O objetivo deste trabalho foi avaliar o efeito inibidor dos sais NaCl, MgCl2 e CaCl2, em mistura e separadamente, nas comunidades microbianas em sistema descontínuos e contínuos para o tratamento de esgoto sanitário de regiões de escassez hídrica. Foi avaliado o estabelecimento da nitrificação e desnitrificação simultânea (NDS) sem o controle da concentração de oxigênio dissolvido mediante à aeração contínua por meio de reatores contínuos de leito fixo estruturado na presença (Reator salino, RS) e ausência (Reator Controle, RC) dos sais de estudo. Nos sistemas descontínuos, um planejamento fatorial e ensaios complementares isotônicos para as pressões osmóticas (PO) de 5, 15 e 30 atm foram realizados para a comunidade nitrificante e para a comunidade desnitrificante afim de investigar os efeitos de inibição, antagonismo, sinergismo e estímulo dos sais em mistura e separadamente. Os efeitos dos sais foram diferentes para as comunidades nitrificante e desnitrificante e as maiores taxas específicas de nitrificação e desnitrificação obtidas foram de 0,11 mgN gSSV-1 min-1 e 0,065 mgN gSSV-1 min-1, respectivamente. Os ensaios em batelada permitiram inferir que o efeito de inibição de cada sal é distinto quando em mistura ou separadamente, independentemente da PO aplicada e que o efeito de toxicidade específica de cada sal é superior ao efeito de inibição da PO do meio, podendo estar relacionado à capacidade de osmoregulação dos microrganismos por solutos compatíveis. Nos reatores contínuos, a NDS foi estabelecida sob aeração contínua com concentrações médias de OD de 6 mg L-1, alcançando eficiências de oxidação de NH4+ e de remoção de N-Total de 60% e 56,3% no RC e 95,9% e 65,8% no RS na Fase 1 com PO de 1,7 atm. Isto evidenciou que a presença dos sais em concentração moderada foi benéfica ao sistema nas condições estudadas e para os inóculos aplicados. A deficiência da remoção de N-total em relação ao que foi nitrificado foi atribuída à falta de doadores de elétrons suficientes para promover a desnitrificação heterotrófica. A remoção de DQO no RS foi pouco afetada pelo aumento da PO, tendo eficiência reduzida de 97,2% para 86,2% com o aumento da PO de 1,7 para 9 atm. Análises de PCR/DGGE indicaram que a diversidade microbiana de ambientes salinos é tão grande ou maior que a de ambientes de água doce e o sequenciamento genético evidenciou que os inóculos possibilitaram a inserção de populações capazes de atuar no ciclo do nitrogênio sob condições de elevada salinidade, destacando-se os gêneros Aeromonas, Rheinhemera, Azospirillum, Thioalkalivibrio e Pseudoalteromonas. / The aim of this research was to evaluate the inhibitory effect of NaCl, MgCl2 and CaCl2 salts, in mixture and separately, on microbial communities in batch and continuous systems for the treatment of wastewater from regions with water scarcity. The establishment of simultaneous nitrification and denitrification process (SND) was assessed without dissolved oxygen control and under continuos aeration in structured bed reactors in the presence (Saline Reactor, SR) and absence (Control Reactor, CR) of the studied salts. In the batch reactors, a factorial design and complementary isotonic experiments for the osmotic pressures (OP) of 5, 15 and 30 atm were performed to investigate the effects of inhibition, antagonism, synergism, and stimulation of the salts alone and in mixture. The salts caused distinct effects on the nitrifying and denitrifying communities and the highest nitrification and denitrification specific rates obtained were 0.11 mgN gSSV-1 min-1 and 0.065 mgN gSSV-1 min-1, respectively. In the studied conditions, the results of the batch experiments showed that the inhibitory effect of each salt is different when they are alone or in mixture, regardless of the applied OP. It was observed that the microbial communities are more negatively affected by the specific toxic effect of each salt than by the unspecific inhibitory effect of the OP, which may be related to the osmoregulation capacity of the microorganisms by compatible solutes system. In the continuous reactors CR and SR, SND was established under continuous aeration at average DO concentrations of 6 mg L-1, achieving NH4+ oxidation and total nitrogen removal efficiencies of 60% and 56.3% in CR, and 95.9 % and 65.8% in SR in Phase 1 with OP of 1.7 atm. This data indicates that the presence of the salts in moderate concentration was beneficial to the system for the studied conditions and inoculum. The total nitrogen removal did not achieve higher efficiencies possibly due to the lack of electron donors, which supports heterotrophic denitrification. The COD removal in the SR was not greatly affected by the increase on OP as the efficiency reduced from 97.2% to 86.2% as the OP increased from 1.7 to 9 atm. PCR/DGGE analyses indicated that the microbial diversity of saline environments is as great as in freshwater ones. Genetic sequencing of the biomass showed the presence of bacterial populations capable of consuming oxidized and reduced forms of nitrogen under high osmotic pressure such as the genus Aeromonas, Rheinhemera, Azospirillum, Thioalkalivibrio and Pseudoalteromonas.

