Development Of Molecular Biosensors And Multifunctional Graphene-Based Nanomaterials

In the first project, a simple, rapid, and reversible fluorescent DNA INHIBIT logic gate has been developed for sensing mercury (Hg2+) and iodide (I-) ions based on a molecular beacon (MB). In this logic gate, a mercury ion was introduced as the first input into the MB logic gate system to assist in the hybridization of the MB with an assistant DNA probe through the thymine–Hg2+–thymine interaction, which eventually restored the fluorescence of MB as the output. With this signal-on process, mercury ions can be detected with a limit of detection as low as 7.9 nM. Furthermore, when iodide ions were added to the Hg2+/MB system as the second input, the fluorescence intensity decreased because Hg2+ in the thymine–Hg2+–thymine complex was grabbed by I- due to a stronger binding force. Iodide ions can be detected with a limit of detection of 42 nM. Meanwhile, we studied the feasibility and basic performance of the DNA INHIBIT logic gate, optimized the logic gate conditions, and investigated its sensitivity and selectivity. The results showed that the MB based logic gate is highly selective and sensitive for the detection of Hg2+ and I- over other interfering cations and anions. In the second project, an ultrasensitive and rapid turn-on fluorescence assay has been developed for the detection of 3’-5’ exonuclease activity of exonuclease III (Exo III) using molecular beacons (MBs). This method has a linear detection range from 0.04 to 8.00 U mL-1 with a limit of detection of 0.01 U mL-1. In order to improve the selectivity of the method, a dual-MB system has been developed to distinguish between different exonucleases. With the introduction of two differently designed MBs which respond to different exonucleases, the T5 exonuclease, Exo III and RecJf exonucleases can be easily distinguished from each other. Furthermore, fetal bovine serum and fresh mouse serum were used as complex samples to investigate the feasibility of the dual-MB system for the detection of the enzymatic activity of Exo III. As a result, the dual-MB system showed a similar calibration curve for the detection of Exo III as in the ideal buffer solution. The designed MB probe could be a potential sensor for the detection of Exo III in biological samples. In the third project, A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5´-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because less affinity with ssDNA than dsDNA. The decrease extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U/mL and good linear correlation between 0.25-1.00 U/mL (R2=0.9896). In this assay, no label was needed for the fluorescence detection. Moreover, the inhibition behaviours of the T4 PNK’s inhibitors were evaluated by this assay. The result indicated a potential of using this assay for monitoring of phosphorylation-related process. In the fourth project, a facile bottom-up method for the synthesis of highly fluorescent graphene quantum dots (GQDs) has been developed using a one-step pyrolysis of a natural amino acid, L-glutamic acid, with the assistance of a simple heating mantle device. The developed GQDs showed strong blue, green and red luminescence under irradiation with ultra-violet, blue and green light, respectively. Moreover, the GQDs emitted near-infrared (NIR) fluorescence in the range 800–850 nm with an excitation-dependent manner. This NIR fluorescence has a large Stokes shift of 455 nm, providing a significant advantage for the sensitive determination and imaging of biological targets. The fluorescence properties of the GQDs, such as the quantum yields, fluorescence life times, and photostability, were measured and the fluorescence quantum yield was as high as 54.5%. The morphology and composites of the GQDs were characterized using TEM, SEM, EDS, and FT-IR. The feasibility of using the GQDs as a fluorescent biomarker was investigated through in vitro and in vivo fluorescence imaging. The results showed that the GQDs could be a promising candidate for bioimaging. Most importantly, compared to the traditional quantum dots (QDs), the GQDs are chemically inert. Thus, the