Plasmid DNA (pDNA) vectors are the current conventional technology driving therapeutic gene transfer, whether for use toward mal/nonfunctional gene replacement, DNA vaccination, or production of therapeutic proteins in mammalian cells. However, the conventional pDNA vector suffers from several safety and efficiency limitations: 1) it imparts adverse immune responses to bacterial sequences required for maintenance and amplification in prokaryotes; 2) its bioavailability can be compromised due to size; and 3) it may be genotoxic due to its potential to integrate into the host chromosome and yield an oncogenic event. In this study we have constructed a robust in vivo bacterial platform for the production of bacterial sequence-free linear covalently closed (LCC) DNA vectors, termed DNA Ministrings, through the manipulation and application of bacteriophage-encoded recombination systems. Phage N15 and PY54 lysogenize their bacterial hosts as a linear plasmid with covalently closed ends (LCC plasmid). LCC morphology is conferred by the phage-encoded telomerase via a single cleaving-joining reaction of the perfect palindrome target site. This system was exploited to generate DNA Ministring vectors, encoding only the gene(s) of interest and necessary complementary eukaryotic expression/enhancement genetic elements that are devoid of unwanted bacterial sequences and are linearized through a single in vivo enzymatic reaction. The tel and telN prokaryotic telomerase (protelomerase) genes were amplified from PY54 and N15 lysates, respectively, and cloned into a bacterial vector that expresses the gene under control of the temperature sensitive bacteriophage ?? CI857 repressor that confers conditional expression from ?? pL/pR promoters. This regulatory circuit was integrated into a RecA+ lacZ+ E. coli K-12 strain via homologous recombination, where successful recombinants were disrupted for the lacZ gene. Recombinant cells are capable of conditional expression of the phage-derived telomerase enzymes by shifting the temperature to >37 ??C. Phage P1-derived Cre recombinase was applied as a positive control, since its functionality in generating DNA minicircle vectors has been previously shown. A multi-purpose 342 bp target site termed Super Sequence (SS) that possesses the Cre, Flp, Tel, and TelN target sites in addition to two flanking SV40 enhancer sequences was cloned into two different sites of a GFP expression eukaryotic pDNA vector. The amplification of this DNA vector through telN / tel or cre expressing Recombinant E. coli cells (R-cells) generated bacterial sequence-depleted (LCC) DNA Ministring and (CCC) Minicircle vectors, respectively, as evidenced by digestion patterns of the purified vector. Transfection efficiency of these vectors was assessed in rapidly dividing human ovarian cancer and in relatively slowly dividing human embryonic kidney cell lines. In vitro experiments with DNA Ministrings in human cells lines resulted in significantly higher transfection efficiency, bioavailability, and cytoplasmic diffusion levels compared to the parental plasmid precursor and isogenic DNA Minicircle counterparts. The safety of the LCC DNA vector conformation, with respect to insertional genotoxicity, was assessed by forcing LCC pDNA vectors into bacterial and human genomic DNA. The integration of LCC DNA into bacterial and human host genomic DNA resulted in chromosomal DNA disruptions at site of integration, loss of genome stability, and subsequent cell death. LCC integration-induced apoptotic cell death and natural elimination of the integrant from human cell population improves the safety profile of DNA Ministrings by eliminating integrants following the potential genotoxic side effects of undesired vector integration into the host genome.