Nos quadros de sepse ocorre o aumento da permeabilidade vascular, translocação e perda de albumina para o espaço extravascular, resultando assim em hipoalbuminemia e redução da pressão coloidosmótica plasmática. Desta forma o objetivo deste estudo foi avaliar a relação da pressão coloidosmótica com a evolução clínica de 41 cadelas com sepse grave ou choque séptico decorrente de piometra que foram submetidas a ovariosalpingohisterectomia. Para tanto, os valores da pressão arterial sistólica, do débito urinário, do lactato, do déficit de base venoso e da pressão coloidosmótica foram avaliados a cada três horas ao longo do período de internação. O momento da mensuração da pressão coloidosmótica foi distinto entre os grupos, sendo grupo I (critério clínico) (n= 21) avaliado após o fim do tratamento e no grupo II (critério quantitativo) (n= 20), as amostras foram avaliadas imediatamente após a colheita. As variáveis clínicas utilizadas como guia a administração de coloide no grupo I não apresentaram correlação com os valores de pressão coloidosmótica baixo. A administração de coloide não apresentou impacto sobre os valores de albumina e pressão coloidosmótica, bem como não interferiu na perfusão tecidual. A pressão coloidosmótica apresentou uma correlação não significativa e inversamente proporcional com o SOFA. Desta forma, a partir dos resultados obtidos é possível concluir que os valores da pressão coloidosmótica não apresentaram correlação com os valores das variáveis de perfusão tecidual; o coloide não contribuiu para a melhora da perfusão tecidual e da manutenção da pressão coloidosmótica após a administração de grandes volumes de solução cristaloide. / Sepsis induces increased vascular permeability, translocation and albumin loss to extracellular space, resulting in hypoalbuminemia and lower plasma colloid osmotic pressure. This study correlate colloid osmotic pressure with the clinical progress of 41 female dogs presenting severe sepsis or sepsis shock due to pyometra, submitted to ovario-salpingo-hysterectomy. The parameters evaluated were: systolic arterial pressure, urinary debt, blood lactate, venous base excess and colloid osmotic pressure. Samples were collected every three hours during the inpatient period. Laboratorial analysis of colloid osmotic pressure was performed in different timings: group I (clinic criteria, n=21), samples analyzed only after the end of the treatment; and group II (quantitative criteria, n=20): samples analyzed immediately after collection. The parameters were a guide to evaluate the necessity of colloid administration. We found no correlation between the parameters evaluate and the values of colloid osmotic pressure. The administration of colloid presented a non-significant and inversely proportional correlation with SOFA. We concluded that colloid osmotic pressure is not directly correlated with tissue perfusion and colloid administration does not improve tissue perfusion or colloid osmotic pressure, even after the administration of substantial volumes of crystalloid solution.

Molecular dynamics simulations are an increasingly valuable tool to biochemical researchers: advances in computational power have expanded the range of biomolecules that can be simulated, and parameters describing these interactions are increasingly accurate. Despite substantial progress in force field parameterization, recent simulations of protein molecules using state-of-the-art, fixed-charge force fields revealed that the interactions among and within protein molecules can be too favorable, resulting in unrealistic aggregation or structural collapse of the proteins being simulated. To understand why these protein-protein interactions are so over-stabilized, I first assessed the ability of simulation force fields to represent accurately the interactions of individual amino acids, employing an osmotic pressure simulation apparatus that enabled direct comparison with experiment. Surprisingly, simulations of most of the amino acids resulted in behavior that was in strong agreement with experiment. A number of amino acids, however—notably those that contain hydroxyl groups and those that carry a formal charge—interacted in ways that were clearly inaccurate. Additionally, some commonly-used force fields failed to accurately represent the interactions of amino acids in a consistent manner. By further investigating the interactions of the functional groups of these amino acids, I was able not only to determine some of the root causes of individual amino acid inaccuracies, but also to implement simple modifications that brought the interactions of these small molecules and amino acids in stronger accord with experiment. These studies have highlighted some of the shortcomings in popular simulation force fields, and have proposed useful modifications to address them. Still, there is additional work that must be—and is being—conducted in order to correctly model the interaction behavior of proteins in simulation.

