Vibrio parahaemolyticus is an opportunistic human pathogen that occurs naturally in a non-pathogenic form in coastal estuarine and marine environments worldwide. Following the acquisition of virulence-associated genes, V. parahaemolyticus has emerged as a significant pathogen causing seafood-borne illnesses. The mechanisms and conditions that promote the emergence of disease causing V. parahaemolyticus strains are not well understood. In addition, V. parahaemolyticus clinical strains isolated from disease-associated samples and environmental strains from sediment, water, and marine organisms have been identified with considerable diversity; however, the evolutionary relationships of disease-causing strains and environmental strains are not known. In the following research, the evolutionary relationships of V. parahaemolyticus clinical and environmental strains are examined. In addition, the contribution of genetic elements and molecular mechanisms such as deficiency of DNA repair to the evolution of V. parahaemolyticus clinical and environmental strains is shown. Molecular analysis of the evolutionary relationships of V. parahaemolyticus clinical and environmental strains demonstrated separate lineages of pathogenic and non-pathogenic strains with the exception of several environmental strains that may represent a reservoir of disease-causing strains in the environment. Sequence characterization of plasmids isolated from diverse environmental Vibrios indicated a role of plasmids in strain evolution by horizontal transfer of housekeeping genes. In addition, analysis of plasmids from V. parahaemolyticus clinical and environmental strains indicated the existence of a plasmid family distributed among V. parahaemolyticus, V. campbellii, and V. harveyi environmental strains. Sequence characterization of a plasmid of this family from a V. parahaemolyticus environmental strain indicated the contribution of these plasmids to the emergence of the clonal pandemic strains. Investigation of the role of molecular mechanisms to the evolution of V. parahaemolyticus strains showed that inactivation of the DNA repair pathway methyl-directed mismatch repair (MMR) increased the accumulation of spontaneous mutations leading to increased nucleotide diversity in select genes. The research findings in the following chapters demonstrate a considerable contribution of genetic elements and molecular mechanisms to the evolution of genetic and phenotypic diversity.

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No description available.

Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.

abstract: Alzheimer's disease (AD) is the most common form of dementia leading to cognitive dysfunction and memory loss as well as emotional and behavioral disorders. It is the 6th leading cause of death in United States, and the only one among top 10 death causes that cannot be prevented, cured or slowed. An estimated 5.4 million Americans live with AD, and this number is expected to triple by year 2050 as the baby boomers age. The cost of care for AD in the US is about $200 billion each year. Unfortunately, in addition to the lack of an effective treatment or AD, there is also a lack of an effective diagnosis, particularly an early diagnosis which would enable treatment to begin before significant neuronal damage has occurred. Increasing evidence implicates soluble oligomeric forms of beta-amyloid and tau in the onset and progression of AD. While many studies have focused on beta-amyloid, soluble oligomeric tau species may also play an important role in AD pathogenesis. Antibodies that selectively identify and target specific oligomeric tau variants would be valuable tools for both diagnostic and therapeutic applications and also to study the etiology of AD and other neurodegenerative diseases. Recombinant human tau (rhTau) in monomeric, dimeric, trimeric and fibrillar forms were synthesized and purified to perform LDH assay on human neuroblastoma cells, so that trimeric but not monomeric or dimeric rhTau was identified as extracellularly neurotoxic to neuronal cells. A novel biopanning protocol was designed based on phage display technique and atomic force microscopy (AFM), and used to isolate single chain antibody variable domain fragments (scFvs) that selectively recognize the toxic tau oligomers. These scFvs selectively bind tau variants in brain tissue of human AD patients and AD-related tau transgenic rodent models and have potential value as early diagnostic biomarkers for AD and as potential therapeutics to selectively target toxic tau aggregates. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2014

