Classical molecular dynamics (MD) simulations are performed to study structural and dynamic properties of water confined within graphite surfaces. The surfaces are separated at distances varying between 7 and 14.5 Å and the water density is held constant at 1g/cc. Results at 298 K show the formation of a well-ordered structure constituted by water layers parallel to the graphite surfaces. The water molecules in the layers in contact with the surface have a tendency to orient their dipole parallel to the surface. Such ice-like structures may have different structural and dynamic properties than those of ice. The calculated mean square displacement reveals that the mobilities of the confined water at a separation of 8 Å become similar to that of low-temperature water (213 K) at the same density, although the structures of water are very different. The temperature at which the mobility of water confined at the separation of 7 Å would become similar to that of bulk low-temperature water was found to be 373K. With respect to the dynamics of confined water, a significant blue shift is observed in the intermolecular vibrational modes associated with the O×××O×××O bending and O×××O stretching of molecules linked by hydrogen bonds. The analysis of the geometry of water clusters confined between two graphite surfaces has been performed using ab initio methods. The ab initio calculations yield two preferential orientations of water molecules which are; 1) one O-H bond points to the surface and the other is parallel; 2) both O-H bonds are parallel to the surface. These orientations agree with those found in our MD simulation results. The calculated energy barriers for proton transfer of the confined H3O+-(H2O) complexes between two graphite model surfaces suggest that the confinement enhances the proton transfer at the separation 6-14.5 Å. When the confinement is high, at a separation of 4 Å, the barrier energies are extremely large. The confinement does not enhance proton transfer when the H3O+-(H2O) complexes are located further from the surfaces by more than 8 Å. As a result, the barrier energies start to increase at the separation of 20 Å.

Two-dimensional (2D) materials, referring to materials being just one atom thick, prove to be attractive not only in fundamental research but also in applications. Graphene, a single layer of carbon atoms arranged in hexagonal rings, is just the first among other materials (including hexagonal boron nitride and molybdenum disulfide) that could be isolated into mono-atomic layers. The presented thesis investigates proton transport through atomically thin two-dimensional materials. While the electronic, optical and mechanical properties of graphene and other 2D materials have been intensely researched over the past decade, much less is known on the interaction of these crystals with protons. It has been reported that most of the defect free two dimensional materials are impermeable to nearly all gases, molecules and ions. Whether proton, the smallest positively charged ion, could transport through two dimensional materials at a low energy level remains unknown. This work investigates proton transport through 2D materials, including graphene, hexagonal boron nitride and molybdenum disulfide, in two different systems: Nafion/Pd solid system and liquid/liquid interface system, both of which provided consistent results. Our results suggest that proton can transport through the interatomic spacings in the lattice of single layer BN and graphene, while single layer MoS2 is impermeable to protons. Single layer BN is the most conductive to protons among the 2D materials investigated in this thesis. Lower proton conductance of graphene is due to its delocalized π electrons while proton impermeability of MoS2 is due to the three atomic layers structure. Moreover, proton transfer is greatly facilitated by the deposition of platinum nanoparticles on the proton conductive 2D membranes to such a degree that platinum decorated BN seems to present negligible resistance to the transfer of protons through its lattice.

Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel provides the principal uptake pathway for protons. A water chain is thought to mediate the relay of protons through the D-channel, but it is interrupted at N139 in all crystallographic structures. Here, free energy simulations are used to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, where reduction and pumping is compromised. A general approach for the calculation of water occupancy in protein cavities is presented and demonstrates that combining efficient sampling algorithms with long simulation times is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of conformational and hydration states of the D-channel is characterized. Results shed light onto the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes.

Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel provides the principal uptake pathway for protons. A water chain is thought to mediate the relay of protons through the D-channel, but it is interrupted at N139 in all crystallographic structures. Here, free energy simulations are used to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, where reduction and pumping is compromised. A general approach for the calculation of water occupancy in protein cavities is presented and demonstrates that combining efficient sampling algorithms with long simulation times is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of conformational and hydration states of the D-channel is characterized. Results shed light onto the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes.