Nos quadros de sepse ocorre o aumento da permeabilidade vascular, translocação e perda de albumina para o espaço extravascular, resultando assim em hipoalbuminemia e redução da pressão coloidosmótica plasmática. Desta forma o objetivo deste estudo foi avaliar a relação da pressão coloidosmótica com a evolução clínica de 41 cadelas com sepse grave ou choque séptico decorrente de piometra que foram submetidas a ovariosalpingohisterectomia. Para tanto, os valores da pressão arterial sistólica, do débito urinário, do lactato, do déficit de base venoso e da pressão coloidosmótica foram avaliados a cada três horas ao longo do período de internação. O momento da mensuração da pressão coloidosmótica foi distinto entre os grupos, sendo grupo I (critério clínico) (n= 21) avaliado após o fim do tratamento e no grupo II (critério quantitativo) (n= 20), as amostras foram avaliadas imediatamente após a colheita. As variáveis clínicas utilizadas como guia a administração de coloide no grupo I não apresentaram correlação com os valores de pressão coloidosmótica baixo. A administração de coloide não apresentou impacto sobre os valores de albumina e pressão coloidosmótica, bem como não interferiu na perfusão tecidual. A pressão coloidosmótica apresentou uma correlação não significativa e inversamente proporcional com o SOFA. Desta forma, a partir dos resultados obtidos é possível concluir que os valores da pressão coloidosmótica não apresentaram correlação com os valores das variáveis de perfusão tecidual; o coloide não contribuiu para a melhora da perfusão tecidual e da manutenção da pressão coloidosmótica após a administração de grandes volumes de solução cristaloide. / Sepsis induces increased vascular permeability, translocation and albumin loss to extracellular space, resulting in hypoalbuminemia and lower plasma colloid osmotic pressure. This study correlate colloid osmotic pressure with the clinical progress of 41 female dogs presenting severe sepsis or sepsis shock due to pyometra, submitted to ovario-salpingo-hysterectomy. The parameters evaluated were: systolic arterial pressure, urinary debt, blood lactate, venous base excess and colloid osmotic pressure. Samples were collected every three hours during the inpatient period. Laboratorial analysis of colloid osmotic pressure was performed in different timings: group I (clinic criteria, n=21), samples analyzed only after the end of the treatment; and group II (quantitative criteria, n=20): samples analyzed immediately after collection. The parameters were a guide to evaluate the necessity of colloid administration. We found no correlation between the parameters evaluate and the values of colloid osmotic pressure. The administration of colloid presented a non-significant and inversely proportional correlation with SOFA. We concluded that colloid osmotic pressure is not directly correlated with tissue perfusion and colloid administration does not improve tissue perfusion or colloid osmotic pressure, even after the administration of substantial volumes of crystalloid solution.

