability of light to entrain the biological clock that is important. Together, the data strongly support the conclusion that the circadian clock plays a role in plant defense regulation.

Le moment, la qualité et la quantité de sommeil dépendent de l'interaction fine entre l'horloge circadienne et la machinerie homéostatique (Borbely A. et al., 1982, Daan S. et al., 1984, Borbely et Achermann, 1999). Au cours des dernières années, l'utilisation de divers organismes modèles a fourni de nouvelles perspectives sur les mécanismes neuronaux et moléculaires de la régulation du sommeil (Miyazaki S. et al., 2017). Cependant, les bases moléculaires de l'homéostasie du sommeil et les circuits neuronaux sous-jacents à son interaction avec le réseau circadien n'ont pas été établis en détail.Dans ce travail de thèse, j'ai utilisé la mouche Drosophile melanogaster comme système modèle pour étudier la fonction d'un sous-ensemble de neurones d'horloge, les DN1ps, dans la mise en place du sommeil. Des études antérieures ont suggéré un rôle de ces neurones circadiens dans la régulation du sommeil (Kunst et al., 2014, Guo et al 2016, Lamaze et al., 2017, Guo et al., 2017). J’ai ainsi démontré que les cellules d'horloge DN1ps DH31 (+) CRY (+) sont impliquées dans la suppression du sommeil. Par ailleurs, j’ai mis en évidence un circuit en aval des DN1ps, qui comprend le groupe dopaminergique postérieur apparié latéral 1 (PPL1) et les neurones dorsaux en forme d’éventail (dFSB), un centre homéostatique récemment décrit pour la régulation du sommeil chez la drosophile (Donlea JM et al., 2011, Liu S. et al., 2012, Ueno et al., 2012, Donlea JM et al., 2014, Pimentel et al., 2016, Qian et al., 2017, Donlea JM. et al., 2018). Nos résultats indiquent que la suppression du sommeil nocturne nécessite la signalisation DH31-R2 dans une sous-population des neurones dopaminergiques PPL1, qui projette au dFSB. Fait intéressant, la perte de sommeil de jour et de nuit médiée par les DN1ps dépend de l'inhibition du dFSB. Néanmoins, nous suggérons que les neurones DN1ps CRY (-) favoriseraient le sommeil, en concordance avec d'autres travaux (Guo et al., 2016; Guo et al., 2017).Ces résultats fournissent de nouvelles données sur le lien entre l'horloge circadienne et l'homéostasie du sommeil, impliqué dans la régulation du comportement sommeil-éveil chez Drosophile melanogaster
The timing, quality and quantity of sleep depend on the fine interaction between circadian clock and homeostatic machinery (Borbely A. et al., 1982; Daan S. et al., 1984; Borbely and Achermann, 1999). In the recent years, the employment of various model organisms has provided new insights into the neuronal and molecular mechanisms of sleep regulation (Miyazaki S. et al., 2017). However, the molecular basis of the sleep homeostat and the neuronal circuitry underlying its interaction with the circadian network haven’t been established in details.In this work, I use the fruit fly Drosophila melanogaster as a model system to investigate the sleep function of a subset of clock neurons, the DN1ps. Previous studies have already suggested a sleep-regulating role for these circadian neurons (Kunst et al. 2014, Guo et al. 2016; Lamaze et al., 2017; Guo et al. 2017). Here, we report the DH31-positive CRY-positive DN1ps as sleep suppressing clock cells. Furthermore, we identify a sleep-relevant circuit downstream of the DN1ps which includes the paired posterior lateral 1 (PPL1) dopaminergic cluster and the dorsal Fan-shaped body projecting (dFSB) neurons, a recently described homeostatic center for sleep regulation in Drosophila (Donlea JM. et al., 2011; Liu S. et al., 2012; Ueno et al., 2012; Donlea JM. et al., 2014; Pimentel et al., 2016; Qian et al., 2017; Donlea JM. et al., 2018). Our results indicate that the night-time sleep suppression requires DH31-R2 signaling in the PPL1-to-dFSB dopaminergic neurons. Interestingly, both day and night-time DN1ps-mediated sleep loss rely on the inhibition of the dFSB. Nevertheless, we suggest the CRY-negative DN1ps as sleep promoting clock neurons, in concordance with other works (Guo et al. 2016; Guo et al. 2017).These findings provide a novel link between circadian clock and sleep homeostat, in the regulation of sleep-wake behavior in Drosophila melanogaster

A vida cotidiana é dividida por intervalos de tempo, assim como, a medida do tempo faz-se necessária para ciência e tecnologia com níveis diferentes de exatidão, pois cada aplicação possui uma exigência da incerteza associada à medida. Atualmente não há outra grandeza física medida com tamanha exatidão quanto à referência de frequência baseadas em transições de níveis de energia atômica, logo, baseando a medida de tempo. Este trabalho visa o estudo e desenvolvimento do sistema de controle e monitoramento de padrões de frequência experimentais. Para isso devemos projetar, construir e caracterizar todo o sistema de controle e aquisição de dados, geração de sinais de controle e processamento do sinal de resposta. O sistema interage com sinais provenientes de uma grande variedade de sensores do padrão de frequência, de modo a detectar possíveis problemas ou efeitos que possam afetar a medida de frequência obtida. Há também a necessidade de armazenagem e tratamento dos dados obtidos para diagnósticos e ações corretivas, para esta tarefa, dispositivos de instrumentação são analisados e desenvolvidos com diferentes abordagens para cada tipo de padrão de frequência, tanto para o chafariz de átomos quanto o padrão portátil. Dessa forma, o sistema de controle e monitoramento do padrão de frequência proporciona a integração entre todos os subsistemas envolvidos na construção de um padrão atômico de frequência.
Everyday life is divided by time intervals, as well as the measurement of time is needed for science and technology with different levels of accuracy, because each application has a requirement of the uncertainty associated with the measure. Currently there is no other physical quantity measured with such accuracy as to the frequency reference transitions based on atomic energy levels, which in turn are based on measurements of time. This work aims to study and develop the system of monitoring and control of experimental frequency standards. For this we must design, build and characterize the whole system control and data acquisition, signal generation control signal processing of response signal. The system interacts with signals from a variety of sensors presents of frequency standard in order to detect possible problems or effects that may affect the frequency measurement obtained. There is also the need for storage and processing of data for diagnostics and corrective actions for this task, instrumentation devices are analyzed and developed with different approaches for each type of pattern frequency to both the fountain and the compact mobile standards. Thus, the control system and monitoring of frequency standard provides integration between all subsystems involved in building an atomic frequency standard.

The hair follicle (HF) is a human mini-organ that autonomously cycles between phases of growth (anagen), regression (catagen) and relative quiescence (telogen). Whilst many molecular controls are now appreciated to influence hair cycle, what ultimately choreographs the switch between each cycle stage is yet to be elucidated. With the increasing link between molecular clock activities in controlling local tissue physiology, we began by studying the hypothesis that the human HF has a functional molecular clock. Utilising human HF organ culture, qRT-PCR and immunofluorescence we found that the HF does indeed have oscillating clock gene expression over 24 and 48 hours in situ, separate from the suprachiasmatic-nucleus. Moreover, core clock proteins BMAL1 and PER1 are expressed in the human HF with PER1 increasing as HFs enter catagen. Next utilising siRNA mediated gene knock-down of either BMAL1 or PER1 in situ, we were able to show that silencing either clock gene leads to anagen prolongation in cultured HFs, demonstrating that the molecular clock modulates the human hair cycle, namely the anagen-catagen switch in situ. As human pigmentation is tightly coupled to the hair cycle and both human HFs and epidermal melanocytes express clock genes/proteins, this led us to investigate the hypothesis that the molecular clock modulates human pigmentation. By silencing BMAL1 or PER1 in HFs an increase in melanin content (Masson-Fontana) was observed in a hair-cycle independent manner. Furthermore, tyrosinase expression/activity as well as TYRP1 and 2 expression, gp100 protein expression, melanocyte dendricity and the number of HF melanocytes were all significantly increased in BMAL1 and/or PER1-silenced HFs. Mechanistically, BMAL1 knockdown reduced PER1 transcription, and PER1 silencing was found to induce phosphorylation of the master regulator of melanogenesis, MITF, thus stimulating human melanogenesis and melanocyte activity. This provides the first evidence that the peripheral molecular clock influences human pigmentation. Finally, the thyroid hormone (T4) has strong links with peripheral clock activity and has been shown to prolong anagen and increase human HF pigmentation. Moreover, T4 is a commonly prescribed treatment for thyroid disorder. As such, we investigated the hypothesis that T4 influences HF clock gene activity. It was observed that transient T4 treatment reduces the amplitude of clock gene oscillations whilst circadian rhythmicity is maintained. Conversely with longer term treatment clock gene activity was significantly increased compared to a scrambled oligo-control. Here we have demonstrated that the human HF has peripheral molecular clock activity which influences the human hair cycle and pigmentation. Finally we were able to uncover a potential novel target, T4, whose pulsatile administration may potentially be used to treat not only hair growth and pigmentation disorders but may be able to modulate circadian activity in peripheral tissues and treat clock-related disease.

Wireless personal area networks have emerged as an important communication infrastructure in areas such as at-home healthcare and home automation, independent living and assistive technology, as well as sports and wellness. Wireless personal area networks, including body sensor networks, are becoming more mature and are considered to be a realistic alternative as communication infrastructure for demanding services. However, to transmit data from e.g., an ECG in wireless networks is also a challenge, especially if multiple sensors compete for access. Contention-based networks offer simplicity and utilization advantages, but the drawback is lack of predictable performance. Recipients of data sent in wireless sensor networks need to know whether they can trust the information or not. Performance measurements, monitoring and control is of crucial importance for medical and healthcare applications in wireless sensor networks. This thesis focuses on development, prototype implementation and evaluation of a performance management system with performance and admission control for wireless sensor networks. Furthermore, an implementation of a new method to compensate for clock drift between multiple wireless sensor nodes is also shown. Errors in time synchronization between nodes in Bluetooth networks, resulting in inadequate data fusion, are also analysed.

