O estresse oxidativo, causado por níveis aumentados de espécies reativas de oxigênio (ROS), pode causar danos celulares. Várias enzimas, como as subunidades da alquil-hidroperóxido redutase (AhpC e AhpF) e as superóxido dismutases (SOD), são responsáveis por remover as ROS. Os mecanismos de regulação da expressão gênica de C. crescentus para os genes ahpC, sodA, sodB e sodC, foram analisados com fusões de transcrição ao gene repórter lacZ, permitindo a quantificação da expressão por ensaios da atividade de b-galactosidase, e RT-PCR quantitativo. As culturas foram cultivadas em meio PYE ou M2 e a expressão de cada gene foi avaliada na presença de peróxido de hidrogênio (H2O2), tert-butil hidroperóxido (tBOOH), paraquat, menadiona, pirogalol, FeSO4 ou DDPi. Em C. crescentus ahpC é induzido por peróxidos e regulado por OxyR. As SODs são induzidas principalmente por superóxidos, sodB e sodC possuem indução de fase na fase estacionária e possivelmente estão sob o controle do sigma J, enquanto o gene sodA é regulado por sigma F e sigma J. / Oxidative stress, caused by increased levels of reactive oxygen species, can lead to damage in all cellular components. Several enzymes, as subunit of alkyl hydroperoxide reductase (AhpC and AhpF) and superoxide dismutases (SOD), are responsible for removing ROS. Mechanisms of gene expression of C. crescentus for genes ahpC, sodA, sodB and sodC, were evaluated with transcription fusions with the lacZ reporter gene were constructed, allowing the quantification of expression by b-galactosidase activity assays, furthermore we analyze of the gene expression by qRT-PCR assay. The cultures were grown in PYE and M2 media, and gene expression was evaluated in the presence of hydrogen peroxide (H2O2), tert-butyl hydroperoxide (tBOOH), paraquat, menadione, pyrogallol, FeSO4 or DPPi. In C. crescentus ahpC is induced by peroxides and is under the control of OxyR. The SODs are mainly induced by superoxide, sodB and sodC are induction in stationary phase and are possibly under the control of sigma J, while the sodA gene is regulated by sigma F and sigma J.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-29T13:28:57Z No. of bitstreams: 1 douglasferreirabrum.pdf: 632780 bytes, checksum: 1da883a558292ba219387c3fdf6f98af (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-29T19:37:42Z (GMT) No. of bitstreams: 1 douglasferreirabrum.pdf: 632780 bytes, checksum: 1da883a558292ba219387c3fdf6f98af (MD5) / Made available in DSpace on 2017-05-29T19:37:42Z (GMT). No. of bitstreams: 1 douglasferreirabrum.pdf: 632780 bytes, checksum: 1da883a558292ba219387c3fdf6f98af (MD5) Previous issue date: 2013-08-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Esse trabalho visa descrever o espaço de Módulos de m-uplas geodésicas complexas distintas em H2c nos casos regular, especial e degenerado. Para tal fim faremos uso da matriz de Gram e dos invariantes (d-invariantes, δ-invariantes, invariante angular e invariantes parabólicos) que descrevem unicamente a classe de congruência de PU(2, 1) de m-uplas ordenadas de geodésicas complexas distintas nos diferentes casos supracitados. / This work aims to describe the Modules space of m-tuples distinct complex geodesics in H2c in the cases regular, special and degenerate. To this end we use the Gram matrix and the invariant (d-invariant, δ-invariants, angular invariant and parabolic invariants) that define uniquely the PU(2,1)-congruence class of ordered m-uplas of distinct complex geodesics in the different cases above.

