Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Four research projects were carried out and they are described in this dissertation. Glycogen synthase kinase-3 beta (GSK3β) plays a pivotal and central role in the pathogenesis of Alzheimer's disease (AD) and protein kinase C (PKC) controls the function of other proteins via phosphorylation and involves in tumor promotion. In pursuit of identifying novel GSK3β and/or PKC inhibitors, substituted quinoline molecules were designed and synthesized based on the structure-activity-relationship studies. Synthesized molecules were evaluated for their neural protective activities and selected molecules were further tested for inhibitory activities on GSK3β and PKC enzymes. Among these compounds, compound 2 was found to have better GSK3β enzyme inhibitory and MC65 cell protection activities at low nanomolar concentrations and poor PKC inhibitory activity whereas compound 3 shows better PKC inhibitory activity. This demonstrates the potential for uses of quinoline scaffold in designing novel compounds for AD and cancer. Pharmacokinetics and distribution profiles of two anti-Alzheimer molecules, CP2 and TP70, discovered in our laboratory were assessed using HPLC/MS. Plasma samples of mice and rats fed with TP70 via different routes over various times were analyzed to quantify the amounts of TP70 in plasma of both species. Distribution profiles of TP70 in various tissues of mice were studied and results show that TP70 penetrated the blood brain barrier and accumulated in the brain tissue in significant amounts. Similarly, the amount of CP2 in plasma of mice was analyzed. The HPLC analysis revealed that both compounds have good PK profiles and bioavailability, which would make them suitable candidates for further in vivo efficacy studies. Nanodelivery of specific dsRNA for suppressing the western corn rootworm (WCR, Diabrotica virgifera virgifera) genes was studied using modified chitosan or modified polyvinylpyrrolidinone (PVP) as nanocarriers. Computational simulation studies of dsRNA with these polymers revealed that nanoparticles can be formed between dsRNA and modified chitosan and PVP polymers. Nanocarriers of hydroxylated PVP (HO-PVP) and chitosan conjugated with polyethylene glycol (PEG) were synthesized, and analyzed using IR spectroscopy. Particle sizes and morphology were evaluated using AFM and encapsulation was studied using UV spectroscopy. However, the formation of stable nanoparticles with dsRNA could not be achieved with either of the polymers, and further efforts are ongoing to discover a better nanocarrier for nanodelivery of siRNA by using chitosan-galactose nanocarrier. In our efforts to discover a novel class of tripeptidyl anti-norovirus compounds that can strongly inhibit NV3CLpro, a set of tripeptidyl molecules were synthesized by modifying the P1 - P3 of the substrate peptide including a warhead. It was found that the replacement of P1 glutamine surrogate with triazole functionality does not improve the inhibitory activities of the compounds. In addition, the synthesis of a known dipeptidyl compound (GC376) was carried out for evaluating its efficacy on feline infectious peritonitis (FIP) in cats.

As bactérias patogênicas em moluscos bivalves podem ser agentes causadores de doenças como a gastroenterite e responsáveis por vários surtos de origem alimentar, representado um risco para os consumidores. Os vírus entéricos são a causa mais comum de surtos de gastroenterites não bacteriana em humanos no mundo e podem ser encontrados nas águas utilizadas no cultivo de moluscos bivalves. Este estudo teve como objetivo avaliar a contaminação de mexilhões (Mytella falcata) e ostras (Crassostrea brasiliana) provenientes do Complexo Estuarino Lagunar de Cananéia-Iguape, Estado de São Paulo, por bactérias (coliformes totais, coliformes termotolerantes, patotipos de Escherichia coli), por astrovírus e norovírus humanos. Um total de 150 amostras de moluscos bivalves (75 ostras e 75 mexilhões) foram coletadas de junho de 2016 a fevereiro de 2017. A estimativa de coliformes totais nos tecidos das ostras variou de 14,1 a 154,5 número mais provável (NMP)/g e de coliformes termotolerantes de 3,0 a 48,6 NMP/g, enquanto que para as amostras de mexilhões, os coliformes totais variaram de 97,4 a 1300 NMP/g e coliformes termotolerantes de 3,6 a 927 NMP/g. E. coli foi detectada em 24 amostras (16%), em concentrações variando entre <3 e >927 NMP/g. Quatro amostras (17%) foram identificadas com Escherichia coli enteropatogênica (EPEC), apresentando o gene eae por PCR (Reação em Cadeia da Polimerase) e RFLP (Polimorfismo no Comprimento de Fragmentos de Restrição), e os amplicons positivos foram sequenciados. As porcentagens de similaridade relativas ao gene phoA de E. coli, para as cinco amostragens realizadas no estudo, apresentaram valores iguais ou superiores a 88,6%. As sequências de EPEC agruparam-se em diferentes clados com outras sequências do Brasil, Suíça e Uruguai, exibindo similaridade de 57,7 e 97,1% quando comparadas umas as outras. Quando comparadas a outras sequências de referência depositadas no GenBank, a similaridade