Orientador: Hélio José Montassier / Banca: Camillo Del Cistia Andrade / Banca: Janete Apparecida Desidério Sena. / Resumo: Anticorpos monoclonais se constituem na base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por "phage display" contra a estirpe vacinal (H120) do VBI, foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01, IBVPR05, isoladas de surtos a campo no Brasil e SE-17, isolada nos Estados Unidos. Após três ciclos de "panning", foi identificado pelo ELISA um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que três desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzido em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus e no estudo de evolução de variantes desse vírus. / Abstract: Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of outbreaks of infectious bronchitis, because these antibodies are homogeneous, highly specific and fully characterized, allowing the improvement of detection of immunological techniques and antigenic characterization of avian infectious bronchitis virus strains (IBV). We used a phage display library prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments reacting with heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning a set of 15 scFv antibodies was expressed in phages and exhibited crossreaction in ELISA with these three viral strains. Western-blotting analysis showed that three of this clone set were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41 strain of IBV. In conclusion, the recombinant fragments of monoclonal antibodies expressed by phage-display technique have a great potential for future use in immunodiagnostic techniques and study the evolution of variant strains of this virus. / Mestre

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Consumption of food contaminated with strains of Staphylococcus aureus can cause diseases, whose signs and symptoms include gastroenteritis, nausea, vomiting, diarrhea, abdominal pain within one to six hours post-consumption of contaminated food. In this way, rapid methods of detection and identification of S. aureus are essential for food quality control and food safety. At this study, objectived to select peptide that binds to S.aureus, through the technique of Phage Display (PD), for development of fast diagnostic tools, of easy handling and low cost. At this study was used bioppaning for selection of peptides that express on the surface filamentous phage peptides that binds to S. aureus. The phage DNA selected was sequenced and subjected to in silico analysis (BioEdit v7.0.9). The sequences obtained were aligned and clones underwent pre-screening (ELISA) for the evaluation of binding specificity to S. aureus. The titles of input and output in biopanning were constant. Nine valid sequences were obtained after sequencing 40 clones selected after 3 rounds of biopanning. The analysis demonstrated that four clones presented reactivity in bacteria, although tests have demonstrated that the peptides exhibited no specific binding capacity in Staphylococcus aureus. Nevertheless, the peptide H06 showed binding specificity in gram-positive bacteria used in the test of reactivity. Furthermore, the in silico analysis showed that the recombinant peptides share chemical characteristics essential the proteins of the bacterial cells. Although the S. aureus specificity had not been observed, the peptide can be used as a method of detecting contamination of food in gram-positive bacteria. In food contamination, fast screening and identification of bacterial groups, allows establish decisions about the marketing and distribution of foods and may prevent outbreaks of food intoxication and ensure food security. / O consumo de alimentos contaminados com cepas de Staphylococcus aureus pode causar doenças, cujos sinais incluem gastroenterites, náuseas, vômitos, diarreia, dor abdominal intensa dentro de uma a seis horas após o consumo do alimento contaminado. Por esta razão, métodos rápidos de detecção de S.aureus são essenciais para o controle da qualidade e da garantia da segurança alimentar. Assim, o presente estudo teve por objetivo selecionar peptídeos ligantes à S.aureus, por meio da técnica de Phage Display (PD), para desenvolvimento de ferramentas diagnósticas rápidas, de fácil manipulação e baixo custo. Neste estudo, foi realizado bioppaning para seleção de peptídeos expressos na superfície de fagos filamentosos que apresentassem peptídeos ligantes a S.aureus. O DNA dos fagos selecionados foi sequenciado e submetido a analise in silico(BioEdit v7.0.9). As sequências obtidas foram alinhadas e os clones foram submetidos à pre-screening (ELISA) para avaliação de especificidade de ligação à S. aureus. Os títulos de entrada e saída obtidos no biopanning foram constantes. Nove sequências válidas foram obtidas após o sequenciamento dos 40 clones selecionados após 3 ciclos de biopanning. A análise de reatividade demonstrou que quatro clones apresentaram reatividade à bactéria, embora os testes de especificidade demonstraram que os peptídeos não exibiram capacidade de ligação específica a S. aureus. Apesar disto, o peptídeo E06 mostrou especificidade de ligação a bactérias do gênero Staphylococcus usadas no teste de reatividade. Além disso, as análises in sílico revelaram que os peptídeos recombinantes compartilham características químicas essenciais a proteínas das bactérias. Embora a especificidade a S.aureus não tenha sido observada, neste estudo o peptídeo pode ser utilizado como um método de detecção a contaminação de alimentos por estafilococos. Nas contaminações de alimentos, a triagem rápida e métodos de identificação de grupos bacterianos permitem estabelecer decisões sobre a comercialização e distribuição e podem prevenir um surto de intoxicação, garantindo a segurança alimentar. / Mestre em Biologia Celular e Estrutural Aplicadas