Mounting concerns about drug-resistant pathogenic bacteria have rekindled the interest in bacteriophages (bacterial viruses). As bacteria’s natural predators, bacteriophages offer a critical advantage over antibiotics, namely that they can be highly specific. This means that phage therapeutics can be designed to destroy only the infectious agent(s), without causing any harm to our microbiota. However, the potential secondary effects on the balance of microbiota through bacteriophage-induced genome evolution remains as one of the critical apprehensions regarding phage therapy. There exists a significant gap in knowledge regarding the direct and indirect effect of phage therapeutics on the microbiota. The aim of this thesis was to: (1) establish an in vivo model for investigation of the evolutionary dynamics and co-evolution of therapeutic phage and its corresponding host bacterium in the gut; (2) determine if phage therapy can affect the composition of the gut microbiota, (3) observe the differences of phage-resistant bacteria mutants evolved in vivo in comparison to those evolved in vitro. We used germ-free mice colonized with a consortium of eight known bacteria, known as the altered Schaedler flora (ASF). The colonizing strain of choice (mock infection) was a non-pathogenic strain E. coli K-12 (JM83) known to co-colonize the ASF model, which was challenged in vivo with T7 phage (strictly lytic). We compared the composition of the gut microbiota with that of mice not subject to phage therapy. Furthermore, the resistant mutants evolved in vivo and in vitro were characterized in terms of growth fitness and motility. / Thesis / Master of Applied Science (MASc) / Bacteriophages are viruses that infect bacteria. After their discovery in 1917, bacteriophages were a primary cure against infectious disease for 25 years, before being completely overshadowed by antibiotics. With the rise of antibiotic resistance, bacteriophages are being explored again for their antibacterial activity. One of the critical apprehensions regarding bacteriophage therapy is the possible perturbations to our microbiota. We set out to explore this concern using a simplified microbiome model, namely germ-free mice inoculated with only 8 bacteria plus a mock infection challenged with bacteriophage. We monitored this model for 9 weeks and isolated a collection of phage-resistant bacterial mutants from the mouse gut that developed post phage challenge, maintaining the community of mock infection inside the gut. A single dose of lytic phage challenge effectively decreased the mock infection without causing any extreme long-term perturbations to the gut microbiota.

<p dir="ltr">Open-access bioinformatics data enables accessible public health research for a variety of stakeholders, including teachers and low-resourced researchers. This project outlines two case studies utilizing open-access bioinformatics data sets and analysis software as proofs of concept for the types of research projects that can be adapted for workforce development purposes. The first case study is a spatial temporal analysis of Lyme disease rates in the United States from 2008 to 2020 using freely available data from the United States Department of Agriculture and Centers for Disease Control and Prevention to determine how urbanization and other changes in land use have impacted Lyme disease rates over time. The second case study conducts a pangenome analysis using bacteriophage data from the Actinobacteriophage Database to determine conserved gene regions related to host specificity.</p>

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

L’exposition aux aérosols microbiologiques présents dans les environnements clos est susceptible de provoquer, chez les occupants, diverses pathologies telles que des infections, des toxi-infections et des allergies. Pour s’en prémunir, diverses stratégies passant notamment par l’emploi de dispositifs épurateurs d’air, ont été développées et commercialisées par les industriels de la ventilation et du traitement de l’air. Cependant, à ce jour, aucune méthodologie d’évaluation y compris normative ne permet d’évaluer la pertinence de ces stratégies. Ce travail de recherche se propose, d’une part, d’appréhender le devenir des aérosols microbiologiques au sein des espaces clos : de la source à l’individu exposé, en explorant le rôle de la ventilation dans ce transport et, d’autre part, d’explorer le gain apporté par les nouvelles technologies de traitement microbiologique de l’air sur l’exposition des occupants. Pour ce dernier point de l’étude, une démarche globale d’évaluation en 3 volets a été adoptée avec l’étude de l’efficacité du ou des principes d’épuration mis en oeuvre, la détermination du rendement intrinsèque en condition dynamique de ces systèmes et l’évaluation du gain apporté par ces derniers sur l’exposition des occupants. Les travaux menés avec les dispositifs épurateurs (filtration et photocatalyse) ont montré que les efficacités intrinsèques des systèmes ne permettent pas de préjuger de leur gain vis-à-vis du niveau de l’exposition des individus lorsqu’ils sont mis en oeuvre en environnement intérieur. Les résultats obtenus ont également mis en évidence que la prise en compte des flux aérauliques et du transport des particules induit par la ventilation et le dispositif épurateur est indispensable à la définition d’une stratégie cohérente de traitement d’air / Exposure to bioaerosols in indoor environments is associated with a wide range of adverse effects on health including infectious diseases, acute toxic effects and allergies. In order to guard against this phenomenon, the ventilation and air treatment industry has developed and marketed many air control strategies. However, at present, there is no methodology adapted to the evaluation of the relevance of these strategies. The aim of this research work was to characterize, in a first time, the progress of microbiological aerosol from the original source, to their eventual inhalation by person exposed, considering their dissemination through the indoor environments. Secondly, the work consisted of determining the efficiency of air cleaner devices applied to control indoor air quality. For this point, a global approach of evaluation in 3 steps was adopted, consisting of studying the efficiency of the epuration principle implemented, determining the intrinsic performance of the systems in dynamic conditions and their impact on the exposure level of the exposed persons. The tests carried out with air cleaner devices (filtration and photocatalysis) have shown that the intrinsic performance wasn’t able to estimate the beneficial impact of these systems on the exposure level of people when there were applied in indoor environments. So the intrinsic performance of devices is not the single impact factor, the airflow promoted by the device is also a factor to consider. Moreover, the characterization of indoor airflows and airborne particles transport is essential to define a coherent strategy of air treatment