Ao longo dos últimos anos, o metabolismo ácido das crassuláceas (CAM) tem sido bem estudado em espécies das famílias Bromeliaceae e, principalmente, Crassulaceae. Essa via fotossintética é caracterizada pelo acúmulo noturno de ácidos orgânicos dentro do vacúolo e pela fixação de CO2 durante a noite pela enzima fosfoenolpiruvato carboxilase (PEPC). No entanto, pouco se sabe sobre a preferência pela atividade da enzima ATPase ou PPiase no transporte de prótons e ácidos orgânicos no interior das vesículas do tonoplasto em espécies CAM. A fotossíntese CAM pode ser induzida em plantas caracterizadas como CAM-facultativas por diversos fatores ambientais, por exemplo, déficit hídrico, termoperíodo, salinidade e deficiência de nutrientes. Contudo, pouco tem sido discutido sobre a influência dos nutrientes na indução do CAM. Esse estudo investigou o transporte de prótons através da membrana do tonoplasto em seis espécies de bromélias CAM e duas espécies de Kalanchoë. Todas as espécies usadas nesse estudo mostrou uma preferência pela ATPase do que pela PPiase para o transporte de prótons e ácidos orgânicos no interior das vesículas do tonoplasto. Nós também observamos uma maior expressão do CAM nas plantas de Kalanchoë laxiflora e Kalanchoë tubiflora mantidas na presença de 2.5 mM de NO3-. Por outro lado, Guzmania monostachia, uma espécie de bromélia epífita, exibiu a maior intensidade do CAM nas folhas mantidas na presença de NH4+ + déficit hídrico. Nessa espécie de bromélia, a maior expressão do gene ALMT9 na porção apical das folhas, seguido pelas maiores taxas de transporte de prótons pela ATPase, acúmulo de açucares solúveis e a ativação das defesas antioxidantes parecem estar relacionados com o aumento da tolerância pelo ajuste osmótico e limitação do dano oxidativo nas folhas mantidas na presença de NH4+ + déficit hídrico. Uma outra explicação para a maior intensidade do CAM no ápice das folhas mantidas em NH4+ + déficit hídrico poderia ser a maior expressão dos genes que codificam aquaporinas, principalmente GmPIPs e GmTIPs, que talvez sejam responsáveis pelo transporte de água das porções basal e mediana para a porção apical das folhas. O maior conteúdo de água conservado na porção apical poderia ajudar a aumentar a intensidade da fotossíntese CAM nessa porção das folhas de G. monostachia mantidas na presença de NH4+ + déficit hídrico / Historically, crassulacean acid metabolism (CAM) has been studied in many families, mainly Bromeliaceae and Crassulaceae. This photosynthetic pathway is characterized by the nocturnal organic acid accumulation in the vacuole as well as CO2 fixation during the night by the phosphoenolpyruvate carboxylase enzyme (PEPC). However, little is known about the preference of ATPase or PPiase activities for the proton and organic acids transport in tonoplast vesicles in CAM species. CAM photosynthesis can be inducted in CAM-facultative species by environmental factors such as, water deficit, thermoperiod, salinity and nutrients deficiency. Although, little has been discussed about the influence of nutrients on CAM induction. This study investigated proton transport in CAM bromeliad species and two CAM Kalanchoë species. All of the species used in this study showed a preference for ATPase rather than PPiase for the proton and organic acids transport into the tonoplast vesicles. We also observed a higher CAM expression in Kalanchoë laxiflora and Kalanchoë tubiflora plants kept in the presence of 2.5 mM of NO3-. On the other hand, Guzmania monostachia plants, an epiphytic tank bromeliad species, exhibited the highest CAM intensity in the leaves kept in the presence of NH4+ + water deficit. In this same bromeliad, a malate transporter gene, ALMT9, showed its highest expression in the apical portion of the leaves and the highest proton transport rates into the vacuole by ATPase. Soluble sugars and antioxidant enzymes activities were also verified in this study in order to observe their influence on increasing the drought tolerance of G. monostachia. In the leaves kept in NH4+ + water deficit the highest antioxidant activities and accumulation of soluble sugars were observed, this suggests that this inorganic nitrogen source seems to increase the drought tolerance by osmotic adjustment and limitation of