Nesta pesquisa, o metodo GPR foi empregado para localizar e mapear urnas funerarias enterradas, visando orientar as escavacoes arqueologicas e auxiliar nas medidas de protecao de sitios arqueologicos na regiao de influencia direta do aproveitamento hidreletrico de Dardanelos, proximo a Aripuana, MT. Um estudo arqueologico previo seria necessario para verificar a presenca de sitios arqueologicos, pois a regiao seria submersa, afetando todo e qualquer possivel artefato presente no sitio. Na area de influencia da usina de hidreletrica ja havia um sitio conhecido, o sitio de Dardanelos, sendo este o objeto da presente pesquisa. Dados GPR obtidos com a antena blindada de 200 MHz foram processados e analisados, e os resultados apresentados na forma de perfis 2D e em 3D na forma de depth-slices. Apos a aquisicao e processamento dos dados foram identificadas as anomalias GPR e interpretadas a fim de identificar os alvos de interesse arqueologico e raizes de arvores, evitando assim, que haja ambiguidade na caracterizacao dos alvos de interesse. A analise 3D gerada a partir dos perfis de reflexao 2D permitiu diferenciar com clareza os alvos de interesse das raizes de arvores, uma vez que nela podemos visualizar um padrao mais alongado ao inves de pontual, como e apresentado quando temos um artefato arqueologico. Ainda, atraves da conversao do tempo de percurso da onda eletromagnetica em profundidade, podemos identificar a profundidade dos alvos. Esta conversao tambem ajuda a esclarecer as ambiguidades, uma vez que as raizes sao mais rasas e os artefatos mais profundos. / In this research, GPR method was used to locate and map buried indigenous urns, aiming to guide and assist the archaeological excavations in order to guide protections acts of archaeological sites in the region directly affected by the hydroelectric of Dardanelos, near to Aripuana, MT. A preliminary archaeological study would be necessary to investigate the presence of archaeological sites, because the area would go underwater, affecting any possible artifact present on the site. In the area of influence of the hydroelectric plant there was already a known site, the site of the Dardanelos, which is the subject of this research. The GPR data obtained with shielded antenna 200 MHz were processed and analyzed, and the results presented as 2D and 3D profiles in the form of depth-slices. After processing the GPR data anomalies were identified and interpreted to identify the targets of archeological interest and roots of trees, thus avoiding ambiguity in the characterization of targets of interest. The 3D analysis generated from the 2D reflection profiles allowed to differentiate clearly the targets of interest from the roots of trees, since they can display a more elongated pattern rather than punctual, as shown when we have an archaeological artifact. Further, by converting the travel time of the electromagnetic wave in depth, we can identify the depth of targets. This conversion also helps to clarify the ambiguities, since the roots are shallower and the artifacts are deeper.

Nesta pesquisa, o metodo GPR foi empregado para localizar e mapear urnas funerarias enterradas, visando orientar as escavacoes arqueologicas e auxiliar nas medidas de protecao de sitios arqueologicos na regiao de influencia direta do aproveitamento hidreletrico de Dardanelos, proximo a Aripuana, MT. Um estudo arqueologico previo seria necessario para verificar a presenca de sitios arqueologicos, pois a regiao seria submersa, afetando todo e qualquer possivel artefato presente no sitio. Na area de influencia da usina de hidreletrica ja havia um sitio conhecido, o sitio de Dardanelos, sendo este o objeto da presente pesquisa. Dados GPR obtidos com a antena blindada de 200 MHz foram processados e analisados, e os resultados apresentados na forma de perfis 2D e em 3D na forma de depth-slices. Apos a aquisicao e processamento dos dados foram identificadas as anomalias GPR e interpretadas a fim de identificar os alvos de interesse arqueologico e raizes de arvores, evitando assim, que haja ambiguidade na caracterizacao dos alvos de interesse. A analise 3D