variou entre 56,2 e 95,4%. Estes resultados são os primeiros a indicar a presença de EPEC em moluscos bivalves no Brasil. Astrovírus não foram identificados nas amostras de moluscos analisadas neste estudo. Norovírus (NoV) foi identificado em 21 (14%) das amostras, sendo 38% de mexilhões e 62% de ostras. As amostras de NoV genogrupo II (GII) foram agrupadas num clado único, juntamente com outras sequências de NoV GII, sendo mais próximas filogeneticamente de sequências originárias do Brasil, Japão e México, com similaridade de 93,8 a 96,6% do que com as outras sequências homólogas. A triagem de moluscos bivalves para coliformes, E. coli e presença de vírus entéricos significativos para a saúde pode ajudar na prevenção de surtos entre os consumidores e contribuir para a melhoria do ambiente estuarino. / The pathogenic bacteria in bivalve molluscs are causative agents of diseases such as gastroenteritis and responsible for several food-borne outbreaks, representing a risk to consumers. Enteric viruses are the most common cause of outbreaks of non-bacterial gastroenteritis in humans in the world and can be found in waters used in the cultivation of bivalve molluscs. The objective of this study was to evaluate the contamination of mussels (Mytella falcate) and oysters (Crassostrea brasiliana) from the estuarine complex Lagunar of Cananéia-Iguape, State of São Paulo, by bacteria (total coliforms, thermotolerant coliforms, Escherichia coli) and by human astroviruses and noroviruses. A total of 150 samples of bivalve molluscs (75 oysters and 75 mussels) were collected from June 2016 to February 2017. The total coliform estimate in oyster tissues varied from 14.1 to 154.5 most probable number (MPN)/g and thermotolerant coliforms from 3.0 to 48.6 MPN/g, whereas for mussel samples, total coliforms ranged from 97.4 to 1300 MPN/g and thermotolerant coliforms from 3.6 to 927 MPN/g. E. coli was detected in 24 samples (16%) at concentrations ranging from <3 to >927 NMP/g. Four (17%) were identified with enteropathogenic Escherichia coli (EPEC), presenting the gene eae by PCR (Polymerase Chain Reaction) and RFLP (Restriction fragment length polymorphism), and the positive amplicons were sequenced. The percentages of similarity relative to the phoA gene of E. coli, for the five samplings carried out in the study, presented values equal or superior to 88.6%. The EPEC sequences were grouped in different clades with other sequences from Brazil, Switzerland and Uruguay, exhibiting similarity of 57.7 and 97.1% when compared to each other. When compared to other reference sequences deposited in GenBank, the similarity ranged from 56.2 to 95.4%. These results are the first to indicate the presence of EPEC in bivalve molluscs in Brazil. Astroviruses were not identified in the mollusk samples analyzed in this study. Norovirus (NoV) was identified in 21 (14%) of the samples, representing 38% of mussels and 62% of oysters. NoV genogroup II (GII) samples were clustered in a single clade, along with other NoV GII sequences, keeping phylogenetically closest to sequences originating in Brazil, Japan and Mexico, with similarity of 93.8 to 96.6% than with the other homologous sequences. The screening of bivalve molluscs for coliforms, E. coli and the presence of enteric viruses significant to health can help preventing outbreaks among consumers and contribute to the improvement of the estuarine environment.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Acute diarrhea is one of the most common infectious diseases and still one of the leading causes of morbidity and mortality in the pediatric world, a global public health problem. Several efforts have been made over the years to reduce infant mortality, despite the commitment of diarrhea still occupy a significant space of malnutrition in children under 5 years. A wide spectrum of enteric pathogens can cause acute childhood diarrhea, among which we can mention Rotavirus, Norovirus and Cryptosporidium spp. Rotavirus is one of the most important. Currently two live attenuated live Rotavirus vaccines have been licensed and are available globally: a human monovalent strain (RV1) (Rotarix®, GlaxoSmithKline Biologicals) and a pentavalent bovine-human rearrangement (RV5) (Rotateq®). Brazil was one of the first countries to integrate the vaccine into its national immunization program (2006), and it was possible to observe significant improvements in reducing infant mortality and hospitalizations for diarrheal diseases in children from Rotavirus. However, diarrhea continues to be a serious health problem, and can be avoidable and treatable. Several etiological agents may be related to these intestinal infections and deserve to be investigated. The objective of this study was to verify the presence of the three etiological agents Cryptosporidium spp., Rotavirus and Norovirus, associated with diarrhea in children aged 0 to 11 years old, attended at Sergipe Emergency Hospital (HUSE). The samples were tested by Elisa for Cryptosporidium spp and Rotavirus for fecal antigen detection and reverse transcription polymerase chain reaction (RT-PCR) for Norovirus