Introduction: Beat-to-beat variability in cycle length exists in spontaneously beating cardiac preparations of varying complexities from the level of the isolated whole heart to the single sinoatrial nodal cell (SANC). The nature of this variability is poorly characterised as are its fundamental physiological origins. Methods: Recordings of spontaneous electrical activity were made from hearts in vivo, during Langendorff-perfusion, and from single SANC. Heart rate variability (HRV) was calculated in the time- and frequency-domains at baseline and in response to pharmacological mediators that interfered with critical processes involved in automaticity (catecholamines, carbachol, ivabradine, zatebradine, ryanodine and thapsigargin). In addition, a novel 2D technique for imaging Ca2+ fluorescence in spontaneously beating, fluo4-AM loaded, patched single sinoatrial nodal cells was developed to investigate the biophysical behaviour of Ca2+ during pacemaking to see if variability in this was responsible for SANC HRV. Results: Under baseline, temperature-stable conditions, levels of HRV were greatest in vivo (human > rat). SANC exhibited slightly lower levels of HRV, whereas HRV levels expressed by Langendorff-perfused hearts were the least (rabbit > rat), although still comprised a significant proportion of the variability witnessed in vivo. Anaesthetising in vivo rabbits decreased HRV to levels similar to those seen in the Langendorff-perfused heart. HRV was decreased by catecholamines and by ryanodine/thapsigargin in the Langendorff heart. Conversely, HRV was increased by carbachol, ivabradine, zatebradine and ryanodine in SANC. Heart rate changes had a marked effect on levels of HRV. 2D Ca2+ imaging of SANC showed that diastolic local Ca2+ releases (LCRs) occurred earlier than previously thought, with early LCRs having characteristics that were distinct from later LCRs. Mean time of occurrence of all the LCRs within a given diastole closely predicted the duration of the cycle. The rate of restitution of the whole cell Ca2+ transient (used as a surrogate for the pumping function of SERCA) in turn closely predicted the mean time of occurrence of LCRs. Tight synchronisation of the electrical activity of the cell with the biophysical behaviour of Ca2+ appeared to predict shorter cycle lengths. Isoprenaline increased LCR amplitude, though did not increase LCR number, size or duration. Isoprenaline caused LCRs to occur earlier, and synchronised their occurrence and the rate of pumping of Ca2+ back into the sarcoplasmic reticulum. Finally, LCRs were found to preferentially recur in certain regions of the cell, dubbed hotspots. Isoprenaline favoured hotspot production. Conclusion: Whilst greatest in vivo, significant HRV exists in spontaneously beating cardiac preparations devoid of a functioning autonomic nervous system. Studies in SANC indicate that the origin of this is likely to be variability in release of LCRs from the SR via ryanodine receptors. This in turn is controlled by SR refilling kinetics via SR Ca2+ pumping. The coupled system of membrane- and Ca2+-pacemaker clocks are so heavily intertwined that myriad factors will come to bear on generating such variability, including the amount of Ca2+ available for pumping and the phosphorylation state of key proteins, to the extent that variability in no one process can take the credit for generating such HRV.

Over fifty structurally diverse, novel fluorinated heteroarenes, have been successfully synthesised by SNAr reaction of a range of fluorinated arenes including pentafluoropyridine, hexafluorobenzene, and methyl pentafluorobenzoate by introduction of a range of groups such as imidazole, triazole, benzimidazole, benzotriazole, and carbazole. Different water solubilising side chains were introduced to some of the successfully synthesised fluorinated heteroarenes to improve water solubility and potential biological activity. X-ray crystal structures of over 10 compounds were obtained including those of two macrocyclic compounds containing 21- and 24-membered rings. The synthesised compounds have been characterized by elemental analysis, IR, 1H and 19F spectroscopy and high resolution mass spectrometry. These compounds have been screened for their biological activities and possible interaction with DNA by methods including UV-visible spectroscopy, fluorescence spectroscopy, co-crystallization for X-ray diffraction analysis, and antimicrobial activity. A number of the fluoroaryl benzimidazole derivatives have been tested against K-562 and MCF-7 cell lines and G361 and HOS cell lines. From the all tested compounds three tethered fluoroaryl benzimidazole derivatives demonstrated micromolar inhibition against K-562 and MCF-7 cell lines. These compounds, in addition to 1-tetrafluoropyrid-4-yl-2-tetrafluoropyrid-4-ylsulfanyl-1H-benzimidazole, also demonstrated micromolar inhibition against G361 and HOS cell lines. Two of the compounds were found to activate caspases leading to apoptosis.

