Dissertations / Theses on the topic 'Pathogenic Organisms'

The research within this thesis sought to determine the ability of various animal derived fats and plant derived oils to support the survival of several pathogenic cocktails over a multitude of storage times. The Salmonella study explored the survival rate of a four strain Salmonella cocktail in beef tallow, pig lard, duck fat, coconut oil, and extra virgin olive oil over seven days at 26˚C and 37˚C storage. The animal fats and the coconut oil supported the survival of the bacteria until the conclusion of the study. The Shiga-toxin producing Escherichia coli study explored the survival rate of a five strain STECs cocktail in extra virgin olive oil over seven days at 26˚C and 37˚C storage. The two Listeria studies explored the survival rate of a four strain Listeria monocytogenes cocktail in extra virgin olive oil over several time periods with different frequencies of sample mixing. In vitro, all genuses showed a 2.5-log cfu/mL to ≥ 7-log cfu/mL reduction in the extra virgin olive oil by the conclusion of the experiments. Extra virgin olive oil was then applied to cooked pork tenderloin, cheddar cheese snack squares, and turkey lunchmeat in hopes of inhibiting the L. monocytogenes cocktail. No reduction was observed.

Thesis (M. Tech.(Biomedical Technology)) - Central University of technology, Free State, 2011
The existence of geophagia from as early as 460 BC up to now, makes it relevant to investigate all aspects related to geophagia. Geophagia is a direct route for potential transmission of pathogens to the human host, through the ingestion of soil. Soil-borne diseases in humans are causing growing concern as sewage disposal, which involve sewage sludge and waste water drainage from these plants, is on the increase. It is estimated that approximately seven million tons of sewage sludge is produced annually and that 54% of this sewage sludge is introduced into soil. Data on enteric infection in humans caused by contamination from soil is limited and need further investigation. The aim of the study was, therefore, to collect information on the microbiological presence in geophagic soil in the Qwa-Qwa district. Objectives included the collecting of information regarding various sampling sites in the Qwa-Qwa district and also soil samples sold by vendors, investigation of the prevalence of known human pathogenic bacteria and fungi in geophagic soil, investigating the culturability of Salmonella enteritidis in geophagic soil in comparison with the viability of these organisms in soil for long periods of time, investigating potential antimicrobial activity of geophagic soil, as some of the geophagists are convinced that the geophagic soils have medicinal properties, and to determine the microbial diversity of geophagic soils, which can not be accomplished by conventional microbial culturing methods.

Traditionally, the hygienic quality of beaches has been determined by monitoring the water for microbial indicators of fecal pollution. Beach sand, which may also be an important medium for the transmission of fecal borne pathogens, has rarely been examined. The aims of this study where to examine the prevalence of fecal indicator organisms in tidally affected beach sand and in dryer upper beach sand, relative to water; identify the potential sources of indicator organisms in beach sand; examine the prevalence of selected eukaryotic microbes at sandy beaches; and investigate the potential health risks related to beach use. Three south Florida Beaches (Ft. Lauderdale Beach, Hollywood Beach, Hobe Beach) were sampled bimonthly for a I year period. Significantly, enterococci, fecal coliform, and E. coli levels were consistently present at higher concentrations in beach sand compared to the seawater at all 3 study beaches. Levels of somatic and F-specific coliphages were also present at higher concentrations in beach sand. Microbial- source tracking analysis by carbon utilization profiling suggested that the predominate sources of enterococci in beach sand were seagulls, and transiently replicating indigenous populations. Acanthamoeba spp. was the most commonly isolated free-living naked amoeba in this study and molecular analysis revealed that 19 of the 20 beach sand clones were genotype T4, the Acanthamoeba keratitis-associated genotype. With respect to salinity, the growth characteristics of beach sand Acanthamoeba isolates were similar to Acanthamoeba isolated from corneal scrapings. Results from the beach survey indicated that beach goers may have an increased risk for acquiring contact related ailments at Hobe Beach. Accordingly, bacterial and viral fecal indicator microbes were detected at the highest frequency and greatest average concentrations from Robe Beach. Reports of enteric and respiratory related symptoms were not higher in beach goers compared to the control cohort.

One of the factors that maintains fruit quality is its microbial flora. Fruit holds a natural non-pathogenic epiphytic microflora but can become contaminated with pathogenic microorganisms during export, causing either postharvest decay or possibly resulting in a food safety risk. In order to study microbial dynamics on fruit surfaces and the environment fruit moves through in the export chain, fruit washings were made, surfaces were sampled and total populations and diversities determined per cm2. Hygiene and safety levels for fruit export environments were hereby determined by sampling various points along the apple export chain, which included two farms and a harbour in South Africa and two harbours, two repacking facilities and two retail centres in Europe. In this first study of its kind, all the surfaces that were sampled exceeded the international standard for cleaning efficacy of food-processing equipment that is <5 cfu/cm2, while several areas exceeded the maximum acceptable index level of microbial air contamination of 22 cfu/h in food industries. Washing of containers on a harbour in South Africa did not have a significant impact on microbial populations. Regarding fruit quality, it was determined that apple microflora fluctuate throughout the export process and that postharvest pathogens that are known to cause great economic losses in the apple industry, proved to be of little significance in this investigation. The presence of six foodborne pathogens i.e. Shigella sonnei, Salmonella muenchen, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus epidermidi/s was monitored throughout the chain. Of these, only S. aureus and E. coli were recorded, although pathogenicity was not confirmed for the latter. Staphylococcus aureus was found in containers and at a retail centre in Europe, and S. aureus and S. epidermidis were recorded on apple surfaces for the first time. Escherichia coli was present in great numbers in fruit washing water on a farm in South Africa. Since the standard for food premises is very stringent and perhaps inapplicable for fresh fruit handling and holding facilities, future research should include development of a more realistic hygiene standard for fresh fruit environments.
Dissertation (MSc(Agric) : Plant Pathology)--University of Pretoria, 2006.
Microbiology and Plant Pathology
unrestricted

Trois études ont été réalisées sur l’adaptation d'une souche aviaire H9N2 de faible pouvoir pathogène sur trois modèles différents : embryons de poulet, poulet de chair puis hamster. La première visait à évaluer l'influence du passage embryonnaire du virus H9N2 sur la stabilité de la séquence d'acides aminés de l'Hémagglutinine 1 (HA1) et sa relation avec la pathogénicité. Elle a montré une augmentation significative de la pathogénicité du virus suite au passage embryonnaire, malgré la présence du même motif au site de clivage. La deuxième partie du travail a consisté à évaluer l'effet de trois passages consécutifs du virus H9N2 chez les poulets de chair sur sa pathogénicité et les variations moléculaires qui en sont responsables. Nous avons montré qu'un passage d'H9N2 faiblement pathogène dans les poulets de chair provoque une augmentation de la pathogénicité, associée à une stabilité de la séquence d'acides aminés de l'Hémagglutinine au site de clivage, et une variabilité dans la séquence de la tige de la neuraminidase. Le risque d'évolution d'une souche peu pathogène vers une souche hautement pathogène a été démontré. La troisième étude, menée sur des hamsters, a mis en évidence l'adaptation de la pathogénicité lors du franchissement de la barrière inter-espèces et a signalé le risque potentiel de maladies zoonotiques dans certains élevages
Three studies were carried out for studying the adaptation of avian H9N2 virus after embryonic, broilers or hamster passaging. The first study have shown that the pathogenicity increased significantly upon passaging in chicken embryos in spite of the presence of the same motif at the HA1 cleavage site. The second study assessed the impact of H9N2 viral passaging in broilers on amino acid sequences of the hemagglutin cleavage site and neuraminidase stalk, and their relatedness to pathogenicity. In that case, we observed that passaging leads to a trand of increase in pathogenic adaptability, associated with a conserved a. A. Sequence of the hemagglutinin cleavage site, and a variability in the sequence of the neuraminidase stalk. The third study demonstrates the impact of avian-H9N2 viral passaging in hamsters on its cross species-pathogenic adaptability, and variability of amino acid sequences of the hemagglutinin and neuraminidase stalk of the original and the differently passaged H9N2 viruses. The adaptation of avian virus to mammalian cells raise a concern of a possible public health threat since the mixing of avian with mammalian animal species is a common practise on farms and petshops of most developing countries

Some yeast strains under certain conditions secrete polypeptide toxins which are inhibitory to sensitive fungal cells into the medium. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component, beta-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. K5 type killer protein was characterized in our labarotory previously. This protein is an exo beta-1,3-glucanase which is stable at pH&
#8217
s and temperatures appropriate for its biocontrol usage. Beta-1,3-glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antifungal agent. According to CLSI methodology, antifungal activity of the K5 type yeast killer protein was tested against 6 fungal strains causing postharvest spoilage in citrus fruits and found to be effective on Botrytis cinerea, Penicillium digitatum, Penicillium italicum whereas non effective on Colletotrichum gloeosporoides, Phythophythora citrophthora, Alternaria citri. The MIC values of the toxin for B.cinerea, P.digitatum, P.italicum were found to be 16 mikrogram/ml while IC 50 values of the toxin were 2.12, 3.31, 2.57 mikrogram/ml respectively. The results showed that K5 type yeast killer protein would be used as a novel and selective agent against B.cinerea, P.digitatum and P.italicum.

In this study, 59 epidemiologically unrelated Salmonella strains isolated from foods in Tü
rkiye and 49 Salmonella strains obtained from National Salmonella Reference Laboratories of Germany were analysed. For the characterization of strains, analyses such as serotyping, phage typing, antibiotyping and molecular biological characterization were done. The strains exhibited 17 different serotypes with S. Enteritidis serotype and PT21 phage type being the most prevalent in Turkish isolates. The highest antimicrobial resistance was observed against NAL for Turkish strains, whereas it was against SUL for strains from German origin. Molecular typing of all strains exhibited different plasmid profiles and PFGE patterns. There were 1-4 plasmids/profile for Turkish strains and 1-7 plasmids/profile for German strains. The PFGE patterns revealed 42 different subgroups, having two major clusters with 44,3% arbitrary homology. Among 72 resistant strains, the most prevalent resistance genotypes were observed as blatem-1 (%56, AMP resistance)
floR (%100, CHL and FFC resistance)
aphA1 (%100, KAN and NEO resistance)
tet(A) (%53, TET resistance)
aadA1 (%82, SPE and STR resistance)
sulI (%78, SUL resistance). The class I integron variable region analyses exhibited 700 bp (1 strain), 1000 bp (37 strain), 1200 bp (16 strain) and 1600 bp (3 strain) integrons.

The withdrawal of water sources concluded the reuse of treated wastewaters, especially for non-potable purposes. Agricultural use of the reclaimed wastewaters is one of the reuse options. However health considerations of the reuse of reclaimed wastewaters for public related purposes are underestimated, since wastewaters contain a variety of microbial pathogens, which may be transmitted to workers and consumers through the crops irrigated. Of these, parasitic eggs have a special place, as they are capable of surviving in the soil for months or even years, depending on environmental conditions. There is insufficient accumulated information on the health related criteria for the reuse of treated wastewaters in Turkey. The aim of this study was therefore to determine the helminthic eggs in raw sewage and in effluents of ASKi municipal wastewater treatment plant in Ankara. The study involved examining to decide whether these organisms exist in the wastewaters at all, and if so in what concentrations. Modified Bailenger&rsquo
s method, which published in the &ldquo
WHO Laboratory Manual of Parasitological and Bacteriological Techniques&rdquo
and &ldquo
U.S.EPA ICR Microbial Laboratory Manual&rdquo
were used in developing the specific methods used in this study.

Although traditional microbiological methods are accepted standard for Salmonella detection, they are labor intensive and time consuming. Therefore, for food industry and public health, finding sensitive and rapid methods is required. As a rapid and reliable tool, Real-Time PCR is one of the most common methods in molecular detection and research area. The aim of the current study is to develop rapid, sensitive and quantitative Salmonella detection method using Real-Time PCR technique based on inexpensive, easy to produce, convenient and standardized plasmid based positive control for the first time. To achieve this, two plasmids were constructed as reference molecules by cloning two most commonly used Salmonella specific target regions &lsquo
invA and ttrRSBC&rsquo
into them. Standard curves were constructed for the plasmids and reproducibility, PCR efficiency, amplification efficiency values were calculated. To illustrate the applicability of the developed method, enriched (as used commonly for Salmonella detection with Real-Time PCR) 105 to 100 CFU/ml level (estimated by standard plate counts before enrichment) S. Typhimurium ATCC 14028 cultures were tried to detect and quantify, also compared with traditional culture method. In addition, detection limits of the developed technique were determined by serial dilution of DNA extracted from 105 CFU/ml level. The results revealed much faster detection ability of the developed plasmid based Salmonella detection method (in comparison to traditional culture method, ISO 6579:2004) allowing quantitative evaluation with perfect reproducibility, sensitivity (except for lower concentrations for invA target), detection limit, PCR efficiency, amplification efficiency for both invA and ttrRSBC targets. The detection and quantification ability of the method developed by using S. Typhimurium ATCC 14028 cultures were tested also with 15 Salmonella species using milk as a representative food. The results also revealed much faster (in comparison to traditional culture method, ISO 6579:2004) quantitative detection ability of the developed method. Thus, the developed method has great potential to be used in food industry for rapid and quantitative Salmonella detection.

