Дисертації з теми "Microbiome engineering"

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
The human gastrointestinal tract is home to a dense and dynamic microbial community. The composition and metabolic output of the human gut microbiota have been implicated in many diseases: from inflammatory bowel disease, colorectal cancer, and diarrheal diseases to metabolic syndromes like diabetes. Treatment of these diseases will likely require targeted therapeutic interventions aimed at modulating the abundance and metabolism of specific commensal microbial species or probiotics. A promising avenue for such interventions is through diet, where the dietary components act as substrates for the species producing beneficial metabolites one wishes to enrich. In this thesis, I focus on a dietary intervention study in healthy individuals. Since the human gut microbiota is known for its highly heterogeneous composition across different individuals, it comes as no surprise that a more personalized approach is preeminent.
We first test effects of multiple micronutrients spiked into a fixed diet. Using a highly controlled diet within the cohort, we identify strong and predictable responses of specific microbes across participants consuming prebiotic spike-ins. However, select macronutrient spike-ins like unsaturated or saturated fat and protein, produce no predictable response. We next investigate prebiotic supplement in diet further as well as its downstream products, short chain fatty acids, in the digestive tract. We look to alleviate the stress of a highly controlled, low complexity diet on participants by testing the effect of different prebiotics simultaneously ex vivo. We show that individuals vary in their microbial metabolic phenotypes (as in they produce different quantities and proportions of short chain fatty acids from the same prebiotic inputs) mirroring differences in their microbiota composition.
Finally, we run a pilot study to elucidate how closely our ex vivo experiment results may reflect the in vivo changes following a short-term dietary fiber supplementation. In addition to obtaining preliminary data on this direct comparison, we also explore different parameters for generating high-throughput data on personalized dietary interventions. Together, these projects provide the framework for building a predicative model for the effect that prebiotic dietary supplementation will have on gut microbiota's composition. Such a prediction model would be equally helpful in both enhancing individuals' gut health and improving gut dysbiosis in cases of disease.
by Le Thanh Tu Nguyen.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
The human microbiome is essential for health and has been implicated in many diseases. DNA sequencing has enabled the detailed characterization of these human-associated microbial communities, leading to a rapid expansion in studies investigating the human microbiome. In this thesis, I describe multiple projects which overcome various data analysis challenges to extract useful clinical insights from microbiome data. In the first project, I present an analysis of lung, stomach, and oropharyngeal microbiomes. I leverage data collected from multiple sites per patient to identify aspiration-associated changes in the relationships between these communities, discovering new properties of the aerodigestive microbiome and suggesting new approaches for treatment. In the second project, I perform a meta-analysis of case-control gut microbiome datasets with standard data processing and analysis methods.
I find consistent patterns characterizing disease-associated microbiome changes and a set of shared associations which could inform clinical treatment and therapeutic development approaches for different microbiome-mediated diseases. Enabled by this work, in the third project I contribute to the development of a method to correct for batch effects in case-control microbiome studies. In the fourth project, I describe a framework for rational donor selection in fecal microbiota transplant clinical trials in which knowledge derived from clinical and basic science research is used to inform which donor is selected for fecal transplants, increasing the likelihood of successful trials. Finally, I present preliminary results analyzing the microbiome and metabolome of residential sewage as a novel platform for community-level public health surveillance.
Together, these projects demonstrate a variety of approaches to mine the human microbiome for clinically-relevant insights and suggests multiple avenues forward for translating findings from microbiome data analyses into clinical and public health impact.
by Claire Marie Noëlle Duvallet.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Host-microbe interactions at the gastrointestinal interface have emerged as a key component in the governance of human health and disease. Advances in micro-physiological systems are providing researchers with unprecedented access and insights into this complex relationship. These systems combine the benefits of microengineering, microfluidics, and cell culture in a bid to recreate the environmental conditions prevalent in the human gut. Here we present the human-microbial cross talk (HuMiX) platform, one such system that leverages this multidisciplinary approach to provide a representative in vitro model of the human gastrointestinal interface. HuMiX presents a novel and robust means to study the molecular interactions at the host-microbe interface. We summarize our proof-of-concept results obtained using the platform and highlight its potential to greatly enhance our understanding of host-microbe interactions with a potential to greatly impact the pharmaceutical, food, nutrition, and healthcare industries in the future. A number of key questions and challenges facing these technologies are also discussed. (C) 2017 THE AUTHORS. Published by Elsevier LTD on behalf of the Chinese Academy of Engineering and Higher Education Press Limited Company. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 86-88).