This Ph.D. thesis deals with principles of microalgae cultivation in laboratory as well as outdoor aquacultures (Chapter 1) using various cultivation systems and photobioreactors (Chapter 2). Case studies illustrate the main research topic as to correlate changes in growth rate with variation of photosynthetic activity, physiological features and biomass composition (Chapter 3). Special attention was paid to elaboration of protocols of chlorophyll a fluorescence techniques for monitoring the physiology and photosynthetic performance of microalgae mass cultures maintained under various growth conditions (Chapter 4).

Chemical sensors capable of detecting a specific atom or molecule under various conditions have been utilized in biological and environmental analyses. Fluorescence based sensors are particularly advantageous in these studies because of their high sensitivity, relative ease in handling, and low technical costs. This dissertation focuses on the detection of two analytes, H+ and hypochlorous acid, which are of interest in biology because the presence of abnormal quantities of these analytes may be indicative of disease. We have established a new platform for which sensitive changes in various regions of pH can be detected using fluorescence. The aminomethylrhodamine (AMR) scaffold is highly versatile, i.e. the pH range in which the sensor is active can be tuned by introducing different substituents on the amine moiety. Overall this systematic approach to the design of pH sensitive fluorophores has allowed for a library of compounds that are responsive over a broad range of pH (pH 3 - 10) by simply changing the substituent on the amino group. We report the synthesis and characterization of a silicon analog of rhodamine for the fluorescence based detection of hypochlorous acid. This fluorophore exhibits a 90 nm bathochromic shift in its absorption and emission, relative to its oxygen counterpart. Hypochlorous acid is a biological agent linked to certain diseases. Therefore, the longer wavelength properties of the this far-red fluorescent sensor will be of significant benefit to imaging experiments of this analyte in biological media and tissue due to its spectral proximity of the so called NIR optical window. Furthermore, the novel synthetic methodology of this sensor possesses a key intermediate, which could potentially lead to future fluorescence based sensors. The characterization of a fluorescent probe designed for the detection of hypochlorous acid (HOCl) using a silicon analog of fluorescein (SiF) was also reported. Over a range of pHs, the probe reacts with a stoichiometric amount of HOCl resulting in a mixture of two pH dependent fluorescent species, a SiF disulfide product and a SiF sulfonate product. The unique colorometric properties of the individual SiF fluorophores were utilized to perform simultaneous detection of HOCl and pH. When an excess of HOCl is present, the SiF fluorophores become chlorinated, via an intermediate halohydrin, resulting in a more pH independent and red-shifted fluorophore. Finally, an attempt was made at developing a pH responsive photodynamic therapy agent. This system was designed to target the relatively low extracellular pH found around tumors. A di-bromohydroxymethylrhodamine system was synthesized and the photophysical properties were characterized. This system absorbs weakly under acidic conditions (ca. pH 3), however was shown to be a moderate photosensitizer under acidic conditions.

The utility of fluorophores for sensing applications in the current state of the art of biological imaging hardly needs to be stated. The use of fluorophores in exploring and determining the internal structure and active dynamics of cellular processes has been pivotal, allowing us to explore areas of study inaccessible through other means. A simple search of fluorophores in Scifinder© demonstrates their popularity, as the number of hits increases year after year, until the year of 2015 when there were 1400+ journal articles published with the phrase. Fluorophore applications range far and wide, from sensing applications related to environmental concerns, to public health, to clinical usage. Fluorophores have been developed to detect explosive residues, to monitor environmental pollutants, and even to detect illicit substances. In cellular applications, a fluorophore needs to be well suited to examining the relevant processes, including participating in the cellular milieu and actively signifying the phenomena that are desired. Chapter I examines the usage of alkynes in fluorescent sensing scaffolds and gives a survey of their applicability in the field. Chapter II demonstrates the utility of disulfide-based macrocyclic scaffolds in the design of supramolecular hosts for chloride anions and their use as solid-state sensors for these anions. Chapter III explores the synthesis and application of an alkyne-based scaffold in the reversible detection of dithiol/disulfide redox flux and a new mode of quantification of dithiol-disulfide redox couples, a classically difficult area of study. Chapter IV focuses on methods utilized to improve the disulfide-based redox sensing capabilities. Chapters V and VI explore the properties of a new fluorophore scaffold discovered during research into another sensing scaffold, demonstrating a new reaction which yields a heretofore underexplored heterocycle with novel photophysical and supramolecular behaviors. This dissertation contains both previously published and unpublished co-authored material.

The purpose of this review is to compile preclinical and clinical results on phthalocyanines (Pcs) as photosensitizers (PS) for Photodynamic Therapy (PDT) and contrast agents for fluorescence imaging. Indeed, Pcs are excellent candidates in these fields due to their strong absorbance in the NIR region and high chemical and photo-stability. In particular, this is mostly relevant for their in vivo activation in deeper tissular regions. However, most Pcs present two major limitations, i.e., a strong tendency to aggregate and a low water-solubility. In order to overcome these issues, both chemical tuning and pharmaceutical formulation combined with tumor targeting strategies were applied. These aspects will be developed in this review for the most extensively studied Pcs during the last 25 years, i.e., aluminium-, zinc- and silicon-based Pcs.

