Background: Brown adipose tissue (BAT) preferentially oxidizes stored triglycerides (TAGs) to generate heat during acute exposure to cold. However, the time course of its activation is not well described as we are currently limited to BAT measurements before and after an acute stimulus. Magnetic resonance imaging (MRI) is a preferred modality to uncover such evidence, as it estimates TAG content via fat fraction (FF), and permits repeat scans in the same subject. As such, serial FF measurements in a defined BAT region of interest during a uniform whole-body temperature challenge is warranted. Objectives: The first objective of this study was to assess the pattern of change in supraclavicular (SCV) BAT and posterior neck subcutaneous adipose tissue (SAT; a region with an unestablished role in non-shivering thermogenesis) FF during a mild cold exposure in adult males. The second objective was to evaluate if indices of body composition were related to the pattern of cold-induced change in SCV BAT FF. The final objective was to assess the influence of warming immediately following cooling on these changes. Methods: Twelve males between the ages of 19 and 28 were recruited to this cross-sectional study. Users of tobacco, nicotine, and/or alcohol, those with contraindications for magnetic resonance imaging (MRI), and diseases, surgeries, and/or medications associated with thermogenesis were excluded. There were two study visits in total. During the initial visit, anthropometric measurements were carried out in triplicate (i.e. height and weight to determine body mass index (BMI), and body composition measurements (i.e. % body total fat and lean mass (kg)) were obtained using Dual Emission X-Ray Absorptiometry. Within 30 days of this initial visit, subjects attended a time course MRI session. At this visit, participants underwent standardized cold (3-hours at 18°C) and subsequent warm (30 minutes at 32°C) exposures using a water-perfused suit while lying in a 3 Tesla MRI scanner, and the temperature of the water entering and leaving the suit was recorded throughout. FF in the SCV region and posterior neck SAT was measured at defined intervals during both temperature challenges. Separate time course plots of the mean reduction in FF from baseline were constructed for the cooling and warming phases. For the first objective, the rate and magnitude of FF changes in SCV BAT and posterior neck SAT over defined time intervals were determined through calculations of slope and area under the curve (AUC), respectively. Identification of the earliest point of change from baseline, and the point at which changes were no longer different from those measured after 3 hours of cooling, were accomplished through paired comparisons using a random-slope linear mixed model with measures at 0 minutes and 180 minutes used as the reference values, respectively. A random-intercept multilevel regression model was used to define the cold-induced change in FF over time. For the second objective, a Spearman rank-order correlation assessed the association between indices of body composition (i.e. BMI and % total body fat) and indices of BAT activity (i.e. AUC and FF reduction) at time points of interest as identified by objective 1. Results: The mean±SD of BMI, LMI, and % total body fat were 24.7±2.8kg/m2, 17.6±1.6kg/m2 and 25.0±7.4%, respectively. Seven of the twelve subjects completed three hours of cold exposure (58.3%), and a further five endured at least one hour. A significant cold-induced reduction in SCV BAT FF was detected at 10 minutes following the onset of cold exposure (mean difference = -1.6%; p=0.005), and changes in FF beyond 30 minutes of cooling were similar to those measured after three hours (p<0.05). Meanwhile, the posterior neck SAT did not experience significant cold-induced changes in FF. A novel attempt at identifying a quadratic model to predict one’s BAT-specific response to a cold challenge was carried out, and the intercept, time, time2, and intraclass correlation coefficient (i.e. parameters which described the relationship between FF and time) were highly significant (p<0.001). Although every participant had a measurable decline in FF, those with a higher BMI and % body fat had a smaller magnitude of change throughout the time