potential toxicity of the intrinsic heavy metal in the traditional QDs would not be a concern for GQDs. In addition, the GQDs possessed an intrinsic peroxidase-like catalytic activity that was similar to graphene sheets and carbon nanotubes. Coupled with 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), the GQDs can be used for the sensitive detection of hydrogen peroxide with a limit of detection of 20 mM. In the fifth project, a general, environmental-friendly, one-pot method for the fabrication of reduced graphene oxide (RGO)/metal (oxide) (e.g. RGO/Au, RGO/Cu2O, and RGO/Ag) nanocomposties was developed using glucose as the reducing agent and stabilizer. The RGO/metal (oxide) nanocomposites were characterized using STEM, FE-SEM, EDS, UV-vis absorption spectroscopy, XRD, FT-IR and Raman spectroscopy. The reducing agent, glucose, not only reduced GO effectively to RGO, but it also reduced the metal precursors to form metal (oxide) nanoparticles on the surface of RGO. Moreover, the RGO/metal (oxide) nanocomposites were stabilized by gluconic acid on the surface of RGO. Finally, the developed nanomaterials were successfully applied to simultaneous electrochemical analysis of L-ascorbic acid (L-AA), dopamine (DA) and uric acid sing RGO/Au nanocomposite as an electrode catalyst. In the sixth project, a reduced graphene oxide/silver nanoparticle (RGO/Ag) nanocomposite using glucose as the environmental-friendly reducing agent was developed. The antibacterial activity of RGO/Ag nanocomposite was carefully investigated using Escherichia coli (E. coli) and Klebsiella pneumoniae (Kp) as bacterial models. We found that, compared with AgNPs, graphene oxide (GO) and RGO, RGO/Ag nanocomposite had higher antibacterial efficiency. Furthermore, under the near-infrared (NIR) irradiation, RGO/Ag nanocomposite demonstrated enhanced synergetic antibacterial activity through the photothermal effect. Almost 100 % of E. coli and Kp were killed by the treatment of 15 µg/mL and 20 µg/mL, respectively, with NIR irradiation. Moreover, the membrane integrity assay and ROS species assay demonstrated that RGO/Ag nanocomposite under NIR irradiation caused the cell membranes disruption and generation of ROS species, providing other possible mechanisms for their high antibacterial activity besides photothermal effect. In the seventh project, a rigid distance spacer, silica shell, was used between GO and dyes in this work to elucidate the quenching ability of GO. First, an organic dye was doped in silica nanoparticles, followed by the modification of another layer of silica shell with a different thickness. Due to the electrostatic interaction between GO and positively charged silica nanoparticles, GO wrapped the silica nanoparticles when they were mixed together. Therefore, the distance between GO and organic dyes was adjusted by the thickness of the silica shell. The quenching efficiency of GO to two different organic dyes, including Tetramethylrhodamine (TAMRA) and Tris(bipyridine)ruthenium(II) chloride (Rubpy), was measured at various distances. This quenching ability investigation of GO to dyes with distance-dependent manner would provide a guideline for the design of the fluorescent functional composite using GO in the future. In the eighth project, we characterized the antibacterial activity of GO in both cell culture and animal models. Klebsiella pneumoniae (Kp) is one of the most common multidrug resistant (MDR) pathogens in causing persistent nosocomial infections and is very difficult to eradicate once established in the host. First, we demonstrated that GO exerted direct killing of Kp in agar dishes and afforded the protection of alveolar macrophages (AM) from Kp infection in culture. We then evaluated the mortality, tissue damage, polymorphonuclear neutrophil (PMN) penetration, and bacterial dissemination in Kp-infected mice. Our results revealed that GO can counteract the invasive ability of Kp in vivo, resulting in lessened tissue injury, significant but subdued inflammatory response, and prolonged mouse survival. These findings indicate that GO may be an alternative agent for controlling MDR pathogens in clinics