[pt] Durante as últimas décadas tem-se acentuado o desenvolvimento de técnicas de fluorescência e suas aplicações em biociências e biotecnologia. Dada sua alta sensibilidade de detecção, tais técnicas têm-se tornado ferramentas importantes em muitas áreas de pesquisa tais como diagnósticos médicos, análise genética, mapeamento estrutural de células, estudos de fármacos, caracterização de complexos de fármacos com metais, análise da interação de fármacos com proteínas, lipídeos e DNA. Os anestésicos locais são fármacos que bloqueiam reversivelmente a condução nervosa quando aplicados a uma região circunscrita do corpo. Entretanto, possuem uma variedade de efeitos nos sistemas biológicos que não somente os efeitos de anestesia. Os antibióticos fluorquinolonas são agentes antimicrobianos sintéticos utilizados clinicamente há mais de 30 anos. Além de sua atividade antibacteriana, algumas fluorquinolonas vêm sendo aplicadas no desenvolvimento de drogas anticancerígenas e anti-HIV. O pH local de muitos meios biológicos tem grande influência nas propriedades espectroscópicas de muitos fármacos. Suas constantes de ionização, pKa, podem ser obtidas experimentalmente através de medidas espectroscópicas tais como absorbância, intensidade de fluorescência e parâmetros da fluorescência resolvida no tempo. Neste trabalho estudamos a partir da técnica de fluorescência estacionária e fluorescência resolvida no tempo os anestésicos locais dibucaína e tetracaína. Estudamos também os antibióticos norfloxacina e levofloxacina e seus respectivos complexos com ouro. Determinamos os tempos de vida de fluorescência e descrevemos ainda como as propriedades espectrais de fluorescência que se alteram em função do pH. Nos estudos de decaimento da fluorescência, desenvolvemos expressões para os fatores pré-exponenciais e as intensidades fracionárias de fluorescência como função do pH para amostras fluorescentes que sofrem transição ácido-base. Mostramos que os valores de pKa são aparentemente deslocados e obtivemos as expressões para o deslocamento. Determinamos as constantes de ionização, pKa, a partir da fluorescência estacionária e de parâmetros associados aos tempos de vida e testamos as expressões teóricas nos resultados experimentais. Os valores obtidos foram coerentes para as constantes de ionização dos fármacos dibucaína, levofloxacina e norfloxacina. Acredita-se que a ação dos anestésicos locais deve-se à interação das moléculas do anestésico com lipídeos e/ou proteínas de membrana. Uma análise desta interação mostra-se necessária para se investigar a difusão do anestésico na membrana e seu efeito na organização e dinâmica dos lipídeos e proteínas que constituem a membrana. Investigamos a interação da dibucaína e tetracaína com membranas enriquecidas em Na,K-ATPase e a incorporação do fármaco à membrana. Determinamos as constantes de associação. O valor obtido para a dibucaína foi de 0.91 × 10(3) M(-1) e para a tetracaína 0.77 × 10(3) M(-1). Estudamos a acessibilidade dos triptofanos por supressão de fluorescência do triptofano pela dibucaína, obtivemos uma acessibilidade de 90% para os triptofanos e uma constante de supressão dinâmica de 4.3 × 10(4) M(-1). / [en] During the last decades the development of fluorescence techniques and their applications in bioscience and biotechnology has been accentuated. Due to high sensitivity, such techniques have become important tools in many research areas such as medical diagnosis, genetic analysis, structural mapping of cells, drug investigation, characterization of drug-metal complexes, analysis of drugs interaction with proteins, lipids and DNA. The local anesthetics are drugs that reversibly block the nervous impulse when applied to a limited region of the body. However, they possess a variety of additional effects in the biological systems. The fluoroquinolone derived antibiotics are synthetic antimicrobial agents that have been used clinically for more than 30 years. In addition to their antibacterial activity, some fluoroquinolones have been applied in the development of anticancer drugs and anti-HIV. The local pH of many biological environments has great influence in the spectroscopic properties of many drugs, including local anesthetics and fluoroquinolones. The ionization constants of the drugs, pKa, can be experimentally obtained via spectroscopic parameters such as absorbance, fluorescence intensity, and time-resolved fluorescence parameters. In this work we studied the local anesthetics dibucaine and tetracaine using steady state and time resolved fluorescence techniques. We also studied the antibiotics norfloxacin and levofloxacin and their complexes with gold. We determined the fluorescence lifetimes and described how the spectral fluorescence properties alter as a function of the pH. In the studies of fluorescence decay, we developed expressions for the pre-exponential factors and the fractional fluorescence intensities as a function of the pH for a fluorophore undergoing an acid-base transition. We showed that the pKa values are apparently shifted and obtained the expressions for the pK shifts. We determined the ionization constants, pKa from steady state fluorescence and from parameters associated with the lifetimes, and we tested the theoretical expressions using the experimental results. The observed pK shifts for dibucaine, levofloxacin and norfloxacin were found to agree with the theoretical expressions. The action of local anesthetics is believed to involve the interaction of the anesthetic molecules with lipids and/or membrane proteins. An analysis of this interaction is necessary to examine the anesthetic diffusion in the membrane and its effect in the organization and dynamics of the membrane lipids and proteins. We investigated the interaction of dibucaine with Na,K-ATPase enriched membranes, and the incorporation of the drug in the membrane. We determined the binding constants. The obtained values were 0.91 × 10(3) M(-1) for dibucaine and 0.77 × 10(3) M(-1) for tetracaine. We also studied the accessibility of the tryptophan residues using the fluorescence quenching by dibucaine. We obtained a dynamic quenching constant of 4.3 × 10(4) M(-1).