Le VIH-1 est l'agent infectieux qui cause le SIDA. De nombreuses thérapies existent pour combattre le SIDA mais aucune ne permet son éradication et des résistances apparaissent. Le développement de nouvelles thérapies est donc nécessaire. Les anticorps simple-domaine (sdAb) de lamas présentent les propriétés idéales pour le développement de molécules neutralisantes. Des lamas ont donc été immunisés avec Vpr et les formes native, ou induite par un miniCD4, du trimère de gp140 (partie extracellulaire de l'enveloppe (Env)). Des banques de sdAbs ont ensuite été construites et des sélections par phage display et par double hybride ont été réalisées. Trois sdAbs se liant au site de liaison du co- récepteur de l'Env et un sdAb se liant au site de liaison du CD4 ont ainsi été sélectionnés. Ces sites sont conservés mais difficile d'accès pour des immunoglobulines conventionnelles. Ces sdAbs ont ensuite été caractérisés par ELISA, SPR et cytométrie de flux pour leur capacité de liaison à différentes Env, et en « single round assay » pour leur capacité de neutralisation d'un large spectre (LS) de pseudovirus. Des protéines multidomaines (plusieurs sdAbs reliés par un linker) ont ensuite été construites et testées pour leur neutralisation. Plusieurs de ces molécules, neutralisant un LS de virus, pourraient être utilisées dans des microbicides. La stabilité caractéristique des sdAbs, même en absence de formation de pont disulfure, par exemple dans un environnement réducteur tel que le cytoplasme, est primordiale dans le développement d'anticorps intracellulaires (intrabodies). / HIV-1 is the infectious agent of AIDS. Numerous therapies exist to fight AIDS, but they are not able to eradicate it, and resistances appear. So, new therapy development is necessary. Single-domain antibodies (sdAb) of llamas have ideal properties to develop neutralizing molecules. So, llamas have been immunized with Vpr and with free or miniCD4 induced trimeric gp140 (extracellular part of the envelope (Env)). SdAb libraries have been built and selections were done by phage display and yeast two hybrid. Three sdAbs targeting the co-receptor binding site of the Env and one sdAb targeting the CD4 binding site have been selected. These sites are conserved but inaccessible by conventional immunoglobulins. These sdAbs have been characterized by ELISA, SPR and FACS for their ability to bind different Env and by single-round assay for their neutralization ability. Multimeric proteins (linked sdAbs) have been built and tested for their neutralization ability. Several of these molecules are able to neutralize a broad spectrum of pseudoviruses. They can be used in microbicides. The characteristic stability of these sdAbs, even without disulfide bound formation, ie into reducing environment, as the cytoplasm, is primordial for intracellular antibody (intrabody) development. One sdAb anti-Vpr has been selected using the Sos Recruitment System (SRS), an yeast two-hybrid system allowing detection of cytoplasmic protein-protein interactions. This sdAb is able to alter the localization of its antigen into eukaryotic cells. It is a proof of concept ot the use of SRS in the selection of intracellularly functional sdAbs.