QC 20120529

La función circadiana es esencial para el crecimiento y adaptación de las plantas a su entorno. La maquinaria molecular responsable de la generación de ritmos circadianos está basada en la expresión rítmica de genes cuyo pico de expresión oscila en diferentes fases durante el día y la noche. Los ritmos de expresión génica se traducen en oscilaciones de procesos fisiológicos y de desarrollo. El crecimiento de las plantas está regulado por una plétora de procesos que en última instancia operan a través del control de la proliferación y diferenciación celular. La proliferación celular depende de la progresión del ciclo mitótico, el cual está dividido en 4 fases: S (Síntesis del ADN), M (Mitosis) y de las interfases G1 y G2 (en inglés Gap 1 y 2) que ocurren antes de las fases S y M respectivamente. El proceso de diferenciación celular coincide con el cambio al endociclo, una variante del ciclo mitótico en la que el ADN genómico se duplica pero sin posterior división, es decir en ausencia de fase M. Aunque la regulación circadiana y el ciclo celular han sido individualmente estudiados en plantas, no se ha demostrado hasta la fecha la posible conexión de ambos ciclos en plantas. El trabajo realizado durante esta Tesis Doctoral se ha centrado en el estudio del papel del reloj circadiano en el control del ciclo celular durante la regulación del crecimiento de la planta. Los resultados obtenidos muestran que plantas con un reloj circadiano de ritmo lento desaceleran la progresión del ciclo celular, mientras que un reloj de ritmo rápido lo acelera. El componente esencial del reloj denominado en inglés TIMING OF CAB EXPRESSION 1 (TOC1) controla la transición de la fase G1 a la fase S, regulando así el ritmo del ciclo mitótico durante los estadios tempranos del desarrollo foliar. Asimismo, TOC1 también controla la ploidía somática característica del endociclo durante estadios tardíos del desarrollo foliar y en las células del hipocotilo. Utilizando técnicas de citometría de flujo y parámetros de cinéticas de crecimiento foliar se pudo determinar que en plantas que sobre-expresan TOC1 la fase S es más corta, lo que se correlaciona con la represión diurna del gen CELL DIVISION CONTROL 6 (CDC6). Este gen codifica un factor esencial en la formación de los complejos de pre-replicación que determinan los orígenes de replicación del ADN. Mediante técnicas de inmunoprecipitación de cromatina encontramos que la represión de CDC6 ocurre a través de la unión directa de TOC1 al promotor de CDC6. Los análisis de interacción genética demostraron que los fenotipos de crecimiento reducido y de ploidía somática alterada observados en plantas que sobre-expresan TOC1, quedaban revertidos al sobre-expresarse también CDC6. Estos resultados confirman que la función de TOC1 en el ciclo celular ocurre en gran medida a través de la represión de CDC6. La desaceleración de la progresión del ciclo celular en plantas que sobre-expresan TOC1 afecta no solo el desarrollo de los órganos de la planta, sino también el desarrollo tumoral en los tallos de las inflorescencias. Por lo tanto, nuestros estudios demuestran que la función de TOC1 es importante en la regulación rítmica de la maquinaria pre-replicativa del ADN para controlar el crecimiento de las plantas en resonancia con el medio ambiente.
The circadian function is essential for plant growth and its adaptation to the environment. The molecular machinery responsible for the establishment of the circadian rhythmicity relies on the rhythmic oscillation of differentially expressed genes with different peaks of expression along the day and night. The rhythms in gene expression are translated into oscillations of physiological and developmental processes. Plant growth is controlled by a plethora of different processes that ultimately work through the control of cell proliferation and differentiation. Cell proliferation relies on the proper progression of the mitotic cycle, which is divided in 4 phases: S (DNA synthesis), M (Mitosis) and two gap phases G1 and G2, that take place before S and M phases, respectively. Cell differentiation coincides with the entry into the endocycle, a variant of the mitotic cycle in which genomic DNA duplicates without further division or mitosis. Even though the circadian clock and cell cycle as separate pathways have been well documented in plants, the possible direct interplay between these two cyclic processes has not been previously addressed. The work performed during this Thesis has focused on the characterization of the role of the circadian clock in the control of the cell cycle during plant growth. We found that plants with slower than Wild-Type circadian clocks slow down the progression of the cell cycle, while plants with faster clocks speed it up. The core clock component TIMING OF CAB EXPRESSION 1 (TOC1) controls the G1 to S-phase transition, thereby regulating the rhythm of the mitotic cycle during the early stages of leaf development. Likewise, TOC1 controls somatic ploidy during later stages of leaf development and of hypocotyl cell elongation. The use of flow cytometry analyses and of leaf growth kinetics showed that in plants over-expressing TOC1, the S-phase is shorter, which correlates with the diurnal repression of the CELL DIVISION CONTROL 6 (CDC6) gene. This gene encodes an essential component of the pre-replication complex, which is responsible for the specification of DNA origins of replication. Chromatin immunoprecipitation assays showed that the diurnal repression of CDC6 most likely relies on the direct binding of TOC1 to the CDC6 promoter. Genetic interaction analyses showeed that the reduced growth and altered somatic ploidy phenotypes observed in plants over-expressing TOC1 were reverted when CDC6 was over-expressed. Thus, our results confirm that TOC1 regulation of the cell cycle occurs through CDC6 repression. The slow cell cycle progression in plants over-expressing TOC1 has an impact not only in organ development but also on tumor growth in stems and inflorescences. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment.

The Periodic Event Synchronization Unit aligns devices without the ability to be triggered by an external source. The primary function of the unit is to align the pattern trigger pulses of two pulse pattern generators which supply four inputs of a multiplexer. The pulse pattern generators lack the ability to start their code according to an external signal. When operating, the designed unit maintains a specific pattern alignment of two binary data streams of 5 gigabits per second as a multiplexer combines them into a data stream of four times the bit rate. In addition to alignment, the unit can introduce offsets of up to 50 nanoseconds to the pattern alignment which corresponds to 250 bits. The unit is designed to allow the alignment of other devices as well, requiring as input the two event signals of the same frequency which need to be aligned. In order to align the devices providing the event pulses, one of the devices must either accept an external clocking source or have the ability to frequency modulate the internal clock. In practice, the test system was able to achieve and maintain the desired signal characteristics from the output of the multiplexer. The unit's robust design is shown by providing alignment of patterns for the full operating range of the pulse pattern generators and allowing a generator pattern to be aligned to a generic event pulse. Use of multiple units allows alignment of additional devices. The development of the Periodic Event Synchronization Unit provided an inexpensive solution to creating very high bit rate signals using preexisting equipment, as no commercial products were found to accomplish the same function.

A trend in design and implementation of modern industrial automation systems is to integrate computing, communication and control into a unified framework at different levels of machine/factory operations and information processing. These distributed control systems are referred to as networked control systems (NCSs). They are composed of sensors, actuators, and controllers interconnected over communication networks. As most of communication networks are not designed for NCS applications, the communication requirements of NCSs may be not satisfied. For example, traditional control systems require the data to be accurate, timely and lossless. However, because of random transmission delays and packet losses, the control performance of a control system may be badly deteriorated, and the control system rendered unstable. The main challenge of NCS design is to both maintain and improve stable control performance of an NCS. To achieve this, communication and control methodologies have to be designed. In recent decades, Ethernet and 802.11 networks have been introduced in control networks and have even replaced traditional fieldbus productions in some real-time control applications, because of their high bandwidth and good interoperability. As Ethernet and 802.11 networks are not designed for distributed control applications, two aspects of NCS research need to be addressed to make these communication networks suitable for control systems in industrial environments. From the perspective of networking, communication protocols need to be designed to satisfy communication requirements for NCSs such as real-time communication and high-precision clock consistency requirements. From the perspective of control, methods to compensate for network-induced delays and packet losses are important for NCS design. To make Ethernet-based and 802.11 networks suitable for distributed control applications, this thesis develops a high-precision relative clock synchronisation protocol and an analytical model for analysing the real-time performance of 802.11 networks, and designs a new predictive compensation method. Firstly, a hybrid NCS simulation environment based on the NS-2 simulator is designed and implemented. Secondly, a high-precision relative clock synchronization protocol is designed and implemented. Thirdly, transmission delays in 802.11 networks for soft-real-time control applications are modeled by use of a Markov chain model in which real-time Quality-of- Service parameters are analysed under a periodic traffic pattern. By using a Markov chain model, we can accurately model the tradeoff between real-time performance and throughput performance. Furthermore, a cross-layer optimisation scheme, featuring application-layer flow rate adaptation, is designed to achieve the tradeoff between certain real-time and throughput performance characteristics in a typical NCS scenario with wireless local area network. Fourthly, as a co-design approach for both a network and a controller, a new predictive compensation method for variable delay and packet loss in NCSs is designed, where simultaneous end-to-end delays and packet losses during packet transmissions from sensors to actuators is tackled. The effectiveness of the proposed predictive compensation approach is demonstrated using our hybrid NCS simulation environment.

Norway spruce (Picea abies L. Karst) is a conifer belonging to the group gymnosperms and is an ecologically and economically important species in several parts of Europe. It is crucial for trees like Norway spruce to adapt timing of events such as bud set and growth cessation to the local environment in order to maximize the growth period while avoiding frost damage. This thesis aims at widening the knowledge about genetic control of annual growth rhythm in Norway spruce and particularly the control of bud set. Using spruce transformants ectopically expressing PaFT/TFL1-LIKE 2 (PaFTL2) the prior hypothesis that PaFTL2 induces bud set is confirmed. This is further supported by spatial and temporal expression patterns in seedlings and adult trees. It is further shown that gymnosperms possess at least two FLOWERING LOCUS T/TERMINAL FLOWER 1 (FT/TFL1)-like genes with TFL1-like function, suggesting the ancestor of FT and TFL1 to be more TFL1-like. PaFTL1 appears to have complementary expression patterns to that of PaFTL2 both spatially and temporally indicating they may act together to control growth in Norway spruce. Since bud set is controlled by photoperiod and circadian clock genes are implicated in this process, putative clock homologs were studied to gain insight into the circadian clock in gymnosperms. Several clock homologs were identified and their expression showed a diurnal pattern but the expression was rapidly damped in constant conditions. Transgenic Arabidopsis expressing putative core clock genes from spruce indicate that at least three genes, PaCCA1, PaGI and PaZTL, appear to have a conserved function between angiosperms and gymnosperms. Taken together these results suggest that gymnosperms have a similar core clock structure as angiosperms even though fundamental differences might exist since the cycling of the clock genes were rapidly damped in free-running conditions. The studies presented in this thesis support substantial conservation of pathway components controlling photoperiodic responses in angiosperms and gymnosperms and identify PaFTL2 as a component of growth rhythm control. However, important changes in these processes are also evident. The results provide a solid basis for future research on molecular mechanisms controlling an adaptive trait in an important non-model organism.

Mammalian circadian rhythms of behaviour are synchronized to external time by daily resetting of the master pacemaker, the suprachiasmatic nucleus (SCN), in response to light, in a process known as light-induced clock entrainment. microRNA-132 (miR-132) is induced by light within the SCN via a MAPK-CREB-dependent mechanism and has the capacity to attenuate the entraining effects of light. However, the identity of the genes that miR-132 regulates and their roles in the light-entrainment process have not yet been characterized. This thesis describes that 2 gene clusters involved in chromatin remodeling (i.e. Mecp2, Ep300, Jarid1a) and translational control (i.e. Btg2, Paip2a) are under the regulation of miR-132 in the SCN and coordinated regulation of these genes underlies miR132-dependent modulation of mPeriod1 (mPer1) and mPeriod2 (mPer2) and the light-entrainment process. I find that the Period genes are bound and transcriptionally modulated by MeCP2. In addition, Paip2a acts as a repressor of Period translation. This work further proposes that miR-132 is enriched for chromatin and translation-associated target genes-and, thus, miR-132 is an important orchestrator of chromatin remodeling and protein translation within the SCN clock, thereby fine-tuning clock entrainment.

Many new fabrication technologies, from nanotechnology and MEMS to printed organic semiconductors, center on constructing arrays of large numbers of sensors, actuators, or other devices on a single substrate. The utility of such an array could be greatly enhanced if each device could be managed by a programmable controller and all of these controllers could coordinate their actions as a massively-parallel computer. Kentucky Architecture nanocontroller array with very low per controller circuit complexity can provide efficient control of nanotechnology devices. This thesis provides a detailed description of the control hierarchy of a digital system needed to build "nanocontrollers" suitable for controlling millions of devices on a single chip. A Verilog design and FPGA prototype of a nanocontroller system is provided to meet the constraints associated with a massively-parallel programmable controller system.

