A leptospirose é uma zoonose de importância mundial, causada por leptospiras patogênicas. Aproximadamente 5 a 10% das infecções humanas cursam com a forma grave. A Doença de Weil é a forma mais comum de doença grave e pode apresentarse com duas formas de evolução, aguda progressiva monofásica ou de curso prolongado. A doença grave se caracteriza por uma combinação de hemorragia, mais comumente pulmonar, icterícia e insuficiência renal, com letalidade de 5 a 15%. O rim é um órgão muito acometido na Leptospirose. Clinicamente o envolvimento renal ocorre de 16 a 40%, com manifestações peculiares como poliúria, hipocalemia, e perda de sódio. A disfunção tubular renal, é característica da leptospirose forma grave, com envolvimento dos transportadores renais de sódio ao longo do néfron, levando às manifestações observadas. A terapêutica antimicrobiana é recomendada na Leptospirose, porém com controvérsias à sua indicação após o quarto dia de doença. Quando da instalação da lesão não haveria benefícios com a utilização de antibióticos. O tratamento pode diminuir a morbidade e letalidade, assim como interferir no envolvimento renal e na expressão dos transportadores renais de sódio. A patogênese pode estar relacionada a efeitos diretos da leptospira ou a resposta inflamatória assim como o estresse oxidativo. A utilização de antioxidantes, pode ser considerada como terapia adjuvante. Nós avaliamos a expressão no túbulo proximal do trocador Na+-H+ (NHE3) e na porção espessa da medula ascendente o cotransportador Na+-K+-2Cl- (NKCC2), no modelo de hamster com as duas formas de evolução de doença grave, mimetizando a doença de humanos realizados em dois experimentos. Os experimentos envolveram animais infectados não tratados e tratados com ampicilina associado ou não ao antioxidante, N-acetilcisteína. A presença de antígenos de Leptospira e a expressão dos transportadores foram avaliadas por imunohistoquímica, e o ácido tiobarbitúrico, marcador de estresse oxidativo, (TBARS) foi quantificado.Hamsters infectados, apresentaram altas quantidades de antígenos nos tecidos-alvo, enquanto que a expressão de ambos os transportadores apresentou-se diminuída. O tratamento com ampicilina esteve associado com mínima detecção ou ausência de antígenos, restabelecimento da expressão dos transportadores nos respectivos locais e redução dos níveis de TBARS. O tratamento precoce e tardio com ampicilina restabeleceu os defeitos tubulares na leptospirose forma grave em ambos experimentos, sem benefícios com a utilização da N-acetilcisteína. / Leptospirosis is a zoonosis of worldwide distribution. About 5-10% of all human infections presents with severe forms. Weil\'s syndrome, the most common presentation of severe forms of leptospirosis, may courses either as a single monophasic disease or as a disease with prolonged course, characterized by a combination of hemorrhage, particularly in the lung, renal failure, and jaundice, with fatality rates ranging from 5 to 15%. The kidney is an important target organ in leptospiral infection. Clinically, renal involvement in leptospirosis occurs in 16% to 40% of cases and is unique because of the atypical presentation of polyuria, hypokalemia, and sodium wasting, suggestive of a special form of tubular dysfunction related to the major renal sodium transporters expressed along the nephron. A wide range of antimicrobial therapy for leptospirosis was described and benefits have been disputed for cases with more than four days of clinical disease, because after a threshold of leptospiremia, the delayed use of antibiotics is unlikely to reduce fatality. Antimicrobial therapy is thought to interfere on fatality, renal involvement, and renal sodium transporters expression during severe disease. The pathogenesis may be related to direct effects of leptospiral compounds or inflammatory response due to oxidative stress. Antioxidant could be considered for adjunctive therapy.We evaluated the expression of proximal tubule type 3 Na+/H+ exchanger (NHE3) and thick ascending limb Na+-K+-2Cl- cotransporter (NKCC2) in infected non treated and treated hamsters reproducing the two forms of clinical human presentations of Weil\'s syndrome divided in two experiments. Animals were treated or not with ampicillin and/or N-acetyl-cysteine (NAC). Leptospiral antigen/s and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen/s in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. Early and late ampicillin treatment rescued tubular defects in leptospirosis severe disease in both experiments, and there was no evidence of benefit from antioxidant therapy.