In this study, two symbiotic fungi of Southern Pine Beetle (SPB), Entomocorticium peryii and Entomocorticium sp.A were evaluated in terms of polyphenol oxidase (PPO) production. The effect of different inhibitors, inducers and assay parameters such as temperature and pH on enzyme activity were investigated and maximum PPO activity was observed at 30°
C, pH 8.0 and when tannic acid was used as an inducer. Copper-chelator salicyl hydroxamic acid (SHAM) and pcoumaric acid, both indicated as inhibitors of tyrosinase and catechol oxidase significantly reduced the activity. For biochemical characterization studies, the enzyme was concentrated by ultrafiltration. To determine type of the enzyme, activity staining after Native-PAGE was carried out. Type of polyphenol oxidase produced by E. peryii and E. sp.A was determined as catechol oxidase by activity staining. However higher activity was observed on hydroquinone (p-diphenol) rather than catechol (o-diphenol). The enzyme obeys Michealis-Menten kinetics with Km and Vmaxvalues being 10.72 mM hydroquinone and 59.44 U/ml for E. peryii and 8.55 mM hydroquinone and 73.72 U/ml for E. sp.A respectively..

Thesis (MScMedSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Mycobacterium tuberculosis is a pathogenic organism that infects a third of the world’s population and causes approximately 2 million deaths per year. Extensive research has been done on this pathogen, however our knowledge of the mechanisms of pathogenicity remain limited. The M. tuberculosis genome contains five ESAT-6 gene cluster regions, ESX-1 to 5, which encode specialized type VII secretion systems. These secretion systems are known to secrete members of the ESAT-6/CFP-10 and PE/PPE protein families, some of which contribute to the pathogenicity and phagosomal escape of the pathogen. ESX-3 has been shown to be essential for in vitro growth and survival of M. tuberculosis. The expression of ESX-3 in M. tuberculosis is regulated by IdeR and Zur, in response to intracellular iron and zinc concentrations, respectively. Interestingly, ESX-3 is not essential for the growth and survival of the saprophytic organism M. smegmatis. In this study, we aimed to identify the subcellular localisation of the individual components of the ESX-3 secretion system in the non-pathogenic, fast-growing organism M. smegmatis. The esx conserved component (ecc) genes from ESX-3 were expressed from the episomal expression vector pDMNI as fusion proteins with green fluorescent protein (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) and MSMEG_0626 (eccE3) were successfully cloned into pDMNI and expression of fusion proteins was confirmed by Western blotting for MSMEG_0615-GFP, MSMEG_0616-GFP and MSMEG_0626-GFP in M. smegmatis. In the M. smegmatis ESX-3 knock-out (with MSMEG_0615 to MSMEG_0626 deleted) expression was confirmed for MSMEG_0615-GFP and MSMEG0626-GFP. Fluorescent microscopy determined that MSMEG_0615-GFP localised to a single mycobacterial pole in both strains. MSMEG_0616-GFP and MSMEG_0626-GFP were found to be membrane associated in M. smegmatis, while MSMEG_0626-GFP was found to be membrane associated in the M. smegmatis ESX-3 knock-out. The unipolar localisation of MSMEG_0615-GFP suggests that the assembled ESX-3 secretion system apparatus is situated at a single pole in M. smegmatis. Therefore, we hypothesize that MSMEG_0615 might act as a recruiter protein that is involved in the assembly of ESX-3 at the mycobacterial pole.
AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis is ‘n patogene organisme wat ‘n derde van die wêreld se bevolking infekteer en eis jaarliks 2 miljoen lewens deur tuberkulose. Ten spyte van uitgebreide navorsing, is daar min kennis oor die meganismes van patogenisiteit van hierdie organisme. Die M. tuberculosis genoom bevat vyf duplikasies van die ESAT-6 geen groep gebiede, ESX-1 tot 5, wat kodeer vir gespesialiseerde Tipe VII sekresie sisteme. Hierdie sekresie sisteme is bekend vir die sekresie van lede van die ESAT-6/CFP-10 en PE/PPE proteïen families, waarvan sommige bydra tot die patogenisieit en fagosomale ontsnapping van hierdie organisme. ESX-3 is noodsaaklik vir die in vitro groei en oorlewing van M. tuberculosis. Die uitdrukking van ESX-3 in M. tuberculosis word gereguleer deur IdeR en Zur in reaksie op intrasellulêre yster en sink konsentrasies, onderskeidelik. ESX-3 word nie benodig vir die groei en oorlewing van die saprofitiese organisme M. smegmatis nie. Hierdie studie was gemik om die sub-sellulêre lokalisering van ESX-3 te identifiseer in die niepatogeniese en vinnig-groeiende organisme, M. smegmatis. Die “esx conserved component” (ecc) gene van ESX-3 is uitgedruk vanaf die episomale uitdrukkingsvektor pDMNI as gekombineerde proteïene met die groen fluoreserende proteïen (GFP). MSMEG_0615 (eccA3), MSMEG_0616 (eccB3), MSMEG_0623 (eccD3) en MSMEG_0626 (eccE3) is suksesvol gekloneer en die uitdrukking van die gekombineerde proteïene is bevestig deur Western oordrag vir MSMEG_0615-GFP, MSMEG_0616-GFP en MSMEG_0626-GFP in M. smegmatis. In die M. smegmatis ESX-3 uitklopmutant (met MSMEG_0615 tot MSMEG_0626 uitgeslaan) is uitdrukking bevestig vir MSMEG_0615-GFP en MSMEG0626-GFP. Fluoresensie mikroskopie het bepaal dat MSMEG_0615-GFP gelokaliseer is by ‘n enkele mikobakteriese pool in beide stamme. MSMEG_0616-GFP en MSMEG_0626-GFP was membraan-geassosieerd in M. smegmatis, terwyl en MSMEG_0626-GFP geassosieer het met die membraan in die M. smegmatis uitklopmutant. MSMEG_0615 het gelokaliseer by ‘n enkele pool in M. smegmatis en dit dui aan dat die saamgestelde ESX-3 sekresie sisteem apparaat slegs by ‘n enkele pool voorkom in M. smegmatis. Ons hipotiseer dat MSMEG_0615 dalk mag optree as ‘n werwer proteïen wat betrokke is by die samestelling van die ESX-3 sekresie sisteem by die mikrobakteriese pool.
Stellenbosch University

Cette thèse porte sur deux aspects complémentaires, le développement et l’implémentation de nouvelles approches de cristallisation et de cristallographie sérielle ainsi que leur mise en œuvre dans l’étude structurale de complexes enzymes : ARNt. La cristallographie est la méthode la plus employée en biologie structurale, mais elle présente encore des points délicats. Plusieurs méthodes avancées ont été déployées dans ce travail pour y pallier qui ont conduit à la résolution de la structure de l’ARNt nucléotidyltransférase du psychrophile Planococcus halocryophilus et à l’étude de son adaptation structurale au froid ; des puces microfluidiques de cristallisation qui ont servi à la résolution de plusieurs structures à température ambiante par cristallographie sérielle ; enfin le Xtal Controller utilisé pour l’étude d’évènements de nucléation et de croissance cristalline dans un but de préparation d’échantillons pour analyse sous rayonnement XFEL. Entre autres systèmes biologiques, cette thèse présente la caractérisation de deux familles d’inhibiteurs visant les aspartyl-ARNt synthétases, notamment du pathogène Pseudomonas aeruginosa
This thesis focuses on two complementary aspects, the development and implementation of new approaches of crystallization and of serial crystallography as well as their use in the structural study of enzymes/tRNA complexes. Crystallography is the most used method in structural biology, but it presents delicate points. Different methods were implemented in this work to overcome these points, which led to the resolution of the structure of the CCA-adding enzyme of the psychrophilic organism Planococcus halocryophilus and to the study of its structural adaptation to the cold; novel microfluidic crystallization chips that have been used for the resolution of several structures by serial crystallography at room-temperature; finally the Xtal Controller used for the study of nucleation and crystal growth events with the purpose of preparing samples for analysis under XFEL radiation. Among other biological systems, this thesis presents the study and characterization of two families of inhibitors targeting aspartyl-tRNA synthetases, including the one of the pathogenic organism Pseudomonas aeruginosa

Afin de caractériser les voies de signalisation du système immunitaire inné, nous étudions l'interaction entre le ver C. elegans et le champignon Drechmeria coniospora. Une des réponses du ver à l'infection consiste en une augmentation de la production de peptides antimicrobiens (PAM) dans l'épiderme. Des vers transgéniques exprimant le gène rapporteur de la GFP sous le contrôle du promoteur d'un PAM, fluorescent vert après infection. Si un gène nécessaire à l'expression des PAM est inactivé, alors les vers transgéniques ne fluorescent plus après infection. Nous avons effectué un crible pour identifier les molécules de signalisation nécessaires à l'expression des PAM en utilisant une approche quantitative et semi-automatique par ARN interference (ARNi). Deux banques d'ARNi couvrant 95% du génome, soit 20 000 gènes, ont été criblées et 360 candidats bloquant l'induction de la GFP après infection ont été obtenus, correspondant à 343 gènes. Une caractérisation phénotypique a permis de placer les candidats dans différentes catégories fonctionnelles et permis d'identifier d'une part un récepteur agissant en amont de la voie de signalisation p38 nécessaire à l'activation des gènes PAM, d'autre part une implication des granules de stress lors de l'infection. Ces analyses sont le fondement pour l'établissement d'une description compréhensive du réseau génétique régulant le système immunitaire inné du ver et permettront de révéler les interactions complexes entre l'immunité et les processus physiologiques au niveau moléculaire, cellulaire et au niveau de l'organisme
To investigate innate immune signaling, we study the interaction of C. elegans with the fungus Drechmeria coniospora. One of the responses of the worm to this infection is the up-regulation of a variety of antimicrobial peptide (AMP) genes in the epidermis. Transgenic worms carrying a GFP reporter gene under the control of an AMP promoter fluoresce green after infection by D. coniospora. If a gene required for AMP gene expression is inactivated, the reporter strain will not turn green upon infection. Using this fluorescent read-out, we have been able to screen for signaling molecules required for AMP gene expression using a quantitative semi-automated RNAi approach. We have screened two RNAi libraries that together cover 95% of the ca. 20,000 genes in the C. elegans genome and we obtained 360 high-confidence candidates that reduced the level of induction of green fluorescence after infection, and correspond to 343 genes. A further phenotypic characterization allowed the candidates to be grouped into distinct functional categories and allowed the identification of both a receptor acting upstream the p38 MAPK pathway necessary for the activation of the AMPs, and the implication of stress granules during infection. Altogether, the screen data and its analysis represent the foundation for the establishment of a comprehensive description of the signaling network regulating the innate immune system of the worm and will shed light on the complex interactions between immunity and other physiological processes at the molecular, cellular and organismal level