Este trabalho descreve a construção de plasmídeos para expressão e ancoragem de proteínas de alta afinidade a íons Hg2+ e As5+. Os genes merR e arsR de C. metallidurans foram inseridos no vetor que contém o sistema para expressão e ancoragem de proteínas heterólogas em bactérias Gram-negativas originando os plasmídeos pCM-Hg e pCM-As. MerR e ArsR foram produzidas sob comando do promotor pan. E. coli recombinantes apresentaram resistência 100% superior a Hg2+ e As5+. C. metallidurans/pCM-As apresentou MIC > Na3As02 1000 mM sendo a Gram-negativa com maior capacidade de sobrevivência a íons As5+. Os plasmídeos elevaram a sobrevivência das bactérias estudadas, podendo ser usados para aumentar índices de sobrevivência e fornecer viabilidade a outras cepas. Células recombinantes apresentaram capacidade de adsorver Hg2+ ou As5+ do meio em níveis superiores às linhagens selvagens. As bactérias descritas são excelentes candidatas para biorremediação. Este trabalho apresenta pela primeira vez a ancoragem da proteína ArsR na superfície celular de um micro-organismo. / This work describes the construction of plasmids for expression and anchoring of high affinity proteins to Hg2+ or As5+ ions. C. metallidurans merR and arsR genes were inserted into the vector which contains the system for expression and anchoring of heterologous proteins in Gram-negative bacterias origining the plasmids pCM-Hg and pCM-As. MerR and ArsR were produced under pan promoter command. Recombinant E. coli showed resistance 100% higher to Hg2+ and As5+. C. metallidurans/pCM-As showed MIC > Na3As02 1000 mM being the most resistance Gram-negative able to survive in As5+ sites. The plasmids increased the studied Gram-negatives bacterial surviving and they can be utilized in other strains to increase the surviving levels and supply viability. Recombinant cells showed ability for adsorption of Hg2+ or As5+ from the media in enhanced levels as compared to the wild type. The described bacterias are excellent candidates for bioremediation. This work presents for the first time the cell surface display of ArsR protein on a microorganism.

L’hème est une molécule essentielle à de nombreuses fonctions bactériennes. Cependant, cette molécule génère des radicaux libres qui lui confèrent des propriétés toxiques. Nous avons caractérisé les mécanismes de l’homéostasie de l’hème impliquant le transporteur d’efflux HrtBA. Chez L. lactis nous avons démontré que HrtBA empêche l’accumulation membranaire et intracellulaire d’hème exogène par un mécanisme ménaquinone dépendant. HrtBA est aussi présent chez de nombreux pathogènes. Chez S. agalactiae, la transcription de HrtBA est régulée par un système à deux composants HssRS. Le senseur HssS reconnait l’hème exogène internalisé. Pour élucider le rôle de l’hème de l’hôte dans la virulence de S. agalactiae, un modèle d’infection systémique chez la souris utilisant la luminescence (lux) et l’imagerie in vivo (IVIS) a été mis en place. Le suivi de la luminescence de bactéries hypersensibles à l’hème (ΔhrtBA) montre que l’hème de l’hôte est toxique et que la capacité de S. agalactiae à assurer son homéostasie est déterminante pour ’linfection. De manière similaire, en montrant que le métabolisme respiratoire est indispensable pour l’infection (ΔcydA), S. agalactiae dépend donc de sa capacité à acquérir l’hème de l’hôte pour être infectieuse. En utilisant le promoteur de HrtBA couplé à l’opéron lux, nous avons étudié la capacité de S. agalactiae à détecter et acquérir l’hème in vivo au cours de l’infection. Nos résultats montrent que l’hème de l’hôte est particulièrement biodisponible dans le foie. Au contraire dans le cerveau, l’hème n’est pas détecté par la bactérie. Nos résultats démontrent que l’hème de l’hôte est un paramètre important des capacités d’adaptation de S. agalactiae à son hôte lors de l’infection. Bloquer HrtBA ou le senseur d’hème HssS pourrait constituer une cible pour la recherche antibiotique chez S. agalactiae ou d’autres pathogènes. Enfin, nous avons démontré chez E. faecalis que l’expression de HrtBA est aussi dépendante d’un système à deux composants. Nous avons utilisé la même stratégie que pour S. agalactiae pour créer un senseur d’hème spécifique qui a permis de démontrer