gerada a partir dos perfis de reflexao 2D permitiu diferenciar com clareza os alvos de interesse das raizes de arvores, uma vez que nela podemos visualizar um padrao mais alongado ao inves de pontual, como e apresentado quando temos um artefato arqueologico. Ainda, atraves da conversao do tempo de percurso da onda eletromagnetica em profundidade, podemos identificar a profundidade dos alvos. Esta conversao tambem ajuda a esclarecer as ambiguidades, uma vez que as raizes sao mais rasas e os artefatos mais profundos. / In this research, GPR method was used to locate and map buried indigenous urns, aiming to guide and assist the archaeological excavations in order to guide protections acts of archaeological sites in the region directly affected by the hydroelectric of Dardanelos, near to Aripuana, MT. A preliminary archaeological study would be necessary to investigate the presence of archaeological sites, because the area would go underwater, affecting any possible artifact present on the site. In the area of influence of the hydroelectric plant there was already a known site, the site of the Dardanelos, which is the subject of this research. The GPR data obtained with shielded antenna 200 MHz were processed and analyzed, and the results presented as 2D and 3D profiles in the form of depth-slices. After processing the GPR data anomalies were identified and interpreted to identify the targets of archeological interest and roots of trees, thus avoiding ambiguity in the characterization of targets of interest. The 3D analysis generated from the 2D reflection profiles allowed to differentiate clearly the targets of interest from the roots of trees, since they can display a more elongated pattern rather than punctual, as shown when we have an archaeological artifact. Further, by converting the travel time of the electromagnetic wave in depth, we can identify the depth of targets. This conversion also helps to clarify the ambiguities, since the roots are shallower and the artifacts are deeper.

The problem of sensing a medium by several sensors and retrieving interesting features is a very general one. The basic framework of the problem is generally the same for applications from MRI, tomography, Radar SAR imaging to subsurface imaging, even though the data acquisition processes, sensing geometries and sensed properties are different. In this thesis we introduced a new perspective to the problem of remote sensing and information retrieval by studying the problem of subsurface imaging using GPR and seismic sensors. We have shown that if the sensed medium is sparse in some domain then it can be imaged using many fewer measurements than required by the standard methods. This leads to much lower data acquisition times and better images representing the medium. We have used the ideas from Compressive Sensing, which show that a small number of random measurements about a signal is sufficient to completely characterize it, if the signal is sparse or compressible in some domain. Although we have applied our ideas to the subsurface imaging problem, our results are general and can be extended to other remote sensing applications. A second objective in remote sensing is information retrieval which involves searching for important features in the computed image of the medium. In this thesis we focus on detecting buried structures like pipes, and tunnels in computed GPR or seismic images. The problem of finding these structures in high clutter and noise conditions, and finding them faster than the standard shape detecting methods like the Hough transform is analyzed. One of the most important contributions of this thesis is, where the sensing and the information retrieval stages are unified in a single framework using compressive sensing. Instead of taking lots of standard measurements to compute the image of the medium and search the necessary information in the computed image, a much smaller number of measurements as random projections are taken. The data acquisition and information retrieval stages are unified by using a data model dictionary that connects the information to the sensor data.