Thesis (MSc Food Sc)--University of Stellenbosch, 2001.
ENGLISH ABSTRACT:Aspalafhus linearis is an indigenous fynbos plant cultivated in the Clanwilliam area of the Western Cape, South Africa. The rooibos tea that is prepared from this plant, has become popular worldwide mainly due to the alleged health properties. Studies on the anti-microbial properties of green, black and oolong teas have shown that these teas have strong anti-microbial activity against a wide range of microbes. No studies have been done on the anti-microbial activity of rooibos tea and the aim of this study was to determine what impact rooibos tea extracts would have on the growth of different food spoilage and potential pathogenic microbes. Water and ethyl acetate extracts of fermented and unfermented rooibos tea were used to determine the inhibitory effect on the growth of an Escherichia coli strain. The E. coli culture was grown in tea-MRS with either added fermented or unfermented rooibos tea extracts. Both the water and ethyl acetate extracts showed a strong inhibitory effect against the E. coli strain in that there was a decrease in the final bacterial cell density (Nmax)(from 0.59 00 to 0.25 00) and the maximum specific growth rate (~max)(from 1.12 h-1 to 0.20 h-1) and an increase in the doubling time (~) (from 0.59 h to 1.80 h) and lag time (tlag)(from 4.81 h to 6.60 h) as the concentration of the soluble solids of the tea extracts was increased from 0.5 to 5.0 g.r1 . Furthermore, it was found that the fermented rooibos tea had a much stronger inhibitory effect (69% decrease in growth at 5.0 g.r1 soluble solids) compared to the unfermented rooibos tea extracts (35.1% decrease in growth at 5.0 g.r1 soluble solids). The resulting data indicated that rooibos tea had a very strong inhibitory effect on the growth of the E. coli strain. It was also found that the water extracts of rooibos tea showed a stronger inhibitory effect on the growth of the E. coli than the ethyl acetate extracts, indicating that the antimicrobial activity of rooibos tea is not exclusively due to the polyphenolic content - individual compounds. It was also determined that the rooibos tea water extracts showed a bacteriostatic action against the E. coli strain in that as soon as the tea is no longer part of the growth medium, the E. coli resumed a normal growth pattern. The data obtained showed that the inhibitory effect of rooibos tea water extracts (69% decrease in growth) against the growth of E. coli was more pronounced than that found when black tea water extracts (25.7% decrease in growth) at the same concentrations were used.Rooibos tea water extracts (0.5 - 5.0 g.r1) of fermented and unfermented tea were also used to determine the inhibitory effect on other food spoilage microbes and potential pathogens. Strains of Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus mutans, Saccharomyces cerevisiae and Zygosaccharomyces rouxii were grown in the presence of fermented and unfermented rooibos tea water extracts. The effect that fermented rooibos tea had on the growth of all the microbes tested was in the following order: Staph. aureus (90.8% decrease in growth) > L. monocytogenes (89.2% decrease in growth) > Strep. mutans (84.1% decrease in growth) > B. cereus (80.3% decrease in growth) > Sacch. cerevisiae (77.7% decrease in growth) > E. coli (69.0% decrease in growth). The rooibos tea clearly had an inhibitory effect on the growth of all the microbes, with the exception of the Z. rouxii strain where the presence of the tea water extracts was found to enhance the growth. The inhibitory effect of rooibos tea on the growth of these microbes was shown by changes in the growth parameters with Nmax and IJmaxshowing decreases, while the ld and tlagincreased as the concentration of the tea soluble solids was increased. As with E. coli, the fermented rooibos tea water extracts showed the stronger inhibitory effect on the growth of the various microbes. The data obtained in this study suggests that rooibos tea is not effective as an anti-microbial agent against all yeast species, but will strongly retard the growth of specific Gram-positive and Gram-negative bacteria. As long as rooibos tea is present, strong anti-microbial activity will be observed at a cup of tea concentration of 2.5 g.r1 soluble solids. These results may be of value to support the health claims associated with rooibos tea and may in the future lead to the use of rooibos tea as a "natural" food preservative.
AFRIKAANSE OPSOMMING:Aspalathus linearis is 'n inheemse fynbosplant wat gekultiveer word in die Clanwilliam area van die Wes Kaap, Suid-Afrika. Rooibostee, wat gemaak word van hierdie plante, het baie gewild geword wereldwyd a.g.v. die gesondheidsaspekte van hierdie tee. Studies toon dat groen, swart en oolong tee sterk anti-mikrobiese aktiwiteit het teen 'n wye reeks mikrobes. Aangesien daar voorheen geen studies gedoen is op die anti-mikrobiese aktiwiteit van rooibostee nie, was die doel van hierdie studie om die effek van rooibostee te bepaal op die groei van verskillende voedselbederwers en potensiele patogeniese mikrobes. Water- en etielasetaat-ekstrakte van gefermenteerde en ongefermenteerde rooibos tee is gebruik om die inhiberende effek op die groei van Escherichia coli te bepaal. Escherichia coli is gegroei in tee-MRS met bygevoegde gefermenteerde of ongefermenteerde rooibostee-ekstrakte. Seide die water- en etielasetaatekstrakte van rooibostee het 'n sterk inhiberende effek gewys teen E. coli en dit word gestaaf deur 'n afname in die finale bakteriese seldigtheid (Nmax)(vanaf 0.59 00 tot 0.25 00) en die maksimum spesifieke groeitempo (lJmax) (vanaf 1.12 h-1 tot 0.20 h-1) en 'n toename in die verdubbelingstyd (~) (vanaf 0.59 h tot 1.80 h) en die sloerfase (tlag)(vanaf 4.81 h tot 6.60 h) 5005 wat die konsentrasie van oplosbare vastestowwe van die tee toeneem van 0.5 tot 5.0 g.r1 . Verder is daar gevind dat die gefermenteerde rooibostee 'n baie sterker inhiberende effek het (69% afname in groei by 5.0 g.r1 oplosbare vastestowwe) in vergelyking met die ongefermenteerde rooibostee-ekstrakte (35.1% afname in groei by 5.0 g.r1 oplosbare vastestowwe). Die resultate van die data dui aan dat rooibos tee 'n baie sterk inhiberende effek het op die groei van die E. coli spesie. Die waterekstrakte van rooibostee het 'n sterker inhibisie getoon teen die groei van E. coli as die etielasetaat-ekstrakte, wat aandui dat die anti-mikrobiese aktiwiteit van rooibostee nie eksklusief toegeskryf kan word aan die polifenoliese samestelling nie. Daar is ook gevind dat rooibostee water-ekstrakte 'n bakteriostatiese effek het teen E. coli, want sodra die tee ekstrakte nie meer teenwoordig is in die groeimedium nie, hervat E. coli normale groei. Die data wys ook dat die inhiberende effek van rooibostee water-ekstrakte (69.0% afname in goei) teen E. coli baie sterker is as die van swart tee water-ekstrakte (25.7% afname in groei) by dieselfde konsentrasies.Rooibostee water-ekstrakte (0.5 - 5.0 g.r1) van gefermenteerde en ongefermenteerde rooibostee is ook gebruik om die inhiberende effek te bepaal teen ander voedselbederwers en potensiele patogene. Spesies van Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus mutans, Saccharomyces cerevisiae en Zygosaccharomyces rouxii is gegroei in die teenwoordigheid van gefermenteerde en ongefermenteerde rooibostee waterekstrakte. Die effek wat gefermenteerde rooibostee het op die groei van die getoetste mikrobes is 5005 volg: Staph. aureus (90.8% afname in groei) > L. monocytogenes (89.2% afname in groei) > Strep. mutans (84.1% afname in groei) > B. cereus (80.3% afname in groei) > Sacch. cerevisiae (77.7% afname in groei) > E. coli (69.0% afname in groei). Rooibostee het 'n duidelike inhiberende effek gehad teen al die organismes, behalwe teen Z. rouxii spes ie, waar die teenwoordigheid van rooibostee die groei van die organisme bevorder het. Die inhiberende effek van rooibostee teen die groei van hierdie mikrobes word ondersteun deur die groei parameters waar die Nmaxen IJmaxafgeneem het terwyl die ~ en tlagtoegeneem het 5005 wat die konsentrasie van die oplosbare vastestowwe toeneem. Die gefermenteerde rooibostee water-ekstrakte het ook 'n sterker inhiberende effek op die groei van die verskillende mikrobes net 5005 met E. coli. Die data wat verkry is van hierdie studie dui aan dat rooibostee nie effektief sal wees as 'n anti-mikrobiese middel teen aile gis spesies nie, maar dit sal die groei van spesifieke Gram-positiewe en Gram-negatiewe bakterie sterk vertraag. So lank as wat rooibostee teenwoordig is, sal sterk anti-mikrobiese aktiwiteit waargeneem word by 'n koppie-tee konsentrasie van 2.5 g.r1 oplosbare vastestowwe. Hierdie resultate kan help om die gesondheidseienskappe geassosieer met rooibostee te ondersteun en help om die gebruik van rooibostee as 'n "natuurlike" preserveermiddel te bevorder. dedicated to my parents

The multibillion dollar agricultural industry is an important part of the United States economy, and the management of factors that affect crop and human health is imperative to maintaining this economic sector. The fungi Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme are the causative agents of several plant diseases and can cause significant crop loss both before and after harvest in commodities such as strawberries, lettuce, citrus, and grains. Fungicides are employed to control these phytopathogens, but the use of these chemicals has led to an increase in fungicide resistance and may negatively affect the environment and human health. In addition to plant pathogens, foodborne pathogens also have a substantial impact on the agricultural industry. Foodborne disease outbreaks involving Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 not only cause considerable economic losses, but can also result in devastating health problems for consumers. The increase in fungicide resistance and number of produce-related foodborne disease outbreaks warrants investigation into additional methods of microbial control for use in the agricultural industry. Many bacterial species, including Lactic Acid Bacteria (LAB) and Bacillus species, produce antifungal and antimicrobial compounds, thus the use of biological control agents pre- and postharvest could augment current methods of pathogen management. The purpose of this study was to screen 22 bacterial isolates for inhibitory activity against the fungal phytopathogens Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme and the foodborne pathogens Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 in vitro, then evaluate antimicrobial efficacy of select isolates against the foodborne pathogens on fresh produce. To evaluate antifungal activity, the bacterial isolates were individually spot-inoculated onto Tryptic Soy Agar, Potato Dextrose Agar, or MRS agar, depending on isolate growth requirements and then a plug of fungal-colonized agar was placed onto the center of the isolate-inoculated plate. Plates were incubated at 24°C for 10 days; fungal growth was evaluated daily, beginning on Day 3. Nine of the 22 isolates screened inhibited all three fungi; inhibition by these isolates ranged from 51-62% for B. cinerea, 60-68% for F. pallidoroseum, and 40-61% for F. moniliforme. Isolates were also screened for biosurfactant activity using the drop-collapse test. Biosurfactant production was detected in seven of the nine isolates. Bacillus megaterium, Bacillus coagulans, Bacillus thuringiensis BT2 and three Bacillus amyloliquefaciens isolates demonstrated strong biosurfactant activity and suppression of all three fungi, and therefore are recommended for further study. Antimicrobial activity of the isolates was assessed using two methods: LAB isolates were screened using a seeded-overlay method and all other isolates were evaluated by spot inoculating the isolate on pathogen-seeded TSA. Three LAB isolates and six Bacillus isolates suppressed L. monocytogenes, Salmonella, and E. coli O157:H7 in vitro. Based on the results of the screening, three LAB isolates—Lactobacillus plantarum, Pediococcus acidilactici, and Pediococcus pentosaceus—were selected for further evaluation and use in challenge studies on fresh produce. The role of organic acids in pathogen inhibition was evaluated by incubating L. monocytogenes, Salmonella, and E. coli O157:H7 cultures in the cell-free supernatant (CFS; pH 3.81-4.27) or the neutralized cell-free supernatant (pH adjusted to 6.5 -7.0) of each isolate. When neutralized, the antimicrobial activity of the CFS of the three LAB isolates was greatly diminished, illustrating the role of lactic acid in the inhibition of pathogen growth. To assess antimicrobial efficacy on Iceberg lettuce, a cocktail of the three LAB isolates (7-8 log CFU/g) was sprayed onto lettuce spot-inoculated with L. monocytogenes (2-3 log CFU/g); lettuce was incubated at 10°C for 14 d. L. monocytogenes levels were 1.84 log lower on LAB-treated lettuce than on untreated lettuce at the end of incubation. Because the LAB cocktail suppressed the growth of L. monocytogenes on lettuce, testing on fresh produce continued using DF1, which was a powdered product comprised of the three LAB isolates and media components. Because DF1 caused substantial browning of Iceberg lettuce after 2 d, Gala apples were chosen to evaluate the antimicrobial activity of DF1 against L. monocytogenes, Salmonella, and E. coli O157:H7. The effect of DF1 on L. monocytogenes, Salmonella, and E. coli O157:H7 on Gala apples was determined by spraying a Gala apple spot-inoculated with pathogen (6-7 log CFU/plug) with approximately 3 mL of a 20% DF1 solution, then incubating at 20°C for 5 d. After 5 d incubation, L. monocytogenes, Salmonella, and E. coli O157:H7 levels on DF1-treated apples were approximately 4, 2, and 2 log higher than the control, respectively. Based on the results of these experiments, DF1 is not the optimal formulation for the biocontrol of foodborne pathogens on fresh produce. This study identified several bacterial isolates with potential for use in the biocontrol of plant and foodborne pathogens. Further investigation is required to assess possible use in the agricultural industry, including characterization of bioactive compounds, optimization of biocontrol product formulation, and evaluation of the commercial viability of the biocontrol product

Les plantes peuvent repondre a l'attaque par des agents pathogenes de type virus, bacterie ou champignon, en induisant une reaction locale hypersensible (rlh), un mecanisme de defense particulierement efficace. L'infiltration dans des feuilles de tabac d'un eliciteur glycoproteique d'origine fongique a permis de caracteriser fonctionnellement cette rlh. La zone traitee developpe une necrose de type hypersensible (zone hr). La zone etroite entourant la zone hr ne contient pas de traces d'eliciteur et est le siege de fortes reponses de defense : elle est appelee zone de resistance locale acquise (lar). La lar est regulee par un signal de plante libere par les cellules en etat de hr et diffusant a faible distance. Ce signal est distinct de l'acide salicylique (sa) et probablement de h#2o#2, deux molecules impliquees dans les processus de signalisation des defenses. Un des modes d'action propose du sa serait d'inhiber l'activite des catalases vegetales, des enzymes degradant h#2o#2. Grace a notre systeme simplifie, nous avons montre que l'activite catalase est en fait regulee via une repression rapide des genes correspondant et non pas via le sa. Le role de h#2o#2 dans la mise en place des defenses a ete examine suivant deux approches complementaires, l'une sur plante entiere et l'autre sur suspensions cellulaires. Les resultats sur cultures de cellules indiquent que, chez le tabac, h#2o#2 n'est ni necessaire ni suffisant pour l'etablissement de la mort hypersensible, pour la stimulation de l'activite phenylalanine ammoniac lyase (impliquee dans la biosynthese de la lignine, d'antibiotiques et du sa), pour l'accumulation de sa et pour la degradation de la scopoletine, une molecule aux proprietes antibiotiques. Les resultats sur plante entiere montrent que h#2o#2 ou d'autres formes reactives de l'oxygene semblent reguler de maniere negative, directement ou indirectement, le signal lar.