oxidative damage. These factors can favor the increase of CAM intensity in the leaves kept under NH4+ + water deficiency. Another explanation for why the highest CAM intensity was observed in the apical portion of the leaves kept in NH4+ + water deficiency is because of the higher expression of aquaporin genes in the basal and middle regions of the leaves in the presence of this N source, mainly GmPIP and GmTIP, which might be responsible for transporting water from the basal and middle portions to the apical portion where these AQP genes are repressed. The fact that the highest water content is conserved in the apical portion might help to explain the increase in the intensity of CAM photosynthesis observed in the leaves of G. monostachia kept in the presence of NH4+ + water deficit

Proton exchange membrane (PEM) fuel cells are at the forefront among different types of fuel cells and are likely to be important power sources in the near future. PEM is a key component of the PEM fuel cells. The objective of this research is to investigate the fundamental aspects of PEM in terms of thermodynamics and proton transport in the membrane, so that the new proton conducting materials may be developed based on the detailed understanding. Since the proton conductivity increases dramatically with the amount of water in PEM, it is important to maintain a high humidification during the fuel cell operation. Therefore, the water uptake characteristics of the membrane are very important in developing fuel cell systems. Thermodynamic models are developed to describe sorption in proton-exchange membranes (PEMs), which can predict the complete isotherm as well as provide a plausible explanation for the long unresolved phenomenon termed Schroeder¡¯s paradox, namely the difference between the amounts sorbed from a liquid solvent versus from its saturated vapor. The sorption isotherm is a result of equilibrium established in the polymer-solvent system when the swelling pressure due to the uptake of solvent is balanced by the surface and elastic deformation pressures that restrain further stretching of the polymer network. The transport of protons in PEMs is intriguing. It requires knowledge of the PEM structure, water sorption thermodynamics in PEM, proton distribution in PEM, interactions between the protons and PEM, and proton transport in aqueous solution. Even proton conduction in water is anomalous that has received considerable attention for over a century because of its paramount importance in chemical, biological, and electrochemical systems. A pore transport model is proposed to describe proton diffusion at various hydration levels within Nafion¢ÃƒÂ§ by incorporating structural effect upon water uptake and various proton transport mechanisms, namely proton hopping on pore surface, Grotthuss diffusion in pore bulk, and ordinary mass diffusion of hydronium ions. A comprehensive random walk basis that relates the molecular details of proton transfer to the continuum diffusion coefficients has been applied to provide the transport details in the molecular scale within the pores of PEM. The proton conductivity in contact with water vapor is accurately predicted as a function of relative humidity without any fitted parameters. This theoretical model is quite insightful and provides design variables for developing high proton conducting PEMs. The proton transport model has been extended to the nanocomposite membranes being designed for higher temperature operation which are prepared via modification of polymer (host membrane) by the incorporation of inorganics such as SiO2 and ZrO2. The operation of fuel cells at high temperature provides many advantages, especially for CO poisoning. A proton transport model is proposed to describe proton diffusion in nanocomposite Nafion¢ÃƒÂ§/(ZrO2/SO42-) membranes. This model adequately accounts for the acidity, surface acid density, particle size, and the amount of loading of the inorganics. The higher proton conductivity of the composite membrane compared with that of Nafion is observed experimentally and also predicted by the model. Finally, some applications of PEM fuel cells are considered including direct methanol fuel cells, palladium barrier anode, and water electrolysis in regenerative fuel cells.