detection. A total of 92 diarrheic stool specimens were analyzed, of which 62% were positive for one of the pathogens studied, 49% positive for Norovirus, 10% for Rotavirus, 4% for Crysptoporidium spp and 37% of diarrheal samples were not positive for any enteropathogen studied. Norovirus was the main cause of childhood diarrhea in the period studied, with predominance (98%) of the GII genotype. The most affected age group were children younger than 24 months. Infections have shown similar symptoms. Complementary studies are needed to discover other etiological agents involved in gastroenteritis in the State of Sergipe, since a significant number of children with diarrhea did not present positivity to any of the pathogens studied. / A diarreia aguda é uma das doenças infecciosas mais comuns e ainda uma das principais causas de morbidade e mortalidade no mundo pediátrico, um problema global de saúde pública. Diversos esforços vêm sendo realizados no decorrer dos anos para redução da mortalidade infantil, apesar do empenho as diarreias ainda ocupam um significativo espaço da desnutrição em menores de 5 anos. Um amplo espectro de patógenos entéricos pode provocar a diarreia infantil aguda, dentre esses podemos citar os Rotavírus, Norovírus e Cryptosporidium spp, sendo o Rotavírus um dos mais importantes. Atualmente duas vacinas orais contra o Rotavírus, de vírus vivos atenuados foram licenciadas e estão disponíveis globalmente: uma cepa monovalente humana (RV1) (Rotarix®, GlaxoSmithKline Biologicals) e um rearranjo bovino-humano pentavalente (RV5) (Rotateq®). O Brasil foi um dos primeiros países a integrar a vacina em seu programa nacional de imunizações (2006), e foi possível observar melhorias significativas na redução da mortalidade infantil e nas hospitalizações por doenças diarreicas em crianças decorrentes de Rotavírus. No entanto, as diarreias continuam sendo um grave problema de saúde, podendo ser evitável e tratável. Diversos agentes etiológicos podem estar relacionados a essas infecções intestinais e merecem ser investigados. O objetivo desse estudo foi verificar a presença dos três agentes etiológicos Cryptosporidium spp., Rotavírus e Norovírus, associados aos quadros de diarreia aguda em crianças de 0 a 11 anos atendidas no Hospital de Urgência de Sergipe (HUSE). As amostras foram testadas por Elisa para Cryptosporidium spp e Rotavírus, para detecção de antígenos nas fezes, e reação de cadeia em polimerase após transcrição reversa (RT-PCR) para detecção de Norovírus. Foram analisadas 92 amostras de fezes diarreicas das quais 62% apresentaram positividade para um dos patógenos estudados, 49% positivos para Norovírus, 10% para Rotavírus, 4% para Crysptoporidium spp e 37% das amostras diarreicas não apresentaram positividade para nenhum enteropatógeno estudado. Norovírus foi o principal causador da diarreia infantil no período estudado, com predominância (98%) do genótipo GII. A faixa etária mais acometida foi de crianças menores de 24 meses. As infecções demonstraram sintomas semelhantes. Estudos complementares são necessários para descobrir outros agentes etiológicos envolvidos em gastroenterites do Estado de Sergipe, visto que uma quantidade significativa de crianças com diarreia não apresentou positividade para nenhum patógeno estudado. / São Cristóvão, SE

This project was designed to develop a method for the collection of environmental samples during prolonged Norovirus (NoV) outbreak investigations, and to develop real-time RT-PCR assays to analyze environmental samples for GI and GII noroviruses. The collection and processing of environmental samples could provide epidemiological data to facilitate investigations of prolonged NoV outbreaks and could guide public health NoV intervention strategies. Real-time RT-PCR assays for the detection of GI and GII NoVs were developed by adapting the State Hygienic Laboratory clinical GI and GII assays to the AB 7500 Fast platform. Analysis of the GI assay performance yielded a dilution curve slope = 3.28, R2 = 0.999 and a calculated amplification efficiency of 102%. The GII assay yielded a dilution curve slope = 3.39, R2 = 0.999 and a calculated amplification efficiency of 97%. Amplification efficiencies determine the sensitivity and the limit of detection of real-time RT-PCR assays. Optimum efficiencies range from 95%-105%, with a 100% efficiency indicating exponential amplification of targeted nucleic acid. To develop a method for the collection of environmental samples, multiple swab types were tested to determine their ability to recover NoV from laboratory spiked environmental surfaces. It was determined that foam swabs moistened with viral transport media were most effective in recovering NoV from spiked surfaces. A field test of the environmental sampling method was conducted by sampling environmental surfaces in four restaurants in one Iowa community. NoVs were not detected in the environmental samples. The collection and processing of environmental samples when conducting an investigation of a prolonged NoV outbreak could provide additional information on the epidemiology of NoV transmission and infection.