Chez les Mammifères, les rythmes circadiens, présentant une rythmicité endogène proche de 24h sont sous le contrôle d'une horloge interne située dans les Noyaux Suprachiasmatiques de l'hypothalamus (NSC). Afin de mieux comprendre la distribution du message temps par les NSC, nous avons étudié spécifiquement le contrôle du rythme journalier de sécrétion de mélatonine, hormone sécrétée de nuit par la glande pinéale, partant du postulat selon lequel les NSC contrôleraient ce rythme en utilisant, pendant la journée, un signal inhibiteur de nature GABAergique pour inhiber la voie polysynaptique reliant les Noyaux paraventriculaires de l'hypothalamus (PVN) à la glande pinéale. Nos premiers travaux ont révélé, par lésions thermiques des différents noyaux intéressés, un rôle de simple relais de l'information pour les PVN, ainsi qu'une action inhibitrice des NSC sur la synthèse de mélatonine de jour associée à une action stimulatrice nocturne. A l'aide de la technique de microdialyse intracérébrale multiple, nous avons ensuite pu confirmer, in vivo, que l'activité neuronale nocturne des NSC était cruciale pour une stimulation nocturne de synthèse de mélatonine. De plus, un rôle important du neurotransmetteur glutamate a pu être montré pour cette action stimulatrice. Nous avons également montré que la chute de sécrétion de mélatonine en fin de nuit était due à une augmentation de sécrétion GABAergique par les NSC associée soit à la disparition du signal stimulateur soit à l'apparition d'un second signal inhibiteur. Par ailleurs, en corrélant l'expression neuronale des gènes de l'horloge Per1 et Per2 et la sécrétion de vasopressine par les NSC, nous avons révélé une régionalisation fonctionnelle des NSC. Ensemble, les résultats de cette thèse ont permis de réactualiser le concept du contrôle du rythme journalier de mélatonine par l'horloge biologique, exemple de moyen de distribution du message temps au reste de l'organisme via le système nerveux autonome
In mammals, circadian rhythms, i. E. Showing an endogenous rhythmicity close to 24h are under the control of a master biological clock, located in the suprachiasmatic nucleus of the hypothalamus (SCN). In order to further understand the mechanisms of time distribution by the SCN, we specifically studied the control of the daily secretion of melatonin, hormone strictly produced at night by the pineal gland, with the initial hypothesis that the SCN controls the daily rhythm of melatonin synthesis by imposing during daytime an inhibitory signal of GABAergic nature onto the polysynaptic pathway connecting the Paraventricular Nucleus of the hypothalamus (PVN) to the pineal gland. By lesioning or removing the different nuclei involved in this pathway, our first study revealed a simple role of information-relay for the PVN, as well as a combined inhibitory and stimulatory role for the SCN during the day and the night respectively. Using the multiple intracerebral microdialysis technique, we were then able to confirm in vivo that the SCN nocturnal activity is crucial for a nocturnal stimulation of melatonin synthesis and we showed as well that glutamatergic transmission is responsible for such a stimulatory action onto the melatonin synthesis. In addition, we revealed that the early morning drop of melatonin synthesis is due to the association of an increased GABAergic secretion derived by the SCN and either the disappearance of the stimularory signal or the appearance of a second inhibitory signal. Furthermore, correlating the neuronal expression of the clock genes Per1 and Per2 and the SCN vasopressin secretion, we revealed a clear functional compartmentalisation of the SCN. Together these results helped re-actualising the concept of the control of the daily rhythm of melatonin synthesis by the biological clock, which is a great example of time distribution to the rest of the organism via the autonomic nervous system

Even though the understanding of cell-cycle regulators in plants has tremendously increased over the last years, still little is known about cell-cycle regulation in response to environmental signals like DNA damage. A ubiquitous stress for any organism is DNA stress that can either be caused by exogenous sources or internal processes like chromatid separation or DNA strands separation during replication. The posttranslational regulation of Cdk1-type kinases through inhibitory phosphorylation through Wee1-type kinases in the so-called P-loop at the residue Tyr15 or the analogous positions has been found to be of pivotal importance for the arrest of the cell cycle after DNA damage in yeast and animals. But this mechanism is apparently not conserved in plants, as suggested by the hypersensitivity analysis of CDKA;1 dephospho-mutants. The first half of this study focus on possible regulation of CDKA;1 through T-loop phosphorylation upon replication stress in Arabidopsis. The positively acting phosphorylation on T161 and analogous residues in the so-called T-loop of the kinase that is required for full CDK activity and serves in substrate recognition. Remarkably, a T-loop phospho-mimicry mutant of CDKA;1, was almost 100% resistant to hydroxyurea (HU) and can partially rescue the hypersensitivity of wee1 to HU. T-loop phosphorylation is catalyzed by CDK activating kinases (CAKs) that are themselves CDKs with typical P- and T-loop regions. Evidence is obtained that WEE1 might inhibit CDKDs (Cdk-activating kinases) that would subsequently result in reduced CDKA;1 activity, and thus, cell-cycle arrest upon DNA damage. It is revealed that dephospho-mimicry mutants of CDKD;2 and 3, which can not be inhibited through WEE1 showed hypersensitivity to HU and not to bleomycin, suggesting their involvement in cell-cycle arrest specifically upon replication stress. Hypersensitivity of cdkd;2cdkd;3 to replication stress suggested possible activation of CDKA;1 through CDKD;1 independent of WEE1. An essential role of CDKDs in stabilizing CDKA;1 kinase activity during gamete development has been suggested. Defects observed in cdka;1VFcdkd mutants during meiosis but not in cdka;1VF mutants emphasize on importance of CDKA;1 T-loop phosphorylation for appropriate meiotic division.In second part of this study interaction between cell-cycle and circadian has been studied. A feedback loop in which the cell cycle could potentially regulate the circadian clock was suggested as a number of circadian genes were found to be deregulated in a microarray experiment with holomorphic CDKA;1 mutants. Thus the circadian gating of cell division of wildtype and cdka;1 mutants was studied under diurnal growth conditions. The altered time of division observed in cell-cycle mutants supported the idea of cell-cycle regulation in a time dependent manner. Expression profile of clock genes were analyzed in cdka;1 mutants through luciferase assay system. An altered period and intensity of expression observed in these mutants compared to wild type plants suggested a direct or indirect effect of CDKA;1 activity on clock gene expression.

Робота присвячена методам й засобам автоматичного спостереження та візуалізації часу в гідродинамічному годиннику власної розробки. Наведено опис конструкції годинника. Запропоновано мікропроцесорну систему визначення поточного часу та декорованої візуалізації часу. Розроблена документація на технічну реалізацію годинника.

A Wireless Sensor Network (WSN) is a collection of tiny devices called sensor nodes which are deployed in an area to be monitored. Each node has one or more sensors with which they can measure the characteristics of their surroundings. In a typical WSN, the data gathered by each node is sent wirelessly through the network from one node to the next towards a central base station. Each node typically has a very limited energy supply. Therefore, in order for WSNs to have acceptable lifetimes, energy efficiency is a design goal that is of utmost importance and must be kept in mind at all levels of a WSN system. The main consumer of energy on a node is the wireless transceiver and therefore, the communications that occur between nodes should be carefully controlled so as not to waste energy. The Medium Access Control (MAC) protocol is directly in charge of managing the transceiver of a node. It determines when the transceiver is on/off and synchronizes the data exchanges among neighbouring nodes so as to prevent collisions etc., enabling useful communications to occur. The MAC protocol thus has a big impact on the overall energy efficiency of a node. Many WSN MAC protocols have been proposed in the literature but it was found that most were not optimized for the group of WSNs displaying very low volumes of traffic in the network. In low traffic WSNs, a major problem faced in the communications process is clock drift, which causes nodes to become unsynchronized. The MAC protocol must overcome this and other problems while expending as little energy as possible. Many useful WSN applications show low traffic characteristics and thus a new MAC protocol was developed which is aimed at this category of WSNs. The new protocol, Dynamic Preamble Sampling MAC (DPS-MAC) builds on the family of preamble sampling protocols which were found to be most suitable for low traffic WSNs. In contrast to the most energy efficient existing preamble sampling protocols, DPS-MAC does not cater for the worst case clock drift that can occur between two nodes. Rather, it dynamically learns the actual clock drift experienced between any two nodes and then adjusts its operation accordingly. By simulation it was shown that DPS-MAC requires less protocol overhead during the communication process and thus performs more energy efficiently than its predecessors under various network operating conditions. Furthermore, DPS-MAC is less prone to become overloaded or unstable in conditions of high traffic load and high contention levels respectively. These improvements cause the use of DPS-MAC to lead to longer node and network lifetimes, thus making low traffic WSNs more feasible.
Dissertation (MEng)--University of Pretoria, 2008.
Electrical, Electronic and Computer Engineering
MEng
Unrestricted

The goal of synchronization is to align/synchronize the time and frequency scales of all nodes within a network. In industrial applications, synchronization enables simultaneous triggering of distributed events and synchronous data acquisition at di erent nodes. For wide distributed systems, such as Internet, clock synchronization is advantageous for maintaining end-to-end Quality of Service (QoS). Ethernet is the technology of choice for the future networks. Its low cost, the ever increasing data rates and low complexity and maintenance are key enablers for adopting it at all geographical scales and applications, ranging from the Network Provider to the industrial level. However, low cost and simplicity that characterizes the legacy Ethernet are only part of its attraction. The challenge is that it was initially conceived as a 'best-e ort' and asynchronous oriented technology, limitations that di cult its adoption to handle, for example, timesensitive applications in the industrial eld, or carrier-class transport of services, from the Network Provider perspective. To better support new applications with tight synchronization requirements, standardization bodies and equipment manufacturers are making considerable e orts to extend its functionalities and release solutions to meet the synchronization requirements of new applications. High accuracy time synchronization is a key enabler for o ering such carrierclass QoS and handling distributed applications with stringent synchronization needs. Today's Ethernet-based approaches that deliver time synchronization rely on timestamped packets that distribute to the network. The acts of timestamping and sending the packet are crucial for achieving high accuracy synchronization, as they are exposed to a number of delay variabilities from the source to the destination node that impair the synchronization accuracy between nodes. As the timestamping is a key component for actual synchronization protocols, the main goal in this work is to evaluate the impact of these sources of inaccuracies of Ethernet layers on the synchronization accuracy between nodes. The followed evaluation method is based on a real prototype utilizing a low-cost platform FPGAs. The inherent complexity of these devices poses an additional challenge to the evaluation process, especially if the addressed synchronization accuracies are at the level of few nanoseconds. Therefore, this work also discusses and proposes methods to overcome platform-dependent limitations. Additionally, this work proposes a di erent perspective for Ethernet technology which consists on envisioning the legacy Ethernet with a time synchronization functionality. We believe that such a new capability would allow Ethernet to better handle time sensitive applications and to be independent and compatible from and with the higher layers while keeping its initial philosophy: low-cost, simplicity and asynchronous technology.
L'objectiu de la sincronització és alinear/sincronitzar les escales de temps de tots els nodes d'una xarxa. En aplicacions industrials, la sincronització permet l'inici simultani d'esdeveniments distribuïts o l'adquisició de dades de forma síncrona als diferents nodes. En grans sistemes distribuïts, com per exemple l'Internet, la sincronització és bene ciosa per mantenir Qualitat de Servei (QdS) entre dos nodes distants entre si. Ethernet és la tecnologia d'el lecció per les xarxes del futur. El seu baix cost, les contínues actualitzacions de velocitat i la baixa complexitat i manteniment són els activadors per adoptar-la a tots els nivells geogrà cs i aplicacions, des de Proveïdors de Xarxa en xarxes metropolitanes, a aplicacions industrials en xarxes locals. No obstant, el baix cost i simplicitat que caracteritzen a Ethernet constitueixen només una part del seu interés. El problema és que aquesta va ser originalment concebuda com una tecnologia de serveis mínims i asíncrona, dues limitacions que di culten la seva adopció en aplicacions amb fortes restriccions de temps, tant en el camp industrial com en el transport de serveis de qualitat d'operadora. Per tal de suportar noves aplicacions amb fortes restriccions de temps, diversos organismes d'estandardització i fabricants d'equipament estan treballant activament per extendre les seves funcionalitats i llançar solucions per tal de complir amb nous requeriments de sincronització. La sincronització de temps d'alta exactitud és clau per oferir serveis d'alt QdS i suportar aplicacions distribuïdes que necessitin fortes restriccions de temps. Les solucions d'avui dia basades en Ethernet que entreguen sincronització de temps es basen en distribuïr paquets amb una marca de temps a la xarxa. Les accions d'inserir la marca de temps i enviar el paquet són decisives per aconseguir sincronització d'alta exactitud ja que estan exposades a un nombre de variabilitats de retard des de l'origen ns el destí que empitjoren l'exactitud de la sincronització entre nodes. Degut a que l'acció d'inserir la marca de temps és un component clau pels protocols de sincronització actuals, l'objectiu principal en aquesta Tesi és avaluar l'impacte d'aquestes fonts d'inexactitud de les capes d'Ethernet en la sincronitzaci ó entre nodes. El mètode d'avaluació està basat en un prototipus real utilitzant plataformes basades en matrius de portes lògiques programables per camp (de l'anglès, Field Programmable Gate Arrays (FPGA)) de baix cost. La inherent complexitat d'aquests dispositius suposa un repte addicional al procés d'avaluació, especialment si s'adreçen exactituds de sincronització de nivells de pocs nanosegons. Aleshores, aquesta Tesi també debat i proposa mètodes per vèncer les limitacions dependents de la plataforma. A més, aquesta Tesi proposa una perspectiva diferent per a la tecnologia Ethernet, la qual consisteix en extendre l'Ethernet inicial amb una funcionalitat de sincronització. Creiem que una funcionalitat com aquesta permetria a Ethernet suportar aplicacions amb fortes restriccions de temps amb independència de, i compatibilitat amb capes més altes tot mantenint la seva loso a inicial: baix cost, simplicitat i tecnologia asíncrona.

