A critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates. Bioelectrochemical techniques offer a promising method to alleviate redox imbalances during the synthesis of biochemicals. Broadly, these techniques reduce intracellular NAD+ to NADH and therefore manipulate the cell’s redox balance. The cellular response to such redox changes and the additional reducing can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can improve product yields and/or productivity. We have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of E. coli. The results elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity.

The increasing prevalence of apicomplexan parasites such as Plasmodium, Toxoplasma, and Cryptosporidium represents a significant global healthcare burden. Treatment options are increasingly limited due to the emergence of new resistant strains. We postulate that parasites have evolved distinct metabolic strategies critical for growth and survival during human infections, and therefore susceptible to drug targeting using a systematic approach. I developed iCS306, a fully characterized metabolic network reconstruction of the model organism Toxoplasma gondii via extensive curation of available genomic and biochemical data. Using available microarray data, metabolic constraints for six different clinical strains of Toxoplasma were modeled. I conducted various in silico experiments using flux balance analysis in order to identify essential metabolic processes, and to illustrate the differences in metabolic behaviour across Toxoplasma strains. The results elucidate probable explanations for the underlying mechanisms which account for the similarities and differences among strains of Toxoplasma, and among species of Apicomplexa.

A critical concern in metabolic engineering is the need to balance the demand and supply of redox intermediates. Bioelectrochemical techniques offer a promising method to alleviate redox imbalances during the synthesis of biochemicals. Broadly, these techniques reduce intracellular NAD+ to NADH and therefore manipulate the cell’s redox balance. The cellular response to such redox changes and the additional reducing can be harnessed to produce desired metabolites. In the context of microbial fermentation, these bioelectrochemical techniques can improve product yields and/or productivity. We have developed a method to characterize the role of bioelectrosynthesis in chemical production using the genome-scale metabolic model of E. coli. The results elucidate the role of bioelectrosynthesis and its impact on biomass growth, cellular ATP yields and biochemical production. The results also suggest that strain design strategies can change for fermentation processes that employ microbial electrosynthesis and suggest that dynamic operating strategies lead to maximizing productivity.

<p>Genetic variation is a natural occurrence in the genome that contributes to the phenotypic differences observed between individuals as well as the phenotypic outcomes of various diseases, including infectious disease and cancer. Single nucleotide polymorphisms (SNPs) have been identified as genetic factors influencing host susceptibility to infectious disease while the study of copy number variation (CNV) in various cancers has led to the identification of causal genetic factors influencing tumor formation and severity. In this work, we evaluated the association between genomic variation and disease phenotypes to identify SNPs contributing to host susceptibility in Staphylococcus aureus (<italic>S. aureus</italic>) infection and to characterize a nervous system brain tumor, known as oligodendroglioma (OD), using the CNV observed in tumors with varying degree of malignancy.</p><p>Using SNP data, we utilized a computational approach, known as in silico haplotype mapping (ISHM), to identify genomic regions significantly associated with susceptibility to <italic>S. aureus</italic> infection in inbred mouse strains. We conducted ISHM on four phenotypes collected from <italic>S. aureus</italic> infected mice and identified genes contained in the significant regions, which were considered to be potential candidate genes. Gene expression studies were then conducted on inbred mice considered to be resistant or susceptible to <italic>S. aureus</italic> infection to identify genes differentially expressed between the two groups, which provided biological validation of the genes identified in significant ISHM regions. Genes identified by both analyses were considered our top priority genes and known biological information about the genes was used to determine their function roles in susceptibility to <italic>S. aureus</italic> infection.</p><p> We then evaluated CNV in subtypes of ODs to characterize the tumors by their genomic aberrations. We conducted array-based comparative genomic hybridization (CGH) on 74 ODs to generate genomic profiles that were classified by tumor grade, providing insight about the genomic changes that typically occur in patients with OD ranging from the less to more severe tumor types. Additionally, smaller genomic intervals with substantial copy number differences between normal and OD DNA samples, known as minimal critical regions (MCRs), were identified among the tumors. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques. </p><p> The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques.</p><p> The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments.</p> / Dissertation

An in silico design algorithm has been developed to design binding ligands for protein targets of known three-dimensional structure. In this method, the binding energy of a candidate ligand is used to ascribe it a probability of binding. A sample of a virtual library of candidate ligands is then used to ascribe implicit weights to all the ligands in the library. These weights are used to obtain virtual sub-libraries which collectively carry a greater probability to bind to the target. This algorithm is presented along with validation studies on the different algorithmic components, demonstrating how optimization of the design method can be best achieved.