O conceito de heterogeneidade vascular é bem aceito pela comunidade cientifica, desempenhando papel essencial em processos fisiológicos e patológicos. Uma vez que os vasos sanguíneos são importantes na organogênese, diferenciação e morfogênese de tecidos e órgãos, torna-se interessante desvendar a diversidade vascular cerebral, identificando novos marcadores moleculares para este órgão tão importante. Utilizando tecnologia combinatorial de phage display in vivo, identificamos um novo motivo peptídico, na qual os aminoácidos FenilalaninaArginina-Triptofano (Phe-Arg-Trp; FRW) predominam. Este motivo peptídico é um ligante seletivo para vasos sanguíneos cerebrais e não se acumula em outros órgãos, incluíndo tecidos como intestinos e gônadas, que também apresentam barreiras endoteliais especificas. No entanto, mais surpreendente foi a observação de que o motivo FRW não se liga aos vasos sanguíneos da retina, o que implica em uma diferença até então desconhecida entre duas barreiras: a barreira hematoencefálica e a barreira hematoretiniana. Combinando phage display in vivo e microscopia eletrônica de transmissão, observamos a presença de partículas de fago ligadas à vasculatura cerebral em um nível supramolecular: aglomerados de fagos filamentosos expressando o motivo FRW foram visualizados ligados às regiões de contato entre as células endoteliais. Por fim, a utilização do peptídeo CFFWKFRWMC permite imageamento in vivo, demonstrando que novas ferramentas para estudar e visualizar o cérebro podem surgir deste motivo. / The concept of vascular heterogeneity is well accepted by the scientific community, playing an essential role in physiological and pathological processes. Since blood vessels are important in organogenesis, differentiation, and morphogenesis of tissues and organs, it becomes interesting to unveil the cerebral vascular diversity, identifying new molecular markers for such important organ. Using in vivo phage display, we show that a new peptide motif that emerged from our combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (Phe-Arg-Trp; FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial in all areas of the brain, including the optic nerve but not in other barrier containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.

O tratamento atual indicado para casos de envenenamentos por peçonhas é a administração intravenosa de antivenenos, produzidos através da hiperimunização de animais. Entretanto, os antivenenos disponíveis podem, algumas vezes, não proteger os pacientes e causar reações de hipersensibilidade. Fosfolipases A2 (PLA2), L-aminoácido oxidases (LAAO), metalo e serinoproteases são os principais componentes de peçonhas ofídicas e contribuem para a neurotoxicidade, hemorragia, hemólise, miotoxicidade, cardiotoxicidade e formação de edemas. Foram empregados ensaios para avaliar as atividades das enzimas presentes na peçonha de serpentes da espécie Lachesis muta rhombeata e aquele para atividade de protease foi otimizado. A tecnologia de Phage display foi empregada para a seleção de fagos-anticorpos capazes de reconhecer a peçonha bruta. Os fagos foram amplificados em Escherichia coli TG1 e usados para infectar E. coli HB2151, a qual produz fragmentos de anticorpos humanos solúveis. Estes foram purificados e utilizados em testes de inibição de alguns dos componentes tóxicos da peçonha. Os testes de atividade para PLA2, protease e Laminoácido oxidase foram padronizados com sucesso e as 3 proteínas mostraram elevada atividade enzimática. Após otimização, a quantidade de peçonha necessária para o ensaio de protease foi reduzida em 25 vezes. A massa molecular de PLA2 foi estimada em 17 kDa e as massas moleculares de proteases foram estimadas em 40, 35 e 24 kDa, através de zimogramas. O método de bio panning foi eficiente para a seleção de fagos-anticorpos contra a peçonha bruta. Diversos fragmentos de anticorpos foram purificados e incubados com a peçonha bruta para testar suas capacidades de neutralização sobre cada enzima. Cinco clones demonstraram-se hábeis em inibir a PLA2 através da inibição da hemólise. O clone 4E inibiu 100% da hemólise durante as duas horas de ensaio quando pré-incubado na proporção 2:1 (scFv:peçonha). Os clones 2C e 4E inibiram 100% durante uma hora quando pré-incubados na proporção 1:1 e os clones 2F e 9F inibiram a hemólise parcialmente. Outros testes serão conduzidos para a seleção de clones capazes de neutralizar as demais enzimas, os quais, juntamente com os clones já selecionados, serão analisados através de ensaios in vivo. Espera-se que eles possam contribuir para a construção de um novo antiveneno capaz de superar algumas das dificuldades associadas às técnicas de imunoterapia convencionais / The current treatment for animal envenoming is the intravenous administration of antivenoms, produced by animal hyperimmunization. Unfortunately, available antivenoms sometimes do not protect patients and may cause hypersensitivity reactions. Phospholipases A2 (PLA2), L-amino acid oxidases, metallo and serine proteases are considered the most important snake venom components and contribute to neurotoxicity, hemorrhage, hemolysis, myotoxicity, edematogeny and cardiotoxicity. Assays for evaluating the enzymes present in Lachesis muta rhombeata venom were developed and the protease one was optimized. Phage display technology was used to select phage antibodies able to recognize the crude venom. Phages were amplified in Escherichia coli TG1 and used to infect E. coli HB2151, which produces human antibody fragments. Inhibition tests aiming the neutralization of some toxic components of the venom were performed using purified antibody fragments. Activity assays for evaluating PLA2, protease and L-aminoacid oxidase were successfully performed and all enzymes showed high activity levels. The molecular mass of PLA2 was estimated in 17 kDa and the molecular mass of proteases were estimated in 40, 35 and 24 kDa, by zymography. After optimizing the conditions for proteolytic assay, it was possible to use 25 times less venom than it was necessary at first. The bio panning method was efficient for selecting specific phage antibodies against the crude venom. Several clones were selected to infect HB2151 and to produce soluble antibody fragments, which were purified and incubated with the venom to test their inhibition capacity over each enzyme. Five clones demonstrated ability to neutralize PLA2 by inhibiting hemolysis. The clone 4E could inhibit 100% of hemolysis for over 2 hours when preincubated at the ratio 2:1 (scFv:venom). Clones 2C and 4E could inhibit 100% for 1 hour when preincubated at the ratio 1:1 and clones 2F e 9F could inhibit partially. Other tests will be performed to select clones able to neutralize other enzymes and, together with the clones already selected, will be evaluated by in vivo experiments. It is expected that they may contribute to the construction of a potential new antivenom able to overcome some of the problems associated with conventional immunotherapy