Orientador: Marcelo Brocchi
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Mestrado
Genetica de Microorganismos
Mestra em Genética e Biologia Molecular

In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and Vero CCL-81, Chlorocebus sp. kidney epithelial cells, which were utilized as a positive control for Golgi expression. N. fowleri and N. lovaniensis whole-cell lysates had indications of a 110 kDa reduced protein, associated with the predicted molecular weights of the beta-COPI subunit of the COPI cis-Golgi vesicular transport complex with further Western immunoblot indication of a weak band around 25 kDa corresponding to rabbit polyclonal antibodies specific for ARF1. Serial Dilutions of Wheat Germ Agglutinin Alexa Fluor 488TM were performed on Vero cells, Naegleria fowleri 30894, and N. gruberi 30540 with 1:100 dilution of recommended stock dilution of WGA 488 determined for utilization in sequential immunofluorescence. Sequential immunofluorescence with Wheat Germ Agglutinin Alexa Fluor 488TM and then blocked with 3% BSA:PBS [wt/vol] dilution with subsequent incubation in rabbit anti-beta-COPI primary 1:250, and 1:1000 of Alexa Fluor 594 goat anti-rabbit secondary antibody exposure showed strong indications of organized cis- and trans-punctate Golgi body markers in close association in individual and dividing cells of Naegleria fowleri and conserved Golgi expression in the positive control Vero cells, but further experiments are necessary to verify this finding with N. fowleri.

Philosophiae Doctor - PhD
In this dissertation three computational approaches are presented that enable optimization of reference-free transcriptome reconstruction. The first addresses the selection of bona fide reconstructed transcribed fragments (transfrags) from de novo transcriptome assemblies and annotation with a multiple domain co-occurrence framework. We showed that selected transfrags are functionally relevant and represented over 94% of the information derived from annotation by transference. The second approach relates to quality score based RNA-seq sub-sampling and the description of a novel sequence similarity-derived metric for quality assessment of de novo transcriptome assemblies. A detail systematic analysis of the side effects induced by quality score based trimming and or filtering on artefact removal and transcriptome quality is describe. Aggressive trimming produced incomplete reconstructed and missing transfrags. This approach was applied in generating an optimal transcriptome assembly for a South African isolate of V. inaequalis. The third approach deals with the computational partitioning of transfrags assembled from RNA-Seq of mixed host and pathogen reads. We used this strategy to correct a publicly available transcriptome assembly for V. inaequalis (Indian isolate). We binned 50% of the latter to Apple transfrags and identified putative immunity transcript models. Comparative transcriptomic analysis between fungi transfrags from the Indian and South African isolates reveal effectors or transcripts that may be expressed in planta upon morphogenic differentiation. These studies have successfully identified V. inaequalis specific transfrags that can facilitate gene discovery. The unique access to an in-house draft genome assembly allowed us to provide preliminary description of genes that are implicated in pathogenesis. Gene prediction with bona fide transfrags produced 11,692 protein-coding genes. We identified two hydrophobin-like genes and six accessory genes of the melanin biosynthetic pathway that are implicated in the invasive action of the appressorium. The cazyome reveals an impressive repertoire of carbohydrate degrading enzymes and carbohydrate-binding modules amongst which are six polysaccharide lyases, and the largest number of carbohydrate esterases (twenty-eight) known in any fungus sequenced to date

Orientador: Arnaldo Yoshiteru Kuaye
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: As moscas são consideradas responsáveis pela transmissão de doenças de origem alimentar ao atuarem como vetores mecânicos de microrganismos patogênicos, como Shigella sp, Salmonella Enteriditis, Escherichia coli O157:H7, Campylobacter jejuni desde o reservatório até os alimentos. Os produtos lácteos, principalmente os produzidos artesanalmente quase sempre em condições inadequadas de higiene, são os mais susceptíveis a estes tipos de vetores, representando sérios riscos à saúde do consumidor. A contagem total de bactérias aeróbias mesofílicas e de coliformes totais e fecais, a presença de microrganismos patogênicos em população de moscas domésticas e o potencial de transmissão dos mesmos para queijos Minas frescal foram estudados neste trabalho. Os resultados confirmaram a presença de Musca domestica em todos os ambientes de fabricação de queijo Minas frescal aqui estudados. As análises microbiológicas das amostras de moscas domésticas, matéria-prima (leite) e produto final (queijo Minas frescal), coletadas em local de fabricação de queijo Minas frescal artesanal, demonstraram uma correlação entre os microrganismos patogênicos presentes no ¿pool¿ de moscas, no leite e no queijo. As análises microscópicas das amostras de leite e queijo artesanal apresentaram matérias prejudiciais à saúde humana como mosca inteira, fragmentos de mosca e pêlos de roedor, indicando condições higiênico-sanitárias precárias nas etapas de obtenção do leite e processamento dos queijos. Foi verificado também o potencial de transmissão dos microrganismos patogênicos (Salmonella spp., Staphylococcus aureus, Listeria monocytogenes e Escherichia coli) das moscas em amostras comerciais de queijo Minas frescal ultrafiltrado. Através do processamento do queijo Minas frescal em planta piloto, pode-se verificar tanto a efetiva eliminação das matérias estranhas pela utilização da etapa de filtração do leite antes da fase de pasteurização, quanto o possível carreamento de microrganismos indesejáveis através do contato com homogeneizado de moscas. A capacidade de transmissão de microrganismos patogênicos de habitat contaminado artificialmente, via Musca domestica, para queijo Minas frescal pode ser verificado, comprovando assim o potencial de risco da presença de moscas domésticas em ambientes de fabricação de produtos lácteos/queijo
Abstract: The flies are considered responsible for the transmission of foodborne diseases origin when acting as mechanical vectors of pathogenic microrganisms, such as Shigella sp., Salmonella Enteriditis, Escherichia coli O157: H7, Campylobacter jejuni from the reservoir to food. The artisan dairy products are produced almost always in a non totally adequate conditions of hygiene. Therefore, the handmade dairy products are the most suitable to these types of vectors, representing serious risks to the consumer¿s health. The total counting of mesophilic microrganisms and total and fecal coliforms, the presence of pathogenic microrganisms in relation to the population of houseflies and the potential of transmission of the same for ¿Minas frescal¿ cheese were studied in this work. The results confirmed the presence of Musca domestica in all environments of manufacturing of ¿Minas frescal¿ cheese studied here. The microbiological analyses of the samples of houseflies, raw material (milk) and last item (¿Minas frescal¿ cheese), collected in places of manufacturing of handmade ¿Minas frescal¿ cheese, demonstrated the direct correlation between pathogenic microrganisms present in ¿pool¿ of flies, in milk and cheese. The microscopical analyses of the milk and handmade cheese, that characterized themselves for presenting harmful material to human health such as the whole fly, fly fragments and rodent hairs, indicating precarious hygienical-sanitary conditions in the stages of the attainment of milk and the processing of cheese. It was verified that there is a real potential of transmission of the pathogenic microrganisms (Salmonella spp., Staphylococcus aureus, Listeria monocytogenes and Escherichia coli) from the flies to a commercial sample of ¿Minas frescal¿ cheese ultrafiltered. Through the processing of the ¿Minas frescal¿ cheese in pilot laboratory, it could be verified the effective elimination of extraneous materials by the use of the stage of filtration of milk before the pasteurization phase, although there is a possible undesirable carrying of microrganisms through the homogeneized contact with flies. The capacity of transmission of pathogenic microrganisms of habitat contaminated artificially, through Musca domestica, to ¿Minas frescal¿ cheese can be verified, thus proving the risk potential of the presence of houseflies in environments of manufacturing of dairy products/cheese
Mestrado
Mestre em Tecnologia de Alimentos

Orientadores: José Roberto Guimarães, Regina Maura Bueno Franco
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo
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Resumo: Neste estudo foi avaliada a eficiência do processo de tratamento por lodos ativados e, a eficiência de um processo oxidativo avançado (POA), a peroxidação assistida por radiação ultravioleta (H2O2/UV) na desinfecção do efluente da estação de esgoto (ETE) Samambaia da cidade de Campinas/SP. A avaliação da eficiência da ETE e dos processos foi feita com base na remoção de coliformes totais (CT), Escherichia coli (EC), Clostridium perfringens (CP), cistos de Giardia spp. oocistos de Cryptosporidium spp., ovos e larvas de helmintos e, na redução dos valores de turbidez, cor (aparente e verdadeira) e carbono orgânico dissolvido (COD). Para tanto, 13 amostras de afluente e 18 amostras de efluente foram coletadas. O efluente foi desinfetado utilizando-se doses UV de 125 a 20.700 mW s cm-2 e concentração de peróxido de hidrogênio de 30 a 93 mg L-1. Os processos de fotólise e peroxidação foram também avaliados de forma isolada. Todas as amostras de afluente e efluente apresentaram as bactérias avaliadas, cistos de Giardia spp. e, larvas e/ou ovos de helmintos. Na avaliação das características temporais do afluente da ETE, verificou-se que alguns dos parâmetros avaliados, tiveram considerável aumento ou redução com relação ao observado em outros períodos. No período de maior incidência de chuvas (primavera, no ano de 2012) observou-se o menor valor para COD (43% menor do que os obtidos para as outras épocas do ano) e, maior valor para a turbidez (aumento de 57%). Na primavera e no inverno detectou-se também oocistos de Cryptosporidium spp., com maior concentração no inverno. Ovos de helmintos tiveram sua quantificação mínima detectada no outono. A peroxidação aplicada isoladamente não promoveu a inativação de nenhum dos organismos avaliados e, nem reduziu os valores das variáveis físicas e químicas. O POA promoveu a redução de mais de 80% do valor de cor aparente e verdadeira (p < 0,05). Os processos foto-assistidos (radiação UV e H2O2/UV) alcançaram a maior inativação de CT e EC com a dose de radiação de 177 mW s cm-2, sendo de 5 log para a fotólise e, o limite <1 NMP/100mL para o POA e, promoveram a inativação de 2 log de CP, independente da dose de radiação UV aplicada. Quanto aos cistos de Giardia spp., os processos avaliados não promoveram a sua redução. Porém o processo de UV e POA gerou alterações nos cistos (p < 0,05) sendo que o POA alcançou alterações (de fluorescência e/ou forma) em 100% dos cistos observados quando utilizada a dose de radiação UV de 1.775 mW s cm-2. Nenhum dos processos reduziu ou danificou ovos de helmintos, independentemente da condição experimental avaliada
Abstract: This study evaluates the effectiveness of activated sludge and the performance of advanced oxidation process (AOP), the peroxidation assisted by ultraviolet radiation (H2O2/UV), used for effluent disinfection at Samambaia Wastewater Treatment Plant (Samambaia WWTP) in Campinas/SP. The evaluation of the WWTP and of the disinfection processes¿ efficiency was based on the inactivation and/or the number of organisms (total coliforms (TC), Escherichia coli (EC), Clostridium perfringens (CP), Giardia spp. cysts, Cryptosporidium spp. oocysts, helminth eggs and larvae) and turbidity, color (apparent and true) and dissolved organic carbon (DOC) reduced. For this evaluation, 13 samples of affluent and 18 of effluent were collected. The effluent was disinfected using UV doses 125 to 20,700 mW s cm-2 and hydrogen peroxide concentration of 30 to 93 mg L-1. The photolysis and peroxidation processes were separately evaluated. Each affluent and effluent sample revealed the bacteria, Giardia spp. cysts and helminth eggs and/or larvae. In the evaluation of affluent seasonality it has been found that some parameters considerably increased or reduced when different periods were compared. In the period with higher incidence of rain (spring of 2012), it was observed that the DCO value was lower (43% lower when compared to other periods) and, turbidity value was higher (an increase of 57%). In the spring and winter Cryptosporidium spp. oocysts were detected with higher concentration in the winter. Helminth eggs had lower quantification during the autumn. The isolated peroxidation process did not promote the inactivation and/or reduction of any evaluated organism, nor reduced the value of any physical or chemical parameter. The AOP promoted the reduction of 80% or higher of apparent and true color (p < 0.05). The photo-assisted processes (UV radiation and AOP) achieved greater inactivation of TC and EC bacteria with UV dose of 177 mW s cm-2, being 5 log of UV radiation and, <1 MPN/100mL of AOP and, promoted the CP inactivation of 2 log, independent on the UV dose applied. The evaluated processes did not promote the Giardia spp. cysts reduction. However UV and AOP processes caused cysts alteration (p < 0.05), as AOP reached 100% cysts alteration when a 1,775 mW s cm-2 UV dose was applied. Helminth eggs were not reduced or damaged for any studied process, independent on the evaluated experimental condition
Doutorado
Saneamento e Ambiente
Doutora em Engenharia Civil

The primary objective of this dissertation is to utilise, adapt and extend current stochastic models and statistical inference techniques to describe the transmission of nosocomial pathogens, i.e. hospital-acquired pathogens, and multiply-resistant organisms within the hospital setting. The emergence of higher levels of antibiotic resistance is threatening the long term viability of current treatment options and placing greater emphasis on the use of infection control procedures. The relative importance and value of various infection control practices is often debated and there is a lack of quantitative evidence concerning their effectiveness. The methods developed in this dissertation are applied to data of methicillin-resistant Staphylococcus aureus occurrence in intensive care units to quantify the effectiveness of infection control procedures. Analysis of infectious disease or carriage data is complicated by dependencies within the data and partial observation of the transmission process. Dependencies within the data are inherent because the risk of colonisation depends on the number of other colonised individuals. The colonisation times, chain and duration are often not visible to the human eye making only partial observation of the transmission process possible. Within a hospital setting, routine surveillance monitoring permits knowledge of interval-censored colonisation times. However, consideration needs to be given to the possibility of false negative outcomes when relying on observations from routine surveillance monitoring. SI (Susceptible, Infected) models are commonly used to describe community epidemic processes and allow for any inherent dependencies. Statistical inference techniques, such as the expectation-maximisation (EM) algorithm and Markov chain Monte Carlo (MCMC) can be used to estimate the model parameters when only partial observation of the epidemic process is possible. These methods appear well suited for the analysis of hospital infectious disease data but need to be adapted for short patient stays through migration. This thesis focuses on the use of Bayesian statistics to explore the posterior distributions of the unknown parameters. MCMC techniques are introduced to overcome analytical intractability caused by partial observation of the epidemic process. Statistical issues such as model adequacy and MCMC convergence assessment are discussed throughout the thesis. The new methodology allows the quantification of the relative importance of different transmission routes and the benefits of hospital practices, in terms of changed transmission rates. Evidence-based decisions can therefore be made on the impact of infection control procedures which is otherwise difficult on the basis of clinical studies alone. The methods are applied to data describing the occurrence of methicillin-resistant Staphylococcus aureus within intensive care units in hospitals in Brisbane and London