La nouvelle technologie innovatrice OLEDs (diodes organiques électroluminescentes) ne cesse de susciter l'engouement des scientifiques ainsi que les recherches à leurs égards. Ces dispositifs à base de matériaux organiques s'appliquent dans quasiment tous les domaines tels que l'éclairage, l'affichage...Une découverte récente vient d'apporter sa pierre à l'édifice en mettant au point une nouvelle troisième génération d'OLEDs. Cette révélation consiste en la réduction du coût des matériaux des OLEDs en utilisant des matériaux peu onéreux dits à fluorescence retardée. C'est dans cette optique que s'inscrit ce travail de thèse qui porte sur l'étude de ce type de matériaux pour les applications dans les OLEDs. Cette thèse est décomposée en deux principales parties. Dans un premier temps, nous avons étudié et synthétisé des matériaux pour cette nouvelle génération d'OLED en se basant sur un modèle de molécules existant. Ce dernier représente des dérivés de triazine-carbazole connus pour leurs propriétés adéquates aux OLEDs. Cette étude a démontré pour la première fois le caractère de fluorescence retardée de ces molécules. Nous avons par la suite caractérisé ces molécules au sein des OLEDs. Les résultats montrent l'efficacité de ces molécules. Dans un deuxième temps nous nous sommes intéressés à l'étude de molécules innovantes. Ainsi, des molécules dotées de fluorescences retardées et innovantes ont été synthétisées. Cette synthèse a été effectuée par une méthode très avantageuse. Par ailleurs, la structure et propriétés de ces molécules indiquent qu'elles sont dotées de fluorescences retardées. Ainsi, il est important de poursuivre ce travail, en évaluant les propriétés de ces molécules synthétisées, ainsi que de les caractériser au sein des OLEDs. / Recently, the synthesis of free metal materials for organic LEDs by Uoyama et al adds a third kind of luminescence, named thermally activated delayed fluorescence (TADF). The added value of this discovery is to lower significantly OLED cost thanks to the metal-free structure of these so-called hyper-fluorescent molecules. This work reports on this recent discovery in OLED's. We first studied the thermally activated delayed fluorescence from an existing molecular model. The latter, fulfil the condition of the delayed fluorescence and are based from triazine-carbazole derivatives. The results revealed for thefirst time the exhibition of the delayed fluorescence from this existing model. In a second part, a novel hyperfluorescent molecule have been synthesized following the design rules for the delayed fluorescence molecules. The synthesis was done by a method based on an attractive process. Furthermore, the structure and properties of these new materials indicate that these molecules are expected to exhibit delayed fluorescent. Thus, it is important to continue this work by evaluating the properties of these molecules and the OLEDs made from these materials.

Lyme disease is the most common vector-borne infection in the Western world with an annual incidence usually in excess of 100 cases per 100 000 people in temperate areas of the United States and Europe. Same as other infectious diseases, Lyme borreliosis wreaks havoc on the host they have invaded. B. burgdorferi, the causative agent of this disease, circulates among wildlife vertebrate hosts and Ixodes tick vectors but may sometimes infect humans. Its natural enzootic cycle usually occurs as follows: The larval/nymphal stage tick feeds on an infected host. During this engorgement, the spirochetes reach the tick gut and stay confined to it. After the tick molts into the next developmental stage, it finds a second host. The new bloodmeal triggers the spirochetes to multiply within the gut and traverse the gut endothelium in a highly organized manner. They finally disseminate through the hemocoel up to the tick salivary glands and into the new host. We studied whether B. burgdorferi is capable of reaching the tick salivary glands during the first infective feeding period in uninfected ticks.

No description available.