Molecular recognition at biomembranes is one of the more poorly understood aspects of fundamental research in physical organic chemistry. Our aim was to improve our understanding of the molecular recognition of polysaccharides at biomembranes, in particular developing synthetic lipids that will recognise and report on the presence of glycosaminoglycans (GAG polysaccharides), like heparin and hyaluronic acid. Elevated levels of hyaluronic acid have been implicated in bladder carcinoma and osteoarthritis, and the use of heparin for medical applications is well documented. We synthesised a boronic acid capped lipid that also bore a fluorinated fluorescent reporter group, which could report on multivalent recognition events at bilayer membranes by fluorescent quenching and changes in the lateral distribution of the reporter groups. These preliminary studies showed these boronic acid capped fluorinated lipids gave a fluorescent signal upon interaction with simple mono- and poly- saccharides, albeit with unexpectedly weak binding to these saccharides. To understand and quantify the weaker binding of saccharides to membrane bound boronic acids a series of novel fluorescent and chromogenic lipids were synthesised that bore the reporter group close to the boronic acid. These studies revealed several underlying factors that had important roles in the recognition of oligosaccharides by boronic acid capped lipids. For the first time the effect of the bilayer on saccharide/boronic acid recognition was quantified, with the membrane weakening the interaction 33-fold. We were able to propose a model for the interaction of saccharides for membrane bound boronic acids that explained many of these unexpected observations.We also devised a parallel approach using GAGs to open or close synthetic membrane channels. Using a GAG to switch on the release of an ion or dye would generate a fluorescent signal that amplifies the original recognition event and improves sensitivity for GAGs. Proof-of-principle studies using palladium ions to open dye-transporting channels were successful and these studies were followed by the synthesis of boronic acid-capped cholates. Incorporation of boronic acid-capped cholates into membranes caused changes in the rate of release of alkali metal ions, which caused an enclosed fluorescent dye to give a signal, in the presence or absence of saccharides. These compounds successfully gave a response to the simple saccharide D-fructose but gave no response to other saccharides tested, including various hyaluronic acids. Although we were not able to develop a selective sensor for GAGs, we have developed a model for saccharide/boronic acid interactions that is a valuable addition to the physical organic chemistry of membranes.

No description available.

Inductively coupled plasma - mass spectrometry (ICP-MS) is the technique of choice worldwide for trace elemental determinations because of its excellent ionization ability, low detection limits and fast analysis speeds. However, the ICP-MS still suffers from some disadvantages, such as spectral overlap and severe matrix effects. Matrix effects or interferences, partly arise from changes in the analyte transmission through the interfacial region between the ICP and mass spectrometer with changes in sample matrix. Better understanding of the transmission behaviors of analyte through the sampling and skimmer cones will provide the insights needed to alleviate matrix interferences and to improve the interface design between the ICP and mass spectrometer. Laser induced fluorescence is a highly sensitive, non-invasive and element specific detection method. The research herein endeavors to explain the transport behaviors of analytes upstream and downstream from the sampling cone in an ICP-MS. The final goal of this research is to improve the consistency and efficiency with which ions are transported from an ICP source to a mass analyzer. Several issues related to analyte transmission through the sampling and skimmer cones have been explored and discussed in this dissertation. First, it is found that the existence of the sampling cone not only disturbs the local thermodynamic equilibrium of the plasma, but also changes the spatial distributions and number densities of analyte species. Second, it has been verified that the spread of analyte species in the first vacuum stage is mass-dependent and can be explained by ambiploar diffusion theory. Finally, the current research suggests that the transmission efficiencies of the skimmer cone are impacted by the nebulizer flow and first vacuum stage pressure of the ICP-MS. To better elucidate the analyte transport behaviors from the plasma to the ion detector in an ICP-MS, more investigation needs to be carried out. Further research, such as the entire measurements of analyte transmission efficiency through the skimmer cone, the variation of doubly charged ions under different plasma operational conditions, and the functions of argon metastable atoms on analyte ionization inside the plasma will require much additional work.

Nanofluidic devices have been of great interest due to their applications in variety of fields, including energy conversion and storage, water desalination, biological and chemical separations, and lab-on-a-chip devices. Although these applications cross the boundaries of many different disciplines, they all share the demand for understanding transport in nanoscale conduits. In this thesis, different elusive aspects of molecule and particle transport through nanofluidic conduits are investigated, including liquid and ion transport in nanochannels, diffusion- and reaction-governed enzyme transport in nanofluidic channels, and finally translocation of nanobeads through nanopores. Liquid or solvent transport through nanoconfinements is an essential yet barely characterized component of any nanofluidic systems. In the first chapter, water transport through single hydrophilic nanochannels with heights down to 7 nm is experimentally investigated using a new measurement technique. This technique has been developed based on the capillary flow and a novel hybrid nanochannel design and is capable of characterizing flow in both single nanoconduits as well as nanoporous media. The presence of a 0.7 nm thick hydration layer on hydrophilic surfaces and its effect on increasing the hydraulic resistance of the nanochannels is verified. Next, ion transport in a new class of nanofluidic rectifiers is theoretically and experimentally investigated. These so called nanofluidic diodes are nanochannels with asymmetric geometries which preferentially allow ion transport in one direction. A nondimensional number as a function of electrolyte concentration, nanochannel dimensions, and surface charge is derived that summarizes the rectification behavior of this system. In the fourth chapter, diffusion- and reaction-governed enzyme transport in nanofluidic channels is studied and the theoretical background necessary for understanding enzymatic activity in nanofluidic channels is presented. A simple analytical expression that describes different reaction kinetics is derived and confirmed against available experimental data of reaction of Trypsin with Poly-L-lysine. Finally, in the last chapter translocation of nanobeads through synthetic nanopores is experimentally investigated using resistive pulse sensing. The emphasis is placed on elucidating the effect of nanobead size on the translocation current and time. The key goals pursued in this study are multiplex detection of different nanobead sizes in a mixture of nanobeads as well as determining the concentration of each component. This problem other than its fundamental significance paves the way for developing new biosensing mechanisms for detection of biomolecules. This thesis further explores the molecule and particle transport in nanoscale conduits and serves for better characterization and development of nanofluidic devices for various applications.