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identifcação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas. / Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease.

A digoxina é um dos medicamentos indicados para o tratamento de falência cardíaca. Possui janela terapêutica estreita, sendo responsável por casos de intoxicação. O único antídoto disponível para a desintoxicação é o anticorpo policlonal DigiFab®, no formato Fab. O seu uso é eficaz, porém de custo elevado. Clones bacterianos produtores de fragmento Fab monoclonal anti-digoxina foram obtidos previamente pelo nosso grupo, pela técnica de phage display. Neste trabalho as variantes Fab dos 4 clones foram expressas em E.coli para estabelecer o método para a purificação. Com a obtenção dos fragmentos Fab purificados, foi caracterizada a sua afinidade ao antígeno e especificidade, em ensaios de inibição por digoxina, digitoxina, digoxigenina e ouabaina. Os parâmetros cinéticos da ligação dos fragmentos Fab dos 4 clones e do DigiFab® foram avaliados por SPR. Nas condições experimentais, não foram verificadas diferenças significativas entre os produtos dos 4 clones e o comercial, demonstrando o potencial dos fragmentos Fab monoclonais obtidos como antídoto à digoxina. / Digoxin is a medication indicated for heart failure treatment. Its therapeutic window is narrow, being responsible for intoxication cases. The only antidote available for the detoxification is a polyclonal antibody - DigiFab® in Fab format. Its use is effective, but costly. Bacterial clones producing anti-digoxin monoclonal Fab fragments were previously obtained by our group using phage display technology. In this work the Fab variants of the 4 clones were expressed in E.coli to establish the purification method. The purified fragments were characterized regarding the affinity to the antigen and the specificity through inhibition assays with digoxin, digitoxin, digoxigenin and ouabain. The binding kinetic parameters of Fab fragments of the 4 clones and the commercial product to Dig-BSA conjugate were assessed by SPR. Under the experimental conditions no significant differences were observed among the 4 clones and the commercial product, demonstrating the potential of monoclonal Fab fragments as an antidote to digoxin.