The primary objective of this dissertation is to utilise, adapt and extend current stochastic models and statistical inference techniques to describe the transmission of nosocomial pathogens, i.e. hospital-acquired pathogens, and multiply-resistant organisms within the hospital setting. The emergence of higher levels of antibiotic resistance is threatening the long term viability of current treatment options and placing greater emphasis on the use of infection control procedures. The relative importance and value of various infection control practices is often debated and there is a lack of quantitative evidence concerning their effectiveness. The methods developed in this dissertation are applied to data of methicillin-resistant Staphylococcus aureus occurrence in intensive care units to quantify the effectiveness of infection control procedures. Analysis of infectious disease or carriage data is complicated by dependencies within the data and partial observation of the transmission process. Dependencies within the data are inherent because the risk of colonisation depends on the number of other colonised individuals. The colonisation times, chain and duration are often not visible to the human eye making only partial observation of the transmission process possible. Within a hospital setting, routine surveillance monitoring permits knowledge of interval-censored colonisation times. However, consideration needs to be given to the possibility of false negative outcomes when relying on observations from routine surveillance monitoring. SI (Susceptible, Infected) models are commonly used to describe community epidemic processes and allow for any inherent dependencies. Statistical inference techniques, such as the expectation-maximisation (EM) algorithm and Markov chain Monte Carlo (MCMC) can be used to estimate the model parameters when only partial observation of the epidemic process is possible. These methods appear well suited for the analysis of hospital infectious disease data but need to be adapted for short patient stays through migration. This thesis focuses on the use of Bayesian statistics to explore the posterior distributions of the unknown parameters. MCMC techniques are introduced to overcome analytical intractability caused by partial observation of the epidemic process. Statistical issues such as model adequacy and MCMC convergence assessment are discussed throughout the thesis. The new methodology allows the quantification of the relative importance of different transmission routes and the benefits of hospital practices, in terms of changed transmission rates. Evidence-based decisions can therefore be made on the impact of infection control procedures which is otherwise difficult on the basis of clinical studies alone. The methods are applied to data describing the occurrence of methicillin-resistant Staphylococcus aureus within intensive care units in hospitals in Brisbane and London

Lettuce is one of the most commonly consumed by Brazilian hardwoods, and has therefore been sought production alternatives that reduce their impact on ecosystems. The objective of this study was to evaluate the productivity and occurrence of pathogens in lettuce cv. Isabela grown under different levels of organic compounds and biofertilizers. The work was conducted at the Experimental Farm Green living, located in the Itabaiana-SE in Acrisol. The experiment was conducted under fiel conditions in randomized complete block randomized in a factorial 4 x 6, ie, four levels of organic compound compost (0, 3, 6 and 9 kg/m2) and six levels of biofertilizer (0, 2, 4, 6, 8 and 10 L/m2), with three replications. Significant effects at doses of organic compost and biofertilizer on variables for interaction among gead dameter, fresh weight of edible leaves, fresh weight. The organic manure incorporated into the soil increases the diameter of the head of lettuce, fresh mass, stem fresh weight, fresh weight of edible leaves, necrotic leaf fresh weight, fresh shoot and total fresh weight. Treatments 6 and 9 kg/m2 of organic compound are better treatments for the development of the lettuce and the biofertilizer reduces the incidence of galls on the roots of cv. Isabela.
A alface é uma das folhosas mais consumidas pelos brasileiros e por isto tem-se buscado alternativas de produção que reduzam o seu impacto nos agroecossistemas. O objetivo deste trabalho foi avaliar a produtividade e ocorrência de organismos patogênicos na alface cv. Isabela cultivada sob diferentes níveis de compostos orgânico e de biofertilizante. O trabalho foi realizado na área experimental da Fazenda Vida Verde, localizada no município de Itabaiana/SE, em Argissolo Vermelho-Amarelo. O experimento foi conduzido em condições de campo no delineamento experimental em bloco casualizado em esquema fatorial 4 x 6, ou seja, quatro níveis de composto orgânico (0, 3, 6 e 9 kg/m2) e seis níveis de biofertilizante (0, 2, 4, 6, 8 e 10 L/m2), com três repetições. Houve efeito significativo para doses de composto orgânico e de biofertilizante em todas as variáveis estudadas para interação entre os fatores diâmetro de cabeça, massa fresca de folhas comestíveis, massa fresca parte aérea e massa fresca total. A adubação orgânica incorporada ao solo aumenta o diâmetro da cabeça da alface, massa fresca radicular, massa fresca do caule, massa fresca de folhas comestíveis, massa fresca folha necrótica, massa fresca parte aérea e massa fresca total. Os tratamentos 6 e 9 kg/ m2 de composto orgânico foram os melhores tratamentos para o desenvolvimento da alface e, o biofertilizante, reduz a incidência de galhas nas raízes da alface cv. Isabela.

Capes
Uma superfície mal higienizada em um ambiente produtivo, somada à capacidade de adesão de um microrganismo, pode se tornar uma fonte potencial de contaminação e levar à formação de biofilmes. Estes, uma vez formados, são de difícil remoção e podem proliferar para a contaminação de alimentos. A preocupação com a segurança dos alimentos é um desafio, visto que problemas a ela relacionados podem comprometer a saúde do consumidor. O objetivo deste trabalho foi isolar microrganismos patogênicos potenciais produtores de biofilme microbiano presentes no processamento industrial de um frigorífico bovino. O desenvolvimento do trabalho se resume em três fases: entrevista com o coordenador de qualidade de um frigorífico da região dos Campos Gerais; diagnóstico de pontos críticos no controle de qualidade do processamento industrial desse frigorífico, por meio de um diagrama decisório e coleta de amostras durante o processo industrial através de swabs, utilizados no isolamento por microbiologia. Em seguida, foi identificado o perfil genético das amostras, por meio do isolamento de DNA, seguida de amplificação por Reação em Cadeia da Polimerase com os primers universais rD1 e fD1. Os dados gerados na primeira fase indicam os programas de controle de qualidade aplicados na indústria frigorífica em estudo. A entrevistada, responsável pelo controle de qualidade da indústria, salientou o uso de Boas Práticas de Fabricação (BPF), Análise de Perigo e Pontos Críticos de Controle (APPCC), Procedimento Padrão de Higiene Operacional (PPHO), Monitoramento de Pragas (MIP) e Folha de Verificação (FV). A partir do diagrama decisório, foram identificados 25 pontos para a coleta de amostras para a identificação de microrganismos patogênicos. Dentre esses, dez pontos amostrais foram isolados por microbiologia convencional com meio de cultura EMB, indicando contaminação de conteúdo gastrointestinal por coliformes fecais. Em dez pontos, nem sempre distintos, houve crescimento em meio de cultura SS – Salmonella Shigella, indicando contaminação durante o abate a partir da manipulação da carne pelos funcionários, uma vez que esses podem ser portadores sadios de microrganismos patogênicos. Para identificação genotípica das amostras sequenciadas, os resultados chegaram a nível de gênero, sendo Escherichia, Proteus, Hafnia e Bacillus, todos pertencentes ao grupo de Enterobactérias, com exceção de Bacillus. Verificou-se através da identificação genotípica, relacionada com os locais de coleta das amostras no fluxograma, que há contaminação cruzada no ambiente produtivo do presente frigorífico, na maioria dos pontos, relacionadas com o manipulador.
An unhygienic surface in a productive environment, added to the adhesion capacity of a microorganism, can become a potential source of contamination and lead to the formation of biofilms. These, once formed, are difficult to remove and can proliferate for food contamination. Concern about food safety is a challenge, as related problems can compromise consumer health. The objective of this work is to select potential pathogenic microorganisms producing microbial biofilms present in the industrial processing of a beef cattle. The development of the work is summarized in three phases: interview with the quality coordinator of a refrigerator in the Campos Gerais region; diagnosis of critical points in the quality control of the industrial processing of this refrigerator, through a decision diagram and sample collection during the industrial process through swabs, used in the isolation by microbiology. Then, the genetic profile of the samples was identified through DNA isolation, followed by amplification by Polymerase Chain Reaction with the universal primers rD1 and fD1. The data generated in the first phase indicated the quality control programs in the refrigeration industry under study. The interviewee, responsible for the quality control of the industry, emphasized the use of Good Manufacturing Practices (GMP), Hazard Analysis and Critical Control Points (HACCP), Standard Operating Procedures (PPHO), Pest Monitoring (IPM) And Verification Sheet (FV). From the decision diagram, 25 points were identified for the collection of samples for the identification of pathogenic microorganisms. Among these, ten sample points were isolated by conventional microbiology with EMB culture, indicating contamination of gastrointestinal contents by fecal coliforms. At ten points, not always distinct, there was growth in the SS - Salmonella Shigella culture, indicating contamination during slaughter from the handling of the meat by the employees, since they may be healthy carriers of pathogenic microorganisms. For genotypic identification of the sequenced samples, the results reached the species level, being Escherichia, Proteus, Hafnia and Bacillus, all belonging to the group of Enterobacteria, except for Bacillus. It was verified through the genotypic identification, related to the sample collection sites in the flowchart, that there is cross contamination in the productive environment of the present refrigerator, in most of the points, related to the manipulator.

Os antibióticos têm sido utilizados como aditivos na alimentação animal para aumentar o desempenho e manter a saúde dos animais, como suínos e frangos, destinadas à produção de alimentos de origem animal. No entanto, desde 2006, a Comunidade Europeia proibiu o uso de antibióticos para esse proposito devido ao desenvolvimento de resistência bacteriana aos antibióticos. Como resultado, várias alternativas foram estudadas e propostas para substituir os antibióticos utilizados na alimentação animal. Os óleos essenciais têm recebido considerável atenção devido às suas propriedades antimicrobianas. Portanto, o objetivo do presente trabalho foi avaliar in vitro a atividade antibacteriana dos óleos essenciais contra a microbiota patogênica e probiótica de ocorrência no trato gastrointestinal de suínos e aves, destinadas à produção de alimentos de origem animal. A atividade antibacteriana seletiva, a qual, significou uma alta atividade antibacteriana contra bactérias patogênicas e reduzida ou nenhuma atividade sobre bactérias probióticas, foi avaliada como característica fundamental dos óleos essenciais com alto desempenho. Esta característica foi avaliada nos óleos essenciais usados individualmente e em combinações binarias. Inicialmente, no Capítulo 2, uma triagem de vinte e oito óleos essenciais (OEs) através do método de difusão em disco mostrou que Eucalyptusglobulus, E. exserta, Pimenta pseudocaryophylllus, Orange Oil Phase Essence, e CitrusTerpens (Os dois últimos OEs foram subprodutos do processamento da laranja para obtenção de suco) tiveram uma atividade antibacteriana seletiva sobre a bactéria patogênica Salmonella Enteritidis e a bactéria probiótica Lactobacillus plantarum. Numa fase posterior, esses cinco óleos foram avaliados individualmente e em misturas binárias, contra cinco bactérias patogênicas e três bactérias probióticas. Os melhores resultados foram observados quando os OEs foram avaliados isoladamente e não em misturas. Assim, Orange Oil Phase Essence e Citrus Terpens destacaram-se por ter a melhor atividade antibacteriana seletiva contra essas bactérias. No Capítulo 3, uma análise mais detalhada da atividade antibacteriana dos óleos essenciais foi realizada utilizando Orange Oil Phase Essence e a mistura composta pelos óleos de E. globulus e P. pseudocaryophyllus. Estes dois óleos foram selecionados com base nos resultados do Capitulo 2 e da disponibilidade de OEs em nosso estoque. Ambos, óleo e a mistura foram avaliados sobre a bactéria patogênica mais resistente, E. faecalis, e a bactéria probiótica menos resistente L. rhamnosus, como observado no Capitulo 2. A avaliação da Concentração Inibitória Mínima e Concentração Bactericida Mínima do Orange Oil Phase Essence e da mistura mostrou que não tiveram um efeito antibacteriano seletivo sobre E. faecalis e L. rhamnosus. Finalmente, no Capitulo 4, foi avaliada a atividade antibacteriana individual e combinada dos óleos de E. globulus e P. pseudocaryophyllus sobre E. faecalis e L. rhmanosus. Os resultados mostraram que a combinação destes dois OEs, avaliadas pelo método checkerboard, não potencializou a atividade antibacteriana seletiva dos dois OEs. Portanto, observou-se que o óleo de E. globulus isoladamente apresentou a melhor atividade antibacteriana seletiva contra E. faecalis e L. rhamnosus. Em conclusão, este trabalho permitiu identificar óleos essenciais com perfil antibacteriano seletivo para eles serem possíveis alternativas botânicas aos antibióticos utilizados na alimentação animal.
Antibiotics have been used in animal feed to maintain health and increase performance, as in the case of pigs and poultry intended for food production of animal origin. However, since 2006 the European Community has banned the use of antibiotics for this purpose due to the emergence and increase of antibiotic-resistant bacteria. As a result, several alternatives have been studied and proposed to substitute antibiotics used in animal feed. Essential oils have received considerable attention due to their antimicrobial properties. Therefore, the objective of this work was to evaluate in vitro the antibacterial activity of essential oils against pathogenic and probiotic bacteria that occur in the gastrointestinal tract of swine and poultry, intended for food production of animal origin. The selective antibacterial activity, which means high antibacterial activity on pathogenic bacteria and reduced or no activity on probiotic bacteria, was evaluated as a fundamental feature of the highest-performance essential oils. This feature was evaluated in essential oils used individually and in binary combinations. Initially, in Chapter 2, the screening of twenty-eight essential oils (EOs) by disk diffusion method showed that Eucalyptus globulus, E. exserta, Pimenta pseudocaryophylllus, Orange Oil Phase Essence, and Citrus Terpens (the last two EOs were by-products of orange juice production) had a selective antibacterial activity against the pathogenic Salmonella Enteritidis and probiotic Lactobacillus plantarum. At a later stage those five oils were evaluated, individually and in binary blends, against five pathogenic bacteria and three probiotic bacteria. Better results were observed when the EOs were checked alone and not in blends. Orange Oil Phase Essence and Citrus Terpens stood out for having the two best selective antibacterial activities against those bacteria. In Chapter 3, a more detailed analysis of essential oil antibacterial activities was perfomed using Orange Oil Phase Essence and the blend composed of E. globulus and P. pseudocaryophyllus. These two oils were selected based on the results of Chapter 2 and from the availability of our EO stock. Both oil and blend were checked on the most resistant pathogenic bacterium, E. faecalis, and on the less resistant probiotic bacterium of the Lactobacillus genus, L. rhamnosus, as observed in Chapter 2. The evaluation of Minimal Inhibitory Concentration and Minimal Bactericidal Concentration for Orange Oil Phase Essence and the blend showed that there was not a selective antibacterial effect against E. faecalis and L. rhamnosus. Finally, in Chapter 4, the individual and combined antibacterial activities of E. globulus and P. pseudocaryophyllus essential oils on E. faecalis and L. rhmanosus were evaluated. The results showed that the combination of two EOs evaluated by checkerboard method did not potentiate the selective antibacterial activity of the two EOs. Therefore, it was observed that the E. globulus essential oil alone had the best selective antibacterial activity against E. faecalis and L. rhamnosus. In conclusion, this work enabled the identification of essential oils with selective antibacterial profile that can become possible botanical alternatives to antibiotics used in animal feed.