The gastrointestinal system plays a vital role in the functioning of the human body, processing food into useable energy, controlling homeostasis, and serving as the front line of the immune system. The intestines are aided in their many functions by the gut microbiome, a collection of 100 trillion anaerobic bacteria cells that live inside the GI tract. Although they play an essential part in the organ system, they remain little-represented in in vitro gastrointestinal models because of the difficulty of replicating the anaerobic conditions of the intestines. We constructed an in vitro model capable of growing aerobic epithelial intestinal cells along with anaerobic microbes in the same bioreactor. A device called the apical flow module seals a 12-well transwell and provides an inlet and outlet port into the apical chamber. Media is deoxygenated using nitrogen bubbles before it is pumped using a nitrogen-actuated pneumatic pump block. Microbes are injected into the anaerobic fluid through a rubber septum injection port before the fluid flows into the sealed transwell. Effluent is collected in sterile tubes at a controlled height so as to regulate the apical side pressure. Oxygen is provided to the basolateral human epithelial cells through basolateral circulation achieved using a pneumatic circulation plate. Preliminary testing confirms our ability to control the oxygen in all parts of the system and to grow cocultures of human and bacteria cells. Epithelial cells grown in our bioreactor show signs of behaving more similarly to cells in vivo when exposed to the conditions present in our system, providing researchers with an oxygen-controlled gastrointestinal in vitro model.
by Steven John Holcomb.
S.M.

The tumor microenvironment is a well-recognized contributor to cancer progression in solid tumors. Cancer cell interactions with abnormal extracellular matrix, tumor associated immune and stromal cells, and aberrant fluid flow all contribute to cancer progression. Breast tumors are often characterized by a dense collagenous stroma and a hypoxic core. A recently identified and little understood component of the breast tumor microenvironment is the breast microbiome. The work described here elaborates on the importance of the tumor microenvironment in cancer progression and demonstrates the importance of studying cancer-microbiome interactions in the context of tumor microenvironmental stimuli.
Master of Science

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
The human gastrointestinal tract is home to a dense and dynamic microbial community. Recent advancements in sequencing technology have revealed numerous relationships between the composition of these communities and human and health and disease. In some cases, researchers have shown causal relationships between the presence or absence of particular microorganisms and disease. These findings offer promise for using microorganisms or microbial communities to modulate health and disease, but to date, we lack tools and mechanistic insight needed for rational engineering of these communities. Understanding how microorganisms enter, colonize, grow, and disperse to new hosts present key considerations for rational engineering of the human gastrointestinal tract. In this thesis, I use experimental studies of the human and murine gastrointestinal tract to address these considerations. In the first study, I examined endospores and other resistant cell types in the gastrointestinal communities of unrelated humans to identify the ecological role of these states in the distribution of bacterial populations in healthy people. I used this information to infer shared roles for these organisms in successional states in the human gut, and identify host- and diet-derived metabolites as environmental signals mediating the growth and colonization of these organisms. In the second study, I examined the potential for using targeted manipulations of diet to favor selective growth and colonization by an introduced bacterium in the murine gastrointestinal tract. I showed that resource exclusivity of this bacterium permits its selective expansion in this environment, and negatively impacts the growth of other commensals. Central to the goal of rational engineering of the gut microbiota, these studies reveal ecological considerations that may promote or inhibit colonization by introduced commensals in this complex ecosystem. This work invites provides a conceptual framework for integrating systems microbial ecology with engineering design to manipulate the composition of the gastrointestinal microbiota.
by Sean M. Kearney.
Ph. D.

Les découvertes récentes sur les communautés microbiennes, ou microbiotes, ont révélé un potentiel biotechnologique considérable dans divers domaines. Ils sont considérés comme essentiels pour accélérer l'innovation dans les systèmes de production alimentaire. Toutefois, les procédés existants ne sont pas adaptés à la culture des microbiotes. La difficulté que représente la culture de microbiotes a notamment pour origine la capacité des microorganismes à interagir par compétition, qui peut conduire à la réduction indésirable de la biodiversité au sein du réacteur de culture. Ce phénomène peut aboutir à l'obtention de communautés qui ne présentent pas les fonctionnalités souhaitées. L'objectif de cette thèse est d'étudier l'influence de la propagation de microbiotes en condition contrôlée sur leur structure et leur fonction. Les travaux de cette thèse ont permis de développer et de déterminer la performance d'un procédé excluant la compétition microbienne pour la culture de communautés bactériennes. La stratégie choisie repose sur le micro-confinement et la ségrégation spatiale des bactéries au sein d'un bouillon de culture structuré en émulsion inverse. Après avoir étudié l'effet de la culture en émulsion inverse sur la croissance de bactéries individuelles, les travaux ont comparé son effet sur la dynamique de communautés propagées selon un régime séquentiel, ou backslopping, avec celui exercé par un système classique non-émulsionné. Les résultats ont montré que l'utilisation d'une émulsion inverse conduit à la génération de nouvelles structures de communautés au cours de la propagation, et que l'utilisation de la culture classique conduit à leur stabilisation. Les comportements différents issus de ces deux systèmes de culture en font des outils complémentaires pour le modelage et la propagation de communautés microbiennes. Enfin, l'effet de la propagation sur la variabilité fonctionnelle de communautés a été étudiée dans un contexte de biopréservation. Le criblage de microbiotes de laits crus propagés a montré qu'ils se différenciaient en termes de robustesse et de reproductibilité de leur activité anti-Listeria, justifiant de tenir compte de la variabilité fonctionnelle des communautés pour leur sélection dans un contexte d'ingénierie de microbiotes