Proton exchange membrane (PEM) fuel cells are an eco-friendly power source that has great potential to reduce our oil dependence for our stationary and transportation applications. In order to make PEM fuel cells an economically viable option, further effort is needed to improve proton conduction under wide operating conditions and reduce the cost of production. Design and synthesis of novel membranes that have superior characteristics require a fundamental molecular-level understanding of the relationship between the polymer chemistry, water content and proton conduction. The performance of a fuel cell is influenced by the electrochemical and molecular/proton transport processes that occur at the catalytic sites in the electrode/electrolyte interface. Therefore, understanding the molecular-level details of proton transport and structure of the multi-phase interfaces is critical. This work is subdivided into two main tasks. The first task is to model membrane/water vapor interfaces and to study their morphology and the transport properties of water and hydronium ions. Classical molecular dynamics simulation is used as the modeling tool for the characterization of the interface. The second task is to model proton transport through the aqueous domains of PEM. Such a model is inherently challenging since proton transport occurs through a combination of structural and vehicular diffusions that are associated with disparate time scales. Toward this end, we have developed and implemented a new reactive molecular dynamics algorithm to model the structural diffusion of proton that involves breaking and forming of covalent bonds. The proton transport through aqueous channels in PEM is governed by acidity and confinement. Therefore, systems in which the acidity and confinement can be independently varied, including bulk water, aqueous hydrochloric acid solutions and water confined in carbon nanotubes are also examined in addition to the application in PEM. We have developed an understanding of how acidity and confinement independently impact proton transport. The correlation between the two components of charge diffusion and their contribution to the total charge diffusion has also been explored for a basic understanding of the proton transport mechanisms. These studies will eventually help us establish the correlation between the morphology of the membrane and proton conduction.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Influenza A (IVA) continues to pose a growing global threat even as current medications are becoming less effective. One of the main avenues of research into new anti-IVA drugs is its homotetrameric Matrix 2 proton channel (M2A), without which the virus would be unable to release its viral RNA into the host cell. The drug amantadine used to bind and block M2A until near-ubiquitous resistance formed as an M2A-S31N mutation, starting around 1995 and proceeding to 2005 when amantadine was disallowed for use as an anti-IVA drug. The standard organic structure currently being used for M2A inhibitor research comprises an adamantyl foot group, a heterocyclic aryl body group, and a cyclic head group. A sample set of compounds with this standard structure was compared and reviewed, focusing on positive and negative moieties and modifications. Modifications on the foot group were all more or less detrimental, body groups with two heteroatoms were advantageous, and larger head groups appeared better. Four other scaffolds known to literature were proposed for further study due to beneficial aspects of each. Where most anti-M2A research deals exclusively with organic compounds, metals and their potential in drugs have been almost entirely ignored due to the increased toxicity they bring. Free copper was found in past research to be the only first-row transition metal to show significant M2A-inhibitory activity, proposed to do so by binding the H37 cluster that acts as a pH-dependent control switch for the channel. Six overall-neutral copper complexes were synthesized as a combination of amantadine, cyclooctylamine, and null scaffolds with two of either acetate or acetamide arms as chelators. The complexes were found to block both M2A-WT and M2A-S31N. Along with CuCl¬¬2, though, they had little to no effect on M2A-H37A, providing confirming evidence that the copper binds at the H37 tetrad. Only one complex, Cu(cyclooctylamineiminodiacetate), outperformed CuCl2 in channel block studies and efficacy against two IVA strains, but all of the complexes were found to have lower cytotoxicity. Because M2-H37 is highly conserved, these complexes show promise for further testing against all strains of influenza A. Five net-divalent copper complexes were then synthesized with multiple aza or amine groups as chelators. The complexes failed to show any significant activity against M2A, however, which was thought to be due to size or polarity rejection or electromagnetic repulsion. One of the ligands, though, an adamantyl derivative of a tetraaza macrocycle, was a novel compound, and its copper complex, along with two others, were unknown to the CCDC database. The three complexes were characterized by X-ray diffraction and discussed.