In this digital age, cryptography is largely built in computer hardware or software as discrete structures. One of the most useful of these structures is finite fields. In this thesis, we explore a variety of applications of the theory and applications of arithmetic and computation in finite fields in both the areas of cryptography and cryptanalysis. First, multiplication algorithms in finite extensions of prime fields are explored. A new algebraic description of implementing the subquadratic Karatsuba algorithm and its variants for extension field multiplication are presented. The use of cy- clotomic fields and Gauss periods in constructing suitable extensions of virtually all sizes for efficient arithmetic are described. These multiplication techniques are then applied on some previously proposed public key cryptosystem based on exten- sion fields. These include the trace-based cryptosystems such as XTR, and torus- based cryptosystems such as CEILIDH. Improvements to the cost of arithmetic were achieved in some constructions due to the capability of thorough optimisation using the algebraic description. Then, for symmetric key systems, the focus is on algebraic analysis and attacks of stream ciphers. Different techniques of computing solutions to an arbitrary system of boolean equations were considered, and a method of analysing and simplifying the system using truth tables and graph theory have been investigated. Algebraic analyses were performed on stream ciphers based on linear feedback shift registers where clock control mechanisms are employed, a category of ciphers that have not been previously analysed before using this method. The results are successful algebraic attacks on various clock-controlled generators and cascade generators, and a full algebraic analyses for the eSTREAM cipher candidate Pomaranch. Some weaknesses in the filter functions used in Pomaranch have also been found. Finally, some non-traditional algebraic analysis of stream ciphers are presented. An algebraic analysis on the word-based RC4 family of stream ciphers is performed by constructing algebraic expressions for each of the operations involved, and it is concluded that each of these operations are significant in contributing to the overall security of the system. As far as we know, this is the first algebraic analysis on a stream cipher that is not based on linear feedback shift registers. The possibility of using binary extension fields and quotient rings for algebraic analysis of stream ciphers based on linear feedback shift registers are then investigated. Feasible algebraic attacks for generators with nonlinear filters are obtained and algebraic analyses for more complicated generators with multiple registers are presented. This new form of algebraic analysis may prove useful and thereby complement the traditional algebraic attacks. This thesis concludes with some future directions that can be taken and some open questions. Arithmetic and computation in finite fields will certainly be an important area for ongoing research as we are confronted with new developments in theory and exponentially growing computer power.

In this digital age, cryptography is largely built in computer hardware or software as discrete structures. One of the most useful of these structures is finite fields. In this thesis, we explore a variety of applications of the theory and applications of arithmetic and computation in finite fields in both the areas of cryptography and cryptanalysis. First, multiplication algorithms in finite extensions of prime fields are explored. A new algebraic description of implementing the subquadratic Karatsuba algorithm and its variants for extension field multiplication are presented. The use of cy- clotomic fields and Gauss periods in constructing suitable extensions of virtually all sizes for efficient arithmetic are described. These multiplication techniques are then applied on some previously proposed public key cryptosystem based on exten- sion fields. These include the trace-based cryptosystems such as XTR, and torus- based cryptosystems such as CEILIDH. Improvements to the cost of arithmetic were achieved in some constructions due to the capability of thorough optimisation using the algebraic description. Then, for symmetric key systems, the focus is on algebraic analysis and attacks of stream ciphers. Different techniques of computing solutions to an arbitrary system of boolean equations were considered, and a method of analysing and simplifying the system using truth tables and graph theory have been investigated. Algebraic analyses were performed on stream ciphers based on linear feedback shift registers where clock control mechanisms are employed, a category of ciphers that have not been previously analysed before using this method. The results are successful algebraic attacks on various clock-controlled generators and cascade generators, and a full algebraic analyses for the eSTREAM cipher candidate Pomaranch. Some weaknesses in the filter functions used in Pomaranch have also been found. Finally, some non-traditional algebraic analysis of stream ciphers are presented. An algebraic analysis on the word-based RC4 family of stream ciphers is performed by constructing algebraic expressions for each of the operations involved, and it is concluded that each of these operations are significant in contributing to the overall security of the system. As far as we know, this is the first algebraic analysis on a stream cipher that is not based on linear feedback shift registers. The possibility of using binary extension fields and quotient rings for algebraic analysis of stream ciphers based on linear feedback shift registers are then investigated. Feasible algebraic attacks for generators with nonlinear filters are obtained and algebraic analyses for more complicated generators with multiple registers are presented. This new form of algebraic analysis may prove useful and thereby complement the traditional algebraic attacks. This thesis concludes with some future directions that can be taken and some open questions. Arithmetic and computation in finite fields will certainly be an important area for ongoing research as we are confronted with new developments in theory and exponentially growing computer power.

Le cycle de division cellulaire et l'horloge circadienne sont deux processus fondamentaux de la régulation cellulaire qui génèrent une expression rythmique des gènes et des protéines. Dans les cellules mammifères, les mécanismes qui sous-tendent les interactions entre le cycle cellulaire et l'horloge restent très mal connus. Dans cette thèse, nous étudions ces deux oscillateurs biologiques, à la fois individuellement et en tant que système couplé, pour comprendre et reproduire leurs principales propriétés dynamiques, détecter les composants essentiels du cycle cellulaire et de l'horloge, et identifier les mécanismes de couplage. Chaque oscillateur biologique est modélisé par un système d'équations différentielles ordinaires non linéaires et ses paramètres sont calibrés par rapport à des données expérimentales: le modèle du cycle cellulaire se base sur les modifications post-traductionnelles du complexe Cdk1-CycB et mène à un oscillateur de relaxation dont la dynamique et la période sont contrôlés par les facteurs de croissance; le modèle de l'horloge circadienne reproduit l'oscillation antiphasique BMAL1/PER:CRY et l'adaptation de la durée des états d'activation et répression par rapport à deux signaux d’entrée hormonaux déphasés. Pour analyser les interactions entre les deux oscillateurs nous étudions la synchronisation des deux rythmes pour des régimes de couplage uni- ou bi-directionnels. Les simulations numériques reproduisent les ratios entre les périodes de l'horloge et du cycle cellulaire, tels que 1:1, 3:2 et 5:4. Notre étude suggère des mécanismes pour le ralentissement du cycle cellulaire avec des implications pour la conception de nouvelles chronothérapies
The cell division cycle and the circadian clock are two fundamental processes of cellular control that generate cyclic patterns of gene activation and protein expression, which tend to be synchronous in healthy cells. In mammalian cells, the mechanisms that govern the interactions between cell cycle and clock are still not well identified. In this thesis we analyze these two biological oscillators, both separately and as a coupled system, to understand and reproduce their main dynamical properties, uncover essential cell cycle and clock components, and identify coupling mechanisms. Each biological oscillator is first modeled by a system of non-linear ordinary differential equations and its parameters calibrated against experimental data: the cell cycle model is based on post-translational modifications of the mitosis promoting factor and results in a relaxation oscillator whose dynamics and period are controlled by growth factor; the circadian clock model is transcription-based, recovers antiphasic BMAL1/PER:CRY oscillation and relates clock phases to metabolic states. This model shows how the relative duration of activating and repressing molecular clock states is adjusted in response to two out-of-phase hormonal inputs. Finally, we explore the interactions between the two oscillators by investigating the control of synchronization under uni- or bi-directional coupling schemes. Simulations of experimental protocols replicate the oscillators’ period-lock response and recover observed clock to cell cycle period ratios such as 1:1, 3:2 and 5:4. Our analysis suggests mechanisms for slowing down the cell cycle with implications for the design of new chronotherapies

Les arbres classiques de distribution du signal d'horloge au sein des microprocesseurs synchrones présentent un certain nombre de limitations : skew, jitter, limitation de la fréquence, influence de perturbations et de dispersions quelles que soient leurs natures. Ces facteurs, critiques pour les microprocesseurs modernes complexes, sont devenus la raison principale qui a poussé à la recherche d'autres types d'architecture de génération et de distribution du signal d'horloge. Un exemple d'un tel système alternatif est le réseau de PLLs couplées, où les PLLs sont géographiquement distribuées sur la puce, et génèrent des signaux d'horloge locaux qui sont ensuite synchronisés, en temps réel, par un échange d'information entre les PLLs voisines et une rétroaction locale réalisé par leur correcteurs. La nature active du réseau de PLLs de génération et de distribution du signal d'horloge, qui peut permettre de surpasser les limitations mentionnées plus tôt, oblige à sortir du cadre classique des outils et des méthodes de la Microélectronique habituellement appliqués à l'étude et à la conception de ce type de systèmes. En effet, les aspects dynamiques de bouclage et de transformation de signaux au sein de tels systèmes complexes rendent leur conception extrêmement difficile voire parfois impossible. La difficulté principale consiste en un changement des propriétés d'un sous-système local indépendant par rapport aux propriétés du même sous-système faisant partie du réseau. Effectivement, il existe beaucoup de méthodes et d'outils de conception d'une PLL isolée garantissant un comportement et des propriétés locales désirés. Néanmoins, ces propriétés désirées locales, selon la topologie d'interconnexion considérée, ne sont pas forcément conservées quand il s'agit d'un réseau de PLLs interconnectées et de son comportement global. Le but principal de cette thèse est ainsi de développer une méthode de synthèse de la loi de commande décentralisée réalisée au sein de chaque sous-système (tel qu'une PLL) assurant le comportement désiré pour le réseau global. Une méthode de transformation du problème de synthèse globale en un problème équivalent de synthèse d'une loi de commande locale est proposée en se basant sur l'hypothèse des sous-systèmes identiques interconnectés en réseau. Le lien entre les propriétés locales et globales est établi grâce aux approches d'Automatique avancée telles que les approches entrée-sortie et la dissipativité. Ce choix de méthode permet non seulement de réduire considérablement la complexité du problème initial mais aussi de ramener le problème de synthèse à une forme proche des méthodes de conception locale utilisées en Microélectronique, ce qui garantit une continuité logique de leur évolution. Ensuite la méthode proposée est combinée avec la commande H∞ et l'optimisation sous contraintes LMIs conduisant au développement d'algorithmes efficaces de résolution du problème posé. Elles sont à la fois particulièrement bien adaptées à l'application considérée, c'est-à-dire à la synchronisation d'un réseau de PLLs, et sont facilement généralisables aux autres types de problèmes de commande de systèmes de grande dimension. Le premier aspect permet une intégration naturelle et aisée de la méthode dans le flux de conception existant en Microélectronique, très riche et mature à ce jour, alors que le deuxième offre une solution à d'autres problèmes de commande de systèmes interconnectés en réseau, un champ d'application aujourd'hui en plein essor.