Recent discoveries about microbial communities, or microbiota, have revealed considerable biotechnological potential in a variety of fields. They are considered essential to accelerate innovation in food production systems. However, existing processes are not adapted to the cultivation of microbiota. One major barrier to community propagation is competition between microorganisms, which can lead to an undesirable reduction in biodiversity within the culture reactor. This phenomenon can lead to communities that lack the desired functionality. The objective of this thesis was to study the influence of microbiota propagation, under controlled conditions, on their structure and function. During this work, a process of microbial culture excluding microbial competition for the propagation of bacterial communities was developed. The chosen strategy is based on the micro-confinement and spatial segregation of bacteria within a broth structured as an invert emulsion. The effect of the invert emulsion culture on the growth of individual bacteria was studied, then the effect of this system on the dynamics of communities propagated according to a sequential regime, or backslopping, as well as that exerted by a conventional non-emulsified system was investigated. The results showed that the use of an inverse emulsion leads to the generation of new community structures during propagation, and that the use of the classical culture leads to their stabilization. The different behaviors of these two culture systems make them complementary tools for the modeling and the propagation of microbial communities. Finally, the effect of propagation on the functional variability of communities was studied in a biopreservation context. The screening of propagated raw milk microbiota showed that they differed in terms of robustness and reproducibility of anti-Listeria activity, emphasizing the need to take into account the functional variability of communities when selecting communities of interest for microbiota engineering

Recent findings, including our own work, demonstrated that intestinal microbiota species produce bioactive metabolites that engage host cellular pathways. Microbiota-derived metabolites have also been detected in circulation and in the, setting up the intriguing possibility that these bacterial products could directly interact with host cellular pathways at distant sites. The study described in this abstract investigates the hypothesis that gut microbiota dysbiosis perturbs the balance of immunomodulatory microbiota metabolites, which exacerbates liver inflammation in steatosis. We utilize a multi-omic approach to identify microbiota-dependent immunomodulatory metabolites and characterize their effects on liver inflammation and metabolic function. In summary, we show that the levels of AAA-derived microbiota metabolites are significantly depleted in a diet model of liver steatosis, and that these metabolite can act directly on hepatocytes to modulate inflammatory pathways. Our results also show that the microbiota metabolites are ligands for the AhR, which could provide a mechanistic link for the observed anti-inflammatory effects. Taken together, our findings support the hypothesis that dysbiosis of the gut microbiota could predispose the liver to inflammation in diet-induced steatosis through an altered microbiota metabolite profile. Prospectively, additional insights into the mechanisms underlying the link between microbiota dysbiosis and NAFLD could provide novel strategies to treat or prevent the progression of fatty liver diseases through the use of probiotics or postbiotics.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 61-65).
Novel sequencing technologies are rapidly advancing studies of microbial community structure and diversity. Sequencing platforms like the Illumina Genome Analyzer II (GAI1) and the Applied Biosystems SOLiD enable experiments that were previously too expensive or time-consuming by providing a very large number of short reads at a significantly lower cost per base pair (bp) than conventional longer-read systems like the Roche-454 GS FLX pyrosequencing instrument. Short-read platforms, however, are not readily amenable to some applications like metagenomics and metatranscriptomics, and therefore pyrosequencing remains the dominant sequencing technique in these fields. The primary reason short-read technologies have not been used for metagenomic analyses is due to the difficulty of confidently assigning phylogeny or putative gene function to short sequences. In an effort to overcome this limitation, a strategy was developed for preparing libraries from sheared genomic DNA with tunable size distributions using solid phase reversible immobilization (SPRI). This size selection captures DNA fragments of the necessary length to enable the generation of overlapping reads when sequenced from both ends. The lower-quality ends of mated reads were then used to produce a high-quality consensus sequence in the region of overlap. The fraction of composite reads that could be assigned to a taxon was similar to those from 454-FLX, despite the slightly shorter average read length of the composite Illumina reads. This technique successfully demonstrates a practical and economical alternative to 454-FLX for metagenomics. In addition, a scalable, fully automated process for creating sequence-ready, barcoded libraries of 16S rDNA for microbial diversity studies was developed for the Illumina platform. This process will enable sequencing of hundreds of environmental samples on a single Illumina flowcell, greatly decreasing the cost per sample while providing thousands of short-reads for microbial ecology studies. The incorporation of error-correcting, short DNA "barcodes" (also called tags or indexes) during polymerase chain reaction (PCR) amplification of the 16S sequence facilitates sample multiplexing. This process also utilizes the SPRI method to replace column-based reaction clean-ups, enabling the library preparation procedure to be performed almost entirely by a robotic liquid handling workstation. Finally, two unique PCR primer systems (primer-clipping and primer-skipping) were engineered to increase the informative read length of 16S sequence by either cutting the known universal tract out of the final-product to be sequenced, or by omitting sequencing of the universal regions using specially-crafted primers designed to be compatible with Illumina platform conditions. By applying both the overlapping-read technique and multiplexed 16S library preparation workflow, a streamlined approach for efficient gene and species discovery has been assembled to accommodate new metagenomic applications for the Illumina sequencing platform.
by Matthew Christopher Blackburn.