An Organic/Inorganic Hybrid Membrane as a Solid “Turn-On” Fluorescent Chemosensor for Coenzyme A (CoA), Cysteine (Cys), and Glutathione (GSH) in Aqueous Media

The preparation of a fluorogenic sensory material for the detection of biomolecules is described. Strategic functionalisation and copolymerisation of a water insoluble organic sensory molecule with hydrophilic comonomers yielded a crosslinked, water-swellable, easy-to-manipulate solid system for water ‘‘dip-in’’ fluorogenic coenzyme A, cysteine, and glutathione detection by means of host-guest interactions. The sensory material was a membrane with gel-like behaviour, which exhibits a change in fluorescence behaviour upon swelling with a water solution of the target molecules. The membrane follows a “turn-on” pattern, which permits the titration of the abovementioned biomolecules. In this way, the water insoluble sensing motif can be exploited in aqueous media. The sensory motif within the membrane is a chemically anchored piperazinedione-derivative with a weakly bound Hg(II). The response is caused by the displacement of the cation from the membrane due to a stronger complexation with the biomolecules, thus releasing the fluorescent sensory moieties within the membraneSpanish Ministerio de Ciencia e Innovación—Feder (MAT2011-22544) and by the Junta de Castilla y León (BU001A10-2

Polímeros sensores: aplicaciones como sensores químicos en detección y cuantificación de analitos

La tesis doctoral describe la preparación de nuevos materiales poliméricos para la detección colorimétrica y/o fluorimétrica de distintos contaminantes y moléculas de interés. Los nuevos polímeros sensores se han diseñado en forma de polímero entrecruzado (membrana, film), así como en forma de polímero lineal (sólido soluble en agua). Estos materiales avanzados cambian de color en presencia de contaminantes como el cianuro, o el hierro (III); y son capaces de generar fluorescencia en presencia de aluminio (III), cromo (VI), mercurio (II) y distintas moléculas de interés biomédico como el coenzima A. Como es natural, los fenómenos de detección en sistemas biológicos se dan en medios acuosos. Por ejemplo, los sitios activos en las enzimas tienen la forma requerida para alojar a la molécula huésped, o parte de ella, y la interacción se basa en enlaces débiles que tienen lugar debido a los dominios hidrofóbicos en un entorno hidrofílico general. En un intento de imitar a la naturaleza, se pueden anclar receptores insolubles en agua, lipofílicos, a cadenas lineales o reticuladas de polímeros hidrofílicos, dando lugar a polímeros sensores solubles en agua o a materiales sensores hinchados en este medio, con comportamiento tipo gel. Esta última aproximación al fenómeno sensor permite el control del hinchamiento por medio del incremento o disminución de la densidad de nudos, denominada relación nominal de entrecruzamiento. Es decir, el carácter hidrofílico del polímero, relacionado con la constitución de los monómeros, se puede controlar no sólo mediante la naturaleza de los comonómeros utilizados en la síntesis del material, sino también a través del porcentaje de entrecruzante empleando en la síntesis, facilitando o disminuyendo la absorción de agua. Así, se puede inducir un carácter hidrofóbico a una estructura polimérica hidrofílica mediante la tensión de la red tridimensional en el proceso de hinchamiento con agua

The development and optimization of microbial molecular biomarkers for the in situ assessment of trace metal toxicity

Merged with duplicate record 10026.1/2607 on 06.20.2017 by CS (TIS)Microorganisms are fundamental components of many geochemical transformations occurring in the aquatic environment. Microbial redox and methylation of metals within the environment can alter metal speciation, mobility and ultimately, toxicity to eukaryotes. It is therefore practical that any environmental monitoring framework advocating the application of `early-warning biomarker system' should incorporate a holistic view of the environment beginning with microbial activity. This thesis describes the development of protocols for assessing the in situ condition of microbial ecosystems within a gradient of metal contaminated sites radiating downstream of the Anaconda Smelter, a USEPA-designated superfund site and within two control sites. Experiments focus on evaluating the incidence (i. e. prevalence and absence) of genes related to general stress and specific metal detoxification reactions. Moreover, a number of selected genes were quantified directly from the environment and statistically correlated with metal concentrations. Furthermore, the influence of metals on structuring microbial communities was also investigated by evaluating temporal communities shifts in response to changing metal concentrations using denaturant gradient gel electrophoresis (DGGE). The data recorded the highest prevalence of all genes was found at the most polluted site directly downstream of the Anaconda Smelter. Furthermore, significant correlations were observed between gene prevalence and metals (arsenic, copper and zinc) (P < 0.05) and organic carbon concentration (P < 0.05). A number of genes were successfully amplified from sediment with significantly higher gene copy number (/ ng DNA) at the more polluted sites when compared to corresponding control sites. Examination of community diversity found that long-term metal-contaminated sediments, adjacent to the Smelter, had microbial communities twice as diverse as corresponding reference sites. In addition, multivariate statistical techniques identified factors important to community structuring, concluding that geographic position and localized geochemistry fundamentally influence the structuring of communities. This thesis represents a significant advance in the use of microorganisms as `early warning systems' of deterioration in ecosystem health, while the application of advanced molecular methods facilitates their intergration within a traditional ecotoxicological framework.Montana State Universit