course. In particular, a strong negative correlation between BMI and AUC FF decline existed as soon as 10 minutes following the onset of cold (rho=-0.786), indicating that those with a lower BMI had a larger magnitude of change in SCV BAT FF at this point. Finally, warming did not visually influence the trajectory of SCV BAT FF. Limitations: Only seven of the twelve participants completed the full 180 minutes of cold exposure, which further limited the already low statistical power of this study. Moreover, complementary measures of BAT activity, such as energy expenditure, and objective measurements of shivering, such as electromyography, could not be evaluated. Conclusions: These findings suggest that significant cold-induced changes in BAT FF occur much sooner than three hours. Thus, a shorter duration of cold exposure may be considered in future studies using MRI to detect BAT activity, as this could increase the feasibility of gathering larger and younger sample populations. / Thesis / Master of Science (MSc)

<p>The inverse problem of modeling biochemical processes mathematically from measured time course data falls into the category of system identification and parameter estimation. Analyzing the time course data would provide valuable insights into the model structure and dynamics of the biochemical system. Based on the types of biochemical reactions, such as metabolic networks and genetic networks, several modeling frameworks have been proposed, developed and proved effective, including the Michaelis-Menten equation, the Biochemical System Theory (BST), etc. One bottleneck in analyzing the obtained data is the estimation of parameter values within the system model.</p> <p>As most models for molecular biological systems are nonlinear with respect to both parameters and system state variables, estimation of parameters in these models from experimental measurement data is thus a nonlinear estimation problem. In principle, all algorithms for nonlinear optimization can be used to deal with this problem, for example, the Gauss-Newton iteration method and its variants. However, these methods do not take the special structures of biological system models into account. When the number of parameters to be determined increases, it will be challenging and computationally expensive to apply these conventional methods.</p> <p>In this research, several methods are proposed for estimating parameters in two classes of widely used biological system models: the S-system model and the linear fractional model (LFM), by utilizing their structure specialties. For the S-system, two estimation methods are designed. 1) Based on the two-term structure (production and degradation) of the model, an alternating iterative least squares method is proposed. 2) A separation nonlinear least squares method is proposed to deal with the partially linear structure of the model. For the LFM, two estimation methods are provided. 1) The separation nonlinear least squares method can also be adopted to treat the partially linear structure of the LFM, and moreover a modified iterative version is included. 2) A special strategy using the separation principle and the weighted least squares method is implemented to turn the cost function into a quadratic form and thus the estimates for parameters can be analytically solved. Simulation results have demonstrated the effectiveness of the proposed methods, which have shown better performance in terms of estimation accuracy and computation time, compared with those conventional nonlinear estimation methods.</p>

<p>The inverse problem of modeling biochemical processes mathematically from measured time course data falls into the category of system identification and parameter estimation. Analyzing the time course data would provide valuable insights into the model structure and dynamics of the biochemical system. Based on the types of biochemical reactions, such as metabolic networks and genetic networks, several modeling frameworks have been proposed, developed and proved effective, including the Michaelis-Menten equation, the Biochemical System Theory (BST), etc. One bottleneck in analyzing the obtained data is the estimation of parameter values within the system model.