S.M.

O aumento da concentração de nutrientes nos corpos receptores, principalmente nitrogênio e fósforo oriundos de efluentes sanitários e industriais pode gerar o fenômeno da eutrofização. Para que isto não ocorra é necessário que este efluente passe por um tratamento adequado, no entanto, o papel desempenhado por diversos grupos de microrganismos encontrados nos sistemas de tratamento de efluentes não é completamente compreendido devido à complexidade das interações. Este trabalho teve como objetivo caracterizar a estrutura e dinâmica da comunidade microbiana (bactérias envolvidas no ciclo do nitrogênio e microfauna) e avaliar a atividade biológica dos reatores aeróbio e anaeróbio de uma indústria de alimentos. Os parâmetros físico-químicos da estação de tratamento foram monitorados, bem como foi feita a avaliação da estrutura e dinâmica da comunidade bacteriana envolvida no ciclo do nitrogênio por meio da técnica de Hibridização in situ Fluorescente. A microfauna do reator aeróbio foi caracterizada e classificada conforme o Índice Biótico do Lodo. A atividade biológica do lodo foi avaliada através do Teste de Respirometria e foram feitas correlações entre a microbiota encontrada no reator aeróbio e parâmetros físico-químicos. Os parâmetros físico-químicos analisados estiveram dentro dos limites permitidos pelas legislações federais e estaduais e os parâmetros Demanda Bioquímica de Oxigênio, Demanda Química de Oxigênio e Nitrogênio Kjeldahl foram reduzidos de 99,8%, 99,6% e 74,9%, respectivamente. Foi possível observar a presença tanto de bactérias oxidadoras de nitrito quanto de amônia em ambos os reatores analisados, bem como em cada ponto de coletas dentro dos reatores. A bactéria Pseudomonas fluorescens também ocorreu em todos os pontos de coleta dos dois reatores. Dentre os grupos que compõem a microfauna do lodo ativado, os ciliados rastejantes foram os mais frequentes, seguido pelas tecamebas, rotíferos, ciliados sésseis, ciliados livre natantes, flagelados e outros invertebrados. Além disso, não houve diferença entre as densidades dos grupos encontradas nos Pontos 1 e 2 do reator aeróbio e o Índice Biótico do Lodo encontrado foi igual a 8 (classe I). A semelhança apresentada entre a Taxa de Consumo de Oxigênio dos pontos 1 e 2, bem como a Taxa de Consumo de Oxigênio específica entre os pontos 1 e 2 sugere que o oxigênio é distribuído de forma homogênea dentro do tanque de aeração, fazendo com que os microrganismos tenham condições semelhantes de crescimento. Os ciliados livre natantes apresentaram correlação positiva com a DQO e DBO5 e os ciliados sésseis apresentaram correlação negativa com a DQO e com a DBO5. Os rotíferos apresentaram correlação negativa com Sólidos Suspensos Voláteis do reator aeróbio. Os ciliados rastejantes, tecamebas e rotíferos apresentaram correlação positiva com a microfauna total encontrada no reator aeróbio. Os ciliados livre natantes apresentaram correlação negativa com os ciliados sésseis, bactérias totais, Nitrobacter e outras bactérias; e correlação positiva com outros invertebrados. Os flagelados apresentaram correlação negativa com as bactérias totais, enquanto as outras bactérias apresentaram correlação positiva. Os outros invertebrados apresentaram correlação negativa com Nitrobacter.