Microbial stress. From sensing to intracellular and population responses

We initially devised this Research Topic (RT) as a valuable initiative to collect high-quality scientific articles from the participants of the 4th European Federation of Biotechnology (EFB) Microbial Stress meeting held in Kinsale, Ireland, April 2018. The scope of the RT is based on the scientific content of that “Microbial Stress: from Systems to Molecules and back” meeting. Indeed, over 40% of the articles eventually accepted for publication were contributed by meeting participants, but notably the remaining 60% was contributed by authors that work in this field. The collection of 22 original research and 2 review articles, contributed by 163 authors collectively, deal with the many different aspects of the microbial responses to biotic and abiotic stresses, relevant to many fields: from host-pathogen interactions to biotechnology, from bioremediation to food processing, from molecular and single-cell to population studies. The RT showcases the rapid developments of the microbial stress research on a range of microorganisms and stress conditions, and confirms that understanding microbial physiology under stress can be a trigger for the development of new methodologies as well as helping to integrate the knowledge from many different microbiological fields of research. The retrospective analysis of the articles contributed to this RT allowed them to be assigned to one of four main sub-topics: (i) impact of weak organic acids and low pH on micro-organisms, from clinical to biotechnological contexts; (ii) adaptive responses in microbial pathogens to abiotic/environmental stress; (iii) oxidative and metal stress, from clinical to bioremediation contexts, and (iv) regulation of transcription and translation under stress, from epigenetic aspects to the role of second messengers and sRNA

Nuevos materiales para la especiación de mercurio: biotransformación de metales en organismos modelo

La contaminación medioambiental representa un problema de gran importancia en la actualidad. Estamos acostumbrados a escuchar por los medios de comunicación los numerosos casos que suceden a nuestro alrededor: la contaminación de ríos, lagos o playas por vertidos residuales; la contaminación de los suelos por utilización indebida de ciertos fertilizantes o por las actividades mineras de la zona; o la contaminación del aire ocasionada por el masivo número de automóviles y zonas industriales en las ciudades provocando las famosas “boinas” de las grandes ciudades. En el caso de los metales pesados o metaloides, pueden acumularse y biomagnificarse a lo largo de la cadena trófica llegando a provocar efectos nocivos en los seres humanos. Los contaminantes clásicos estudiados en esta tesis engloban el mercurio, arsénico, cadmio y plata. Entre todos ellos el mercurio y el arsénico son los que mayor repercusión medioambiental representan. El mercurio es un metal cuya toxicidad es muy elevada pero su repercusión en los efectos provocados en los seres vivos depende de la forma química en la que se encuentre en el medio. Las dos especies predominantes son mercurio inorgánico (Hg2+) y metilmercurio (CH3Hg+). El arsénico es un metaloide donde sus compuestos inorgánicos presentan mayor toxicidad. La contaminación de arsénico en agua y suelos supone un problema que afecta a millones de personas en Asia y en la zona Andina. El cadmio al igual que ocurre con el mercurio, es un elemento con gran tendencia a bioacumularse en la cadena trófica, estando relacionado con el cáncer de pulmón, próstata o renal. La exposición puede ser a través de la dieta o por inhalación de vapores; los fumadores incrementan esta exposición en un 50%. La plata es un elemento que encontramos en multitud de aplicaciones, principalmente en la energía solar, en el campo de la fotografía y en algunos medicamentos. Una exposición elevada provoca argiria en la piel u ojos. Existen otros contaminantes, los denominados emergentes, que son aquellos que debido a un aumento de las aplicaciones nanotecnológicas y del uso de nanopartículas, los podemos encontrar en el medio ambiente y sus efectos aún no están definidos, siendo actualmente objeto de estudio, como las nanopartículas de quantum dots (QDs), constituidas por seleniuro de cadmio principalmente. La toxicidad de estos QDs depende del recubrimiento de la nanopartícula, del tamaño y de la carga..