Dans les systèmes temps réel et répartis, la synchronisation de tâches fait souvent référence au temps réel. Cependant, cette synchronisation ne peut être cohérente que si tous les nœuds ont la même perception du temps. La synchronisation des horloges locales des nœuds s'impose alors pour que l'ensemble des nœuds aient une vue globale cohérente du temps qu'ils manipulent. Dans cette thèse nous nous intéressons à ce problème de synchronisation d'horloges notamment sous les aspects suivants : proposition d'une approche permettant d'étudier les structures des algorithmes de synchronisation d'horloges en utilisant les mêmes modèles. Cette partie fait suite à la thèse de J. He soutenue en 1993. Analyse des performances temporelles (la précision d'accord et l'exactitude) et de complexité des algorithmes en termes de nombre de messages. Analyse des fautes traitées par les algorithmes de synchronisation d'horloges et proposition d'une démarche d'analyse de la tolérance aux fautes dans les algorithmes de synchronisation d'horloges. Étude d'implantation des algorithmes de synchronisation d'horloges. On s'intéresse notamment aux performances obtenues en plaçant la synchronisation des horloges au niveau M. A. C. (Medium Access Control) des réseaux

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Bacteria are becoming resistant to a growing number of conventional antibiotics at a worrisome rate. Therefore, there is an increasing demand for new antimicrobial therapeutics. Antimicrobial peptides (AMPs) are one of promising alternatives for conventional antibiotics and are thought to be less likely to induce resistance. AMPs have been coupled to molecular scaffolds for biomedical applications such as hydrogels for wound dressings and covering implants. Here AMPs were chemically conjugated to the CR4 hydrophilic polymers. The CPF_C1 is a short peptide isolated from Xenopus clivii, the original sequence was modified one called LCPF_C1, aiming to create some distance between the first glycine and the azide added on the N terminus (on the coupled sequence). The conjugation between the AMP and the CR4 polymer used a click chemistry reaction with two steps, dependent of a hetero crosslinker (DBCO???PEG4???NHS Ester). Dynamic light scattering (DLS) and fluorimeter assays were used to evaluate the efficiency of coupling. MALDI-ToF analysis showed 3 molecules of LCPF_C1 peptide for each CR4 polymer. Moreover, microrheology showed changes on hybrids increasing the viscosity. Finally, the compounds were evaluate against four different bacteria: Staphylococcus aureus, Klebsiela pneumoneae carbapenemases (Kpc), Escherichia coli and Pseudomonas aeruginosa. It was possible to observe MIC???s against P. aeruginosa of 11 mM by using the peptide (LCPF_C1) and 55 mM for the original sequence. When the hibrids were compared to the free polymer was not found MIC values against K. pneumoneae (CR-Kp). On the other hand, the hibrids showed three times less activity than the free polymer against P. aeruginosa. No MIC values were found aganst S. aureus. Finally, against E. coli was observed a MIC value of 1000 mM for the free CR4 and 250 mM for the hibrids. On this way, the present work showed the possibility to functionalize biopolymers by using bioactive molecules coupled to biopolimers, changing the physical-chemical characteristics and increasing they applicability against bacterial infections.
Uma taxa alarmante de bact??rias tem se tornado resistente a um grande n??mero de antimicrobianos convencionais. Desta forma, a demanda por novas terapias antimicrobianas tem aumentado proporcionalmente. Assim, o uso de pept??deos antimicrobianos (PAMs) consistem em uma promissora alternativa para antibacterianos convencionais, particularmente podendo ser acoplado a estruturas moleculares para aplica????es biom??dicas, como hidrog??is para curativos e cobertura de implantes. No presente trabalho, PAMs foram quimicamente conjugados ao pol??mero hidrof??lico inspirado nas prote??nas naturais col??geno e seda CR4. O pept??deo CPF_C1 consiste em um pept??deo contendo 17 res??duos de amino??cidos isolado de Xenopus clivii, com atividade comparada ?? bact??rias Gram-negativas e -positivas. Para este estudo, a sequ??ncia original do CPF-C1 foi modificada e intitulada LCPF_C1, objetivando aumentar a dist??ncia entre a primeira glicina e a azida adicionada na por????o N-terminal. A conjuga????o entre o PAM e o pol??mero CR4 foi realizada por meio de click chemistry reaction em dois passos, dependentes do hetero crosslinker (DBCO???PEG4???NHS Ester). Ensaios de espalhamento de luz din??mico (DLS) e fluorimetria foram utilizados para avaliar a efici??ncia de acoplamento e demonstraram a presen??a do pept??deo acoplado ao pol??mero. An??lises de MALDI-ToF demonstraram 3 mol??culas do pept??deo LCPF-C1 para cada mol??cula do pol??mero CR4. Al??m disso, dados de microrreologia demonstraram mudan??as nos h??bridos como aumento de viscosidade. Finalmente, os compostos foram avaliados contra quatro bact??rias diferentes: Staphylococcus aureus, K. pneumoneae carbapenemase (Kpc), Escherichia coli e Pseudomonas aeruginosa. Foi poss??vel observar para P. aeruginosa MICs de 11 mM utilizando o pept??deo (LCPF_C1) e 55 mM para a sequ??ncia original. Quando comparados aos h??bridos em rela????o a atividade do pol??mero livre n??o foi encontrado valor de MIC contra K. pneumoneae (CR-Kp). Por outro lado, os h??bridos demonstraram um MIC cerca de tr??s vezes menor que o pol??mero livre contra P. aeruginosa. Nenhum valor de MIC foi encontrado contra S. aureus. Finalmente, contra E. coli observou-se MIC de 1000 mM para o pol??mero CR4 e 250 mM para os h??bridos. Desta forma o presente trabalho demonstrou a possibilidade de funcionalizar biopol??meros por meio do acoplamento de mol??culas bioativas, alterando suas caracter??sticas f??sico-qu??micas e aumentando sua aplicabilidade contra infec????es bacterianas.

Streamciphers are common cryptographic algorithms used to protect the confidentiality of frame-based communications like mobile phone conversations and Internet traffic. Streamciphers are ideal cryptographic algorithms to encrypt these types of traffic as they have the potential to encrypt them quickly and securely, and have low error propagation. The main objective of this thesis is to determine whether structural features of keystream generators affect the security provided by stream ciphers.These structural features pertain to the state-update and output functions used in keystream generators. Using linear sequences as keystream to encrypt messages is known to be insecure. Modern keystream generators use nonlinear sequences as keystream.The nonlinearity can be introduced through a keystream generator's state-update function, output function, or both. The first contribution of this thesis relates to nonlinear sequences produced by the well-known Trivium stream cipher. Trivium is one of the stream ciphers selected in a final portfolio resulting from a multi-year project in Europe called the ecrypt project. Trivium's structural simplicity makes it a popular cipher to cryptanalyse, but to date, there are no attacks in the public literature which are faster than exhaustive keysearch. Algebraic analyses are performed on the Trivium stream cipher, which uses a nonlinear state-update and linear output function to produce keystream. Two algebraic investigations are performed: an examination of the sliding property in the initialisation process and algebraic analyses of Trivium-like streamciphers using a combination of the algebraic techniques previously applied separately by Berbain et al. and Raddum. For certain iterations of Trivium's state-update function, we examine the sets of slid pairs, looking particularly to form chains of slid pairs. No chains exist for a small number of iterations.This has implications for the period of keystreams produced by Trivium. Secondly, using our combination of the methods of Berbain et al. and Raddum, we analysed Trivium-like ciphers and improved on previous on previous analysis with regards to forming systems of equations on these ciphers. Using these new systems of equations, we were able to successfully recover the initial state of Bivium-A.The attack complexity for Bivium-B and Trivium were, however, worse than exhaustive keysearch. We also show that the selection of stages which are used as input to the output function and the size of registers which are used in the construction of the system of equations affect the success of the attack. The second contribution of this thesis is the examination of state convergence. State convergence is an undesirable characteristic in keystream generators for stream ciphers, as it implies that the effective session key size of the stream cipher is smaller than the designers intended. We identify methods which can be used to detect state convergence. As a case study, theMixer streamcipher, which uses nonlinear state-update and output functions to produce keystream, is analysed. Mixer is found to suffer from state convergence as the state-update function used in its initialisation process is not one-to-one. A discussion of several other streamciphers which are known to suffer from state convergence is given. From our analysis of these stream ciphers, three mechanisms which can cause state convergence are identified.The effect state convergence can have on stream cipher cryptanalysis is examined. We show that state convergence can have a positive effect if the goal of the attacker is to recover the initial state of the keystream generator. The third contribution of this thesis is the examination of the distributions of bit patterns in the sequences produced by nonlinear filter generators (NLFGs) and linearly filtered nonlinear feedback shift registers. We show that the selection of stages used as input to a keystream generator's output function can affect the distribution of bit patterns in sequences produced by these keystreamgenerators, and that the effect differs for nonlinear filter generators and linearly filtered nonlinear feedback shift registers. In the case of NLFGs, the keystream sequences produced when the output functions take inputs from consecutive register stages are less uniform than sequences produced by NLFGs whose output functions take inputs from unevenly spaced register stages. The opposite is true for keystream sequences produced by linearly filtered nonlinear feedback shift registers.