pour la première fois que E. faecalis rencontre et utilise l’hème du tractus digestif. / Heme is a redox-reactive molecule with essential function in bacterial metabolism. However, this molecule generates reactive oxygen species responsible for its toxicity. We characterized the mechanism of heme homeostasis involving the efflux transporter HrtBA. In L. lactis, we demonstrated that HrtBA prevents from membrane and intracellular accumulation of internalized exogenous heme thanks to a menaquinone dependent mechanism. HrtBA is also present in several pathogens. In S. agalactiae, the transcription of HrtBA is regulated by a two-component system HssRS. The HssS sensor recognizes internalized exogenous heme. To clarify the role of heme of the host in the virulence of S. agalactiae, a systemic infection model in mice using luminescence (lux) and in vivo imaging (IVIS) has been set up. The monitoring of luminescence generated by heme hypersensitive (ΔhrtBA) bacteria shows that heme of host is toxic and that the capability of S. agalactiae to control heme homeostasis is crucial for infection. In the same way, by demonstrating that respiratory metabolism is crucial for infection (ΔcydA), we demonstrated that S. agalactiae depends on its capacity to acquire the heme of the host to become infectious. By using the HrtBA promoter coupled with lux operon, we studied the capacity of S. agalactiae to detect and to acquire heme in vivo during the infection. Our results show that host heme is especially biodisponible in the liver. On the contrary, heme is not detected by bacteria in the brain. Our results prove that heme of the host is an important parameter for the adaptation of S. agalactiae to its host during infection. Blocking HrtBA or heme sensor HssS could so be a target for antibiotic research against S. agalactiae and other pathogens. Finally, we show in E. faecalis that HrtBA expression also depends on a two-component system. We used the same strategy as in S. agalactiae to create a specific heme sensor that allowed us to demonstrate for the first time that E. faecalis meets and uses heme in the digestive tract.

Les défaut de la vision sont analysés et classés à partir des caractéristiques mathématiques du front d’onde de l’oeil considéré. Après avoir présenté la méthode actuelle basée sur la décomposition du front d’onde dans la base orthonormale de Zernike ainsi que certaines de ses limitations, on propose ici une nouvelle base de décomposition. Celle-ci repose sur l’utilisation del’espace des fronts d’onde polynomiaux de valuation supérieure ou égale à L + 1 (où L est un entier naturel) et permet de décomposer de manière unique un front d’onde polynomial en la somme d’un front d’onde polynomial de bas degré (inférieur ou égal à L) et un front d’onde polynomial de haute valuation (supérieure ou égal à L + 1). En choisissant L = 2, une nouvelle décomposition est obtenue, appelée D2V3, où le front d’onde polynomial de haut degré ne comporte pas de termes de degré radial inférieur ou égal à deux. Cette approche permet de dissocier parfaitement les aberrations optiques corrigibles ou non par le port de lunettes. Différents cas cliniques présentés dans la dernière section permettent de mettre en évidence l’intérêt de cette nouvelle base de décomposition. / The eye vision defaults are analyzed and classified by studyingthe corresponding eye wavefront. After presenting the orthogonal basis, called the Zernike basis, that is currently used for the medical diagnosis, a new decomposition basis is built. It is based on the use of the space of polynomials of valuation greater or equal to L+1 (for L a natural integer). It allows to uniquely decompose a polynomial wavefront into the sum of a polynomial of low degree (lesser or equal to L) and a polynomial of high valuation (greater or equal to L +1). By choosing L = 2, a new decomposition, called D2V3, is obtained where the polynomial wavefront of high degree does not include terms of radial degree lesser or equal to 2. In particular, it allows to quantify perfectly the aberrations that can be corrected by eyeglasses or not. Various clinical examples clearly show the interest of this new basis compared to a diagnosis based on the Zernike decomposition.