</p> <p>As most models for molecular biological systems are nonlinear with respect to both parameters and system state variables, estimation of parameters in these models from experimental measurement data is thus a nonlinear estimation problem. In principle, all algorithms for nonlinear optimization can be used to deal with this problem, for example, the Gauss-Newton iteration method and its variants. However, these methods do not take the special structures of biological system models into account. When the number of parameters to be determined increases, it will be challenging and computationally expensive to apply these conventional methods.</p> <p>In this research, several methods are proposed for estimating parameters in two classes of widely used biological system models: the S-system model and the linear fractional model (LFM), by utilizing their structure specialties. For the S-system, two estimation methods are designed. 1) Based on the two-term structure (production and degradation) of the model, an alternating iterative least squares method is proposed. 2) A separation nonlinear least squares method is proposed to deal with the partially linear structure of the model. For the LFM, two estimation methods are provided. 1) The separation nonlinear least squares method can also be adopted to treat the partially linear structure of the LFM, and moreover a modified iterative version is included. 2) A special strategy using the separation principle and the weighted least squares method is implemented to turn the cost function into a quadratic form and thus the estimates for parameters can be analytically solved. Simulation results have demonstrated the effectiveness of the proposed methods, which have shown better performance in terms of estimation accuracy and computation time, compared with those conventional nonlinear estimation methods.</p>

As genes with similar expression pattern are very likely having the same biological function, cluster analysis becomes an important tool to understand and predict gene functions from gene expression profi les. In many situations, each gene expression profi le only contains a few data points. Directly applying traditional clustering algorithms to such short gene expression profi les does not yield satisfactory results. Developing clustering algorithms for short gene expression profi les is necessary. In this thesis, two novel methods are developed for clustering short gene expression pro files. The fi rst method, called the network-based clustering method, deals with the defect of short gene expression profi les by generating a gene co-expression network using conditional mutual information (CMI), which measures the non-linear relationship between two genes, as well as considering indirect gene relationships in the presence of other genes. The network-based clustering method consists of two steps. A gene co-expression network is firstly constructed from short gene expression profi les using a path consistency algorithm (PCA) based on the CMI between genes. Then, a gene functional module is identi ed in terms of cluster cohesiveness. The network-based clustering method is evaluated on 10 large scale Arabidopsis thaliana short time-course gene expression profi le datasets in terms of gene ontology (GO) enrichment analysis, and compared with an existing method called Clustering with Over-lapping Neighbourhood Expansion (ClusterONE). Gene functional modules identi ed by the network-based clustering method for 10 datasets returns target GO p-values as low as 10-24, whereas the original ClusterONE yields insigni cant results. In order to more speci cally cluster gene expression profi les, a second clustering method, namely the protein-protein interaction (PPI) integrated clustering method, is developed. It is designed for clustering short gene expression profi les by integrating gene expression profi le patterns and curated PPI data. The method consists of the three following steps: (1) generate a number of prede ned profi le patterns according to the number of data points in the profi les and assign each gene to the prede fined profi le to which its expression profi le is the most similar; (2) integrate curated PPI data to refi ne the initial clustering result from (1); (3) combine the similar clusters from (2) to gradually reduce cluster