Neste trabalho aplicou-se a metodologia diferencial para o cálculo do equilíbrio osmótico dentro da abordagem de Lewis-Randall para sistemas multisolventes. Nessa abordagem, são obtidas equações diferenciais que relacionam pressão e composição do sistema na condição de potencial químico constante dos solventes: o estado de equilíbrio osmótico é calculado integrando-se as equações, obtendo-se a curva de pressão osmótica em função da concentração do soluto. Essas equações não têm solução analítica, mas foram numericamente integradas para sistemas cuja não idealidade seja descrita pelo modelo UNIQUAC. A metodologia foi aplicada na análise de sistemas em que ocorre equilíbrio de fases líquido-líquido, o que em princípio corresponde ao teste mais severo a que pode ser submetida. A comprovação da eficácia da metodologia foi feita por meio da verificação das relações de equilíbrio nos passos intermediários e pela equação de Gibbs-Duhem. Os resultados mostraram que a abordagem é bastante confiável, e que o equilíbrio é corretamente calculado para uma variedade de situações. Foi possível discernir os casos em que é admissível utilizar um pseudo-solvente na descrição do equilíbrio osmótico (sistemas cujos solventes apresentam estruturas semelhantes, sem interação preferencial), e situações em que o cálculo pode não levar a resultados adequados (sistemas em que os solventes apresentem miscibilidade parcial). Os programas desenvolvidos são genéricos e, portanto, podem ser usados para o cálculo do equilíbrio osmótico em qualquer sistema descrito pela equação UNIQUAC. / A differential approach to the calculation of osmotic equilibrium of multisolvent systems within the Lewis-Randall framework is presented in this work. Differential equations relating pressure and composition at constant solvent chemical potential are developed, through whose integration the osmotic equilibrium is calculated. The curves of osmotic pressure as functions of the solute concentration cannot be analytically calculated, and were obtained through numerical integration. The methodology was used to calculate osmotic equilibrium for systems described by the UNIQUAC equation presenting liquid-phase split, which presumably corresponds to the most severe test. The approach was verified by checking the equilibrium conditions at intermediate points and also by checking the Gibbs-Duhem equation. The results show that the methodology is reliable and that the osmotic equilibrium can be correctly calculated for many situations. It was possible to identify the circumstances wherein a pseudo-solvent can be defined (systems whose solvents show similar structures, without presenting preferential interactions), and wherein the calculation may lead to unreliable results (systems wherein the solvents present partial miscibility). The computer programs developed are not specific, and can therefore be used to calculate osmotic equilibrium for any systems described by the UNIQUAC equation.

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Cationic polymers have shown potential as gene delivery vectors due to their ability to condense DNA and protect it from cellular and restriction nucleases. Dendrimers are hyperbranched macromolecules with precisely defined molecular weights and highly symmetric branches stemming from a central core. The nanosize, tunable surface chemistries and ease of surface functionalization has made dendrimers an attractive alternative to conventional linear polymers for DNA delivery applications. The commercially available, cationic dendrimer poly(amidoamine) or PAMAM is the most widely studied dendrimer for use as a gene delivery vector. The aim of this dissertation is to provide an increased understanding of the packaging and forces within PAMAM–DNA complexes. In Chapter 4, we will discuss the effect of molecular chain architecture on DNA-DNA intermolecular forces by examining DNA condensed by low generation (G0 & G1) PAMAM and comparing them to comparably charged linear arginine peptides. Using osmotic stress coupled with X-ray scattering, we are able to determine the structure and forces within dendrimer-DNA complexes, or dendriplexes. We show that PAMAM–DNA assemblies display significantly different physical behavior than linear cation–DNA assemblies. In Chapter 5, we examine the role of pH on condensation in these same low generation PAMAM-DNA complexes. PAMAM dendrimers have both terminal primary amines and internal tertiary amines with different pKas of approximately 9 and 6, respectively. We show changes in the pH at condensation greatly influence the resulting packaging as well as the resulting phase behavior for PAMAM dendriplexes. In Chapter 6, we examine the packaging of DNA by G4 PAMAM as a function of the percent zwitterionic modification. Many cationic polymers, including PAMAM, have shown high transfection efficiency in cell culture and potential for in vitro and in vivo applications, but its development is hindered by cytotoxicity in many cell lines and tissues. We hypothesize that zwitterionic PAMAM (zPAMAM) represent a new means to tune polymer-DNA interactions through PAMAM surface charge potentially enhancing intracellular unpackaging while reducing cellular toxicity. These zPAMAM complexes are currently under investigation for their potential as safer and more efficient materials for DNA delivery.