The increasing concentration of nutrients in receiving water bodies, especially nitrogen and phosphorus originating from domestic and industrial effluent discharges can cause eutrophication. In order to avoid that, the effluents must be properly treated for nutrients removal in wastewater treatment plants prior discharge. However, the role played by various groups of microorganisms found in wastewater treatment systems is not completely understood due to the complexity of interactions. This study aimed to characterize the structure and dynamics of microbial community (with focus on bacteria involved in the nitrogen cycle and microfauna) and evaluate the biological activity of aerobic and anaerobic reactors for wastewater treatment operated at a food industry. The physical and chemical parameters of the treatment plant were monitored. At the same time, hybridization in situ fluorescent assessed the structure and dynamics of bacterial community involved in the nitrogen cycle. The microfauna in the aerobic reactor were characterized and classified according to the Sludge Biotic Index. The sludge biological activity was assessed by respirometry assays and correlations were made between the microbiota found in the aerobic reactor and selected physicochemical parameters. The physical and chemical parameters analysed complied with the limits allowed by the federal and state regulations and parameters Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD) and Kjeldahl nitrogen were reduced 99.8%, 99.6% and 74.9%, respectively. It was found the presence of both nitrite-oxidizing and ammonia-oxidizing bacteria in both reactors and in each sampling point within the reactors. Pseudomonas fluorescens bacteria also occurred in all collection points of both reactors. Among the microorganism groups observed in the activated sludge, crawling ciliates were the most frequent, followed by tecamoebians, rotifers, ciliates sessile, free natant ciliates, flagellates and other invertebrates. In addition, there was no difference between the densities of the groups found in Points 1 and 2 in the aerobic reactor and the Sludge Biotic Index was found equal to 8 (class I). The similarity between the presented Oxygen Consumption Rate of items 1 and 2 as well as the Oxygen Consumption Rate particularly between points 1 and 2 suggest that oxygen is distributed evenly within the aeration tank, causing the microorganisms to have similar growth conditions. The free natant ciliates were positively correlated with COD and BOD5 and sessile ciliates showed a negative correlation with the COD and the BOD5. Rotifers were negatively correlated with Suspended Volatile Solids in the aerobic reactor. Crawling ciliates, rotifers and the tecamoebians were positively correlated with the total microorganisms found in the aerobic reactor. The free natant ciliates showed a negative correlation with the sessile ciliates, total bacteria, Nitrobacter and other bacteria and a positive correlation with other invertebrates. The flagellates were negatively correlated with the total bacteria, while other bacteria were positively correlated. The other invertebrates showed a negative correlation with Nitrobacter.

This thesis reports the development of BioMe, a co-culture microplate platform that enables high-throughput, real-time quantitative growth dynamics measurements of interacting microbial batch cultures. The primary BioMe components can be 3D-printed, allowing ease of fabrication and DIY accessibility in the microbiome community. A pairwise 3D-printed iteration of the BioMe device was used in diffusion and co-culture experiments. Genetically engineered Escherichia Coli lysine and isoleucine auxotroph strains were used to characterize the diffusion of amino acids across the porous membranes. Results demonstrated a nonlinear relationship between growth rate and pore size and also distinct diffusion behavior for lysine and isoleucine. Pairwise syntrophic co-culture experiments demonstrated synergistic but repressed interaction between these two paired auxotrophs. Investigation of the effect of varying initial amino acid conditions on growth dynamics demonstrated that small changes in initial media condition can consistently affect patterns of yield and growth rate of constituent microbial species.
2020-08-30T00:00:00Z

abstract: The Human Gut Microbiome (GM) modulates a variety of structural, metabolic, and protective functions to benefit the host. A few recent studies also support the role of the gut microbiome in the regulation of bone health. The relationship between GM and bone health was analyzed based on the data collected from a group of twenty-three adolescent boys and girls who participated in a controlled feeding study, during which two different doses (0 g/d fiber and 12 g/d fiber) of Soluble Corn Fiber (SCF) were added to their diet. This analysis was performed by predicting measures of Bone Mineral Density (BMD) and Bone Mineral Content (BMC) which are indicators of bone strength, using the GM sequence of proportions of 178 microbes collected from 23 subjects, by building a machine learning regression model. The model developed was evaluated by calculating performance metrics such as Root Mean Squared Error, Pearson’s correlation coefficient, and Spearman’s rank correlation coefficient, using cross-validation. A noticeable correlation was observed between the GM and bone health, and it was observed that the overall prediction correlation was higher with SCF intervention (r ~ 0.51). The genera of microbes that played an important role in this relationship were identified. Eubacterium (g), Bacteroides (g), Megamonas (g), Acetivibrio (g), Faecalibacterium (g), and Paraprevotella (g) were some of the microbes that showed an increase in proportion with SCF intervention.
Dissertation/Thesis
Masters Thesis Electrical Engineering 2020

abstract: Machine learning (ML) has played an important role in several modern technological innovations and has become an important tool for researchers in various fields of interest. Besides engineering, ML techniques have started to spread across various departments of study, like health-care, medicine, diagnostics, social science, finance, economics etc. These techniques require data to train the algorithms and model a complex system and make predictions based on that model. Due to development of sophisticated sensors it has become easier to collect large volumes of data which is used to make necessary hypotheses using ML. The promising results obtained using ML have opened up new opportunities of research across various departments and this dissertation is a manifestation of it. Here, some unique studies have been presented, from which valuable inference have been drawn for a real-world complex system. Each study has its own unique sets of motivation and relevance to the real world. An ensemble of signal processing (SP) and ML techniques have been explored in each study. This dissertation provides the detailed systematic approach and discusses the results achieved in each study. Valuable inferences drawn from each study play a vital role in areas of science and technology, and it is worth further investigation. This dissertation also provides a set of useful SP and ML tools for researchers in various fields of interest.