La mucoviscidose est une maladie génétique létale induite par des mutations du canal ionique CFTR, provoquant une perte de sa fonctionnalité au niveau des tissus épithéliaux de divers organes. Le poumon est particulièrement touché et devient sujet à des infections bactériennes chroniques. Dans le but de traiter la maladie, nous avons développé des « stabilisateurs » de la protéine CFTR : il s’agit de peptides inhibant l’interaction de la protéine CFTR avec le médiateur-clé de sa demi-vie à la membrane apicale des cellules épithéliales, la protéine CAL. En particulier, le peptide iCAL36 a démontré une hausse de fonctionnalité de la protéine CFTR mutée. Le but de cette thèse a été de renforcer cet effet biologique en améliorant ses caractéristiques pharmacologiques : pénétration cellulaire (vectorisation), stabilité métabolique et affinité pour la protéine CAL.Le premier axe d’optimisation a été l’internalisation du peptide iCAL36 par 7 différents peptides vecteurs (CPP). Les conjugués correspondants ont été évalués suivant leur cytotoxicité, leur efficacité d’internalisation et leur capacité à maintenir cette efficacité en présence de sérum. Le mécanisme d’entrée des deux meilleurs conjugués a ensuite été étudié. Divers biais couramment rencontrés lors de l’analyse de l’efficacité d’internalisation de peptides vecteurs par des méthodes de fluorescence ont également été identifiés et expliqués. La séquence du peptide iCAL36 a ensuite été modulée par inclusion d’acides aminés non-naturels. Le criblage des interactions peptide/protéine a été réalisé par une procédure optimisée dans le cadre de cette thèse (méthode PIPEPLUS) et a permis d’identifier 32 analogues prometteurs de la séquence d’iCAL36 incluant différentes substitutions. En particulier, une des séquences identifiées (iCAL-Q27) a démontré une affinité 70 fois supérieure à celle du peptide iCAL36 pour la protéine CAL, indiquant une inhibition plus complète de l’interaction CAL/CFTR.Ces résultats majeurs permettent dans leur ensemble de développer des « stabilisateurs » peptidiques de seconde génération pouvant avoir un effet biologique accru dans le contexte de la mucoviscidose. / Cystic fibrosis is a lethal disease induced by genetic mutations of the CFTR chloride channel, leading to a loss of its function in the epithelial tissues of various organs. The lung is particularly affected and becomes a target for chronical bacterial infections. To cure the disease, we developed so-called CFTR “stabilizers”, which are peptides inhibiting the interaction between the CFTR protein and the key mediator of its half-life at the apical membrane of epithelial cells, the CAL protein. In particular, the iCAL36 peptide showed an increase of the functionality of the mutated CFTR protein. The aim of this thesis was to increase this biological effect by improving its pharmacological parameters: cellular internalization (vectorization), metabolic stability and affinity for the CAL protein.The first axis of optimization was the internalization of the iCAL36 peptide by 7 different cell-penetrating peptides (CPP). The corresponding conjugates were evaluated upon their cytotoxicity, their uptake efficiency and their capacity to maintain this efficiency in the presence of proteases. The mechanism of entry of the two best candidates was then studied. Various bias frequently encountered during the analysis of CPP uptake efficiency by fluorescence methods were also identified and explained. Afterwards, the iCAL36 sequence was modulated by inclusion of non-natural amino acids. The screening of the peptide/protein interactions was performed by a method optimized during this thesis (PIPEPLUS process) and allowed the identification of 32 promising analogues of the iCAL36 sequence including several substitutions. In particular, one of these sequences (iCAL-Q27) showed an affinity 70 times stronger for the CAL protein compared to iCAL36, hinting a more complete inhibition of the CAL/CFTR interaction.Overall, these major results grant the access to second-generation “stabilizers” potentially showing an improved biological effect in the context of cystic fibrosis.

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1–2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.

No description available.