Mycobacterium tuberculosis (Mtb) is one of the most virulent and prevalent bacterial pathogens across the world. As Mtb infects millions of people a year, it remains essential to study its physiology with the goal of developing new therapeutic interventions. A critical part of the bacteria’s ability to propagate is through successful cell division. Although the process of bacterial cell division and the key proteins therein are well understood in Escherichia coli, much remains to be understood about division in mycobacteria. Genetic and cell biological approaches have recently begun to identify key divisome components in Mycobacterium smegmatis. However, questions remain regarding the role and function of one divisome protein in particular, the DNA translocase FtsK. In this dissertation, I investigated the necessity of FtsK for the growth of mycobacteria. Using an inducible knockdown of FtsK, I present evidence that complete loss of FtsK is required to inhibit growth in both Mtb and M. smegmatis, and that these orthologs share a homologous function. Additional work suggests extended loss of FtsK may be lethal to bacteria. These observations support that FtsK is an essential member of the divisome in mycobacteria, facilitating the processes of growth and division.

Globally, investigations into food-borne illnesses show that the majority of cases involve poor hand hygiene of the food handler. The challenge of providing safe food therefore requires new strategies for evaluating cross-contamination of pathogenic micro-organisms on the food handler's hands, which might be detrimental or hazardous to the health of the patient Although food-borne diseases may be multifactorial in aetiology, no standards or evaluation systems, such as an occupational health surveillance programme, are available to monitor and ensure that food is free of pathogens. The formulation and implementation of standards may contribute to ensuring that food handlers comply with hand hygiene practices during food handling. Such practices guarantee that food reaching the patient is safe. The objectives in this research project originated from the occupational health practice and gave direction of the empirical research project. The literature was reviewed to discover what is currently known concerning the food handlers' hand hygiene during food handling and food-borne illnesses and the theoretical framework gave direction and guidance to the survey design of the empirical research, which was quantitative, explorative, descriptive and contextual in nature. The food handlers from the food preparation sections of the four major healthcare services in Potchefstroom, in the North West Province, South Africa, were the target population and the sampling method was all-inclusive (n=110). Eighty (75.47%) food handlers participated in the research project. The design entailed three steps. The first was conducted with a questionnaire, to identify the food handlers' compliance with hand hygiene during food handling. The second step involved determining the prevalence of Escherichia coli and Staphylococcus aureus on the food handlers' hands. The results were used for the formulation of standards for the hand hygiene of food handlers. Finally, recommendations for practice, education and research were made. The implementation of these recommendations could contribute knowledge to the body of nursing and promote good hand hygiene practices in the healthcare service.
Thesis (M.Cur.)--North-West University, Potchefstroom Campus, 2009.

Genetically modified crops are submitted to strict regulation to ensure the safety of consumers and the environment. To complement the comparison between GM plants and their counterparts, in the present Thesis, we evaluated the possible unexpected effects of the transgene on the host plant, by means of transcriptomic technologies. More exactly, we studied three pathogen-resistant GM rice lines: S-afp, expressing constitutively the antifungal protein AFP; and S-bp217 and S-bp213, expressing undecapeptide BP100 derivatives, which were developed in the UdG in the context of this Thesis. Although the high phytotoxicity of the BP100 derivatives on the host plant the transcriptional changes observed in S-afp, S-bp217 and S-bp213 compared to the conventional line Senia were similar that those observed in other GM crops, of other species and with different transgenes, and only the half of them was attributed to the insertion and/or expression of the transgene.
Les plantes modificades genèticament (MG) destinades a comercialització estan sotmeses a estricta legislació per garantir la seguretat del consumidor i del medi ambient. Per complementar la comparativa entre plantes MG i convencionals, en aquesta tesi s’ha abordat l’avaluació dels possibles efectes no esperats del transgèn sobre la planta hoste, mitjançant tècniques de transcriptòmica. Concretament s’han estudiat línies d'arròs MG que presenten fenotips de resistència a patògens: S-afp, que expressa constitutivament la proteïna antifúngica AFP, i S-bp213 i S-bp217, que expressen derivats de l’undecapèptid BP100, desenvolupat a la UdG, que s’han obtingut també en el marc d’aquesta tesi. Malgrat l’elevada fitotoxicitat dels derivats de BP100 enfront la planta hoste, els canvis transcripcionals de S-afp, S-bp213 i S-bp217 respecte la línia convencional Senia són similars als observats en altres events MG, de diferents espècies i amb diferents transgens; i només la meitat d’ells s’ha atribuit a la presència o expressió del transgèn.

Greywater is domestic household wastewater without inputfrom the toilet, i.e. wastewater from sinks, the shower,washing machine and dishwasher in a home. Source separation ofgreywater can be a strategy to enhance recirculation of plantnutrients and/or improve water use. The risk for transmissionof disease when reusing greywater is largely dependent on thecross-contamination by faeces. High levels of faecalindicators, mainly thermotolerant coliform bacteria, have beenreported in greywater, indicating substantial faecal pollution.However, growth of indicator bacteria within the system leadsto an overestimation of thefaecal input and thus the hygienerisk. The faecal input of the greywater in Vibyåsen,Sollentuna, North of Stockholm, was estimated to be 0.04 ±0.02 g faeces person-1 day-1 from the quantification of thefaecal sterol coprostanol, compared to 65 g, 5.2 g and 0.22 gp-1 d-1 using E. coli, enterococci and cholesterolrespectively.

Prevalence of pathogens in the population and the faecalload based on coprostanol concentrations were used to form thebasis of a screening-level quantitative microbial riskassessment (QMRA) that was undertaken for rotavirus, Salmonellatyphimurium, Campylobacter jejuni, Giardia intestinalis andCryptosporidium parvum, looking at the treatment required to bebelow an acceptable level of risk (10-3) for reuse or dischargeof the greywater. The different exposure scenarios simulated–groundwater recharge, direct contact, irrigation andrecreational water–showed that a reduction of 0.7–3.7 log was needed for rotavirus, with the measured level offaecal load in Vibyåsen. The other pathogen of concern wasCampylobacter, where a 2.2 log reduction was needed forgroundwater recharge. The infectious dose of Salmonella is highand the excretion numbers of Giardia cysts and Cryptosporidiumoocysts low, resulting in no treatment requirements for theseorganisms under these circumstances. Pathogen input fromcontaminated food via the kitchen sink had a minor effect onthe microbiological quality of the greywater. Studies on virusoccurrence in greywater as well as validation of the faecalload of greywater at another site would give valuable input forfuture QMRAs.

Greywater treatment efficiency studies, especially on virusremoval, are scarce and more investigations are warranted.Active sludge may not be a suitable technique for greywater dueto the low carbon content in this flow. Chemical precipitationhas the advantage of removing phosphorus as well as virusesefficiently and it is suggested as one possible method fortreating greywater. Otherwise the most common practice forgreywater treatment in Sweden is soil infiltration. However, itis suggested that the recommendations for wastewaterinfiltration also be observed for greywater, despite the lowfaecal load, due to the simulated results on virus reductionneeded.

Key words:greywater, greywater reuse, greywatertreatment, microbial risk assessment, groundwater recharge,irrigation, recreational water, faecal contamination, indicatorbacteria, index organisms, faecal sterols, bacteriophages,enteric pathogens, rotavirus, Salmonella, Campylobacter,Giardia, Cryptosporidium, Legionella

Orientador: Jose Luiz Pereira
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Abstract: The abstract is available with the full electronic document
Doutorado
Doutor em Ciência de Alimentos

La détection et l’identification de micro-organismes pathogènes est un réel enjeu de santé public tant au niveau alimentaire, que clinique ou d’intérêt national tel qu’illustré en biodéfense. Dans tous ces domaines, il est important d‘avoir des méthodes d’identification et de détection qui soient à la fois rapide, sensible et robuste. Cette thèse a pour objectif de contribuer au développement d’une approche rapide d’identification de micro-organismes sans a priori par spectrométrie de masse en tandem. Cette approche innovante, appelée phylopeptidomique, repose sur l’alliance de la peptidomique, i.e. analyse à large échelle des peptides provenant de la digestion enzymatique d’un échantillon biologique, et de la phylogénie des organismes cellulaires. Après extraction des protéines présentes dans l’échantillon à ausculter, des peptides sont générés et analysés par spectrométrie de masse en tandem. La déconvolution des signaux MS/MS à l’aide du logiciel « µOrg.ID » développé en propre au laboratoire permet d’identifier et quantifier les organismes présents dans l’échantillon en fonction des organismes indexés dans les bases de données. L’étude du protéome de Bacillus atrophaeus, agent simulant de l’anthrax, sous forme sporulée et végétative a permis d’illustrer une méthode d’identification de biomarqueurs protéiques permettant de déterminer le ratio entre les deux formes dans un échantillon. La limite de détection de la phylopeptidomique dans des échantillons purs et des échantillons en mélange équimolaire a été établie sur des bactéries modèles d’intérêts médical et environnemental. La limite de détection de spores de Bacillus atrophaeus en présence de 14 matrices interférentes (alimentaires, environnementales et autres) a permis de mettre en évidence les avantages et limitations de l’approche. Enfin, un mélange artificiel standardisé de 24 organismes a été développé afin d’évaluer les outils de bio-informatique en métaprotéomique
The detection and identification of pathogenic microorganisms is a real public health issue for the food industry and the clinics or national interest as illustrated in the biodefense field. Thus, it is important to have identification and detection methods that are fast, sensitive and robust. This PhD thesis aims at contributing to the development of a rapid approach to identify microorganisms without any a priori by tandem mass spectrometry. This innovative approach, called phylopeptidomics, is based on the combination of peptidomics, i.e. large scale analysis of peptides derived from the enzymatic digestion of a biological sample, and the phylogeny of cellular organisms. After extraction of the proteins from the sample of interest, peptides are generated and analyzed by tandem mass spectrometry. The deconvolution of MS/MS signals using the "μOrg.ID" software developed in the laboratory enables the identification and quantification of organisms present in the sample according to the organisms indexed in generalist databases. The study of the proteome of Bacillus atrophaeus, a simulant agent of anthrax, in sporulated and vegetative form, has provided an illustration of a new method of identification of protein biomarkers, which allows determining the ratio between both forms. The limit of detection of phylopeptidomics in pure samples and equimolar mixtures was established with model bacteria of medical and environmental interests. The limit of detection of B. atrophaeus spores in the presence of 14 interfering matrices (food, environmental and others) has highlighted the advantages and limitations of the approach. Finally, a standardized artificial mixture of 24 organisms was developed in order to evaluate bioinformatics tools in metaproteomics