Dissertation/Thesis
Doctoral Dissertation Electrical Engineering 2018

Synthetic biology, a field that sits between Biology and Engineering disciplines, has come into its own in the last decade. The decreasing cost of DNA synthesis has lead to the creation of larger and more complex synthetic gene networks, engineered with functional goals rather than simple demonstration. While many methods have been developed to reduce the time required to produce complex networks, none focus upon the considerable tuning needed to turn structurally correct networks into functional gene networks. To this end, we created a Plug-and-Play synthetic gene network assembly that emphasizes character-driven iteration for producing functional synthetic gene networks. This platform enables post-construction modification and easy tuning of networks through its ability to swap individual parts. To demonstrate this system, we constructed a functional bistable genetic toggle and transformed it into two functionally distinct synthetic networks. Once these networks have been created and tuned at the bench, they next must be delivered to bacteria in their target environment. While this is easy for industrial applications, delivering synthetic networks as medical therapeutics has a host of problems, such as competing microbes, the host immune system, and harsh microenvironments. Therefore, we employed bacteriophage technologies to deliver functional synthetic gene networks to specific bacterial strains in various microenvironments. We first sought to deliver functional genetic networks to bacteria present in the gut microbiome. This allows for functionalization of these bacteria to eventually sense disease states and secrete therapeutics. As a proof of concept a simple circuit was created using the Plug-and-Play platform and tested before being moved into the replicative form plasmid of the M13 bacteriophage. Bacteriophage particles carrying this network were used to infect gut bacteria of mice. Infection and functionality of the synthetic network was monitored from screening fecal samples. Next, we employed phagemid technologies to deliver high copy plasmids expressing antibacterial networks to target bacteria. This allows for sustained expression of antibacterial genes that cause non-lytic bacterial death without reliance upon traditional small molecule antibiotics. Phagemid particles carrying our antibacterial networks were then tested against wild type and antibiotic-resistant bacteria in an in vitro and in vivo environment.

The gut microbiome is an integral component of the human body that plays a role in many physiological processes. Dysbiosis, an imbalance of the microbiome, has been associated with disease states including inflammatory bowel disease, type II diabetes, and obesity, and moreover, contributes to the pathogenesis of these states. Understanding the functional mechanisms governing microbial ecology and microbe-host interactions is essential to understanding the microbiome’s role in health and disease. However, at present, functional genetic studies of diverse natural mammalian gut microbiomes remain challenging, due to a lack of genetic tools for bacteria outside of a handful of well-studied model organisms. Altering the metagenome of a complex microbial community requires novel platform technologies for genetic engineering which can operate in a generalized fashion across many different host organisms. In this thesis, I present two novel genetic tools designed for genetic modification of bacterial communities. The first, the Cas-Transposon platform, is a host-independent targeted genome editing tool that utilizes programmable, targeted transposases to mediate site-specific gene insertions into user-defined loci. The Himar1 transposase naturally inserts transposases into random TA dinucleotides in a genome, but when fused to the dCas9 RNA-guided, DNA-binding protein, the fusion protein Himar1-dCas9 targets transposon insertions to a single TA site. The activity of Himar1-dCas9 was characterized using in vitro experiments, demonstrating that site-specific transposition is dependent on guide RNA (gRNA) orientation relative to the target site and the sequence surrounding the target site, but robust to variations in DNA and protein concentration, presence of background DNA, and temperature. We additionally showed that the Cas-Transposon platform is capable of performing site-specific transposition into a plasmid in vivo in E. coli, although further optimization of the system may be necessary to effect site-specific transposition into a genomic locus. The Himar1-dCas9 protein is the first example of a transposase that inserts transposons into locations programmable by an RNA, making it a novel tool for gene insertion and knockout in potentially any organism, without relying on DNA repair by a host cell. Metagenomic Alteration of Gut microbiome by In situ Conjugation (MAGIC) is an approach to directly modify gut bacteria in their native habitat by harnessing naturally occurring horizontal gene transfer activity to deliver engineered DNA. Because many gut bacteria are difficult to cultivate and thus difficult to genetically manipulate in the laboratory, MAGIC uses donor bacteria, delivered directly into the gut environment, to conjugate mobile vectors bearing engineered genetic payloads. Using payloads with selectable markers, we identified organisms across 4 major phyla of gut bacteria that were amenable to genetic modification with libraries of conjugative vectors we created. Using a lab-adapted E. coli strain as a donor, we achieved transient expression of the engineered payload in the microbiome. We also demonstrated that engineered native gut bacteria containing conjugative vectors could be deployed back into the gut to stably recolonize and mediate secondary transfer of the payload into other microbes, potentially enabling long-term infiltration of the payload into the metagenome. The results from this study suggest that both short-term and long-term genetic alteration of the metagenome are possible by choosing different donors, and that the MAGIC platform could enable development of more diverse microbial chasses for synthetic biology applications. MAGIC could also be used to create personalized engineered probiotics for diagnostic or therapeutic applications. In Chapter 4 of this thesis, we explored the targeted use of MAGIC to genetically modify Segmented Filamentous Bacteria, a gut commensal that is important for immune regulation but recalcitrant to in vitro cultivation. The Cas-Transposon and MAGIC technologies expand our capabilities in the areas of targeted genome editing and gene delivery into bacteria, respectively. Together, they form a suite of complementary approaches to genetically engineer undomesticated gut commensal bacteria and probe the functional genetic networks in the gut microbiome, which will enhance our understanding of microbiome ecology and host-microbiome interactions. In addition, the expanded range of genetic manipulations made possible by these tools may enable production of more diverse, perhaps personalized, probiotics containing engineered functions, such as sensing disease markers or drug delivery.