numbers by a hierarchical clustering method. The PPI-integrated clustering method is evaluated on 10 large scale A. thaliana datasets using GO enrichment analysis, and by comparison with an existing method called Short Time-series Expression Miner (STEM). Target gene functional clusters identi ed by the PPI-integrated clustering method for 10 datasets returns GO p-values as low as 10-62, whereas STEM returns GO p-values as low as 10-38. In addition to the method development, obtained clusters by two proposed methods are further analyzed to identify cross-talk genes under fi ve stress conditions in root and shoot tissues. A list of potential abiotic stress tolerant genes are found.

Orientador: Carlos Alexandre Costa Crusciol / Banca: Rogério Peres Soratto / Banca: Munir Mauad / Banca: Salatier Buzeetti / Banca: Takashi Muraoka / Resumo: Com a ampliação da demanda da indústria ricinoquímica e, mais recentemente, a possibilidade do uso do óleo de mamona como biocombustível, há necessidade de realizar-se estudos referentes à absorção e acumulação de nutrientes nas diferentes fases de desenvolvimento da mamona, o que permite determinar as épocas em que os elementos são mais exigidos durante o desenvolvimento da cultura e a distribuição desses nas diferentes estruturas da planta possibilitando um manejo adequado da adubação, principalmente a nitrogenada de cobertura. Em função do exposto, objetivou-se estudar a marcha de absorção de nutrientes em dois híbridos de mamona (Híbrido Lyra, Híbrido Savana) em sistema de plantio direto conduzidos em safra e safrinha no ano agrícola de 2006 na Fazenda Experimental Lageado pertencente à Faculdade de Ciências Agronômicas da UNESP, em Botucatu-SP. O delineamento experimental utilizado foi em blocos casualizados com quatro repetições. Foram realizadas as seguintes avaliações: matéria seca acumulada, quantidade acumulada de nutrientes (N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Mo e Zn) nos caules, folhas e estruturas reprodutivas e produtividade de grãos. No cultivo de safra concluiu-se que: a) A produtividade do híbrido Lyra foi de 2.995 kg ha-1 e do Savana 2.582 kg ha-1; b) o acúmulo de matéria seca total pelo Lyra foi de 8.629 kg ha-1 e pelo Savana foi de 6.904 kg ha-1; c) Os valores em quilogramas por hectare (kg ha-1) de macronutrientes acumulados na matéria seca total pelo Lyra foram: N=180; P=61,6; K=78; Ca=77,5; Mg=33; S=30,9. No híbrido Savana foram: N=160; P=51,2; K=64; Ca=55,5; Mg=25; S=29. d) Os valores em gramas por hectare (g ha-1) de 2 micronutrientes acumulados na matéria seca total pelo Lyra foram: B=75; Cu=18; Fe=98; Mn=166; Mo=2; Zn=190. No híbrido Savana foram: B=58; Cu=11,3; Fe=93,7; Mn=95; Mo=1,2;... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The increasing demand of oil industry with the possibility of using castor bean as biofuel, requires more reseach in nutrient uptake and accumulation in all stages of castor bean growth which can lead to an appropriate fertilizer management, mainly nitrogen cover. This study aimed at determining the uptake rate in two castor bean cultivars (Lyra hybrid and Savana Hybrid) in no-tillage system in crop and no season crop in 2006. The experiment was carried out at Lageado Experimental farm from School of Agriculture - UNESP in Botucatu / São Paulo State. The experimental design was randomized blocks with four replications. Accumulated dry matter, nutrient content ( N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Mo, and Zn) in stems, leaves and flowers, yield were evaluated. The crop results showed that: a) Lyra yield was 2.995 kg ha-1 and Savana's 2.582 kg ha-1. b) The total dry matter acumulated by Lyra was 8.629 kg ha-1 and 6.904 kg ha-1 by Savana. c) The macronutrients accumulated in total dry matter by Lyra was: N= 180.0; P=61.6; K=78.0; Ca=77.5; Mg=33.0; S=30.9 kg ha-1. By Savana hybrid was: N= 160.0; P=51.2; K=64.0; Ca=55.5; Mg=25.0; S=29.0 kg ha-1. d) The micronutrients accumulated in total dry matter by Lyra was: B=75.0; Cu=18.0; Fe=98.0; Mn=166.0; Mo=2.0; Zn=190 g ha-1. By Savana hybrid was: B=58.0; Cu=11.3; Fe=93.7; Mn=95.0; Mo=1.2; Zn=100 g ha-1 . The no season crop show that: a) Lyra yield was 1.201 kg ha-1 and Savana's 494 kg ha-1 b) The accumulated dry matter by Lyra was 2.230 kg ha-1 and Savana was 1.140 kg ha-1; c) The macronutrients accumulated in total 4 dry matter by Lyra was: N= 66.5; P=22.1; K=27.7; Ca=16.0; Mg=8.7; S=12.7 kg ha-1. By Savana hybrid was: N= 32.0; P=8.4; K=13.4. Ca=11.5; Mg=4.2; S=7.3 kg ha-1. d) The micronutrients accumulated in total dry matter by Lyra was: B=48.0; Cu=18.7; Fe=1.026; Mn=151;... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:30:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-25Bitstream added on 2014-06-13T21:01:03Z : No. of bitstreams: 1 nascimento_ms_dr_botfca.pdf: 914604 bytes, checksum: 2887ca4922c6af0c8d8f9c4f190a3ce8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Com a ampliação da demanda da indústria ricinoquímica e, mais recentemente, a possibilidade do uso do óleo de mamona como biocombustível, há necessidade de realizar-se estudos referentes à absorção e acumulação de nutrientes nas diferentes fases de desenvolvimento da mamona, o que permite determinar as épocas em que os elementos são mais exigidos durante o desenvolvimento da cultura e a distribuição desses nas diferentes estruturas da planta possibilitando um manejo adequado da adubação, principalmente a nitrogenada de cobertura. Em função do exposto, objetivou-se estudar a marcha de absorção de nutrientes em dois híbridos de mamona (Híbrido Lyra, Híbrido Savana) em sistema de plantio direto conduzidos em safra e safrinha no ano agrícola de 2006 na Fazenda Experimental Lageado pertencente à Faculdade de Ciências Agronômicas da UNESP, em Botucatu-SP. O delineamento experimental utilizado foi em blocos casualizados com quatro repetições. Foram realizadas as seguintes avaliações: matéria seca acumulada, quantidade acumulada de nutrientes (N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Mo e Zn) nos caules, folhas e estruturas reprodutivas e produtividade de grãos. No cultivo de safra concluiu-se que: a) A produtividade do híbrido Lyra foi de 2.995 kg ha-1 e do Savana 2.582 kg ha-1; b) o acúmulo de matéria seca total pelo Lyra foi de 8.629 kg ha-1 e pelo Savana foi de 6.904 kg ha-1; c) Os valores em quilogramas por hectare (kg ha-1) de macronutrientes acumulados na matéria seca total pelo Lyra foram: N=180; P=61,6; K=78; Ca=77,5; Mg=33; S=30,9. No híbrido Savana foram: N=160; P=51,2; K=64; Ca=55,5; Mg=25; S=29. d) Os valores em gramas por hectare (g ha-1) de 2 micronutrientes acumulados na matéria seca total pelo Lyra foram: B=75; Cu=18; Fe=98; Mn=166; Mo=2; Zn=190. No híbrido Savana foram: B=58; Cu=11,3; Fe=93,7; Mn=95; Mo=1,2;... / The increasing demand of oil industry with the possibility of using castor bean as biofuel, requires more reseach in nutrient uptake and accumulation in all stages of castor bean growth which can lead to an appropriate fertilizer management, mainly nitrogen cover. This study aimed at determining the uptake rate in two castor bean cultivars (Lyra hybrid and Savana Hybrid) in no-tillage system in crop and no season crop in 2006. The experiment was carried out at Lageado Experimental farm from School of Agriculture - UNESP in Botucatu / São Paulo State. The experimental design was randomized blocks with four replications. Accumulated dry matter, nutrient content ( N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, Mo, and Zn) in stems, leaves and flowers, yield were evaluated. The crop results showed that: a) Lyra yield was 2.995 kg ha-1 and Savana’s 2.582 kg ha-1. b) The total dry matter acumulated by Lyra was 8.629 kg ha-1 and 6.904 kg ha-1 by Savana. c) The macronutrients accumulated in total dry matter by Lyra was: N= 180.0; P=61.6; K=78.0; Ca=77.5; Mg=33.0; S=30.9 kg ha-1. By Savana hybrid was: N= 160.0; P=51.2; K=64.0; Ca=55.5; Mg=25.0; S=29.0 kg ha-1. d) The micronutrients accumulated in total dry matter by Lyra was: B=75.0; Cu=18.0; Fe=98.0; Mn=166.0; Mo=2.0; Zn=190 g ha-1. By Savana hybrid was: B=58.0; Cu=11.3; Fe=93.7; Mn=95.0; Mo=1.2; Zn=100 g ha-1 . The no season crop show that: a) Lyra yield was 1.201 kg ha-1 and Savana’s 494 kg ha-1 b) The accumulated dry matter by Lyra was 2.230 kg ha-1 and Savana was 1.140 kg ha-1; c) The macronutrients accumulated in total 4 dry matter by Lyra was: N= 66.5; P=22.1; K=27.7; Ca=16.0; Mg=8.7; S=12.7 kg ha-1. By Savana hybrid was: N= 32.0; P=8.4; K=13.4. Ca=11.5; Mg=4.2; S=7.3 kg ha-1. d) The micronutrients accumulated in total dry matter by Lyra was: B=48.0; Cu=18.7; Fe=1.026; Mn=151;... (Complete abstract click electronic access below)