Abstract Species of Lactobacillus (L.) are important starter and probiotic lactic acid bacteria used in the dairy industry. Industrial fermentation processes are prone to phage infections, which can cause severe economic losses. The main objective of this thesis was to examine in more depth the genetic variation and evolution of L. delbrueckii and L. rhamnosus phages. Aspects of interactions and co-evolution of a phage and its host have also been included in this study. In this study, the complete genomic DNA sequences of four Lactobacillus phages were determined and analyzed in detail. Specific phage genes and genetic elements were identified and studied in more depth. The L. delbrueckii phage JCL1032 was found to be a temperate phage which is able to integrate into two distinct genes of L. delbrueckii, but with exceptionally low frequency. The isolated JCL1032-lysogenic bacteria expressed a complex phage resistance against several L. delbrueckii phages. The rarely reported coexistence of phage adsorption resistance and immunity could not be explained by lysogenic conversion. Instead, the spontaneously induced JCL1032 may have provided a selective advantage to adsorption resistant lysogens. The biological activity of two group I introns residing within the terminase large subunit and tape measure genes of the JCL1032 genome (49,433 bp) was demonstrated. The diversification of L. delbrueckii phages is mainly due to insertions, deletions and recombination, as was demonstrated by comparative analyses of the LL-Ku and c5 genomes of 31,080 bp and 31,841 bp, respectively. Interestingly, both phages have possible autonomous transcription units of genes within their genomes. It seemed that evolution of the 36,366-bp genome of the L. casei phage Lc-Nu has been fuelled by deletions as well. The lytic phage Lc-Nu has an imperfect lysogeny module and the phage is genetically closely related to L. casei prophages. This clearly demonstrated that Lc-Nu has a recent temperate origin. This study provides genetic tools, genes, and regulatory elements for biotechnological applications and for developing starter strains with enhanced phage resistance properties. / Tiivistelmä Hapatteina ja probiootteina käytetyt Lactobacillus-maitohappobakteerit (L. ) ovat merkittävässä asemassa meijeriteollisuudessa. Teolliset käymisprosessit ovat alttiita faagi-infektioille, jotka voivat aiheuttaa tuotantolaitoksille huomattavia taloudellisia tappioita. Tämän tutkimuksen päätavoitteena oli syventää tietoa L. delbrueckii ja L. rhamnosus -bakteereita infektoivien faagien geneettisestä muuntelusta ja evoluutiosta. Tutkimuksessa käsitellään myös faagin ja isäntäbakteerin välistä vuorovaikutusta sekä yhteisevoluutiota. Tutkimuksessa määritettiin neljän Lactobacillus-faagin genominen DNA-sekvenssi, identifioitiin faagigeenejä ja muita geneettisiä elementtejä sekä tutkittiin niiden toimintaa. L. delbrueckiin JCL1032-faagi osoittautui tutkimuksessa temperaatiksi. JCL1032-genomi voi integroitua kahteen eri geeniin isäntäbakteerin kromosomissa, joskin lysogeniafrekvenssi on hyvin alhainen. Tutkimuksessa eristetyt JCL1032-lysogeeniset bakteerikannat olivat resistenttejä useille Lactobacillus-faageille. Osassa lysogeenisia bakteereita resistenssi ilmeni jo faagin adsorptiovaiheessa. Vastaavanlainen ilmiö on kuvattu vain harvoin aiemmin. Havaittua kompleksista resistenssiä ei voitu selittää lysogeenisella konversiolla. Sen sijaan ilmiön taustalla voi olla JCL1032-profaagien spontaani indusoituminen bakteerin kromosomista, mikä voi antaa valintaetua adsorptioresistenteille lysogeenisille bakteereille. JCL1032-genomissa (49 433 emäsparia) osoitettiin olevan kaksi biologisesti aktiivista intronia terminaasin suurta alayksikköa ja hännän mittaproteiinia koodaavissa geeneissä. LL Ku- ja c5-faagien genomien (31 080 ja 31 841 emäsparia) vertailu osoitti L. delbrueckii -faagien evoluution olevan pääasiassa seurausta insertioista, deleetioista ja rekombinaatiosta. Kummassakin genomissa oli mahdollisesti päällekkäisiä ja itsenäisesti transkriptoituvia geenialueita. Deleetiot ovat muokanneet myös L. casein lyyttisen Lc- Nu-faagin genomia (36 466 emäsparia). Faagin lysogeniamoduuli sisälsi vain osan lysogeeniseen elinkiertoon tarvittavista geeneistä. Lc-Nu on geneettisesti läheistä sukua L. casei -profaageille, mikä myös viittaa siihen, että Lc-Nu on kehittynyt temperaatista faagista. Tutkimustuloksia faagien geeneistä ja säätelyelementeistä voidaan hyödyntää hapatebakteerien faagiresistenssiominaisuuksien kehittämisessä sekä erilaisissa bioteknologisissa sovelluksissa.