A hemostasia é um evento biológico passível de ser avaliado e estudado, assim como seus distúrbios. Há situações durante a anestesia que podem cursar com alterações hemostáticas culminando com aumento do sangramento ou até mesmo hemorragias graves. Os tempos de coagulação têm recebido especial atenção tendo-se em vista os diferentes contratempos hemostáticos que pode ocorrer durante o procedimento anestésico-cirúrgico. Sendo assim, nesse estudo buscou-se estabelecer os valores padrões para tempo de protrombina (TP) e de tromboplastina parcial ativada (TTPA) para cães submetidos a diferentes procedimentos cirúrgicos. Foram estudados 50 cães hígidos para padronização dos valores de TTPA e TP utilizando aparelho automático. Os valores de TTPA estavam dentro dos valores de normalidade da literatura (6,9 a 17,6 segundos) e valores de TP discretamente maiores (de 6,65 a 12,8 segundos). Foram estudadas 20 cadelas classificadas como ASA I e 18 cães ASA II e III. Nestes animais observaram-se aumentos significativos de valores de TTPA (de 12,04 para 14,29 segundos em ASA I, com P<0,0378; e de 13,4 para 15,11 segundos nos cães ASA II e III, P<0,0067) e de TP (de 8,36 para 9,7 segundos em ASA I, P<0,0323; e de 8,32 para 9,34 segundos nos caes ASA II e III, P< 0,0084) entre os momentos pré e pós-anestésicos Estes aumentos acompanham quedas da pressão coloidosmótica, indicando que o processo de anestesia, cirurgia e fluidoterapia causam hemodiluição, e conseqüente aumento nos tempo de coagulação. / Hemostasis is a biological event that could be evaluated and studied, just like yours disturbs. There are situations in to anesthetic procedures that could curse with hemostatics disturbs causing bleeding increase or even critical blood loss. The blood clotting times have been received special attention because different hemostatic setbacks that could occur during the anesthetic and surgical procedures. This way, this study look for establish the values for reference to protrombine time (PT) and to activated partial tromboplastin time (APTT) for dogs. Were studied 50 healthy dogs to standardize the values of PT and APTT utilizing an automatic instrument. The APTT values was in agreement with the literature values (6,9 to 17, 6 seconds), and the PT values was discret increased when compared with literature (from 6,65 to 12,8 seconds). Was studied 20 bitches classifieds as ASA I and 18 dogs classifieds as ASA II and III. In this animals observed significant increase values of APTT (from 12,04 to 14,29 seconds in the ASA I, P<0,0378; and from 13,4 to 15,11 seconds in the dogs ASA II and III, P<0,0067) and of PT (from 8,36 to 9,7 seconds in ASA I, P<0,0323; and from 8,32 to 9,34 seconds in the ASA II and III dogs) between the pre and post anesthetic moments. These increases attendance decease in the colloid osmotic pressure, indicating that the anesthetic, surgical and fluid therapy procedures can cause hemodilution, and consequent increase in the blood clotting times.

Hydroxyethyl starches (HES) are the most routinely used synthetic colloids during fluid resuscitation and have reported effects on coagulation. The overall goal of the investigation in this thesis was to evaluate the effects of tetrastarch administration on hemodynamic, laboratory, and hemostatic variables in healthy dogs and dogs with systemic inflammation. The objectives were to compare hemodynamic and laboratory variables in dogs receiving an isotonic crystalloid (0.9% NaCl) or tetrastarch during health and after induction of systemic inflammation; to compare the hemostatic effects of an isotonic crystalloid (0.9% NaCl) and synthetic colloid (tetrastarch) in healthy dogs and dogs with induced systemic inflammation; to compare two different protocols for TEG® activation and to determine the correlation between TEG® variables and traditional coagulation test results. Sixteen adult purpose-bred Beagles were randomized into one of two groups receiving fluid resuscitation with either 40 mL/kg IV isotonic crystalloid (0.9% NaCl) or synthetic colloid (tetrastarch) after administration of lipopolysaccharide (LPS; 5 μg/kg, IV) or an equal volume of placebo (0.9% NaCl, IV). Blood samples, for analysis, were collected at 0, 1, 2, 4, and 24 hours from the time of fluid resuscitation. After a 14-day washout period, the study was repeated such that dogs received the opposite treatment (LPS or placebo) and the same resuscitation fluid. Resuscitation with equal volumes of 0.9% NaCl and tetrastarch caused similar changes in hemodynamic and laboratory variables in dogs with LPS-induced systemic inflammation; however, larger increases in HR and blood pressure were seen within the first 2 hours following tetrastarch administration compared to 0.9% NaCl. Tetrastarch administration increased COP in all dogs, despite a decrease in TS. Tetrastarch bolus administration to dogs with LPS-induced systemic inflammation also resulted in a transient hypocoagulability characterized by a prolonged PTT, decreased clot formation speed and clot strength, and acquired type 1 von Willebrand disease. Considering the limited additional benefit of tetrastarch administration on hemodynamic variables demonstrated, as well as the transient adverse hemostatic effects of tetrastarch administration, the increased cost associated with the use of tetrastarch likely negates its use as a first line treatment during fluid resuscitation in dogs. / Pet Trust Fund