Tese no âmbito do Doutoramento em Biociências, especialização em Bioquímica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Chronic metabolic diseases related to obesity and lifestyle are imposing an ever-increasing burden on healthcare worldwide. Incidences of non-alcoholic fatty liver disease (NAFLD) and Type-2 diabetes (T2D) have been rising particularly rapidly in Western Societies, showing strong associations with sedentary lifestyle and excessive caloric intake in relation to daily expenditure. A significant fraction of this hypercaloric intake is accounted by sugar, in particular fructose, present in processed food and sweetened beverages. This diet promotes weight gain, insulin resistance and dyslipidemia thereby providing the foundations for NAFLD and T2D development. In addition to directly disrupting the control of systemic carbohydrate and lipid metabolism, such diets may also mediate these effects indirectly via the gut microbiome. The gut microbiome is a complex ecosystem that is normally in a symbiotic relationship with the host and having a key role in nutrient processing and energy harvest. In obesity-related diseases such as NAFLD and T2D, dysbiotic alterations in the microbiome composition, its metabolic endproducts, and its interactions with host tissues, are implicated in their pathogenesis. The first part of this work aimed to study the effects of a diet high in simple sugars on the composition of the intestinal microbiome and its metabolite products in mouse models. It was hypothesized that dietary glucose and fructose have distinct effects on these parameters with focus on the relative abundance of Gram-negative species and alterations in SCFA levels. Three groups of mice were fed over 10 weeks with standard, high glucose or high fructose chow. Fecal samples were periodically collected and the microbiome was profiled by real-time quantitative polymerase chain reaction (qPCR). Fecal metabolites were analyzed by proton nuclear magnetic resonance (1H NMR). On the metabolites content significant differences were also observed with a reduction in beneficial SCFA like butyrate, to be induced by fructose. For the mice fed the high fructose diet, unmetabolized fructose was found in the feces indicating that the intestinal absorption capacity had been saturated and that fructose was available to the entire intestinal microbiome. Mice fed the high fructose diets also showed a shift in microbiome species towards Gram-negative bacteria and decreased fecal levels of butyrate relative to acetate and propionate. An increased abundance of Gram-negative bacteria could promote visceral inflammation through increased abundance and leakage of endotoxin from the intestinal lumen into surrounding tissues. In addition, alterations in microbiome metabolic endproducts such as lactate and short-chain fatty acids (SCFA) can have an impact on intestinal as well as hepatic and peripheral metabolism through direct substrate effects as well as by activation of substrate-specific receptors such as the GPR family. In the second part of the Thesis, the specific contributions of dietary glucose and fructose to hepatic and adipose tissue lipogenesis were determined by integrating 2H-enrichment of triglyceride from deuterated water to provide estimates of de novo lipogenesis (DNL) and glycerol synthesis from all sources with 13C-triglyceride (TG) enrichment from 13C-glucose and fructose tracers to provide specific contributions of each exogenous sugar. In liver, exogenous fructose contributed significantly more to both DNL and glycerol synthesis compared to exogenous glucose. Moreover, fructose promoted the synthesis of saturated fatty acids to a greater degree than that of oleate whereas glucose did not. Fructose also contributed to mesenteric adipose tissue TG synthesis, albeit to a lesser degree than glucose while only glucose contributed to subcutaneous adipose tissue TG synthesis. In conclusion, this dissertation provided new insights on the role of the intestine and visceral adipose tissue in the metabolism of dietary glucose and fructose and has opened two important new lines of research. First, the potential for overflow of fructose into the lower intestine: to what extent does this happen in humans in the Western diet setting and how does this influence microbiome composition and metabolism? In this context, there is evidence that fructose malabsorption in the human population may be more widespread than initially thought. Second, the lipogenic utilization of fructose carbons by mesenteric adipose tissue begs the question of how this sugar became available to these adipocytes. Was it via direct absorption or was it converted beforehand to glucose by intestinal gluconeogenesis? If it was by direct absorption, this implies that mesenteric adipocytes have a capacity for fructose uptake and metabolism. Also, it suggests that mesenteric adipose tissue has some degree of privileged access to nutrients that are absorbed by the intestine.