Antibiotic resistance in multidrug resistant bacteria cause high mortality rates worldwide, where there has been over 1,000,000 deaths reported as of the year 2019. Antibiotics were thought to be the cure for fighting infectious diseases and preventing further spreading of infection. This became a major problem due to bacteria evolving and developing mechanisms for resistance. The purpose of this review was to see if there are differences in the resistance mechanism of gram negative and gram positive bacteria, focusing mainly on the six most common multidrug resistant pathogenic bacteria; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae and Streptococcus pneumoniae. The results show that there is a difference in the resistance mechanism between gram positive and gram negative multidrug resistant bacteria. The difference in resistant mechanisms is due to the cell wall compositions of gram negative and gram positive bacteria. The main difference as to why the gram negative bacteria have more resistance is due to the outer membrane. Antibiotics have a hard time to diffuse through and into the cell, that is they can easily decrease their outer membrane permeability. Gram positive bacteria lack an outer membrane which makes them become more susceptible to antibiotics. The most common antibiotic resistance mechanisms in gram negative bacteria are outer membrane mechanisms such as lipid A and lipopolysaccharide modification as well as mutations in porin channels. On the other hand, the most common resistance mechanisms for gram positive bacteria are point mutations especially in penicillin binding proteins as well mutations in the rpoB gene. One important gram positive bacteria is Methicillin resistant Staphylococcus aureus, which developed a new mechanism against antibiotics, a missense mutation and mutation on the promoter region in penicillin binding protein 4. Recently new research has come forward showing that N-chlorotaurine (NCT) inhibits resistance in both gram positive and gram negative multidrug resistant bacteria. The research on NCT is still fairly new and only time will tell if this method of inhibiting resistance will be used in the future. This review highlights the importance and concern of multidrug resistance bacteria, especially due to bacteria being able to rapidly evolve when antibiotics are used incorrectly. It is important to understand the differences in resistance between gram negative and gram positive bacteria and how resistance spreads. This knowledge can be used to develop antibiotics that treat infections. It is however still a challenge to overcome resistance in multidrug resistant bacteria due to evolutionary adaptation especially through horizontal gene transfer, where resistant bacteria can adapt to changing conditions. / Antibiotikaresistens hos multiresistenta gramnegativa och grampositiva bakterier orsakar hög dödlighet över hela världen, där det har rapporterats över 1,000,000 dödsfall för år 2019. Antibiotika ansågs vara botemedlet för att bekämpa infektionssjukdomar och förhindra ytterligare spridning av infektioner. Detta blev ett stort problem på grund av att bakterier utvecklades mekanismer för resistens, vilket gör att de kan överleva när de behandlas med antibiotika. Syftet med denna studien är att se om det finns skillnader i resistensmekanismener för gramnegativa och grampositiva bakterier, med fokus på de sex vanligaste multiresistenta bakterierna; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae och Streptococcus pneumoniae. Resultaten visar att det finns en skillnad i resistensmekanismen mellan grampositiva och gramnegativa multiresistenta bakterier. Skillnaden i resistenta mekanismer beror på cellväggssammansättningen av gramnegativa och grampositiva bakterier. Den största skillnaden till varför de gramnegativa bakterierna har mer resistens beror på det yttre membranet. Antibiotika har svårt att penetrera genom och in i cellen genom att minska deras yttre membranpermeabilitet. Grampositiva bakterier saknar ett yttre membran som gör att de blir mer mottagliga för antibiotika. De vanligaste antibiotikaresistensmekanismerna hos gramnegativa bakterier är yttre membranmekanismer som lipid A och lipopolysackaridmodifiering samt mutationer i porinkanaler. De vanligaste resistensmekanismerna för grampositiva bakterier är punktmutationer, särskilt i penicillinbindande proteiner samt mutationer i rpoB genen. En viktig grampositiv bakterie är Meticillin-resistent Staphylococcus aureus, som utvecklade en ny mekanism mot antibiotika, en missense-mutation och mutation på promotorregionen i penicillinbindande protein 4. Nyligen har ny forskning kommit fram som visar att N-klorotaurin (NCT) hämmar resistens i både grampositiva och gramnegativa multiresistenta bakterier. Forskningen om NCT är fortfarande ny och bara tiden kommer att utvisa om denna metod för att hämma resistens kommer att användas i framtiden. Den här studien belyser vikten och oron för multidresistena bakterier, särskilt på grund av att bakterier snabbt kan utveckla antibiotikaresistens när antibiotika används på fel sätt. Det är viktigt att förstå skillnaderna i resistens mellan gramnegativa och grampositiva bakterier och hur resistens sprids inom resistenta bakterier. Denna kunskap kan användas för att utveckla antibiotika som behandlar infektioner orsakade av både gramnegativa och grampositiva bakterier. Det är fortfarande en utmaning att övervinna resistens hos multiresistenta bakterier på grund av evolutionär anpassning särskilt genom horisontell genöverföring, där resistenta bakterier kan anpassa sig till förändrande förhållanden.