In this study a biosensor based on real time quartz crystal microbalance (QCM) monitoring is optimized and characterized for the application in the Norosensor. This biosensor is aimed to recognise, capture and amplify Norovirus (NoV). In an initial step a simplified bioassay was developed that focuses on the latter parts of the assay which consists of DNA-guided probing and amplification of the captured virus and includes the development of an amplification model assay directly to the functionalised crystal surface. A padlock probe with matching sequence to the conjugated oligonucleotide on the quartz crystal surface is used as target in the model assay. Although a number of studies have been carried out based on padlock probe ligation and rolling circle amplification (RCA) based QCM sensing, these studies utilize the entire crystal surface to capture and amplify the biomolecule. In this research work the QCM monitoring is explored on a centrally functionalised electrode surface through conjugation only at the centre of the electrode for increased mass sensitivity. Thus, allowing capture and amplification of the padlock probe only at the centre of the quartz crystal. A 14mm diameter, thermoncompensated AT-cut, nonpolished quartz crystal with a 10mm diameter gold surface coating acting as electrode was utilized for QCM measurements. The detection system is based on mass binding and amplification on the QCM to produce a negative frequency shift in the fundamental frequency of the vibrating quartz crystal. The amplification products were additionally fluorescently labelled and fluorescent microscopy images were also obtained at the end of every experiment to verify the presence or absence of DNA capture and amplification. Experimental findings show that the current flow chamber with a 15ul capacity is able to detect a specific padlock probe concentration of 1nM on a conjugated region of ~2.5mm diameter. RCA amplified the mass with an average frequency shift of -80Hz in 60mins RCA incubation time. Further, the specificity and sensitivity of the QCM system was explored. However, the system has limitations where sensor binding of reaction proteins, such as DNA ligase and BSA, to some extent is observed. The storage stability of the functionalized self-assembled monolayer (SAM) on the QCM is also observed to deteriorate and thus, is of concern. Nevertheless the combination of RCA based amplification with QCM real-time monitoring has the potential for rapid and simple, low cost detection of the Norovirus. / I det här arbetet har vi optimerat och karateriserat en biosensor för detektion av Norovirus som orsakar häftiga utbrott av kräksjuka under vinterhalvåret vilket leder till både försämrad vård samt stora ekonomiska förluster för samhället. Målet inom EU projektet “Norosensor” är att utveckla ett snabbtest som kan tillämpas efter ett utbrott på till exempel en vårdavdelning och som ska mäta mängden virus i luften vilket kan fungera som riktlinje för om en avdelning är säker att användas eller ej. Tekniskt är målet med testet att fånga in viruspartiklar från luften som specifikt binds till sensorytan. Därefter ökar vi känsligheten från bundna partiklar genom en DNA-baserad amplifiering. Detta genererar specifik, viruskorrelerad massa som mäts med en kvartskristall mikrovågs sensor. När massan ökar minskar frekvenser vid vilken kristallen vibrerar och detta mäts i realtid. Det här arbetet har inte behandlat infångande eller inbindning av virus utan har fokuserat på den senare delen av protokollet som omfattar amplifieringen på sensorytan. En modell-assay har därför utvecklats där viruspartikeln istället representeras av en så kallad “padlock probe” (hänglås probe). Då sensorn är mycket känslig har först olika protokoll testats för effektiv rengöring av ytan med hjälp av ultraljud. I nästa steg har ytan funktionaliserats med thiol-modifierade syntetiska DNA molekyler som används för infångningen av målmolekylen på sensorytan (virus eller i detta fall padlock proben). Det har tidigare uppskattats att för att få maximal känslighet i massmätningen så är det fördelaktigt att binda viruset endast i mitten på en mycket liten yta av