As doenças metabólicas crónicas associadas a obesidade e estilo de vida representam um encargo significativamente crescente na saúde mundial. Nas sociedades ocidentais, as incidências de fígado gordo não alcoólico (NAFLD) e diabetes tipo 2 (T2D) têm aumentado consideravelmente correlacionando-se com sedentarismo e consumo excessivo de calorias perante o consumo diário. Nestas dietas hipercalóricas, o açúcar é maioritário em especial a frutose, presente em concentrações elevadas nos alimentos processados. Estas condições estimulam o aumento de peso, resistência à insulina e dislipidémia, que estão na base do desenvolvimento de NAFLD e T2D. Para além destas dietas promoverem distúrbios diretos no metabolismo de lípidos e hidratos de carbono, podem derivar indiretamente os mesmos efeitos, através do microbioma intestinal. Este é um ecossistema complexo que normalmente se encontra em simbiose com o hospedeiro, o que é fundamental para o processamento de nutrientes e extração de energia. Alterações às populações microbianas e perda de simbiose, produtos metabólicos dessas espécies e a respetiva interação com vários tecidos do hospedeiro estão na origem das patologias referidas. A primeira parte deste trabalho visou estudar, em modelos de ratinho, o efeito de dietas ricas em açúcares simples, na composição do microbioma intestinal e dos seus produtos metabólicos. A hipótese propunha que a glicose e frutose produzem efeitos distintos, com foco para alterações na abundância relativa de espécies Gram-negativas e nos níveis de ácidos gordos de cadeia curta (SCFA). Três grupos foram alimentados por um período de 10 semanas com 3 tipos de dieta: standard, rica em glucose ou rica em frutose. Periodicamente, recolheram-se amostras fecais e o perfil microbiológico analisado por real-time quantitative polymerase chain reaction (qPCR). Os metabolitos fecais foram analisados por ressonância magnética nuclear de protão (1H NMR). Observaram-se diferenças significativas no conteúdo em metabolitos com uma redução, induzida por frutose, em SCFA benéficos como o butirato. Nos animais alimentados com elevados níveis de frutose, moléculas não metabolizadas deste açúcar foram encontradas nas fezes, indicando uma saturação da absorção intestinal que resulta em maior disponibilidade de frutose para o microbioma. No mesmo grupo de ratinhos verificou-se uma alteração na composição microbiana com maior tendência para espécies Gram-negativas e um decréscimo nas concentrações de butirato em relação a acetato e propionato. A prevalência destas bactérias pode promover inflamação visceral pelo seu crescimento e libertação de endotoxinas do lúmen intestinal para os tecidos circundantes. Adicionalmente, variações nos metabolitos bacterianos como lactato e SCFA podem alterar o metabolismo intestinal, hepático e periférico através de modificações diretas por substratos, como ativação específica de recetores sensíveis a esses, tais como os da família GPR. Na segunda parte desta Tese, foram determinadas as contribuições específicas da glucose e frutose nas dietas, para a lipogénese no fígado e tecido adiposo, através da integração do enriquecimento nos triglicerídeos (TG) em 2H proveniente de água deuterada para quantificação da lipogénese de novo (DNL) e síntese de glicerol gerais, com o enriquecimento nos mesmos TG em 13C proveniente de marcadores 13C-glucose e 13C-frutose para analisar as contribuições específicas destes açúcares exógenos. No fígado, a frutose deteve um contributo significativamente superior para a DNL e síntese de glicerol quando comparada com glucose. Neste contexto, a frutose promoveu a produção de ácidos gordos saturados em maior escala, efeito não verificado com a glucose. No tecido adiposo mesentérico a frutose contribuiu para a síntese de TG embora em menor porção que a glicose. No tecido adiposo subcutâneo apenas a glicose contribuiu para a lipogénese. Em nota conclusiva, esta dissertação apresenta novas evidências no papel do intestino e tecido adiposo visceral no metabolismo da glicose e frutose e estabelece bases para duas novas linhas de investigação. A primeira reporta à saturação de frutose no intestino grosso: em que medida este fenómeno ocorre em humanos sob dietas ocidentais e a influência que terá na composição do microbioma e metabolismo? Existem evidências de que a malabsorção de frutose em humanos poderá ser transversal a um grupo maior de populações que inicialmente esperado. A segunda refere-se à utilização dos carbonos da frutose para lipogénese no tecido adiposo mesentérico em que se questiona por que processo este açúcar fica disponível aos adipócitos. Por via de absorção direta ou através de gluconeogénese intestinal por conversão prévia a glicose? Se se verificar a absorção direta, será demonstrado que os adipócitos mesentéricos são capazes de assimilar e metabolizar frutose inferindo que o tecido adiposo mesentérico possui, em alguma medida, um acesso privilegiado a nutrientes absorvidos pelo intestino.