This thesis is devoted to designing and applying advanced algorithmical and statistical tools for analysis of NGS data related to cancer and infection diseases. NGS data under investigation are obtained either from host samples or viral variants. Recently, random peptide phage display libraries (RPPDL) were applied to studies of host's antibody response to different diseases. We study human antibody response to breast cancer and mouse antibody response to Lyme disease by sequencing of the whole antibody repertoire profiles which are represented by RPPDL. Alternatively, instead of sequencing immune response NGS can be applied directly to a viral population within an infected host. Specifically, we analyze the following RNA viruses: the human immunodeficiency virus (HIV) and the infectious bronchitis virus (IBV). Sequencing of RNA viruses is challenging because there are many variants inside population due to high mutation rate. Our results show that NGS helps to understand RNA viruses and explore their interaction with infected hosts. NGS also helps to analyze immune response to different diseases, trace changing of immune response at different disease stages.

Standard purification processes in large scale antibody production are largely dependent on Protein A chromatography. Protein A binds specifically to many subclasses of IgG with high affinity. However, in order to elute the proteins, the pH needs to be lowered. Since lowering of the pH can be detrimental to some antibodies, a milder purification process is of great interest. A variant of Protein A, called ZCa, has previously been engineered to bind to IgG in a calcium-dependent manner. The antibody binds to ZCa when calcium is present and releases when calcium is removed. For the IgG1 subclass, the elution still requires a slight lowering of the pH, which is why there is room for improvement of the molecule. A phage display selection has been performed with the aim to obtain calcium-dependent IgG1 binders that are able to release the antibodies upon calcium depletion at neutral pH. In addition, an attempt on increasing the alkaline stability of the binders was made. Sequence analysis of the selection output showed almost no indications of increased alkaline stability. Instead, M13K07 helper phages were exposed to new selections for increased alkaline tolerance which might be useful in future phage display selections. Even though the binders selected for in this project did show some promising characteristics, none of them were able to elute upon calcium depletion at neutral pH as aimed for. However, one of the variants did show promising results during most of the performed characterizations. Most interestingly, the elution properties of this variant could indicate a higher calcium-dependence in the interaction with the target than that of ZCa, although further characterizations need to be performed in order to draw any conclusions about possible improvements of this protein domain.