Thesis (MScEng (Electrical and Electronic Engineering))--University of Stellenbosch, 2003. / No abstract available

L'interférence ARN est un processus biologique permettant la dégradation d'un ARN messager par un ARN double brin de courte taille spécifique de cet ARNm. Elle a un potentiel d'application en thérapie antivirale pour peu que les ARN interférents (ARNi) soient délivrés efficacement in vivo. Dans le genre Morbillivirus, on trouve des pathogènes importants en santé publique et vétérinaire tels que le virus de la rougeole et les virus de la peste des petits ruminants (PPR) et de la peste bovine. Il n'existe aucun traitement contre les infections à morbillivirus. L'objectif de ce travail était d'évaluer la possibilité d'administrer in vivo un ARNi actif contre le virus PPR in vitro. Une formulation basée sur des liposomes complexés avec des ARNi ou un adénovirus non réplicatif exprimant des ARN courts en tête d'épingle (shARN) ont été testés chez des chèvres dans un modèle d'épreuve infectieuse avec une souche virulente de PPR. Les différences observées n'étaient cependant pas significatives au plan statistique. Pour améliorer la délivrance par vecteur viral, nous avons comparé un autre vecteur de type baculovirus qui s'est avéré plus efficace in vitro que l'adénovirus précédent. Par ailleurs, nous avons testés in vitro également deux peptides capables de pénétrer dans les cellules. L'un d'entre eux, le Perfect 6 (PF6) a presque complètement inhibé l'expression du gène de la nucléoprotéine par le virus PPR. En revanche, l'autre (PF14) a été moins efficace mais a relativement mieux résisté à l'inhibition de son activité par la présence de fortes concentrations de sérum dans le milieu. Dans le but d'évaluer in vivo ces nouveaux systèmes de délivrance en s'affranchissant du modèle chèvre lourd et couteux à mettre en œuvre, nous avons initié une stratégie de mise au point d'un modèle non infectieux de suivi dynamique de l'interférence ARN chez la souris par imagerie in vivo. Dans ce travail, nous montrons qu'il est possible de mesurer et de standardiser l'expression d'un gène rapporteur comprenant une séquence du virus PPRV et ensuite de quantifier le niveau de dérégulation de l'expression induit par un ARNi dirigé contre le virus PPR. Après calibration, ce modèle est désormais pour tester différents systèmes de délivrance de siRNA chez la souris / RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner. RNAi has a potential of developing into an effective and specific antiviral therapy if small interfering RNAs (siRNAs) can be efficiently delivered in vivo. Morbillivirus genus includes important pathogens of humans and animals, which include measles virus, peste des petits ruminants virus (PPRV) and rinderpest virus. No treatment exists for morbillivirus diseases. The aim of this work was the in vivo delivery of siRNA against PPRV infection. The delivery of siRNA by a liposome and short hairpin RNA (shRNA) by means of a replication deficient adenovirus was tested in goats which were later challenged with PPRV. However, significant therapeutic effects were not obtained. To find more efficient vectors, the PPRV inhibition efficiency of recombinant replication deficient adenovirus and a baculovirus expressing shRNA against nucleoprotein of PPRV were compared in vitro. The baculoviral vector was found to be more efficient. Similarly, two cell penetrating peptides (CPPs) were also compared and PepFect6 (PF6) could deliver siRNA NPPRV1 effectively in vitro resulting in an almost complete inhibition of N gene expression by PPRV. Another CPP, the PF14 though with lower transfection efficiency in vitro, was found to be relatively serum resistant compared to PF6. A small animal model for PPRV infection does not exist. Due to economic, ethical, and biosecurity issues involved with use of small ruminants, a strategy based on the use of a non-infectious mouse model and a dynamic follow up of siRNA treatment by live imaging was developed. We show in this work that it is possible to measure and standardize the expression of a bioluminescent reporter gene containing a PPRV sequence and thus, to quantify a down-regulation of such gene by siRNA against PPRV. After some calibration, siRNA delivery can now be tested in this mouse model for comparing various delivery vectors in vivo.