Les pesticides appartiennent à la grande famille des polluants organiques. Ils sont destinés à lutter contre les parasites des cultures au sens large.La diffusion des pesticides dans la nature engendre une pollution des différents compartiments de la biosphère (eau, sol et atmosphère) et peut induire des effets toxiques aigus sur les êtres vivants de la biomasse terrestre et aquatique. Il est aujourd'hui démontré que certains pesticides sont des perturbateurs endocriniens et présentent en particulier des effets cancérogènes et mutagènes chez l'être humain. Les pesticides peuvent subir différents processus de transformation dans le cycle biologique naturel (biodégradation, volatilisation, irradiation solaire...) ou suite aux traitements appliqués dans les filières de potabilisation des eaux naturelles et dans les stations de retraitement des eaux usées. La présence de produits de dégradation de pesticides dans notre environnement est d'autant plus alarmante que leurs structures et leurs toxicités potentielles demeurent généralement inconnues. Des molécules qui appartiennent à deux familles de pesticides ont été choisies pour cette étude : les herbicides, représentés par le metholachlore, et les fongicides, représentés par la procymidone, le pyrimethanil et le boscalid. Le premier volet de la thèse a porté sur le développement d'une stratégie analytique permettant de caractériser les structures de composés issus de la dégradation par photolyse de ces pesticides. Le deuxième volet a porté sur l'estimation de la toxicité des produits de dégradation à l'aide d'une base de test in silico. L'identification des produits de dégradation a été réalisée grâce à deux techniques d'analyses complémentaires : la chromatographie en phase gazeuse couplée à un spectromètre de masse "multi étapes" (GC-MSn) et la chromatographie en phase liquide couplée à un spectromètre de masse en tandem (LC-MS/MS). L'estimation de la toxicité des produits de dégradation a été réalisée grâce au programme T.E.S.T. QSAR récemment développés pour tenter de prévoir la toxicité des molécules. La stratégie d'élucidation structurale des produits de dégradation des pesticides étudiés est basée sur l'étude des mécanismes de fragmentation des molécules mères des produits de dégradation. La masse molaire des molécules mères a été précisée par l'identification des masses sur charge (m/z) des ions pseudo moléculaires obtenus à l'aide des différentes modes d'ionisations en spectrométrie de masse : ionisation électronique (EI) et ionisation chimique (CI) en GC-MS et ionisation par électronébulisation (ESI) en LC-MS. La stratégie utilisée pour l'élucidation structurale des produits de dégradation est avérée très efficace, la plupart des structures chimiques des produits formés ayant été élucidées. Une étude cinétique a été réalisée pour visualiser l'apparition et la disparition des produits de dégradation durant la photolyse et pour donner une idée sur le taux de disparition de la molécule mère pour chaque pesticide. Les résultats de photolyse ont montré que le méthalochlore, le procymidone et le boscalid ont été dégradés sous irradiation après un certain temps en libérant des produits de dégradation avec des structures proches ou différentes des molécules mères. Tandis que le pyrimethanil a présenté une grande stabilité pendant 8 heurs d'irradiation (quantité restante après irradiation est de 60%) et a donné des produits de dégradation avec des quantités modérées. Concernant l'estimation de la toxicité, la plupart des produits de dégradation identifiés présentent des toxicités équivalentes ou plus importantes que celles des molécules d'origine. Les méthodes chimiques d'analyses utilisées et l'estimation de toxicités des produits de dégradations identifiés sont avérées complémentaires et indispensables pour mettre en évidence la présence d'autres produits polluants toxiques qui émergent dans l'environnement sans vrai contrôle.

Alzheimer’s disease (AD) is a progressive, degenerative neurological disorder for which there is no cure. The causative agent is ?-amyloid (A?) which becomes neurotoxic upon conformational change from ?-helix to ?-sheet. In silico methods have been used to indentify endogenous small molecules of the brain that are capable of binding to A? to inhibit conformational changes; this is a novel approach to the disease. Through the use of computational methods, several small molecules that are endogenous to the brain, such as phosphoserine, have been identified as being capable of binding to the monomeric forms of A?; in vitro studies support their role as anti-aggregants. One of the small molecules identified through these in silico methods, 3-hydroxyanthranilic acid (3HAA) has been developed through the use of Quantitative Structure-Activity Relationship (QSAR) studies to design more potent analogues. These in silico studies have also examined the capacity of synthetic compounds (structurally similar to endogenous molecules) to bind to both A? and other proteins affiliated with AD. Results indicate the potential for a single molecule to bind “promiscuously” to multiple proteins bearing a common BBXB (where B is a basic amino acid) motif affiliated with AD. This will allow for the development of molecules to target AD in a multifaceted approach. Furthermore, these small molecules can be selected through the use of “physinformatics” to bind with equal efficacy to the HHQK and LVFF regions (which play a role in the misfolding process) of A?; this will prevent conformational changes of the protein. A novel diagnostic imaging agent for AD has also been developed through computational methods; solapsone (formerly used to treat leprosy) has been identified as being structurally similar to species that bind to A? to initiate conformational changes. Results show that solapsone can chelate gadolinium, used in MRI, and bind to the soluble forms of A?, allowing for imaging of the toxic species in the human brain, and thus a definitive diagnosis of AD (which is not currently possible with living patients). Computational methods have proved useful in developing a new approach to treating AD, and designing a novel imaging agent.