DEVELOPMENT OF MOLECULAR SYSTEMS FOR THE DETECTION OF ENVIRONMENTAL ARSENIC

Arsenic is an ubiquitous toxic metalloid naturally present in the soil, water and air that adversely affects human health. The abundance of arsenic in the environment has guided the evolution of multiple defence strategies in almost all microorganisms which must therefore sense the metalloid and regulate the transcription of genes coding for resistance proteins. In this sense microorganisms participate to the geochemical cycling of arsenic in their living environments, promoting or inhibiting arsenic release from sediment material. The aim of this thesis has been the characterization at molecular level of the mechanisms of arsenic resistance in T. thermophilus and the realization of “Cell-Based” and “Enzyme-Based” biosensors for the detection of arsenic species in soils and waters. The thermophilic gram negative bacterium Thermus thermophilus HB27 is able to grow in the presence of both arsenate and arsenite in a range of concentrations which are lethal for other microorganisms. The putative resistance genes have not been found in a single resistance operon but associated to chromosomal genes apparently not functionally related. In particular we found a gene coding for a thioredoxin-coupled arsenate reductase (TtArsC) which catalyzes the reduction of pentavalent arsenate to trivalent arsenite; two genes (TTC1447, TTC0354) coding putative ArsB-like transporters; and a gene coding for a transcriptional repressor (TtSmtB) sensitive to arsenic, belonging to the ArsR/SmtB family of transcriptional regulators. TtsmtB is part of an operon containing putative internal promoters upstream of genes with no obvious functional relationship. The purified recombinant protein is a dimeric DNA binding protein able to bind in-vitro to target sequences and to dissociate upon arsenate and arsenite binding. Inactivation of the TtsmtB gene, in a T. thermophilus TtsmtB- mutant strain, induces the expression of the ars genes among which TtarsC and a putative efflux protein. These results prove that TtSmtB has a functional role in the regulation of the arsenic resistance. Analysing the TTC0351, TTC0353 and TTC0354 promoter activities in-vivo, through β-galactosidase reporter systems, it has been developed the first whole-cell arsenic biosensor based on the use of the thermophilic microorganism T. thermophilus. The biosensor response could be measured with reliability within 30 minutes of arsenate or arsenite addition, and have a minimum detection limit of 0.1 mM for both arsenate and arsenite. An intriguingly feature of this biosensor rely on its thermophilic nature, hence, despite not having a higher arsenic detection limit it could be more versatile, stable and strong in case of highly contaminated waters. Moreover, it has been developed an enzyme-based biosensor to screen for the presence of arsenic using TtArsC as biomolecular probe. TtArsC has been adsorbed on gold nanoparticles (AuNPs) and nanobiocomplexes demonstrating stability and the capacity to strongly bind the toxic ions. Interestingly, TtArsC-AuNPs interaction with arsenic can be followed by naked eye, since solutions completely change their colors. Therefore, a straightforward application in fast and low-cost screening of water can be envisaged. Finally, Geobacillus stearothermophilus has been isolated from a geothermal area near Naples known as Pisciarelli, and has been identified as a new arsenic tolerant microorganism. Our results made G. stearothermophilus a novel model of study for the development of new arsenic biosensing and bioremediation techniques and confirm the possibility of using Thermus thermophilus as biological systems (cellular or enzymatic) for the traceability of pollutants after a thorough molecular, structural and functional characterization of the components involved and their interactions

Synthetic biology enabled cellular and cell-free biosensors for environmental contaminants

Cell-based biosensors have great potential to detect various toxic and pathogenic contaminants in aqueous environments. However, frequently they cannot meet practical requirements due to insufficient sensing performance, inadequate sensing platforms and biosafety issues. Here, I investigated a novel, comprehensive and modular methodology for optimising cell-based biosensors to address these challenges, and to enable them for their practical applications. In particular, this methodology combines multiple synthetic biology strategies, which can systematically and significantly improve sensors’ sensing performance in a predictable manner. It first optimises a sensor’s sensitivity by regulating its intracellular receptor densities, then further improves its output by applying a multi-layer transcriptional amplifier cascade, and finally regulates its leakiness by combining promoter structure engineering and post-translational regulation. Exemplary bacterial cell-based arsenic and mercury sensors were used to demonstrate this methodology, and their detection limits and outputs were improved up to 5,000-fold and 750-fold respectively. Facilitated by this methodology, I developed easy-to-interpret sensing platforms for cost-effective and portable field testing, where the analytes were easily quantified by simple visualisation. Physical entrapment methods, i.e., agarose gel entrapment and microfluidic biodisplay, were applied to the sensing platforms to mitigate and minimise the biosafety concerns. To further eliminate the biosafety issues, the arsenic and mercury sensors were transferred into a crude cell extract-based cell-free system (CFS). To adapt the sensors to the CFS, aforementioned methodology combined with additional tuning methods were applied, such as tuning the sensors’ DNA concentration and their receptor to promoter ratio, introducing transcriptional amplifiers and promoter engineering. A similar paper-based sensing platform could be generated based on these optimisation methods. A mercury sensor with colorimetric output was adapted to a paper-based CFS, where the sensor’s response to 2 ppb mercury could be easily visualised by the naked eye. Overall, the verified signal amplifying methodology along with the cellular and CFS-based sensing platforms can be widely applicable to many other cell-based sensors, paving the way for their real world applications in the environment and healthcare