Orientadores: Bruno Coraucci Filho, Roberto Feijo de Figueiredo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo
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Resumo: O objetivo deste projeto foi estudar as valas de filtração como um método de baixo custo aplicável a residências isoladas e pequenas ou médias comunidades brasileira, analisar as valas de filtração como unidades de pós-tratamento de efluente anaeróbio de um sistema fossa filtro, na remoção natural de nutrientes e microrganismos patogênicos. No presente estudo foram estudadas três valas de filtração em escala piloto com 1 m de comprimento e 0,50 m de largura, com altura da camada de areia igual a 0,25, 0,50, e 0,75 m, foi utilizado tubo de drenagem em PEAD - polietileno de alta densidade ¿ com 0,10 m de diâmetro, as valas de filtração foram alimentadas por efluente de um sistema fossa filtro, foram empregadas 4 taxas hidráulicas (40, 60, 80, e 100 L/m2.dia) cada taxa foi aplicada na freqüência de 6, 12, 18, e 24 hora/dia. O esgoto bruto, afluente, e efluente das valas de filtração, foram analisados semanalmente permitindo constatar que os resultados obtidos no estudo das valas de filtração no emprego de altas taxas hidráulicas proporcionavam uma remoção superior a 82,5% DBO e adequação de coliformes e a ausência de microrganismo patogênico. Quanto aos compostos nitrogenados, ocorreu uma grande nitrificação. A concentração de fósforo também foi muito reduzida, adequando o efluente das valas de filtração a legislação brasileira, em todas as taxas empregadas
Abstract: The aim of this work was to study filtration ditches as a method of low cost applicable the isolated homes and the small Brazilian municipalities for the posttreatment of anaerobic effluent from a system septic tank with anaerobic unit in the removal of natural nutrients and pathogenic microorganisms. For the research were analyzed three filtration ditches on pilot-scale with 1 m length and 0,5 m width, with height of the layer of sand equal 0,25, 0,50, and 0,75 m was used a drainage pipe in HDPE - High Density Polyethylene - with 0,10 m in diameter. The of filtration ditches were fed by effluent from a system septic tank with anaerobic unit were used four high hydraulic rates (40, 60, 80, and 100 L/m2.day) each rate was applied in the frequency of 6, 12, 18, and 24 hours/day. The municipal wastewater, affluent and the effluent of the filtration ditches were weekly analyzed allowing that the results obtained in the study removal of organic matter exceeded 82,5% BOD and adequacy of coliform, absence of pathogenic microorganisms. In terms of nitrogen compounds, a high nitrification occurred. The phosphorus concentration also was very reduced, the effluent of the three filtration ditches being in compliance with Brazilian laws at all the rates used.
Mestrado
Saneamento e Ambiente
Mestre em Engenharia Civil

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Analyse comparative du comportement de 2 souches de p. F. De pathogenicite differente. L'etude clef du pouvoir entomopathogene a lieu au niveau de la reconnaissance de la cuticule par l'apex du tube germinatif. Il existe des sites preferentiels tegumentaires d'infection. Le genre pathogene est reconnu par les hemocytes, mais l'activite cytotoxique du champignon empeche la formation de granulone. La contamination de blessures hemorragiques par l'isolat non pathogene entraine une cicatrisation par les hemocytes qui peut bloquer l'infection fongique. L'analyse de l'ultrastructure des hemocytes des insectes sains conduit a identifier divers types cellulaires de p. B. Et a elaborer une hypothese sur le role des inclusions cytoplasmiques chez les hemocytes granuleux

The conversion of carbon dioxide (CO2) to bicarbonate (HCO3-) and protons (H+) is a physiologically relevant reaction in all life kingdoms. The uncatalyzed hydration-dehydration reaction CO2 + H2O ⇋ HCO3- + H+ is slow at physiological pH and thus, in biological systems, the reaction is accelerated by enzymatic catalysts, called carbonic anhydrases (CAs, EC 4.2.1.1). CA isozymes have been found in virtually all mammalian tissues and cell types, where they function in CO2 transport and other physiological processes. In the present thesis, it has been carried out a wide study concerning the inhibition profiles of CAs identified in the genome of pathogens causing disease in humans, such as Vibrio cholerae, Plasmodium falciparum, Porphyromonas gingivalis and Malassezia globosa CAs from the pathogens aforementioned were biochemically characterized and an extensive inhibition profile was carried out using the classical CAs inhibitors such as sulfonamides and anions. These studies have contributed to the search of antibiotics with a novel mechanism of action. Additionally, the present thesis has contributed to the discovery of the η-CA, a new genetic families of CAs; to the introduction of a new technique, named protonography, useful for the identification of CA activity on a polyacrylamide gel; and to the phylogenetic analysis of the α-, β- and γ-CAs identified in the genome of the Gram-positive and Gram-negative bacteria.