No / A library of 53 benzimidazole derivatives, with substituents at positions 1, 2 and 5, were synthesized and screened against a series of reference strains of bacteria and fungi of medical relevance. The SAR analyses of the most promising results showed that the antimicrobial activity of the compounds depended on the substituents attached to the bicyclic heterocycle. In particular, some compounds displayed antibacterial activity against two methicillin-resistant Staphylococcus aureus (MRSA) strains with minimum inhibitory concentrations (MICs) comparable to the widely-used drug ciprofloxacin. The compounds have some common features; three possess 5-halo substituents; two are derivatives of (S)-2-ethanaminebenzimidazole; and the others are derivatives of one 2-(chloromethyl)-1H-benzo[d]imidazole and (1H-benzo[d]imidazol-2-yl)methanethiol. The results from the antifungal screening were also very interesting: 23 compounds exhibited potent fungicidal activity against the selected fungal strains. They displayed equivalent or greater potency in their MIC values than amphotericin B. The 5-halobenzimidazole derivatives could be considered promising broad-spectrum antimicrobial candidates that deserve further study for potential therapeutic applications.

Bacterial infections are a major cause of morbidity and mortality in both the community and nosocomial settings, particularly among the elderly and chronically ill. Sepsis is the body’s response to antigens and toxins released by the invasive pathogenic organisms that cause infection. When infection is not effectively controlled, sepsis may develop and progress to severe sepsis and septic shock. Early diagnosis and treatment is pivotal for survival in severe sepsis and particularly, septic shock. Our research focuses on developing a novel treatment strategy for septic shock by using single, ultra-high doses of aminoglycosides. In this project, the effect of a single, ultra-high dose of gentamicin in clearing bacteria from the blood and reducing the bacterial burden in vital organs was evaluated in a rat model of E. coli (Bort strain) induced peritonitis with severe sepsis/septic shock. Serum cytokine levels and serum lactate levels were serially measured. Further, the potential adverse effects of ultra-high dosing of aminoglycoside antibiotics in a short-term (9 h) invasive study and long term (180 days) non-invasive study were assessed. Neuromuscular paralyses due to ultra-high doses of aminoglycosides were assessed. In addition, renal injury markers such as serum creatinine and urinary Neutrophil Gelatinase Associated Lipocalin (NGAL) were assayed. The auditory and vestibular function was also assessed after ultra-high dosing of aminoglycoside in the long-term study. We conclude that animals can tolerate ultra-high doses of aminoglycosides with appropriate support. Animals were under neuromuscular paralysis for 28 – 50 minutes and were on ventilator support after single ultra-high doses (80 and 160 mg/kg) of aminoglycoside antibiotics (gentamicin and tobramycin). There was no significant acute or delayed renal or ototoxicity associated with the single, ultra-high dose aminoglycoside therapy. Histology studies of the kidneys and the cochlea of single, ultra-high aminoglycoside dosed animals and untreated control animals were performed after 180 days (6 months). Results indicated that there were no morphological differences between the treated and untreated control animals. Terminal deoxy-nucleotidyl transferase dUTP nick end labeling (TUNEL) assay of kidney tissue indicated that there was no apoptosis of endothelial cells in the tubular and glomerular regions with single, ultra- high dose of aminoglycosides consistent with an absence of ultrahigh dose induced nephrotoxicity. In the septic shock model, the E. coli Bort was below the limit of detection from the blood of the animals within minutes after single, ultra-high dose aminoglycoside administration. After necropsy, bacterial load was determined from all the vital organs and peritoneal fluid (site of infection). The bacterial levels were below the detection limit from the kidneys and there was a significant reduction in bacterial counts from all the remaining organs compared to the infected control animals. A decrease in serum cytokine and serum lactate levels compared to baseline was observed after ultra-high doses of aminoglycosides in the septic shock animals. Our studies have indicated that the ultra-high dose gentamicin is well tolerated by rats. It is highly effective in clearing E. coli Bort from the blood and reducing the bacterial burden in the organs in an experimental model of bacterial peritonitis/septic shock. Further studies in larger animals such as rabbits, sheep, pigs or dogs are required to confirm these results. If these findings are replicated in larger animals, this therapy may be developed further from ‘lab to bedside’ to treat septic shock patients in intensive care units (ICUs). / October 2015