Les contaminations des surfaces constituent un problème majeur de santé public rencontré dans plusieurs secteurs tels que les milieux hospitaliers et l’industrie alimentaire. Cette contamination consiste en l’adhésion de bactéries pathogènes ou opportunistes qui peuvent former des biofilms. Ces biofilms sont potentiellement des sources de développement et de prolifération des bactéries. Une voie efficace de lutte contre la contamination microbienne est l’élaboration de surfaces bactéricides visant à empêcher ou diminuer l’adhésion bactérienne. Basé sur le savoir-faire du laboratoire de Chimie des Substances Naturelles dans le domaine des polysaccharides, nous avons entrepris d’élaborer des supports antibactériens en fixant de manière covalente des molécules aux propriétés antibactériennes sur des fibres lignocellulosiques de pâte à papier (la pâte Kraft). Le lien triazole formé est stable vis-à-vis de l’hydrolyse acide ou basique, et subsiste dans des conditions oxydantes et réductrices. Nous avons choisi de nous intéresser dans un premier temps à la fixation de manière covalente d’un antibactérien commercial connu, le triclosan, sur des fibres lignocellulosiques de la pâte à papier ; puis dans une deuxième approche nous nous sommes orientés vers la fixation de porphyrines sur ces supports. Enfin dans une troisième approche, nous avons fixés des molécules aromatiques qui n’acquièrent leur potentiel antimicrobien qu’après greffage sur le support via le lien triazole. L’étude de l’effet antimicrobien des différents matériaux a montré pour une activité bactéricide du matériau Pâte Kraft-triclosan vis-à-vis notamment de trois souches fréquemment trouvées en milieu hospitalier: Pseudomonas aeruginosa, Bacillus cereus et Escherichia coli ; Dans le cas du greffage de photosensibilisateurs, seul le matériau pâte Kraft-Porphyrine neutre a permis une photoinactivation des souches après irradiation
Surface contamination by pathogens constitutes a major public health problem encountered in many areas such as hospitals, environment and food industry. This contamination consists in the adhesion of pathogenic or opportunistic bacteria that can attach to a biotic or abiotic surface and lead to the formation of biofilm. An effective way to fight against microbial contamination is the development of antibacterial surfaces, in order to prevent or reduce bacterial adhesion. Based on the expertise of the Laboratoire de Chimie des Substances Naturelles in the field of polysaccharides, we have undertaken the development of antibacterial materials by grafting through covalent bonds molecules presenting antibacterial properties onto lignocellulosic fibers (in this case Kraft pulp fibers). Triazoles are resistant to acid and basic hydrolysis, reductive and oxidative conditions. This moiety is also relatively resistant to metabolic degradation and is not posing particular toxicity problems. The study of the antibacterial effect has shown a bactericidal activity of the triclosan-Kraft pulp sheet against three strains frequently found in hospitals: Pseudomonas aeruginosa, Bacillus cereus and Escherichia coli. In the case of grafting photosensitizers, only the neutral porphyrin-Kraft pulp sheet material displayed a strong photobactericidal activity after irradiation

[Truncated abstract] This thesis is split into two parts. In part one; A 50 K dual mode oscillator, the aim of the project was to build a 50 K precision oscillator with frequency stability on the order of 1014 from 1 to 100 seconds. A dual-mode temperature compensation technique was used that relied on a turning point in the frequency-temperature relationship of the difference frequency between two orthogonal whispering gallery modes in a single sapphire crystal. A cylindrical sapphire loaded copper cavity resonator was designed, modelled and built with a turning point in the difference frequency between an E-mode and H-mode pair at approximately 52.5 K . . . The frequencies and Q-factors of whispering spherical modes in the 3-12 GHz range in the fused silica resonator are measured at 6, 77 and 300 K and the Q-factor is used to determine the loss tangent at these temperatures. The frequency and Q-factor temperature dependence of the TM2,1,2 whispering gallery mode at 5.18 GHZ is used to characterise the loss tangent and relative permittivity of the fused silica from 4-300 K. Below 22 K the frequency-temperature dependence of the resonator was found to be consistent with the combined effects of the thermal properties of the dielectric and the influence of an unknown paramagnetic impurity, with a spin resonance frequency at about 138 ± 31 GHz. Below 8 K the loss tangent exhibited a 9th order power law temperature dependence, which may be explained by Raman scattering of Phonons from the paramagnetic impurity ions. A spherical Bragg reflector resonator made from multiple concentric dielectric layers loaded in a spherical cavity that enables confinement of field in the centre of the resonator is described. A set of simultaneous equations is derived that allow the calculation of the required dimensions and resonance frequency for such a resonator and the solution is confirmed using finite element analysis. A spherical Bragg reflector resonator is constructed using Teflon and free-space as the dielectric materials. A Q-factor of 22,000 at 13.87 GHz was measured and found to compare well with the design values.

La synthèse contrôlée par la cible sous contrôle cinétique (Kinetic Target-Guided Synthesis, KTGS) est une approche relativement peu explorée, alternative à la chimie combinatoire traditionnelle,dans laquelle la protéine cible participe à la synthèse du ou de ses propres ligands. Ainsi, les travaux présentés dans la première partie de cette thèse ont pour principal objectif d'élargir l'éventail des réactions actuellement disponibles en KTGS grâce à la réaction d'aldolisation voire d'amidation, et ce en utilisant la β-sécrétase (BACE-1) comme cible biologique, qui est une enzyme étroitement impliquée dans la maladie d'Alzheimer. La seconde partie de cette thèse a été consacrée à la synthèse de marqueurs de masse fluorescents bioconjugables basés sur l'association d'un noyau coumarinique et d'une fonction phosphonium. Les deux générations présentées dans ce manuscrit ont entre autre permis de synthétiser une sonde FRET permettant de détecter l'activité enzymatique de la BACE-1, qui pourrait par ailleurs être un outil intéressant pour l'analyse des bruts réactionnels des réactions de click in situ,et diminuer les quantités d'enzyme engagées dans ces expériences. Enfin dans la dernière partie de cette thèse nous décrivons la mise au point de nouvelles réactions de conjugaison bio-orthogonale pour le marquage de molécules comportant une fonction aldéhyde. Nous avons ainsi développé d'une part une réaction trois composants via une séquence de condensation/Mannich/lactamisation et d'autre part une réaction d'oléfination de Wittig
The kinetic target-guided synthesis (KTGS), is an underexplored alternative approach to combinatorial chemistry, in which the biological target is able to assemble its own inhibitors from a pool of fragments. Thus, the first part of this thesis aimed at extending the scope of the reactions available for the KTGS, by investigating the aldolisation and amidation reaction, using the β-secretase (BACE-1) as biological target, which is an enzyme narrowly involved in the Alzheimer's disease. The second part of this thesis was dedicated to the synthesis of bioconjagatable fluorophores containing a phosphonium group as mass tag associated to a coumarin core. Both generations presented in this manuscript allowed us, among other things, to synthesize a FRET probe that proved suitable for the determination of BACE-1 enzymatic activity. The utility of such a fluorogenic tool could be leveraged to facilitate the analysis of crude mixtures obtained during KTGS experiments, and lessen the amount of enzyme required in these experiments. Finally, in the last part of this thesis, we describe the development of two new bioorthogonal reactions allowing the selective labeling of molecules containing an aldehyde moiety : 1) a three component reaction involving a condensation/Mannich/lactamisation procedure, between an amine, an aldehyde and an enol partner; 2) a Wittig ligation between an aldehyde and a phosphonium bearing an active methylene

Today, the growing and aging population, and the rise of new global threats on human health puts an increasing demand on the healthcare system and calls for preventive actions. To make existing medical treatments more efficient and widely accessible and to prevent the emergence of new threats such as drug-resistant bacteria, improved diagnostic technologies are needed. Potential solutions to address these medical challenges could come from the development of novel lab-on-chip (LoC) for point-of-care (PoC) diagnostics. At the same time, the increasing demand for sustainable energy calls for the development of novel approaches for energy conversion and storage systems (ECS), to which micro- and nanotechnologies could also contribute. This thesis has for objective to contribute to these developments and presents the results of interdisciplinary research at the crossing of three disciplines of physics and engineering: electrokinetic transport in fluids, manufacturing of micro- and nanofluidic systems, and surface control and modification. By combining knowledge from each of these disciplines, novel solutions and functionalities were developed at the macro-, micro- and nanoscale, towards applications in PoC diagnostics and ECS systems. At the macroscale, electrokinetic transport was applied to the development of a novel PoC sampler for the efficient capture of exhaled breath aerosol onto a microfluidic platform. At the microscale, several methods for polymer micromanufacturing and surface modification were developed. Using direct photolithography in off-stoichiometry thiol-ene (OSTE) polymers, a novel manufacturing method for mold-free rapid prototyping of microfluidic devices was developed. An investigation of the photolithography of OSTE polymers revealed that a novel photopatterning mechanism arises from the off-stoichiometric polymer formulation. Using photografting on OSTE surfaces, a novel surface modification method was developed for the photopatterning of the surface energy. Finally, a novel method was developed for single-step microstructuring and micropatterning of surface energy, using a molecular self-alignment process resulting in spontaneous mimicking, in the replica, of the surface energy of the mold. At the nanoscale, several solutions for the study of electrokinetic transport toward selective biofiltration and energy conversion were developed. A novel, comprehensive model was developed for electrostatic gating of the electrokinetic transport in nanofluidics. A novel method for the manufacturing of electrostatically-gated nanofluidic membranes was developed, using atomic layer deposition (ALD) in deep anodic alumina oxide (AAO) nanopores. Finally, a preliminary investigation of the nanopatterning of OSTE polymers was performed for the manufacturing of polymer nanofluidic devices.

QC 20140509

Rappid
NanoGate
Norosensor

Conselho Nacional de Desenvolvimento Científico e Tecnológico
A hiperatividade locomotora e as alterações nos ritmos circadianos têm sido descritas em roedores e humanos expostos ao etanol durante o desenvolvimento. Considerando que a atividade locomotora em camundongos é conhecida por variar ao longo das fases do ciclo claro escuro, é possível que o fenótipo hiperativo resultante da exposição precoce ao etanol também varie em função da hora do dia. Além disso, é possível que a hiperatividade apresentada pelos indivíduos expostos ao etanol durante o desenvolvimento esteja associada com distúrbios no sistema de controle do ritmo circadiano. Neste estudo, avaliamos estas duas possibilidades realizando uma análise circadiana da atividade locomotora e da expressão dos genes de relógio de camundongos adolescentes expostos ao etanol durante o período de surto de crescimento cerebral. Para tanto, camundongos suíços criados e mantidos em um ciclo claro/escuro de 12h (luzes acesas às 2:00h, apagadas as 14:00h) foram injetados com etanol (5g/kg ip, grupo ETOH) ou um volume equivalente de solução salina (grupo SAL) em dias alternados do segundo ao oitavo dias pós-natais. No 30 dia pós-natal, os animais foram testados em campo aberto por 15 minutos em diferentes momentos do ciclo claro/escuro: durante a fase clara entre 6:00 e 7:30h e entre12:00 e 13:30h; durante a fase escura entre 18:00 e 19:30h e entre 0:00 e 01:30h. Durante a fase escura os testes foram realizados sob iluminação com luz vermelha. Após os testes comportamentais, alguns animais foram randomicamente selecionados para as análises de imunofluorescência da expressão dos genes PER 1, 2 e 3 no núcleo supraquiasmático. Ao longo dos seis primeiros minutos, a atividade locomotora dos animais testados durante o período claro não mudou significativamente ou apresentou um leve aumento e a dos animais testados no período escuro apresentou uma marcante redução. Além disso, o grupo de animais testados entre 00:00 e 1:30h apresentou a maior atividade locomotora e o grupo dos animais testados entre 12:00 e 13:30h apresentou a menor atividade locomotora. De modo importante, a exposição neonatal ao etanol promoveu hiperatividade locomotora apenas no grupo de animais testados entre 00:00 e 1:30h. Em relação aos genes de controle do ritmo circadiano, a exposição precoce ao etanol afetou apenas a expressão do gene Per1 que foi menor entre 18:00 e 19:30h. O fato de que a expressão dos genes de controle do ritmo circadiano foi alterada no meio da fase escura e que a hiperatividade locomotora foi observada apenas no final da fase escura é compatível com a hipótese de que a hiperatividade induzida pelo etanol pode estar associada com as perturbações de controle do ritmo circadiano.
Locomotor hyperactivity and alterations of circadian rhythmicity have been described both in rodents and in humans exposed to ethanol during development. Considering that spontaneous locomotor activity in mice is known to vary as a function of the time of the day, it is conceivable that the expression of the locomotor hyperactivity phenotype resulting from developmental ethanol exposure also varies throughout the day. In addition, it is possible that the hyperactivity presented by individuals early exposed to ethanol is associated with a dysfunction in the control system of the circadian rhythm. In this study, we tested these two possibilities by performing a circadian analysis of the locomotor activity and the expression of clock genes in adolescent mice exposed to ethanol during the brain growth spurt period. Subjects were Swiss mice that were bred and maintained in our laboratory on a 12:12h light/dark cycle (lights on: 2:00, lights off: 14:00). From postnatal day 2 (PN2) to PN8, litters either received ethanol (5 l/g i.p., 25% in saline solution) or an equivalent volume of saline solution every other day. At PN30, locomotor activity was automatically assessed for 15 min in the open field test. During the light period, the animals were tested between 6:00 and 7:30 h or between 12:00 and 13:30 h whereas during the dark period the tests were performed between 18:00 and 19:30 h or between 0:00 and 1:30 h. During the dark period, the tests were conducted under red dim light illumination. After the behavioral tests, a sample of animals was randomly selected for the analysis by immunofluorescence of the expression of genes PER 1, 2 and 3 in the suprachiasmatic nucleus. Throughout the first 6 min, the locomotor activity of animals tested during the light period did not change or only increased slightly, while animals tested during the dark period presented a marked reduction. Furthermore, animals tested between 00:00 and 1:30 h presented the highest activity while animals tested between 12:00 and 13:30 h presented the lowest locomotor activity. Importantly, neonatal exposure to ethanol caused locomotor activity only in those animals tested between 00:00 and 1:30 h. As for the the clock genes, neonatal exposure to ethanol only affected the PER 1 expression, which was lowest between 18:00 and 19:30. Our data is in line with the idea that locomotor activity varies as a function of the time of the day. The fact that the expression of a clock gene was altered in then middle of the dark cycle and that locomotor hyperactivity was observed only at the end of this period is compatible with the hypothesis that the hyperactivity observed as result of ethanol exposure is associated with alterations in the control of the circadian rhythm.