No description available.

No description available.

The present set of studies examines the roles of two ion-motive enzymes, vacuolar-type H+-ATPase (VA) and Na+/K+ ATPase (NKA), in energizing transepithelial ion transport across the larval caecum and midgut epithelia of Drosophila melanogaster and Aedes aegypti. Even though both VA and NKA are expressed in insect epithelia, VA was considered the more important enzyme until the early 2000 because the ion transport was unaffected by the NKA inhibitor ouabain in many insect epithelia, a phenomenon termed the ‘ouabain paradox’. This paradox was resolved by the discovery of an organic anion transporter (OATP) that is colocalized with NKA and prevents the actions of ouabain on NKA. Since the resolution of the ouabain paradox, this is the first set of studies that investigates the role of NKA in energizing ion transport across the caeca and midgut of insects. First, I show that both VA and NKA are expressed in the caecum and the midgut. Moreover, the ATPase enzyme activities of VA and NKA are quantitatively similar within each region of the gut that was studied, suggesting that both ATPases may be important for establishing favourable electrochemical gradients for transport of ions across the gut. I used ATPase inhibitors to demonstrate that cation transport is dependent on the actions of both VA and NKA. Furthermore, this is the first set of studies that provides an insight into the ion transport mechanisms of the gastric caecum, an organ that is understudied in insects. In Aedes aegypti, I show that 5-hydroxytryptamine regulates the VA-rich cells of the gastric caecum, and therefore the rates of ion transport of these cells. Additionally, I also show that rearing salinity conditions for Aedes aegypti larvae alters the expression patterns of VA and NKA in the gastric caecum. In freshwater, increased activity of VA and NKA energizes transport of ions into the lumen of the caecum that likely maintains fluid volumes and ionic composition at levels appropriate for digestion and absorption. Overall, these studies provide novel information for caeca and midgut-specific actions of VA and NKA in insects, and present a number of new avenues for future research. / Thesis / Doctor of Philosophy (PhD) / This thesis focuses on investigating the roles of two enzymes, vacuolar-type H+-ATPase (VA) and Na+/K+ ATPase (NKA), which utilize energy to transport electrically charged atoms (ions) across the cells of the insect gut. Although VA was considered the more important of the two enzymes until the early 2000s, I have demonstrated that NKA also plays a role in maintaining insect gut function in fruit flies and mosquito larvae. Furthermore, the activities of both enzymes are dependent on the salinity of the medium in which mosquito larvae are reared, suggesting that they play a role in maintaining the ionic composition of the gut fluids in freshwater larvae. Additionally, I have also demonstrated that a neurochemical, serotonin, can modulate the activity of gut cells in mosquito larvae. Overall, this thesis provides novel information on the actions of VA and NKA in the insect gut, and presents a number of new avenues for future research.

Indiana University-Purdue University Indianapolis (IUPUI) / Pediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the Tmem67P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-andglucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using in vitro models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease.

In the 1970s it was discovered that organic polymers, a class of materials otherwise best know as insulating plastics, could be made electronically conductive. As an alternative to silicon semiconductors, organic polymers offer many novel features, characteristics, and opportunities, such as producing electronics at low costs using printing techniques, using organic chemistry to tune optical and electronic properties, and mechanical flexibility. The conducting organic polymers have been used in a vast array of devices, exemplified by organic transistors, light-emitting diodes, and solar cells. Due to their softness, biocompatibility, and combined electronic and ionic transport, organic electronic materials are also well suited as the active material in bioelectronic applications, a scientific and engineering area in which electronics interface with biology. The coupling of ions and electrons is especially interesting, as ions serve as signal carriers in all living organisms, thus offering a direct translation of electronic and ionic signals. To further enable complex control of ionic fluxes, organic electronic materials can be integrated with various ionic components, such as ion-conducting diodes and transistors. This thesis reports a background to the field of organic bioelectronic and ionic devices, and also presents the integration of ionic functions into organic bioelectronic devices. First, an electrophoretic drug delivery device is presented, capable of delivering ions at high spatiotemporal resolution. The device, called the organic electronic ion pump, is used to electronically control amyloid-like aggregation kinetics and morphology of peptides, and offers an interesting method for studying amyloids in vitro. Second, various ion-conducting diodes based on bipolar membranes are described. These diodes show high rectification ratio, i.e. conduct ions better for positive than for negative applied voltage. Simple ion diode based circuits, such as an AND gate and a full-wave rectifier, are also reported. The AND gate is intended as an addressable pH pixel to regulate for example amyloid aggregation, while the full-wave rectifier decouples the electrochemical capacity of an electrode from the amount of ionic charge it can generate. Third, an ion transistor, also based on bipolar membranes, is presented. This transistor can amplify and control ionic currents, and is suitable for building complex ionic logic circuits. Together, these results provide a basic toolbox of ionic components that is suitable for building more complex and/or implantable organic bioelectronic devices.