Haemophilus influenzae tipo b (Hib) é uma patógeno causador de doenças como meningite. O polissacarídeo capsular (PRP) é usado como antígeno vacinal. O alto custo da vacina provém da instabilidade do PRP. Este trabalho tem por finalidade otimizar o processo de produção do PRP. Primeiro, foi escolhido um modelo matemático para a descrição da cinética de Hib. Em seguida, foi realizado um desenho experimental para avaliar os efeitos de pH e temperatura sobre a cinética. Tanto a produtividade de PRP quanto sua massa molecular foram máximas em pH 7,1, porém em temperaturas mais altas a produtividade foi maior e a massa molecular menor. As condições otimizadas foram definidas como pH 7,1 e 30 °C. Nestas condições, a massa molecular foi cerca de 78 % maior que nas condições da literatura de 37°C e pH 7,5, com uma produção de PRP equivalente. Isto resultou em maior recuperação na primeira etapa de purificação, comprovando que a condição de cultivo otimizada pode contribuir em melhores rendimentos durante o processo e assim impactar na redução do custo final da vacina. / Haemophilus influenzae type b (Hib) is a pathogen responsible for deseases like meningitis. The capsular polysaccharide (PRP) is used as antigen in vaccines. The high cost of the vaccine is due to instability of PRP. The goal of this work is to optimize the production of PRP. First, a mathematical model was selected to describe Hib kinetics. Then, an experimental design was carried out to evaluate the effects of pH and temperature. Both PRP productivity and molecular mass were optimal in pH 7.1, but at higher temperature productivity increased and molecular mass decreased. The optimal conditions were set at pH 7.1 and 30 °C. In these conditions, the molecular mass was 78 % higher, with the same amount of PRP produced. This resulted in higher recovery at the first step of purification, ensuring that the improved conditions may contribute with processes efficiency and enable reduction of the final cost of the vaccine.

Cette thèse aborde la question du traitement correct et complet de la couleur selon les contraintes métrologiques. Le manque d’approches adaptées a justifié la reformulation principaux outils de traitement d’images que sont le gradient, la détection et la description de points d’intérêt. Les approches proposées sont génériques : indépendantes du nombre de canaux d’acquisition (de la couleur à l’hyper-spectral), de la plage spectrale considérée et prenant en compte les courbes de sensibilité spectrales du capteur ou de l’œil.Le full-vector gradient nait de cet objectif métrologique. La preuve de concept est effectuée sur des images couleurs, multi et hyper-spectrales. L’extension développée pour l’analyse de la déficience visuelle ouvre également de nombreuses s perspectives intéressantes pour l’analyse du système visuel humain. Ce gradient est au cœur de la proposition d’un détecteur de points d’intérêt, lui aussi générique. Nous montrons la nécessité d’un choix mathématiquement valide de la distance entre attributs et l’importance de la cohérence de la paire attribut/distance. Une paire attribut/distance complète l’ensemble.Pour chaque développement, nous proposons des protocoles objectifs de validation liés à des générateurs d’images de synthèse explorant toute la complexité spatio-chromatique possible. Notre hypothèse est que la difficulté d’extraction du gradient/des points d’intérêts… est liée à la complexité de discrimination des distributions couleur dans la zone de traitement. Une confrontation aux approches courantes du domaine a été également mise en œuvre. / The PhD thesis objective is to study a colour’s correct and complete processing, respecting metrological constraint. The lack of compatible approaches justified that we reformulate the main image processing tools that are gradient, key point detector and descriptor. The proposed approaches are generic: channel count independent and taking the sensor’s or eye’s sensitivity curves into account.The full-vector gradient is born from this metrological objective. Proof of concept was realised on colour, multi and hyper-spectral images. The extension developed for human vision deficiency opens interesting perspectives to study of the human vision system. This gradient is the centre of the key point detector proposition, also generic.We also showed how necessary was a mathematically valid choice of distance between features. We revealed the importance of the pair feature/distance and completed the work with a pair: RC2O/Kulback-Leibler divergence based on colour differences.For each development, we propose unbiased validation protocols linked to synthetic images generators exploring the most spatial-chromatic complexity possible. Our hypothesis being that the extraction difficulty comes from the discrimination complexity between colour distributions in the processing area. We also compared our proposition to state of the art approaches in recurring datasets/protocols.