kristallen. Den här avhandlingen har därför fokuserat på att utveckla protokoll för detta där ytan först funtionaliserats i mitten innan resten av ytan blockats för att undvika ospecific inbindning. Resultaten visar att vi kan generera en centrerad funtionalisering och att vi får låg ospecifik binding. Protokollet består av flera biokemiska reakionssteg såsom (i) inbindning och lingering av padlock probe och (ii) amplifiering av den ligerade proben genom “rolling circle amplification”. För att kunna verifiera att vi fått amplifieringsprodukter på ytan har vi dels mätt frekvensändringen på grund av ökad massa men också märkt in dem med fluorescerande molekyler och detekterat dem i microskop. Under arbetets gång har ett flertal olika typer av kristaller testats. Det visade sig att om en polerad yta används (1μm grovhet) så migrerade molekylerna iväg från mitten när vi oscillerade kristallen medan vi fick bättre resultat om något grövre (3μm) ytor användes. Vi testade även ett flertal olika flödesceller av olika material och med olika reaktionsvolymer. Eftersom kristallen är mycket känslig så påverkar faktorer som flödeshastigheter och eventuella luftbubblor frekvensen. Vi optimerade därför detta och körde mätningarna vi6konstant flöde men med alternerande, låga hastigheter när vi tillsatte nya reagens eller inkuberade reaktionerna. Vi förvärmde även reaktionsmixarna för att minska ospeficika effekter och konstaterade att den funktionaliserade ytan påverkades av lagring över tid. I våra försök såg vi att protein såsom ligeringsenzymet och albumin, vilka har förhållandevis stor massa, hade effekter på frekvensen redan i sig genom att binda till ytan. Ytterligare optimeringar måste därför göras framöver för att minska denna inbinding bland annat genom bättre tvättsteg. Vi kunde dock påvisa linjär massökning med ökad amplifieringstid och har bevisad hög specificitet. Slutligen utvecklades ett litet mjukvaruprogram för att automatisera analysen och minska bruset. Sammanfattingsvis har vi lyckats utveckla ett enkelt och snabbt system för specifik massamplifering av Norovirus.

No description available.

Microbubbles are small gas-filled bubbles with diameters ranging from 50 to 1 μm, and less than 200 nm are called nanobubbles. Their small sizes and large specific surface area result in a high gas dissolution rate and long lifetime in liquid. Ozone is a strong oxidant that destroys microorganisms and only produces oxygen as the final by-product in fresh water. However, due to the poor stability of aqueous ozone, critical gas waste happens during treatments which leads to a high economic loss. Microbubbles have shown promising enhancement of ozone treatment. In previous studies, ozone microbubbles exhibited excellent efficacy in the removal of organic contaminants and inactivation of microorganisms including bacteria, spores, and fungi, but few articles discuss the virus inactivation of ozone microbubbles treatment. Human noroviruses (NoVs) are the primary cause of foodborne illnesses in the US, and the development of effective inactivation methods is crucial. Because of the absence of suitable in vitro cultivation methods for NoVs and the constraints of the available infectivity models for these viruses, most of the studies about inactivation use surrogate viruses that are similar to NoVs in genetics and structure. Tulane virus is a NoV surrogate that can identify the same putative co-factor. This study focuses on the influence of treatment time, disinfectant air exposure, and the presence of organic contaminants on the inactivation efficacy of ozone microbubbles or millimeter bubbles. The results demonstrate that more than one log10 reduction was produced when the Tulane virus was exposed to ozone millimeter bubbles and ozone microbubbles for a short period of time, even in the presence of high organic load (FBS), and the protective effect of the organic load was shown when the disinfectant induced volume increased. The findings indicate that conducting further research on ozone microbubbles in aqueous applications in food-related applications is useful.

No description available.

No description available.

No description available.