碩士
國立交通大學
資訊工程研究所
82
Owing to the rapid development of hardware, we are able to integrate services which are provided by dedicated networks at the old days and to provide the more popular multimedia services. In Broadband Integrate Service Digital Network (B- ISDN), a universal network interface can be provided to users. In this the construction cost of networks can be reduced. However, the traffic characteristics of multimedia services vary dramastically. Besides, the real time constraint of services has to be met. It has been the main goal of efficient congestion control for Asynchronous Transfer Mode (ATM) network to satisfy the Quality of Service (QOS) requested by users and maximize the utilization of network. A number of works on congestion control for ATM network have been published recently. However, they are either without practical algorithm or lack of flexibility and efficiency. In this thesis, we propose a virtual clock based congestion control. In addition to reserving the features of virtual clock algorithm such as efficiency and flexibility, we propose a method to solve the unbounded delay problem. It is shown by simulation that our scheme is able to bound the end-to-end delay and to perform as expected at other aspects like cell loss rate, queue length, throughput.

Transporting continuous bit rate (CBR) or real-time periodic traffic is one of the major services that ATM-based B-ISDN technology is promising to provide. This service requires the receiver to preserve the original inter-cell spacing. However, statistical multiplexing and buffering in the ATM transport networks can introduce significant jitter in the inter-arrival period of the cell stream, thus degrading the quality, of the cell play-back at the receiver. An additional complication in plesiochronous networks is the absence of the transmitter clock frequency at the receiver. Therefore, the receiver must be capable of extracting the frequency of the transmitter clock and removing the jitter from the arriving cell stream. This thesis provides a thorough treatment of the clock recovery and jitter removal problems for CBR traffic in ATM networks, and proposes a new practical design of a receiver unit for handling multirate CBR traffic. The design proposed complies with the ATM standards. Our design employs a number of control parameters that can be varied to optimize the operation of the receiver and to provide high adaptability to rapidly changing input cell traffic. The proposed scheme is based on monitoring the fluctuation in the receiver buffer occupancy to derive a jitter free receiver clock. The hardware design has been specified and simulated extensively using VHDL (a hardware description language), and the simulation results show that our design is robust and very effective in removing cell delay jitter and restoring the original CBR stream.

碩士
國立中央大學
電機工程學系
103
In recent year, according to rapid development of process and computers, the data bandwidth increases progressively. The serial data transmission is widely used for bus instead of parallel data transmission, for example, DisplayPort, SATA, USB, and PCI-E. This study presents a clock and data recovery (CDR), and takes the DisplayPort specification as reference material. In this thesis, a CDR with adaptive gain control is proposed. The adaptive gain control circuit measures the jitter of recovered clock to detect the input data implied high-frequency or low-frequency jitter at the moment. By adjusting the bandwidth of data recovery loop, the clock and data recovery circuits can improve jitter tolerance at high-frequency and low-frequency. At 5.4 Gbps data rate, CDR jitter tolerance improvement is 60.9 percent at high-frequency, and 81.6 percent at low-frequency. This proposed was implemented by TSMC 90 nm (TN90GUTM) 1P9M process with 1 V supply voltage. When input data rate is at 5.4 Gbps, the recovered clock rate is 2.7 GHz. The period jitter of the output recovered clock is 23.11 ps (p-p). The power consumption of the CDR is 24.8 mW. The chip area is 1260 1178 um2 and the core area is 323 329 um2.

碩士
國立暨南國際大學
電機工程學系
97
Clock and data recovery (CDR) circuit plays an important role in the receivers of communication applications. In recent years, high data rate and low power consumption become the main research issues of CDR. In this thesis, we proposed a 1.8V 1.25Gbps full rate CDR with full linear control range VCO. The delay cell of the VCO is modified to extend the linear controlled voltage range from 0V to 1.8V. Hence it is very suitable for low voltage operation. The simulation result shows that the oscillating frequency range of VCO is from 0.88GHz to 1.63GHz. The VCO power consumption is 19.61mW without buffer and 47.75mW with buffer. The phase noise of the VCO is -108dBc/Hz. The jitter of the CDR is 310ps. The circuit is implemented using TSMC 0.35μm 2P4M CMOS technology. The chip core area is 271.15μm 199.95μm. For testing purpose, the chip is also including a 1.25GHz PLL and a PRBS. Besides, we used MATLAB Simulink to establish the model for analyzing the stability and feasibility of the circuit.

碩士
國立暨南國際大學
電機工程學系
97
Clock and data recovery (CDR) circuit is a key component used in Ethernet, optical network and serial link. High-speed and low-power CDR circuits find their applications in a variety of communication systems. The thesis introduces the architectures and some design issues of clock and data recovery circuits. A low voltage and high speed CDR is then designed and implemented. Behavior model built by using Matlab Simulink is used to simulate and analyze the function and stability of the CDR. A 1.2V 2.5Gbps CDR circuit has been implemented in TSMC 0.18m 1P6M CMOS process. A half-rate CDR circuit using 1.25GHz VCO is presented. The VCO uses a modified delay cell to extend the linear control range to 0~1.2V for low voltage operation. The linearity and gain of the VCO transfer characteristics are much improved. The modified VCO works at a tuning range from 0.76 GHz to 1.5GHz, and its peak-to-peak jitter at 1.25 GHz is 30ps. The phase noise is -100.7dBc /Hz at 1MHz offset from a 1.25GHz center frequency. The power consumption of the core circuit is 1.8mW, with buffer is 3.21mW. The clock and data recovery circuit consumed 49.5mW at 1.2V power supply, and its jitter is 175ps. The power consumption of the half-rate phase detector is 12.7 mW. The circuit buffers consumed 33mW. For testing purpose a 2.5GHz PLL and a 215-1 PRBS generator are included in the chip. The whole chip area is 920m 920m. The CDR circuit area is 322.4m 403.3m.