L'utilisation des oligonucléotides antisens PMO ou PNA, pour corriger les erreurs d'épissage par blocage stérique, constitue une nouvelle stratégie prometteuse pour réguler l'expression génétique. Ces ON peuvent mener au traitement de maladies comme la β-thalassémie, la dystrophie musculaire de Duchenne (DMD) ou les cancers. Cependant leur développement clinique requiert un système de délivrance efficace. Les peptides cationiques (CPPs) sont caractérisés par leur capacité à s'internaliser dans les cellules eucaryotes. Cependant leur efficacité à promouvoir la délivrance cytoplasmique et nucléaire des ON est limitée par leur séquestration dans des vésicules d'endocytose, ce qui est à l'origine de la dégradation du matériel internalisé. Nous avons contribué à l'étude du trafic intracellulaire et de l'activité dans un essai de correction d'épissage de plusieurs familles de CPPs capables de délivrer efficacement des analogues d'ON à des doses non toxiques et en absence d'agents endosomolytiques. Nos études mécanistiques indiquent que ces constructions (covalentes ou non covalentes) CPP-ON sont endocytées par la voie clathrine, que la ségrégation dans les endosomes reste une limitation et qu'il existe une bonne corrélation entre leur activité biologique et leur capacité à déstabiliser les membranes endosomales. / The use of antisense oligonucleotides PMO or PNA to correct splicing errors by steric- block represents a new promising therapeutic strategy. These ONs lead to the treatment of diseases such as β-thalassemia, Duchenne muscular dystrophy (DMD) or cancers. However their functional success requires efficient delivery. Cationic cell penetrating peptides (CPPs) are characterized by their ability to be internalized in eukaryotic cells. However their efficiency in promoting cytoplasmic and nuclear delivery of ON has been hampered by endocytic sequestration and subsequent degradation of internalized material in endocytic vesicles, which is responsible for the degradation of internalized material. We have contributed to the study of intracellular trafficking and activity (using splicing correction assay) of several families of CPPs capable of delivering effective analogs ON at nontoxic doses and in the absence of agents endosomolytic. Our mechanistic studies indicate that these constructs (covalent or noncovalent) CPP-ON are internalized through clathrin, that segregation in endosomes remains a limitation and that there is good correlation between biological activity and their ability to destabilize endosomal membranes.

O desenvolvimento de resistência antimicrobiana e a consequente seleção de microrganismos multirresistentes consolidam-se como grandes ameaças à saúde global. Neste contexto, a busca por novas drogas antimicrobianas/microbicidas é fundamental e compostos como os peptídeos antimicrobianos (AMPs) tornaram-se alvos atraentes. Os AMPs são compostos químicos de massa molar média e grande diversidade estrutural, produzidos por todos os seres vivos e com capacidade de inibir o crescimento de e/ou matar microrganismos. O AMP Cheferina I (Chef I) foi isolado das raízes de Capsella bursa-pastoris e é resultado da proteólise de uma proteína da família das proteínas ricas em glicina, que em plantas estão relacionadas às funções de defesa e cicatrização. O nosso grupo de pesquisa foi pioneiro no desenvolvimento e estudo de análogos truncados amidados deste AMP atípico rico em glicina (67,9%) e histidina (28,6%), que se mostraram ativos frente às diferentes cepas de Candida e a S. cerevisiae pela internalização/ação celular acompanhada de manutenção da integridade da membrana plasmática; o análogo amidado (Chef Ia) e o análogo marcado com 5(6)-carboxifluoresceína/FAM (FAM-Chef Ia) tiveram as suas atividades antifúngicas potencializadas por íons Zn2+. Este trabalho deu continuidade ao estudo do efeito dos íons metálicos divalentes Zn2+, Cu2+, Ca2+ e Mg2+ nas atividades anticandida/fungistática e candidacida/fungicida a diferentes pHs e forças iônicas, estruturas e localizações intracelulares destes análogos. Os resultados na ausência de íons em pH 5,1 revelaram maior atividade do análogo fluorescente em relação à do não fluorescente. Neste mesmo pH, as atividades anticandida e candidacida de Chef Ia foram influenciadas negativamente pelos íons Ca2+ e Mg2+ (2-4 vezes) enquanto que, na presença de