Le travail de cette thèse s’inscrit dans le cadre du projet ANR SUSoSTRESS qui a pour objectif la compréhension des mécanismes moléculaireset génétiques sous-jacents à la variabilité individuelle de réponses de stress et a collecté des données longitudinales à plusieurs niveaux biologiquessur une population d’étude porcine (race Large White). La thèse est organisé en deux partie. La première partie s’articule autour de l’analyse de données cliniques et transcriptomiques collectées à plusieurs pas de temps avant et après application de deux types de stress : injection d’ACTH et de LPS. Dans cettepartie, on cherche à développer d’un modèle fonctionnel permettant de décrire et d’intégrer au mieux l’ensemble des sources de variation génétique du fonctionnement de l’axe corticotrope et plus généralement des réponses de stress dans notre population d’étude. Plus précisément, il s’agit d’élaborer un modèle (au sens biologique du terme) décrivant les différentes réponses biologiques de stress et l’influence des variations génétiques (simples et en interaction), dans le but de prédire les leviers les plus efficaces en fonction de l’objectif de sélection. Ce travail a mis en évidence une liste de 65 gènes différentiellement exprimé au cours des réponses au stress, dont un ensemble de 8 gènes liés au au cortisol (l’hormone principale du stress) par NR3C1, le récepteur aux glucocorticoides. Ces gènes sont des biomarqueurs potentiels pouvant être fournis aux éleveurs en tant que leviers de sélection permettant un meilleur équilibre entre amélioration des caractères de production et des caractères de robustesse. La deuxième partie de ce travail s’articule autour du développement d’un outil d’analyse statistiques adapté à l’intégration de données ’omiques longitudinalesavec une variable cible d’intérêt.Nous proposons la «multiway-SIR », qui étend la méthode dual-STATIS, une méthode d’analyse de données cubiques non supervisée, au cadre de la SIR, une méthode de régression semi-paramétrique pouvant être utilisée à des fins exploratoires. Cette méthode est appliquée sur les données cliniques de l’expérience d’ACTHet permet d’y explorer l’influence de la variabilité de la réponse du cortisol à une injection d’ACTH. / This PhD thesis is part of the SUSoSTRESS project. This ANR funded project aims at improving the knowledge about molecular and genetic mechanisms underlying inter-individual variability in stress responses. Longitudinal data were collected at several biological levels on a porcine population (Large White). This work is structured in 2 parts. The first part is built around clinical and transcriptomic longitudinal data analyses collected before and after 2 types of stress factors : ACTH and LPS injection. The aim of this contribution is to develop a functional model describing all sources of genetic variation in the HPA axis activity and in stress responses in our study population.More precisely, it aims at defining a model describing the different biological stress responses and the influence of genetic variations in order to identify the most efficient selection levers according to selection goals. This work allowed for the identification of 65 differentially expressed genes during stress responses. Among them, 8 genes were highly linked to cortisol (the main stress hormone) through NR3C1 (glucocorticoid receptor (GR)). These genes are potential biomarkers and can be communicated to breeders as selection levers for a better trade-off between production and robustness traits in farmanimals. The second part is built around the development of a statistical tool suited for the data integration of repeated omicmeasurements with a real target variable.We introduce the "multiway-SIR" approach which extends the dual-STATIS (an approach to study 3-way datasets) method to the SIR framework (a semi-parametric regression model that can be used in an exploratory way). This method is illustrated on clinical data from the ACTH experiment. It allows for the exploration of the link between clinical variable response over time and inter-individual variability in the cortisol response to an ACTH injection.

No description available.