Tese de doutoramento em Biologia Experimental e Biomedicina, na especialidade de Neurociências e Doença, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra
To maintain organism homeostasis, organisms developed a system of endogenous molecular clocks to anticipate the momentum of the day and couple several physiological processes. A dysfunctional clock may be involved in several diseases, including metabolic syndromes and neurological disorders. Alzheimer’s disease (AD) patients display abnormal circadian rhythms, well before the appearance of cognitive deficits. There is evidence that: (i) a dysfunctional clock is responsible for metabolic syndromes and alterations in glucose and insulin levels, (ii) insulin affects β-amyloid levels, one the AD-associated peptides and (iii) AD patients have an altered circadian clock. Altered brain metabolism, namely impaired glucose utilization and energy metabolism, is observed early in AD progression. This occurs prior to a major hallmark of AD, the deterioration of cognitive functions. Therefore, energy failure appears to be one of the earliest symptoms of AD. Interestingly, disruption of the circadian system has been considered to be an early event in AD as well. Aging underlies a decline in many capabilities and body functions. The molecular clock also slowly loses its pace. This lost is a progressive process and seems to be a derivative of aging. Importantly, many of those processes are orchestrated by the circadian clock, which ‘times’ the homeostasis, cell cycle, as well as memory and learning. The body may also lose the ability of accurately tracking time and coordinate body functions, such as metabolism in all its different forms, namely lipidogenesis, adipogenesis, glucose and insulin metabolism. Thus, the repercussions on the synchrony of metabolic processes are implicit and might be of pivotal importance in the onset of age-related neurodegenerative diseases such as AD. The circadian system is a design model consisting of an oscillator responsible for the generation of the daily rhythm. It relates to input pathways, by which the environment and other components of the nervous system feedback information to the clock, and also to output routes, by which the oscillator provides temporal information to a wide range of physiological and behavioral processes. When cells are removed and maintained in a brain slice, they generate 24-hour rhythms of electrical activity, secretion, and gene expression. The amplitude of the electrical activity, gene expression and hormone‑secreting activity decline in the aging brain. Impairment of circadian clock synchronization displays major alterations throughout the body. These include changes in memory formation, as well as hormonal and neurochemical changes, with consequences for neuroplasticity. The triple transgenic (3xTg-AD) mouse model of AD displays abnormalities in circadian rhythmicity prior to AD pathology, making this mouse model instrumental to investigate the interplay between circadian clock, metabolism and the link to AD pathogenesis. Thus, in this study we aimed to unveil the impact of AD in biological rhythms relevant in pathology, as well as to gain a deeper understanding of the physiopathology. Initially, this study evaluated the impact of the circadian profile on cognitive performance in 24 week-old 3xTg-AD mice versus age-matched control (Non-Tg) mice, explored how this correlated with hippocampal long-term potentiation (LTP) and how mitochondrial function, an indicator of brain metabolism, was impacted by circadian biology. Our analysis was performed at Zeigeber (ZT) 04 (4 hours after lights on) and ZT 16 ( 4 hours after lights off). The diurnal variation, in hippocampus-dependent learning tasks of 3xTg-AD and Non-Tg animals was assayed. The Morris water maze (MWM) results showed that, independently of the time of day, cognitive deficits were observed in 3xTg-AD; indeed, a correlation between ZT’s and genotypes was only possible when making use of the Spatial Reverse learning task. These results suggested that the pattern of circadian variation of memory performance depends on the type of task and the impact of circadian clock on AD pathology is better outlined in more complex tasks. The amplitude of hippocampal LTP, an electrophysiological correlate of memory related processes, i.e. electrical signals recorded extracellularly at Schaffer fibers/CA1 pyramid synapses, exhibited a circadian profile in Non-Tg animals, which was not observed in 3xTg-AD mice. Disturbances in mitochondrial function and oxidative stress have been implicated in the pathophysiology of AD. Therefore, mitochondrial membrane potential (ΔΨm) was measured in cortical synaptosomes; exposure to hydrogen peroxide (H2O2) revealed increased mitochondrial depolarization at Zt16, suggesting a ZT-dependent susceptibility to oxidative stimulus. Simultaneously, intracellular calcium (iCa) levels were measured, revealing an inability of 3xTg-AD synaptosomes to maintain iCa levels at Zt16; H2O2 stimulus did not show significant changes between ZT´s or genotypes. Data suggest that, when challenged/stressed, the mitochondria unravel to be dysfunctional in the AD mouse model. These findings are in agreement with the oxidative stress hypothesis for AD. We concluded that, at this stage, there is impairment of the circadian rhythm on key traits of AD pathogenesis; the study further outlines the prevalence of diurnal variation and pinpoints the need to better characterize the neurobiology of disease and integration of circadian rhythms as a crucial variable. Moreover, it supported the idea of mitochondrial dysfunction and increased susceptibility to oxidant stimulus. NMR has been largely used as a method for longitudinal studies of metabolites and biochemical pathways. Therefore, metabolic analysis was also performed using this technique. This technique allows to measure, under baseline conditions, the direct pathway (hepatic glycogen synthesis from intact hexose) and indirect pathway (hepatic glycogen synthesis from gluconeogenic processing). The administration of deuterated water (2H2O) leads to positional 2H-enrichment of hepatic glycogen, rendering the effect of a disease (e.g. type 2 diabetes or other interventions on these fluxes (circadian disruptions) accessible to in vivo evaluation. At the age of 6 months (24 wks) the pathology in 3xTg-AD mice has been described to be established. Thus, in the second chapter we proved that at this age the cognitive decline, electrophysiological and mitochondrial dysfunction were present. Then, we posed a question regarding the circadian rhythm: Are biological rhythms impaired in AD? Data outlined the relevance of the diurnal variation studies in pathology. We showed that AD impairs biological rhythms, similarly to a circadian disruption. Animals were given intraperitoneally a bolus of 1% 2H2O 24h prior to euthanazia and the liver used. mRNA and metabolites were extracted and analyzed, by QPCR and NMR, respectively. Biological samples of 3xTg-AD and Non-Tg mice were collect at ZT 04 & ZT 16. Our analysis showed alterations in already validated biomarkers at specific ZT’s. Metabolic alterations perceived in AD samples were accompanied by changes in gene expression. Moreover, rate limiting enzymes presented a diurnal variation profile in Non-Tg animals, while 3xTg-AD animals displayed alterations in the circadian pattern of expression in some metabolic genes. Analysis of metabolic fluxes by NMR depicted changes in lipid metabolism, and differences in gluconeogenesis, which were coincident with metabolic alterations seen in type 2 diabetes mellitus, obesity or metabolic syndrome. Finally, analysis of canonical circadian genes revealed an alteration in the diurnal variation of core clock genes in the 3xTg-AD mice. In addition, AD animal model showed alterations in diurnal variation profile of metabolic networks. Those changes correlate with a chronodysfunction at the peripheral level. In suma, in this thesis we show that AD-related pathology in the 3XTg-AD mouse model is linked to cognitive decline (Chapters 4.1 & 4.2) and metabolic alterations in brain and peripheral tissues (Chapter 4.2 & 4.3). In addition, this thesis ends by underpinning an AD-related chronodysfunction, thus raising the question of circadian dysfunction as a culprit for neuropathology.
De modo a manter a homeostasia, os organismos desenvolveram um sistema de relógios moleculares endógenos com vista a antecipar o momento do dia e assim sincronizar vários processos fisiológicos. Um relógio disfuncional pode estar envolvido em várias doenças, incluindo síndrome metabólica e doenças neurológicas. Pacientes com a doença de Alzheimer (AD) exibem ritmos circadianos anormais que precedem o aparecimento de défices cognitivos. Há evidências de que: (i) um relógio disfuncional é responsável por síndromes metabólicas e alterações nos níveis de glucose e de insulina, (ii) insulina afecta os níveis de β-amilóide, um dos péptidos associados com AD e (iii) pacientes com AD possuem uma alteração do relógio circadiano. Alterações no metabolismo, nomeadamente alterações na utilização de energia e no metabolismo da glicose, precedem a deterioração das funções cognitivas. Estas alterações parece ser um dos primeiros sintomas de ADDA. O envelhecimento é acompanhado de um declínio varias funções corporais. O relógio circadiano também perde lentamente o seu ritmo. Este é um processo progressivo e parece ser um derivado do processo de envelhecimento. É importante salientar que muitos desses processos são orquestrados pelo relógio circadiano, que regula temporalmente a homeostase, o ciclo celular, bem como os processos de memória e aprendizagem. O corpo também pode perder a capacidade de controle temporal e de coordenacão e sincronização das funções corporais, tais como o metabolismo em todas as suas diferentes formas, nomeadamente lipidogenesis, adipogênese, metabolismo da glicose e insulina. As repercussões da falta de sincronia dos processos metabólicos são implícitos e parece ser fundamental no aparecimento de doenças neurodegenerativas em que a idade e um factor de risco, tais como DA. O sistema circadiano é um relógio molecular, que consiste de um oscilador responsável pela geração do ritmos biológicos diários. Através de vias de entrada, o ambiente e outros componentes do sistema nervoso enviam informação para o relógio, e também para as rotas de saída, através da qual o oscilador fornece informação temporal para uma grande variedade de processos fisiológicos e comportamentais. Quando as células são removidas e mantidas em uma fatia do cérebro, eles geram ritmos de 24 horas de actividade eléctrica, de secreção e da expressão do gene. A amplitude da actividade eléctrica, a expressão dos genes e a actividade hormona-secretora declina com o envelhecimento cerebral. A dessincronização do relógio circadiano apresenta grandes alterações em todos os sistemas biológicos. Estes incluem mudanças na formação da memória, bem como alterações hormonais e neuroquímicas, com consequências para a neuroplasticidade. O modelo animal triplo transgénico (3xTg-AD) da AD exibe abnormalidades na ritmicidade circadiana que precedem alterações patológicas, tornando este modelo de doença instrumental para investigar a interação entre o relógio circadiano, o metabolismo e as possíveis conexões com a patologia. Sendo assim, este estudo teve como objetivo revelar o impacto de AD nos ritmos biológicos relevantes na patologia, bem como obter uma compreensão mais profunda da fisiopatologia. Inicialmente, este estudo avaliou o impacto do perfil circadiano no desempenho cognitivo em murganhos 3xTg-AD de 24 semanas de idade versus animais controle da mesma idade (Non-TG), explorou também como estas alterações são correlacionadas com a potenciação de longa duração (LTP)no hipocampo e como a função mitocondrial, um indicador do metabolismo cerebral, e alterada pela biologia circadiano. A nossa análise foi realizada no Zeitgeber (ZT) 04 (4 horas após a luz acesa) e ZT 16 (4 horas após a luzes apagadas). A variação diurna, em tarefas de aprendizagem dependentes do hipocampo nos 3xTg-AD e nos Non-Tg foi avaliada. Os resultados do “Morris water maze” (MWM) mostraram que, independentemente da a hora do dia existiam défices cognitivos observáveis nos 3xTg-AD; uma correlação entre ZT e os genótipos só foi possível quando se realizou tarefa de aprendizagem espacial reversa no MWM. Estes resultados sugerem que o padrão de variação circadiana do desempenho da memória depende do tipo de tarefa e o impacto do relógio circadiano em patologia é mais visível em tarefas mais complexas. A amplitude da LTP no hipocampo, uma correlação eletrofisiológica com processos de memória relacionada, ou seja os sinais elétricos, registrados extracelularmente em fibras de Schaffer/CA1 sinapses pirâmide, exibiu um perfil circadiano em animais Non-Tg, o que não foi observado em animais 3xTg-AD. Perturbações na função mitocondrial e do stress oxidativo têm sido implicados na patofisiologia da AD. Por conseguinte, o potencial de membrana mitocondrial (ΔΨm) foi medida em sinaptossomas corticais; exposição ao peróxido de hidrogênio (H2O2) revelou aumento de depolarização mitocondrial em ZT 16, sugerindo uma susceptibilidade ZTdependente ao estímulo oxidativo. Simultaneamente, os níveis de cálcio intracelular (ICA) foram medidos, revelando uma incapacidade dos sinaptossomas de 3xTg-AD para manter os níveis iCa ao ZT 16; a exposição a estímulo não mostraram alterações significativas entre ZT's ou genótipos. Os dados sugerem que, quando desafiado / estressado, as mitocôndrias apresentam um comportamento disfuncional no modelo animal de DA. Estes resultados estão de acordo com a hipótese do stress oxidativo na DA. Concluiu-se que, nesta fase, há comprometimento dos ritmos circadianos em características-chave na patogénese da DA; o estudo destaca ainda mais a prevalência de variação diurna e aponta a necessidade de caracterizar melhor a neurobiologia da doença e a integração de ritmos circadianos como uma variável crucial. RMN tem sido largamente utilizado como um método para os estudos longitudinais de metabolitos e vias bioquímicas. Esta técnica permite medir, em condições basais, a via direta (síntese de glicogênio hepático de hexose intacta) e via indireta (síntese de glicogênio hepático do processamento gliconeogênica). A administração de água deuterada (2H2O) leva ao enriquecimento posicional 2H- de glicogénio hepático, tornando o efeito de uma doença (por exemplo, diabetes tipo 2 ou outras intervenções nestes fluxos (dessincronia de ritmos circadianos), acessíveis para avaliação. Com a idade de 6 meses (24 semanas) a patologia em ratinhos 3xTg-AD foi descrito como estando estabelecida. Assim, no segundo capítulo, mostramos que a esta idade o declínio cognitivo, disfunção eletrofisiológico e mitocondrial estavam presentes. Em seguida, perguntamo-nos sobre o ritmo circadiano: estão os ritmos biológicos dessincronizados na DA? Os dados sublinham a relevância dos estudos de variação diurna em patologia. Mostrámos que da prejudica os ritmos biológicos, de forma semelhante a uma dessincronização do ritmo circadiano. Os animais receberam por via intraperitoneal um bolus de 1% 2H2O 24 horas antes de serem euthanaziados e o fígado removido. RNA e metabolitos foram extraídas e analisadas, por qPCR e RMN, respectivamente. As amostras biológicas de 3xTg-AD e ratos Non-Tg foram coletados ao ZT 04 & ZT 16. A análise revelou alterações em biomarcadores já validados em ZT’s específicos. AS alterações metabólicas percebidos em amostras de AD foram acompanhadas por mudanças na expressão gênica. Além disso, as enzimas limitantes apresentam um perfil de variação diurna em animais Non-Tg, enquanto nos animais 3xTg-AD apresentaram alterações no padrão circadiano de expressão em alguns genes metabólicos. Análise de fluxos metabólicos por RMN apresentam alterações no metabolismo lipídico e diferenças na gliconeogênese, que eram coincidentes com alterações metabólicas descritas em diabetes mellitus tipo 2, obesidade ou síndrome metabólica. Finalmente, a análise dos genes circadianos canônicos revelou uma alteração na variação diurna desses genes nos animais 3xTg-AD. Além disso, o modelo animal de DA mostrou alterações no perfil de variação diurna das redes metabólicas. Essas alterações parecem se correlacionar com um cronodisfunção ao nível periférico. Em suma, nesta tese demonstramos que patologia relacionada com a AD no modelo 3XTg-AD está correlacionada com o declínio cognitivo (capítulos 4.1 e 4.2) e alterações metabólicas no cérebro e tecidos periféricos (Capítulo 4.2 e 4.3). Além disso, esta tese termina sustentando um cronodisfunção relacionadas com a AD, sugerindo a questão da disfunção circadiana como um possível culpado para esta neuropatologia.
FCT - SFRH/BD/51667/2011