íons Zn2+ as atividades anticandida de ambos os análogos foram aumentadas (Chef Ia: 8-64 vezes; FAM-Chef Ia: 4-32 vezes). Os íons Cu2+ aumentaram a atividade anticandida de Chef Ia (2-4 vezes), mas não a do análogo fluorescente, mas as atividades candidacidas de ambos foram melhoradas (Chef Ia: 2-8 vezes; FAM-Chef Ia: 2 vezes). Em pH 5,1, os íons Zn2+ mantiveram a atividade anticandida de Chef Ia em alta força iônica, mas só FAM-Chef Ia exibiu atividade candidacida. Em pH 7,4 ambos análogos foram inativos em baixa e alta forças iônicas na ausência e presença de Zn2+ ou Cu2+. As maiores porcentagens de folhas-β-antiparalelas e dobras foram observadas no espectro de DC de Chef Ia em pH 7,4, sendo que aqueles registrados em pH 5,1 e 7,4 em presença de íons Zn2 e Cu2+ indicaram a formação de quelatos estruturalmente distintos. Ambos os peptídeos são bioquelantes em potencial, sendo as proporções peptídeo: íon obtidas as seguintes: FAM-Chef Ia = 1:2 para Cu2+, 1:10 para Zn2+; Chef Ia = 1:1 para Cu2+. A análise da internalização celular de FAM-Chef Ia permitiu a suposição de dois mecanismos de internalização (translocação direta e endocitose), sendo que nas células vivas a presença de Zn2+ afetou negativamente a translocação direta (p 0,0343) e potencializou a endocitose (p 0,0002). / The development of antimicrobial resistance and the consequent selection of multiresistant microorganisms have become major threats to global health. In this context, the search for new antimicrobial/microbicidal drugs is crucial and the antimicrobial peptides (AMPs) have been seen as attractive targets. AMPs are chemical compounds of medium molecular mass and high structural diversity produced by all living beings, capable of inhibiting the growth of microorganisms and killing them. The AMP Shepherin I (Shep I) was isolated from the roots of Capsella bursa-pastoris, being a bioactive peptide encrypted in a glycine-rich protein from a family that in plants are strictly related to defense and healing functions. Our research group has pioneered the development and study of amidated truncated analogues of this atypical glycine- (67.9%) and histidine-rich (28.6%) AMP, which has shown activity against different strains of Candida and S. cerevisiae through cellular internalization with maintenance of the plasma membrane integrity. The amide analogue (Chef Ia) and its fluorescent analog labeled with 5 (6) - carboxyfluorescein / FAM (FAM-Chef Ia) had their antifungal activities potentiated by Zn2+ ions, so the present work continued examining the effect of the divalent metallic ions Zn2+, Cu2+, Ca2+ and Mg2+ on the anticandidal/fungistatic and candidacidal/fungicide activities at different pHs and ionic forces, structures and intracellular locations of these analogues. The results in the absence of those ions at pH 5.1 revealed that the fluorescently labelled analog was more potent than the nonfluorescent. At the same pH, Shep Ia anticandidal and candidacidal activities were negatively influenced by Ca2+ and Mg2+ ions (2-4 fold), whereas in the presence of Zn2+ ions the anticandidal activities of both analogues were increased (Shep Ia: 8-64 fold, FAM- Shep Ia: 4-32 fold). Cu2+ ions increased Shep Ia anticandidal activity (2-4 fold) but not that of FAM-Shep Ia, nevertheless, the candidacidal activities of both analogues were increased (Shep Ia: 2-8 fold, FAM-Shep Ia: 2 fold). Also at pH 5.1, the Zn2+ ions helped retaining the anticandidal activity of Shep Ia at high ionic strength, although only FAM-Shep Ia exhibited candidacidal activity. At pH 7.4 both analogues were inactive at low and high ionic strengths in the absence or presence of Zn2+ or Cu2+. The highest percentages of antiparallel β-sheet and turns were observed in Shep Ia CD spectrum at pH 7.4, while those recorded at pH 5.1 and 7.4 in the presence of Zn2+ or Cu2+ ions indicated the formation of structurally different chelates. Both peptides are potential biochelates, with the following peptide:ion ratios: FAM-Shep Ia = 1: 2 for Cu2+, 1:10 for Zn2+; Shep Ia = 1: 1 for Cu2+. The analysis of the cellular internalization of FAM-Chef Ia allowed the assumption of two mechanisms of internalization (direct translocation and endocytosis) and in the living cells the presence of Zn2+ negatively affected the direct translocation (p 0.0343) and potentiated endocytosis (p 0.0002).