The availability of completely sequenced genomes for a number of organisms provides an opportunity to understand the molecular basis of physiology, metabolism, regulation and evolution of these organisms. Significant understanding of the complexity of organisms can be obtained from the functional characterization of repertoire of proteins encoded in their genomes. Computational approaches for recognition of function of proteins of unknown function encoded in genomes often rely on ability to detect well characterized homologues. Homology searches based on pair-wise sequence comparisons can reliably detect homologues with sequence identity more than 30%. However, detecting homologues characterized by sequence identity below 30% is difficult using these methods. Distant homology relationship can be established using profiles or position specific scoring matrices, which encapsulate information about structurally and functionally conserved residues. These conserved residues imply high constraints at a particular amino acid residue site due to their involvement in structural stability, enzymatic activity, ligand binding, protein folding or protein–protein interactions. In addition, information on three dimensional structures of proteins also aid in detection of remote homologues, as tertiary structures of proteins are conserved better than the primary structures of proteins. The gross objective of the work reported in this thesis is to employ various sensitive remote homology detection methods to recognize relevant functional information of proteins encoded mainly in pathogenic organisms. Since proteins do not work in isolation in a cell, it has become essential to understand the in vivo context of functions of proteins. For this purpose, it is essential to have an understanding of all molecules that interact with a particular protein. Thus, another major area of bioinformatics has been to integrate protein-protein interaction information to enable better understanding of context of functional events. Protein-protein interaction analysis for host-pathogen can lead to useful insight into mode of pathogenesis and subsequent consequences in host cell. Chapters 2-6 of the thesis discuss the sequence and structural characteristics along with remote evolutionary relationships and functional implications of uncharacterized proteins encoded in genomes of following pathogens: Helicobacter pylori, Plasmodium falciparum and Leishmania donovani. The Chapters 6-8 discuss mainly various sequence, structural and functional aspects of protein kinases encoded in genomes of various prokaryotes and viruses. Chapter 1 discusses background information and literature survey in the areas of homology detection and prediction of protein-protein interactions. The growth of genomic data and need for processing genomic data to infer context of various functional events have been highlighted. Different approaches to recognize functions of proteins (experimental as well as computational) have been discussed. Various experimental and computational approaches to detect/predict protein-protein interactions have been mentioned. Chapter 2 discusses recognition of non-trivial remote homology relationships involving proteins of Helicobacter pylori and their implications for function recognition. H. pylori is microaerophilic, Gram negative bacterial pathogen. It colonizes human gastric mucosa and is a causative agent of gastroduodenal disease. The pathogen infects about 50% of the human population. It can lead to development of Mucosa-associated lymphoid tissue lymphoma. About 10% of the infected population develop gastric or duodenal ulcer and approximately 1% develop gastric cancer. H. pylori has been classified as class I carcinogen by WHO. Pathogen is characterized by type IV secretion system. The complete genomic sequences of three widely studied strains including 26695, J99 and HPAG1 of Helicobacter pylori are available. According to the genome analysis, the number of predicted open reading frames in strain 26695, J99 and HPAG1 are 1590, 1495 and 1536 respectively. Out of predicted H. pylori proteins from 26695, J99 and HPAG1 strains, numbers of proteins with no functional domain assignments in Pfam database (Protein family database) are 453, 357 and 400 respectively. There are proteins in different strains of H. pylori genomes where one part of the protein is associated with at least one protein domain of known function and hence preliminary indication of their functions is available whereas rest of the region is not associated with any function. There are 772, 803 and 790 such segments in proteins from strains 26695, J99 and HPAG1 respectively with at least 45 residues with no functional assignment currently available. Sensitive remote homology detection methods have been employed to establish relationships for 294 amino acid sequences and results have been grouped into 4 categories. Results of homology detection have been further confirmed by studying conservation of amino acid residues which are important for functioning of the proteins concerned. (i) Remote relationship has been established involving protein domain families for which no bonafide member is currently known in H. pylori. For example: DNA binding protein domain (Kor_B) has been assigned to a H. pylori protein at sequence identity of 20%. Study involving secondary structure prediction and conservation of amino acid residues confirms the results of homology detection methods. (ii) Remote relationship has been established involving H. pylori hypothetical proteins and protein domain families, for which paralogous members are present in Helicobacter pylori. For example, Cytochrome_C, an electron transfer protein domain could be associated with a Helicobacter pylori protein sequence which shows a sequence identity of 14% with sequences of bonafide cytochrome C. (iii) “Missing” metabolic proteins of H. pylori have also been recognized. For example, Aspartoacylase (EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartic acid to produce acetate and L-aspartate. This enzyme in aspartate metabolism pathway has not been reported so far from H. pylori. A remote evolutionary relationship between a H. pylori protein and Aspartoacylase domain has been established at sequence identity of 17% thus filling the gap in this metabolic pathway in the pathogen. (iv) New functional assignments for domains in H. pylori sequences with prior assignment of domains for the rest of the sequences have been made. For example, DNA methylase domain has been assigned to C-terminal region of H. pylori protein which already had Helicase domain assigned to the N-terminal region of the protein. All these information should open avenues for further probing by carrying out experiments which will impact the design of inhibitor against this pathogen and will result in better understanding of pathogenesis of this organism in human. Chapter 3 describes prediction of protein–protein interactions between Helicobacter pylori and the human host. A lack of information on protein-protein interactions at the host-pathogen interface is impeding the understanding of the pathogenesis process. A recently developed, homology search-based method to predict protein-protein interactions is applied to the gastric pathogen, Helicobacter pylori to predict the interactions between proteins of H. pylori and human proteins in vitro. Many of the predicted interactions could potentially occur between the pathogen and its human host during pathogenesis as we focused mainly on the H. pylori proteins that have a transmembrane region or are encoded in the pathogenic island and those which are known to be secreted into the human host. By applying the homology search approach to protein-protein interaction databases DIP and iPfam, in vitro interactions for a total of 623 H. pylori proteins with 6559 human proteins could be predicted. The predicted interactions include 549 hypothetical proteins of as yet unknown function encoded in the H. pylori genome and 13 experimentally verified secreted proteins. A total of 833 interactions involving the extracellular domains of transmembrane proteins of H. pylori could be predicted. Structural analysis of some of the examples reveals that the predicted interactions are consistent with the structural compatibility of binding partners. Various probable interactions with discernible biological relevance are discussed in this chapter. For example, interaction between CFTR protein (NP_000483) and multidrug resistance protein (HP1206) has been predicted. The structure of the CFTR intracellular domain is known in the homomeric form and consists of five AAA transport domains in tandem (PDB code 1XMI). Out of the five identical subunits, two subunits (the B chain and the E chain in the PDB structure) have been selected. The structure of multidrug resistance protein of the pathogen based on the B chain (sequence identity 32%) of the template has been modeled. This exercise suggests that interface residues in the model are congenial for interaction. This makes the structural complex feasible in in vitro conditions and suggests that the pathogen protein may compete for occupancy with the host protein. Chapter 4 describes recognition of Plasmodium-specific protein domain families and their roles in Plasmodium falciparum life cycle. Malaria in humans is caused by the parasites of intracellular, eukaryotic protozoan of apicomplexan nature belonging to the genus Plasmodium. Out of five species of Plasmodium, namely, P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi which infects human, P. falciparum causes lethal infection. P. falciparum proteins have diverged extensively during the course of evolution. Pathogen genome is rich in A+T composition which larger than the homologous proteins from other organisms due to presence of low complexity regions. Organism specific families are important as they play roles in peculiar life style of an organism. If the organism is a pathogen, then these family members may play roles in pathogenesis. Inhibiting these specific proteins is unlikely to interfere with host system as no homolog may be present in host. In the present work we identify Plasmodium specific protein families and their role in different stages of life cycle of the pathogen. A total of 5086 amino acid sequences (full length sequences/fragments of proteins) show homology only with amino acid sequences from Plasmodium organisms and hence are Plasmodium-specific. These Plasmodium-specific amino acid sequences cluster into 106 Plasmodium-specific families (≥2 members per family). 14 Plasmodium-specific protein domain families with known physico-chemical properties are observed. These Plasmodium-specific protein domain families are involved in various important functions such as rosetting and sequestering of infected erythrocytes, binding to surface of host cell and invasion process in life cycle of pathogen. Also, 89 new Plasmodium-specific protein domain families have been recognized. Analysis of various aspects of members of Plasmodium-specific proteins domain families such as their potential to target apicoplast, protein-protein interaction, expression profile and domain organization has been performed to derive relevant information about function. New Plasmodium specific domain families for which no function can be associated could provide some insight into much diverged Plasmodium species. These proteins may play role in parasite-specific life style. Experimental work on these Plasmodium-specific proteins might fill the gaps of less understood physiology of this parasite. Chapter 5 presents genome-wide compilation of low complexity regions (LCR) in proteins. An indepth analysis of the nature, structure, and functional role of the proteins containing low complexity regions in Plasmodium falciparum, was undertaken given the high prevalence of LCRs in the proteome of this organism. Low complexity regions and repeat patterns have been recognized in proteins encoded in 986 genomes (68 archaea, 896 prokaryotes and 22 eukaryotes). Low complexity regions have been classified into following three categories: a) Composition of LCRs: (i) LCRs can be stretches of homo amino acid residues (ii) LCRs can be stretches of more than one amino acid residue type b) Periodicity of amino acids in LCRs: Certain amino acid residues can be observed at certain specific periodicity in proteins. c) Repeat patterns: Certain motif of amino acid residues are repeated in protein. 850 Plasmodium falciparum proteins are observed to have at least one repeat pattern where the repeating unit is at least 5 amino acid residues long. Statistical analysis on single amino acid residue repeats indicate that occurrence of stretches of homo amino acid residues is not a random event. Studies on recognition of functions, protein protein interactions and organization of tethered domain(s) in proteins containing LCR suggest that these proteins are part of variety of functional events such as signal transduction, enzymatic processes, cell differentiation, pyrimidine biosynthesis, fatty acid biosynthesis and chromosomal replication. Representations of low complexity regions of Plasmodium falciparum in protein data bank suggest that LCRs can take conformation of regular secondary structure (apart from disordered regions) in 3-D structures of proteins. Chapter 6 describes sequence analysis, structural modeling and evolutionary studies of Leishmania donovani hypusine pathway enzymes. Leishmania is an eukaryotic kinetoplastid protozoan parasite which causes leishmaniasis in humans. Hypusine is a non standard polyaminederived amino acid Nε-(4-amino-2-hydroxybutyl) lysine and is named after its two structural components, hydroxyputrescine and lysine. The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein containing hypusine. Synthesis of hypusine is critical for the function of elF5A and is essential for eukaryotic cell proliferation and survival. Formation of hypusine is the result of a two step post-translational modification process involving enzymes (i) deoxyhypusine synthase (DHS) (ii) deoxyhypusine hydroxylase (DOHH). DHS, the first enzyme involved in hypusine pathway catalyzes the NAD-dependent transfer of the butylamino moiety of spermidine (substrate) to the ε-amino group of a specific lysine residue of eIF5A precursor and generates deoxyhypusine containing intermediate. DOHH, the second enzyme in same pathway catalyzes the hydroxylation of deoxyhypusine-containing intermediate, generating hypusine-containing mature eIF5A. Two putative deoxyhypusine synthase (DHS) sequences DHS34 and DHS20 have been identified in Leishmania donovani, by Professor Madhubala and coworkers (Jawaharlal Nehru University, New Delhi) with whom the work embodied in this chapter was done in collaboration. Detailed comparison of DHS34 sequence from Leishmania with human DHS protein indicated conservation of functionally important residues. 3D structural modeling studies of protein suggested that residues around the active site were absolutely conserved. NAD binding regions are located spatially closer, however, one NAD binding region was observed in a large (225 amino acid residues long) insertion. Based on these observations, DHS34 was predicted to have enzymatic activity. Experimental studies done by our collaborators confirmed preliminary results of computational analysis. Based on sequence and structural analysis of DHS20 and DOHH proteins, DHS20 and DOHH were proposed to be catalytically inactive and active respectively. Experimental studies on these proteins supported results of computational analysis. Deoxyhypusine synthase (DHS) and Deoxyhypusine hydroxylase (DOHH) are key proteins conserved in the hypusine synthesis pathways of eukaryotes. Because they are highly conserved, they could be coevolving. Comparison of the genetic distance matrices of DHS and DOHH proteins reveals that their evolutionary rates are better correlated when compared to the rate of an unrelated protein such as Cytochrome C. This indicates that they are coevolving, further serving as an indicator that, even non-interacting proteins that are functionally coupled, experience correlated evolution. However, this correlation does not extend to their tree topologies. Chapter 7 provides a classification scheme for protein kinases encoded in genomes of prokaryotic organisms. Overwhelming majority of the Ser/Thr protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Ser/Thr protein kinases prokaryotic Ser/Thr protein kinases. Traditional sequence alignment and phylogenetic approaches have been used to identify and classify prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence databases, it is recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly, subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, it was also possible to identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a degree of complexity in protein-protein interactions and the signaling pathways in these microbes. Chapter 8 focuses on recognition, classification of protein kinases encoded in genomes of viruses and their implications in various functions and diseases. Protein kinases encoded by viral genomes play a major role in infection, replication and survival of viruses. Using traditional sequence homology detection tools, sequence alignment methods and phylogenetic approaches, protein kinases were recognized. 646123 protein sequences from 35799 viral genomes (including strains) have been used in this analysis. Protein kinases are identified using a combination of profile-based search methods such as PSI-BLAST, RPS-BLAST and HMMER approaches. Based upon sequence similarity over the length of catalytic kinase domains, 479 protein kinase domains recognized in 244 viral genomes have been clustered into 46 subfamilies with minimum sequence identity of 35% within a subfamily. Viral protein kinases are encoded in genomes of retro-transcribing viruses or viruses which possess double stranded DNA as genetic material. Based on the available functional information present for one or more members of a subfamily, a putative function has been assigned to other members of the subfamily. Information regarding interaction of viral protein kinases with viral/host protein has also been considered for enhancing understanding of function of kinases in a subfamily. Out of 46 subfamilies, 14 subfamilies are characterized by various functions. Kinases belonging to UL97, US69, UL13 and BGLF subfamilies are virus specific. For 7 subfamilies, nearest neighbors are from well characterized eukaryotic protein kinase groups such as AGC, CAMK and CDK. Out of 25 new uncharacterized subfamilies observed in this analysis, 13 subfamilies are virus specific. Different subfamilies have been characterized by various functions which are crucial for viral infection such as synthesis of structural unit, replication of genetic material, modification of cellular components, alteration in host immune system, competing with cellular protein for efficient usage of host machinery. Also, many viral kinases share very high sequence identity (~97%) with their eukaryotic counterpart and represent disease state. For example, a protein kinase encoded in Avian erythroblastosis virus shares 97% sequence identity with catalytic domain of human epidermal growth factor receptor tyrosine kinase. Leucine at position 861 in human protein is substituted by Gln in cancer conditions; the viral protein kinase sequence possesses Gln at corresponding position and thus represents disease state. Chapter 9 provides study of dependency on the ability of 3-D structural features of comparative models and crystal structures of inactive forms of enzymes to predict enzymes by considering protein kinases as case study. With the advent of structural genomics initiatives, there is a surge in the number of proteins with 3-D structural information even before functional features are understood on many of these proteins. One of the useful annotations of a protein is the demarcation of a protein into an enzyme or non-enzyme solely from the knowledge of 3-D structure. This is facilitated by the identification of active sites and ligand binding sites in a protein. In this work, which was carried out in collaboration with Dr Jim Warwicker of Manchester University, UK, an approach developed by Warwicker and coworkers has been used. In the 3D structure of proteins, the largest clefts are generally considered to be ligand binding sites. This feature along with other sequence alignment independent properties such as residue preferences, fraction of surface residues and secondary structure elements have been considered to differentiate enzymes from non-enzymes. Electrostatic potential at the active site is one of the key properties utilized in this respect. Active sites in enzymes are generally associated with ionizable groups which can take part in catalysis. In addition to the feature of large clefts in enzymes, active site residues are in buried environments and show larger deviation in pKa values than surface residues. The method proposed by Warwicker and co-workers distinguish proteins in to enzymes and non-enzymes considering the electrostatic features at clefts along with the sequence profile of the protein concerned. Conformation of the inactive state of an enzyme is not congenial to the catalytic function. In an ideal situation, a method should be capable of predicting an enzyme irrespective of whether determined structure corresponds to active or inactive state. Peak potential values have been calculated by using Warwicker program for a set of 15 protein kinases for which 3-D structures are present in active as well in inactive conformations. Comparison of peak potential values calculated for active and inactive conformations suggests that algorithm can differentiate between active and inactive conformations as value for active conformations are generally higher than corresponding values for inactive conformations. However, the peak potential values are high enough for even the inactive conformations to be predicted as enzyme. Peak potential values calculated for generated homology models of protein kinases (for which crystal structures are already available) at different sequence identities with template sequences predict protein kinases as enzymes and their peak potential values are comparable to corresponding values for X-ray structures. This suggests that proteins for which there are no crystal or NMR structures yet available and no good template with high sequence identity are present, peak potential values for models generated at low sequence identity can still give insight into probable function of protein as an enzyme. The enzyme/non-enzyme prediction algorithm was also found to be useful in confirming enzyme functionality using 3-D models of putative viral kinases. Initially, putative function of kinase has been assigned to these viral proteins based solely upon their sequence characteristics such as presence of residues/motifs which are important for activity of the protein. The enzyme recognition method which is not directly sensitive to these motifs confirmed that all the analyzed putative viral kinases are enzymes. Chapter 10 presents conclusions of work embodied in the entire thesis. Very briefly, various computational approaches have been used to analyze and understand structural and functional properties of repertoire of proteins of pathogenic organisms. Analysis of uncharacterized protein domain families has helped to understand the functional implications of constituent proteins. Experimental validation of these results can further facilitate unraveling of functional aspects of proteins encoded in various pathogenic organisms. Apart from studies embodied in the thesis, author has been involved in two other studies, which are provided as appendices. Appendix 1 describes comparison of substitution pattern of amino acid residues of protein encoded in P. falciparum genome with substitution pattern of corresponding homologous proteins from non-Plasmodium organisms. Salient differences have been highlighted. Appendix 2 discusses study of bacterial tyrosine kinases with an objective of recognition of all putative protein tyrosine kinases in E. coli. Computational study suggests that protein SopA can be a potential tyrosine kinase and this conclusion is being tested experimentally in collaborator’s laboratory.

博士
國立臺北科技大學
化學工程與生物科技系化學工程博士班
107
To prevent disease outbreaks and for general food safety, food products are constantly tested for infectious pathogen contamination; thus, a rapid, accurate, and user-friendly molecular diagnostic tool is urgently needed in the food analysis field. The current gold standards for microbial detection are culture-based methods; however, these methods are time-consuming and laborious, making these methods unsuitable for rapid diagnosis during a potential outbreak. This study has developed a novel, integrated, real-time electrochemical nucleic acid platform for pathogen rapidly detection. Our electrochemical platform has many advantages, including size, portability, high specificity, and high sensitivity, while also being a user-friendly device that provides stable signals. In this research, the nucleic acid detection platform consists the five major directions: 1. Testing of novel electrochemical nucleic acid detection device; 2. Evaluation and electrochemical analysis of high-efficient nucleic acid intercalating probes; 3. Evaluation and comparison of nucleic acid amplification methods; 4. Realization of rapid isothermal electrochemical nucleic acid detection platform; 5. Application of the platform to clinical and food pathogens for real-time quantitative analysis. We successfully developed an electrochemical nucleic acid amplification platform for real-time detection of pathogenic organisms. Through the results of real-time LAMP of Salmonella in a turbid environment, the detection limit of 10 copies without pretreatment was possible using the electrochemical method only. Our platform was used for bacterial detection in 150 turbid food samples, including juice, milk, and soy milk. The new electrochemical platform demonstrates the best performance for bacterial detection in the rapid contaminated food or clinical detection category.

Fresh produces, fruit juices and herbal teas used in our regular diet may have importance in the protective treatment of some infectious diseases. In this study, dietary produces were investigated for their antioxidant activities and antimicrobial activities against group A ß
-haemolytic streptoccoci. Streptococcus pyogenes, a member of the group A ß
-haemolytic streptococci, is a very dangerous pathogen, which may cause diseases such as tonsillopharyngitis, meningitis, rheumatic arthritis. Fruits and vegetables
onion, radish, carrot, plum, fruit juices
orange, peach, pomegranate, grape and teas
sage, anise, rosehip, chamomile were chosen as samples of regular daily diets. Dry extracts were obtained either by lyophilizing or fractionating in ethyl acetate. Antioxidant activities of extracts were examined by total phenolic content determination, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) methods. Antimicrobial activities of extracts were studied by disk diffusion test, minimum inhibitory and bactericidal concentration methods. Sage, plum, onion and radish displayed high radical scavenging activity with EC50 values of 0.043, 0.049, 0.148 and 0.414 mg/mL, respectively. Plum, sage, onion and radish were found high in total phenolic contents with &
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g gallic acid equivalent of 50.506, 48.299, 44.427 and 13.135 in mg extract, respectively. High antimicrobial activities were obtained by onion, radish, anise, carrot and peach extracts as tested by disk diffusion method with respective 20, 16, 16, 14 and 14 millimeters clear growth inhibition zones. Carrot, onion and radish extracts were found as effective bacteriostatic and bactericidal agents with minimum inhibitory and bactericidal respective concentrations of 0.008, 0.125, 0.250 mg/mL and 0.06, 0.5, 1 mg/mL.