Targeting human aquaporin function : physiological and chemical approaches

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2018Aquaporins (AQPs) are a group of small membrane proteins belonging to a highly conserved family of membrane proteins called MIPs (Major Intrinsic Proteins) that are responsible for the bidirectional transport of wate (orthodox aquaporins) and also small uncharged solutes (aquaglyceroporins) across cell membranes, in response to osmotic or solute gradients. Rapid water flux across membranes is crucial to maintain the water homeostasis in many epithelia and endothelia involved in fluid transport. In addition, due to the unique ability of aquaglyceroporins to transport glycerol in addition to water, they have important roles in glycerol metabolism and skin hydration in non-fluid transporting tissues such as skin, fat and liver. The thesis introduction (Chapter 1) presents an overview of aquaporins structure, their main biological functions and related pathologies, with special emphasis on the so far described mechanisms of regulation. In the first part of this thesis (Chapter 2), we report the discovery of a new role for Aquaporin-5 (AQP5, an orthodox aquaporin) in adipocyte biology, where Aquaporin-7 (AQP7, an aquaglyceroporin) has been the mainly characterized protein in adipose tissue responsible for glycerol efflux. A better understanding of aquaporin regulation and gating would allow manipulation of their activity facilitating the identification of new putative modulators. A cellular model optimized to assess the function of aquaporins and discriminate individually each isoform, instead of mammalian cells where more than one isoform is usually expressed, is a useful tool to study aquaporin regulation. The second part of this thesis (Chapter 3) is dedicated to the functional characterization of different mammalian aquaporin isoforms (AQP3, AQP5, AQP7 and AQP10), using a yeast heterologous expression system devoided of endogenous aquaporins, a background where analysis is unlikely to be compromised by the co-expression of other aquaporin isoforms. Using the stopped-flow technique to evaluate the channel permeability for water and for glycerol, we were able to disclose gating mechanisms of aquaporin isoforms, being given special emphasis to the regulation by pH and phosphorylation. In the third part of this thesis (Chapter 4), a screening of several small gold compounds as inhibitors for Aquaporin-3 (AQP3, an aquaglyceroporin) was performed aiming at identifying new modulators with potential therapeutic use.A água possui um papel crucial para a vida devido às suas propriedades únicas. Todos os processos bioquímicos e fisiológicos de um organismo dependem da presença de água, sendo esta o componente fundamental na manutenção da homeostase celular. Nas células eucarióticas, a água encontra-se distribuída pelos vários compartimentos intracelulares separados entre si por membranas intracelulares e do meio extracelular pela membrana plasmática. Estas membranas de composição bilipídica são normalmente impermeáveis à maioria dos solutos polares e/ou carregados, cuja passagem é facilitada através de canais membranares específicos. No entanto estas membranas são bastante permeáveis à água, sendo então propostas três vias de transporte: por difusão simples, por transporte passivo associado ao transporte de iões e solutos e por canais específicos para a água. Atualmente sabe-se que a maioria das células de um organismo possui proteínas específicas – as aquaporinas – que conferem à membrana uma permeabilidade à água de cerca de 5 a 10 vezes superior às membranas que não possuem estas proteínas. Devido às suas características estruturais, as aquaporinas permitem um rápido transporte bidirecional de água, seletivo e regulado, em resposta a gradientes osmóticos, ao mesmo tempo que previnem a passagem de protões e iões através da membrana plasmática. Em mamíferos, são conhecidas até à data treze isoformas (AQP0-AQP12) que são classificadas em três grupos de acordo com a sua sequência primária, localização celular e seletividade em 1) aquaporinas ortodoxas, primariamente seletivas à água; 2) aquagliceroporinas, para