No description available.

The increasing prevalence of apicomplexan parasites such as Plasmodium, Toxoplasma, and Cryptosporidium represents a significant global healthcare burden. Treatment options are increasingly limited due to the emergence of new resistant strains. We postulate that parasites have evolved distinct metabolic strategies critical for growth and survival during human infections, and therefore susceptible to drug targeting using a systematic approach. I developed iCS306, a fully characterized metabolic network reconstruction of the model organism Toxoplasma gondii via extensive curation of available genomic and biochemical data. Using available microarray data, metabolic constraints for six different clinical strains of Toxoplasma were modeled. I conducted various in silico experiments using flux balance analysis in order to identify essential metabolic processes, and to illustrate the differences in metabolic behaviour across Toxoplasma strains. The results elucidate probable explanations for the underlying mechanisms which account for the similarities and differences among strains of Toxoplasma, and among species of Apicomplexa.

Human cytomegalovirus (CMV) is a significant infectious agent causing disease in immunocompromised HIV-infected patients, transplant recipients, and neonates. The current antiviral therapeutic strategy against CMV is limited in its utility due to the inherent toxicity and lack of bioavailability of currently available anti-CMV agents, ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS). The development of the prodrug of GCV, valganciclovir (val-GCV), has vastly improved the bioavailability profile of GCV. However, val-GCV demonstrates limited effectiveness against tissue-invasive CMV diseases as side effects involved with traditional intravenously administered GCV such as haematologic and reproductive toxicities remain. In addition, the emergence of antiviral resistant CMV mutant strains due to prolonged treatment with currently available antivirals necessitates the development of novel anti-CMV agents with reduced toxicity and improved bioavailability. In this study, select groups of novel compounds were analysed for their potential for further development as anti-CMV agents. Three groups of compounds were identified based on two screening methods which included the computer simulated screening process of compounds known as in silico screening and the traditional method of random screening. The first group of compounds (CATi) were identified by in silico screening against the CMV DNA polymerase catalytic aspartate triad, resulting in the identification of 31 compounds with the potential for inhibitory activity against CMV. The second group of compounds (PRO-i) were identified through in silico screening against the CMV protease, identifying a total of 18 lead compounds exhibiting structural complementarity with CMV protease. The third and final group of compounds (TPEX) were identified through random screening and consisted of plant extracts purified from tropical plants. All three compounds were initially screened for cytotoxicity against human fibroblasts. Plaque reduction assays were performed using compounds with acceptable levels of cytotoxicity to determine the ability of the compounds to inhibit the replication of the laboratory antiviral sensitive CMV strain, Towne. Two of the PRO-i compounds demonstrated good antiviral activity against CMV. Eleven percent (2/18) of the PRO-i compounds inhibited CMV replication, with PRO-i-43 and PRO-i??-44 displaying mean 50% inhibitory concentrations (IC50) of 4.8 ?? 1.2 ??M and 8.04 ??M, respectively. PRO-i-43 and PRO-i-44 are thus good candidates for further development as novel antiviral agents against CMV. The majority of CATi and TPEX compounds displayed significant cytotoxicity against human fibroblasts and compounds with acceptable levels of cytotoxicities did not significantly inhibit CMV replication. However, the identification of compounds with low cytotoxicities provides a good foundation for further development of novel anti-CMV agents with superior antiviral activity. In silico screening against three-dimensional viral protein models is a useful strategy for the identification of novel antiviral agents with the potential for inhibitory activity against CMV. Structural modification to produce potent derivatives of the identified anti-CMV compounds (PRO-i-43 and PRO-i-44) is a good option for the further development of novel antiviral agents against CMV. Such further examination of the identified compounds with anti-CMV activity is required to investigate their activity against not only antiviral sensitive CMV strains but also resistant CMV strains. Further investigations will yield new insights into their target, allowing further identification of compounds with potential anti-CMV activity with pharmaceutical application.