La tesis aborda el análisis estadístico de series simples y múltiples de experimentos de "Time Course Microarray" (TCM). El trabajo se centra en el desarrollo, aplicación y evaluación de métodos estadísticos específicos que consideran la problemática de este tipo de datos, tanto desde el punto de vista de selección de genes como del análisis funcional. Las técnicas desarrolladas se comparan con otros métodos del estado del arte actual evaluando las diferentes metodologías en términos de eficiencia y significado biológico de los resultados. En la tesis se incluye la descripción del funcionamiento de la tecnología de "microarrays" así como una revisión crítica de los métodos estadísticos aplicados a este tipo de datos mostrando los inconvenientes que surgen al aplicar métodos generales a series temporales de "microarrays" y justificando la necesidad de desarrollar nuevas técnicas para el análisis de TCM. La primera técnica desarrollada es maSigPro ("microarray Significant Profile") que usa análisis de regresión lineal para modelar la expresión génica y lleva a cabo una estrategia en dos pasos para seleccionar los genes diferencialmente expresados. La aplicación de la técnica multivariantes ASCA (ANOVA "Simultaneous Component Analysis") a datos de TCM da como resultado el método ASCA-genes que combina la exploración multivariante de datos con un procedimiento de selección para identificación de genes con cambios relevantes. El método ASCA es también usado para crear una estrategia de filtrado de datos de gran utilidad para eliminar el alto nivel de ruido estructural de los datos de microarrays. Por último, se desarrollan métodos estadísticos para una evaluación directa e integrada de las alteraciones que pueden sufrir las funciones génicas en TCM. Para este propósito, se ha adaptado las técnicas maSigPro, ASCA y PCA incorporándoles información funcional obteniendo las metodologías maSigFun, PCA-maSigFun y ASCA-functional. / Nueda Roldán, MJ. (2009). Statistical methods for time course microarray data [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/6061

In view of the increasing global challenge of bacterial resistance, there exists an urgent need for the rationale development of antibacterial compounds with either novel or multiple mechanisms of action. Two chalcone-derivatives, F1 and F23, demonstrated MICs within the range of 16 to >512 μg/ml against two plant pathogens (P. caratovoram and C. michiganensis subsp. michiganensis) as well as important clinical bacterial species. Both compounds displayed an MIC of 32 μg/ml against quinolone-resistant S. aureus. Whilst possessing weak activities individually, each semi-synthetic agent displayed notable synergistic action with polymyxin B against S. aureus, C. violaceum, E. coli and Ps. aeruginosa, thereby recording FICs within the range of <0.093 to 2 that indicated the existence of synergism in some instance. These chalcone compounds applied with polymyxin B displayed a notable FICindex of <0.093 against the Neisseriaceae C. violaceum, and a potential noteworthy capacity to extend the spectrum of activity of the latter antibiotic to include Gram-positive S. aureus species. F1 inhibited staphylococcal replication in broth and the combination of either of both chalcone-derivatives with polymyxin B instituted a metabolic blockage in S. aureus and other bacterial species as determined through a modified MTT reduction assay. The combined agents inflicted major disruptions to the S. aureus cytoplasmic membrane bilayer as evidenced by the release of intracellular potassium as well as the influx of Sytox Green fluorescent stain. Notable levels of cell membrane potential dissipation, leakage of intracellular potassium ions and blockage of reducing enzymes activities occurred within the first 30 minutes, well in advance of significant loss in cell viability that was recorded usually after 4 – 8 hours, suggesting these activities were prerequisites to cell death. In erythrocyte lysis assay, the synergistic combinations of 128 μg/ml of either of both chalcone derivatives with 128 μg/ml polymyxin B displayed the lowest degree of haemolysis, followed by that occurring with 32 μg/ml of the chalcone-derivatives combined with 256 μg/ml of the polypeptide antibiotic. In conclusion, further structure activity modifications aimed at improving the aqueous solubility of these chalcone-derivatives as well as the antibacterial activity recorded for certain combination concentrations of polymyxin B with either of these semi-synthetic agents may be required before considerations are made for the possibility for potential external formulations. Such preparations may include antiseptic creams, lotions, ointments, as well as aerosols that can be applied with nebulizers in targeted delivery for such cases like cystic fibrosis.