além de água também transportam pequenos solutos neutros, como glicerol e ureia; e 3) super-aquaporinas, que são encontradas em membranas intracelulares e possuem menor homologia. No entanto, a lista de substâncias que são capazes de permear as diferentes aquaporinas tem aumentado ao longo do tempo. Recentemente, para além de água e glicerol, foi também descrito o transporte facilitado através de algumas isoformas de arsenito, amoníaco e peróxido de hidrogénio. Devido à grande diversidade de tecidos onde são encontradas as aquaporinas, o seu papel de facilitar o transporte de água e/ou solutos através das membranas plasmáticas é importante em vários processos fisiológicos, tais como: secreção de fluido glandular, mecanismo de concentração urinária, excitabilidade neuronal, metabolismo dos lípidos, hidratação epidérmica e balanço de água no cérebro. A observação do fenótipo de ratinhos geneticamente modificados com knock-out de determinadas aquaporinas revelou funções fisiológicas muito importantes no aparecimento e desenvolvimento de várias patologias, como epilepsia, edema cerebral, glaucoma, cancro e obesidade. No Capítulo 1 é apresentada uma introdução geral que visa proporcionar um conhecimento abrangente sobre as principais funções das aquaporinas humanas e patologias associadas, dando especial atenção aos diferentes mecanismos de regulação já conhecidos. Na primeira parte dos resultados desta tese (Capítulo 2), através da construção de linhas celulares de pré adipócitos de ratinho 3T3-L1 com diferentes níveis de expressão da Aquaporin-5 (cenário de ganho e perda de função) foi possível estabelecer um novo e determinante papel desta aquaporina na diferenciação dos adipócitos. Na segunda parte dos resultados desta tese (Capítulo 3), pretendeu-se usar um sistema de expressão heteróloga em Saccharomyces cerevisiae (S. cerevisiae) para permitir avaliar de forma individual a função de cada aquaporina. A levedura S. cerevisiae é considerada um valioso sistema de expressão heteróloga para estudar inúmeras proteínas devido à elevada homologia funcional entre esta e os eucariontes superiores, incluindo mamíferos. Pelo facto de existir uma grande biblioteca de estirpes disponíveis, ser de fácil manipulação genética, ser pouco dispendioso em comparação com as culturas de células animais e poderem ser testadas uma variedade de condições externas, este sistema oferece condições experimentais ótimas para estudar a especificidade e regulação das aquaporinas. Após a expressão e confirmação da sua localização celular, procedeu-se à caracterização da função de cada isoforma, utilizando a técnica de interrupção brusca de fluxo, seguindo a variação de volume celular por fluorescência quando se introduz uma perturbação no meio extracelular. Os fluxos de água através da membrana celular causados por gradientes de pressão osmótica (de solutos impermeantes ou permeantes) provocam alterações transitórias de volume, até se atingir um novo volume final de equilíbrio osmótico. A velocidade com que as alterações de volume ocorrem e o tempo que a célula leva a re-estabelecer o seu novo equilíbrio osmótico dependem diretamente das características intrínsecas de transporte da membrana, em particular da quantidade de canais específicos para a água e para o soluto em questão. No Capítulo 3 foram estudadas quatro isoformas diferentes (AQP3, AQP5, AQP7 e AQP10) e os seus mecanismos de regulação por pH e fosforilação foram revelados pela primeira vez. Vários esforços têm vindo a ser feitos com o intuito de desenvolver possíveis fármacos para tratamento das aquaporinopatias, mas até agora nenhum composto se revelou qualificado para estudos in vivo, quer pela sua fraca solubilidade quer pela sua baixa capacidade de inibição. Na terceira parte dos resultados desta tese (Capítulo 4), deu-se especial atenção à descoberta de novos compostos organometálicos, inibidores da função da Aquaporina-3, que poderão ser usados para benefício clínico na prevenção ou tratamento das várias patologias associadas.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/52384/2013, Programa de Doutoramento Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD