abstract: Dendrites are the structures of a neuron specialized to receive input signals and to provide the substrate for the formation of synaptic contacts with other cells. The goal of this work is to study the activity-dependent mechanisms underlying dendritic growth in a single-cell model. For this, the individually identifiable adult motoneuron, MN5, in Drosophila melanogaster was used. This dissertation presents the following results. First, the natural variability of morphological parameters of the MN5 dendritic tree in control flies is not larger than 15%, making MN5 a suitable model for quantitative morphological analysis. Second, three-dimensional topological analyses reveals that different parts of the MN5 dendritic tree innervate spatially separated areas (termed "isoneuronal tiling"). Third, genetic manipulation of the MN5 excitability reveals that both increased and decreased activity lead to dendritic overgrowth; whereas decreased excitability promoted branch elongation, increased excitability enhanced dendritic branching. Next, testing the activity-regulated transcription factor AP-1 for its role in MN5 dendritic development reveals that neural activity enhanced AP-1 transcriptional activity, and that AP-1 expression lead to opposite dendrite fates depending on its expression timing during development. Whereas overexpression of AP-1 at early stages results in loss of dendrites, AP-1 overexpression after the expression of acetylcholine receptors and the formation of all primary dendrites in MN5 causes overgrowth. Fourth, MN5 has been used to examine dendritic development resulting from the expression of the human gene MeCP2, a transcriptional regulator involved in the neurodevelopmental disease Rett syndrome. Targeted expression of full-length human MeCP2 in MN5 causes impaired dendritic growth, showing for the first time the cellular consequences of MeCP2 expression in Drosophila neurons. This dendritic phenotype requires the methyl-binding domain of MeCP2 and the chromatin remodeling protein Osa. In summary, this work has fully established MN5 as a single-neuron model to study mechanisms underlying dendrite development, maintenance and degeneration, and to test the behavioral consequences resulting from dendritic growth misregulation. Furthermore, this thesis provides quantitative description of isoneuronal tiling of a central neuron, offers novel insight into activity- and AP-1 dependent developmental plasticity, and finally, it establishes Drosophila MN5 as a model to study some specific aspects of human diseases. / Dissertation/Thesis / Ph.D. Neuroscience 2012

O Distrito Mineiro de Salto do Jacuí (DMSJ) compreende a maior jazida de ágata em geodos do RS. O DMSJ está inserido na Supersequência Gondwana III (da Bacia do Paraná) onde são posicionadas a rochas vulcânicas da Formação Serra Geral (Cretáceo) e os arenitos da Formação Botucatu (Jurássico). Observa-se ainda a interação entre as lavas e os sedimentos (intertraps arenosos) formando feições como diques de arenito, fraturas e geodos preenchidos por sedimentos além de diversos tipos de brechas, sempre associados com a mineralização de ágata. Ideias que abrangem desde uma origem magmática a altas temperaturas como também uma possível formação a temperaturas mais baixas tem sido apresentadas, mas até o momento não há um consenso sobre a gênese deste mineral. Neste trabalho são aplicadas técnicas estratigráficas, químicas e isotópicas na análise da mineralização de ágata com objetivo de caracterizar e propor um modelo para a sua formação. As rochas vulcânicas, as sedimentares e amostras de ágata foram analisadas por técnicas petrográficas, por difratometria de raios X (DRX), por microscopia eletrônica de varredura (MEV) e análises químicas (em amostras de rocha total) e isotópicas (relações isotópicas do Sr, Nd, Nd e O). Em relação às rochas vulcânicas ficou caracterizada a presença de derrames de basalto e de dacito. Os arenitos dos intertraps são semelhantes ao arenito Botucatu. No entanto, localmente, foi descrita uma fácies mais fina, argilosa e micácea interpretada como possível ambiente do tipo interduna. A ágata tem diferentes características petrográficas, químicas e isotópicas o que mostra a complexidade dos processos envolvidos na sua gênese. O modelo de formação da ágata destaca a formação de um fluido silicoso a partir da lixiviação da sílica da matriz vítrea dos dacitos por águas meteóricas. A solução saturada em sílica gerada migra através do sistema permo-poroso onde predominam as fraturas e microfraturas. Esta solução ao encontrar um espaço maior, os geodos, precipita observando a sequência: opala – calcedônia – ágata – quartzo macrocristalino. Como a ágata normalmente é encontrada nas proximidades dos geodos com sedimentos (interduna??) e também por evidências isotópicas, pode-se supor que no modelo proposto, secundariamente houve influência de água/nível freático associada com a facies fina do intertrap. Assim, com base em diferentes técnicas analíticas foi possível propor um modelo genético de baixa temperatura para a mineralização de ágata do DMSJ. / Salto do Jacuí Mining District (DMSJ) comprises the largest deposit of agate geodes in the Rio Grande do Sul State, Brazil. The DMSJ is included in the Gondwana III Supersequence (Paraná Basin) where the volcanic rocks of the Serra Geral Formation (Cretaceous) are positioned, as well as the sandstones of the Botucatu Formation (Jurassic). Also, interaction between lavas and sediments (sandstone intertraps) is present, creating features such as sandstone dikes, fractures and geodes filled by sediment. Several types of breccias are observed too and they are always associated with the agate mineralization. Until now, there is no consensus about the genesis of this mineral and propositions include the influence of a magmatic source to high temperatures, as well as formation at lower temperatures. In the present work, we applied stratigraphic, chemical and isotopic techniques in the analysis of agate mineralization in order to characterize and propose a model for its formation. The agate samples, the volcanic and sedimentary rocks were analyzed with the help of petrographic techniques, X-ray diffraction (XRD), scanning electron microscopy (SEM), chemical analyses (in whole rock samples) and isotopic analyses (isotopic ratios of Sr Nd, Nd and O). The volcanic rocks were characterized by the presence of basaltic flows and dacite. The intertrap sandstones are similar to those observed in the Botucatu sandstone. However, locally, it has been described a finer, clay-rich and micaceous facies, interpreted as possible interdune environment. Agate displayed different petrographic, chemical and isotopic characteristics, showing the complexity of the processes involved in its genesis. The model of agate formation indicates that a siliceous fluid is generated from the leaching of silica of the dacites glass matrix by rainwater. The saturated silica solution migrates through the permo-porous system where fractures and microfractures predominate. When this solution flows into a larger space, like geodes, it precipitates according to the following sequence: opalchalcedony- agate-quartz. Usually, agate is found in the surroundings of geodes containing sediments (interdune?), but also by isotopic evidence. Taking these facts into account, we can assume that the proposed model was secondarily influenced by water/groundwater level associated with the thin facies of intertrap. Thus, based on different techniques, it was possible to propose a genetic model for the low temperature agate formation in DMSJ.

O Distrito Mineiro de Salto do Jacuí (DMSJ) compreende a maior jazida de ágata em geodos do RS. O DMSJ está inserido na Supersequência Gondwana III (da Bacia do Paraná) onde são posicionadas a rochas vulcânicas da Formação Serra Geral (Cretáceo) e os arenitos da Formação Botucatu (Jurássico). Observa-se ainda a interação entre as lavas e os sedimentos (intertraps arenosos) formando feições como diques de arenito, fraturas e geodos preenchidos por sedimentos além de diversos tipos de brechas, sempre associados com a mineralização de ágata. Ideias que abrangem desde uma origem magmática a altas temperaturas como também uma possível formação a temperaturas mais baixas tem sido apresentadas, mas até o momento não há um consenso sobre a gênese deste mineral. Neste trabalho são aplicadas técnicas estratigráficas, químicas e isotópicas na análise da mineralização de ágata com objetivo de caracterizar e propor um modelo para a sua formação. As rochas vulcânicas, as sedimentares e amostras de ágata foram analisadas por técnicas petrográficas, por difratometria de raios X (DRX), por microscopia eletrônica de varredura (MEV) e análises químicas (em amostras de rocha total) e isotópicas (relações isotópicas do Sr, Nd, Nd e O). Em relação às rochas vulcânicas ficou caracterizada a presença de derrames de basalto e de dacito. Os arenitos dos intertraps são semelhantes ao arenito Botucatu. No entanto, localmente, foi descrita uma fácies mais fina, argilosa e micácea interpretada como possível ambiente do tipo interduna. A ágata tem diferentes características petrográficas, químicas e isotópicas o que mostra a complexidade dos processos envolvidos na sua gênese. O modelo de formação da ágata destaca a formação de um fluido silicoso a partir da lixiviação da sílica da matriz vítrea dos dacitos por águas meteóricas. A solução saturada em sílica gerada migra através do sistema permo-poroso onde predominam as fraturas e microfraturas. Esta solução ao encontrar um espaço maior, os geodos, precipita observando a sequência: opala – calcedônia – ágata – quartzo macrocristalino. Como a ágata normalmente é encontrada nas proximidades dos geodos com sedimentos (interduna??) e também por evidências isotópicas, pode-se supor que no modelo proposto, secundariamente houve influência de água/nível freático associada com a facies fina do intertrap. Assim, com base em diferentes técnicas analíticas foi possível propor um modelo genético de baixa temperatura para a mineralização de ágata do DMSJ. / Salto do Jacuí Mining District (DMSJ) comprises the largest deposit of agate geodes in the Rio Grande do Sul State, Brazil. The DMSJ is included in the Gondwana III Supersequence (Paraná Basin) where the volcanic rocks of the Serra Geral Formation (Cretaceous) are positioned, as well as the sandstones of the Botucatu Formation (Jurassic). Also, interaction between lavas and sediments (sandstone intertraps) is present, creating features such as sandstone dikes, fractures and geodes filled by sediment. Several types of breccias are observed too and they are always associated with the agate mineralization. Until now, there is no consensus about the genesis of this mineral and propositions include the influence of a magmatic source to high temperatures, as well as formation at lower temperatures. In the present work, we applied stratigraphic, chemical and isotopic techniques in the analysis of agate mineralization in order to characterize and propose a model for its formation. The agate samples, the volcanic and sedimentary rocks were analyzed with the help of petrographic techniques, X-ray diffraction (XRD), scanning electron microscopy (SEM), chemical analyses (in whole rock samples) and isotopic analyses (isotopic ratios of Sr Nd, Nd and O). The volcanic rocks were characterized by the presence of basaltic flows and dacite. The intertrap sandstones are similar to those observed in the Botucatu sandstone. However, locally, it has been described a finer, clay-rich and micaceous facies, interpreted as possible interdune environment. Agate displayed different petrographic, chemical and isotopic characteristics, showing the complexity of the processes involved in its genesis. The model of agate formation indicates that a siliceous fluid is generated from the leaching of silica of the dacites glass matrix by rainwater. The saturated silica solution migrates through the permo-porous system where fractures and microfractures predominate. When this solution flows into a larger space, like geodes, it precipitates according to the following sequence: opalchalcedony- agate-quartz. Usually, agate is found in the surroundings of geodes containing sediments (interdune?), but also by isotopic evidence. Taking these facts into account, we can assume that the proposed model was secondarily influenced by water/groundwater level associated with the thin facies of intertrap. Thus, based on different techniques, it was possible to propose a genetic model for the low temperature agate formation in DMSJ.

O Distrito Mineiro de Salto do Jacuí (DMSJ) compreende a maior jazida de ágata em geodos do RS. O DMSJ está inserido na Supersequência Gondwana III (da Bacia do Paraná) onde são posicionadas a rochas vulcânicas da Formação Serra Geral (Cretáceo) e os arenitos da Formação Botucatu (Jurássico). Observa-se ainda a interação entre as lavas e os sedimentos (intertraps arenosos) formando feições como diques de arenito, fraturas e geodos preenchidos por sedimentos além de diversos tipos de brechas, sempre associados com a mineralização de ágata. Ideias que abrangem desde uma origem magmática a altas temperaturas como também uma possível formação a temperaturas mais baixas tem sido apresentadas, mas até o momento não há um consenso sobre a gênese deste mineral. Neste trabalho são aplicadas técnicas estratigráficas, químicas e isotópicas na análise da mineralização de ágata com objetivo de caracterizar e propor um modelo para a sua formação. As rochas vulcânicas, as sedimentares e amostras de ágata foram analisadas por técnicas petrográficas, por difratometria de raios X (DRX), por microscopia eletrônica de varredura (MEV) e análises químicas (em amostras de rocha total) e isotópicas (relações isotópicas do Sr, Nd, Nd e O). Em relação às rochas vulcânicas ficou caracterizada a presença de derrames de basalto e de dacito. Os arenitos dos intertraps são semelhantes ao arenito Botucatu. No entanto, localmente, foi descrita uma fácies mais fina, argilosa e micácea interpretada como possível ambiente do tipo interduna. A ágata tem diferentes características petrográficas, químicas e isotópicas o que mostra a complexidade dos processos envolvidos na sua gênese. O modelo de formação da ágata destaca a formação de um fluido silicoso a partir da lixiviação da sílica da matriz vítrea dos dacitos por águas meteóricas. A solução saturada em sílica gerada migra através do sistema permo-poroso onde predominam as fraturas e microfraturas. Esta solução ao encontrar um espaço maior, os geodos, precipita observando a sequência: opala – calcedônia – ágata – quartzo macrocristalino. Como a ágata normalmente é encontrada nas proximidades dos geodos com sedimentos (interduna??) e também por evidências isotópicas, pode-se supor que no modelo proposto, secundariamente houve influência de água/nível freático associada com a facies fina do intertrap. Assim, com base em diferentes técnicas analíticas foi possível propor um modelo genético de baixa temperatura para a mineralização de ágata do DMSJ. / Salto do Jacuí Mining District (DMSJ) comprises the largest deposit of agate geodes in the Rio Grande do Sul State, Brazil. The DMSJ is included in the Gondwana III Supersequence (Paraná Basin) where the volcanic rocks of the Serra Geral Formation (Cretaceous) are positioned, as well as the sandstones of the Botucatu Formation (Jurassic). Also, interaction between lavas and sediments (sandstone intertraps) is present, creating features such as sandstone dikes, fractures and geodes filled by sediment. Several types of breccias are observed too and they are always associated with the agate mineralization. Until now, there is no consensus about the genesis of this mineral and propositions include the influence of a magmatic source to high temperatures, as well as formation at lower temperatures. In the present work, we applied stratigraphic, chemical and isotopic techniques in the analysis of agate mineralization in order to characterize and propose a model for its formation. The agate samples, the volcanic and sedimentary rocks were analyzed with the help of petrographic techniques, X-ray diffraction (XRD), scanning electron microscopy (SEM), chemical analyses (in whole rock samples) and isotopic analyses (isotopic ratios of Sr Nd, Nd and O). The volcanic rocks were characterized by the presence of basaltic flows and dacite. The intertrap sandstones are similar to those observed in the Botucatu sandstone. However, locally, it has been described a finer, clay-rich and micaceous facies, interpreted as possible interdune environment. Agate displayed different petrographic, chemical and isotopic characteristics, showing the complexity of the processes involved in its genesis. The model of agate formation indicates that a siliceous fluid is generated from the leaching of silica of the dacites glass matrix by rainwater. The saturated silica solution migrates through the permo-porous system where fractures and microfractures predominate. When this solution flows into a larger space, like geodes, it precipitates according to the following sequence: opalchalcedony- agate-quartz. Usually, agate is found in the surroundings of geodes containing sediments (interdune?), but also by isotopic evidence. Taking these facts into account, we can assume that the proposed model was secondarily influenced by water/groundwater level associated with the thin facies of intertrap. Thus, based on different techniques, it was possible to propose a genetic model for the low temperature agate formation in DMSJ.

Many papers have been presented on the study of change points detection.　Nonetheless, we would like to point out that in dealing with the time series with switching regimes, we should also take the characteristics of change periods into account.　Because many patterns of change structure in time series exhibit a certain kind of duration, those phenomena should not be treated as a mere sudden turning at a certain time. In this paper, we propose procedures about change periods detection for nonlinear time series.　One of the detecting statistical methods is an application of fuzzy classification and generalization of Inclan and Tiao’s result.　Moreover, we develop the genetic-based searching procedure, which is based on the concepts of leading genetic model.　Simulation results show that the performance of these procedures is efficient and successful.　Finally, two empirical applications about change periods detection for Taiwan monthly visitors arrival and exchange rate are demonstrated.

L'autisme est un trouble du développement du système nerveux central défini par des altérations des interactions sociales et de la communication, et par des comportements restreints et répétitifs. Sa prévalence est actuellement évaluée jusqu'à 1% dans la population générale. L'autisme est caractérisé par une grande hétérogénéité sur les plans phénotypiques et génétiques. À ce jour, plus de 300 gènes candidats ont été caractérisés soit par des variations du nombre de copies (CNV) et/ou des variations nucléotidiques (SNV). Leur identification a permis de mettre en évidence une contribution significative de mutation de novo, ainsi que l'implication de voies physiopathologiques cibles, en particulier la densité post-synaptique (PSD). / Autism is a developmental disorder of the central nervous system defined by impairments in social interaction and communication, and by restricted and repetitive behavior. Its prevalence is currently estimated at around 1% in the general population. Autism is characterized by a wide heterogeneity at both phenotypic and genetic level. To date, more than 300 candidate genes were characterized either by copy number variations (CNV) and/or nucleotide variations (SNV). Their identification has highlighted a significant contribution of de novo mutations, as well as the involvement of targeted pathophysiological pathways, particularly post-synaptic density (PSD).

近幾年來，非線性時間數列轉折點的研究愈來愈受到重視，學者們也提出許多關於轉折點的偵測及檢定方法。若考慮實際資料走勢轉變的情形，“轉折區間”的概念更可以解釋結構改變的現象。但文獻中對於如何找尋時間數列結構改變之轉折區間的研究並不多。本文擬以時間數列統計模式及模糊學理論的角度來研究，並結合遺傳演算的規則而提出主導模式的概念，來架構出時間數列遺傳模式，再藉由轉折區間決策法則來找出數列的轉折區間。其中，我們以統計模式為遺傳演化過程中的染色體，而以候選模式之隸屬度函數為衡量染色體適應能力的指標。最後，我們舉出臺灣股價收盤指數之實例，分別以我們所提出的方法及其他方法找出數列的轉折區間及轉折點，並做比較。 / For recent years, the research of change point in nonlinear time series has been considered to be more and more important. Scholars have proposed a lot of detecting and testing methods about change points.If considering the trend of real situation, the concept of change period will show the phenomena of structure change.But there are not many researches about how to find change period in time series.My paper is based on the points of time series models and fuzzy theory.Besides,it combines the rules of genetic algorithm and provides the concepts of leading model to construct time seriep genetic model and to find out change period by decision rule.ln this paper, we use time series statistical models as chromosome in procedure of genetic evolution, and we also use membership function of selected models as pereformance: index of chromosome.Finally, the empirical application about change periods and change points detecting by our method and other's for Taiwan stock closing prices is demonstrated and make a comparision with these results.

Mechanical forces have been observed to affect various aspects of life, for example, cell differentiation, cell migration, locomotion and behavior of multicellular organisms etc. Such forces are generated either by external entities such as mechanical touch, fluid flow, electric and magnetic fields or by the living organisms themselves. Study of forces sensed and applied by living organisms is important to understand the interactions between organisms and their environment. Such studies may reveal molecular mechanisms involved in mechanosensation and locomotion. Several techniques have been successfully applied to measure forces exerted by single cells and cell monolayers. The earliest technique made use of functionalized soft surfaces and membranes as substrates on which cell monolayers were grown. The forces exerted by the cells could be measured by observing deformation of the substrates. Atomic Force Microscope (AFM) is another sensitive instrument that allows one to exert and measure forces in pico-Newton range. Advances in micromachining technology have enabled development of miniature force sensors and actuators. Latest techniques for mechanical force application and measurement use micromachined Silicon cantilevers in single as well as array form and micropillar arrays. Micropillar arrays fabricated using soft lithography enabled the use of biocompatible materials for force sensors. Together, these techniques provide access to a wide range of forces, from sub micro-Newton to milli-Newton. In the present work, types of forces experienced in biological systems and various force measurement and actuation techniques will be introduced. This will be followed by in depth description of the two major contributions of this thesis, 1) ―Colored polydimethylsiloxane micropillar arrays for high throughput measurements of forces applied by genetic model organisms‖. Biomicrofluidics, January 29, 2015. doi: 10.1063/1.4906905 2) ―Air microjet system for non-contact force application and the actuation of micro-structures‖. Journal of micromechanics and microengineering, December 15, 2015. doi: 10.1088/0960-1317/26/1/017001 Device developed for force measurement consists of an array of micropillars made of a biocompatible polymer Poly Dimethyl Siloxane (PDMS). Such devices have been used by researchers to measure traction forces exerted by single cells and also by nematode worm Caenorhabditis elegans (C. elegans). C. elegans is allowed to move in between the micropillars and the locomotion is video recorded. Deflection of the micropillar tips as the worm moves is converted into force exerted. Transparent appearance of C. elegans and PDMS poses difficulties in distinguishing micropillars from the worm, thus making it challenging to automate the analysis process. We address this problem by developing a technique to color the micropillars selectively. This enabled us to develop a semi-automated graphical user interface (GUI) for high throughput data extraction and analysis, reducing the analysis time for each worm to minutes. Moreover, increased contrast because of the color also delivered better images. Addition of color changed the Young‘s modulus of PDMS. Thus the dye-PDMS composite was characterized using hyper-elastic model. The micropillars were also calibrated using commercial force sensor. Analysis of forces exerted by wild type and mutant C. elegans moving on an agarose surface was performed. Wild type C. elegans exerted a total average force of 7.68 µN and an average force of ~1 µN on an individual pillar. We show that the middle of C. elegans exerts more force than its extremities. We find that C. elegans mutants with defective body wall muscles apply significantly lower force on individual pillars, while mutants defective in sensing externally applied mechanical forces still apply the same average force per pillar compared to wild type animals. Average forces applied per pillar are independent of the length, diameter, or cuticle stiffness of the animal. It was also observed that the motility of the worms with mechanosensation defects, lower cuticle stiffness, and body wall muscle defects was reduced with worms that have defective body wall muscle having the largest degree. Thus, we conclude that while reduced ability to apply forces affects the locomotion of the worm in the micropillar array, the reduced motility/locomotion may not indicate that the worm has reduced ability to apply forces on the micropillars. We also used the colored micropillar array for the first time to measure forces exerted by Drosophila larvae. Our device successfully captured the peristaltic rhythm of the body wall muscles of the larva and allowed us to measure the forces applied on each deflected pillar during this motion. Average force exerted by 1st instar wild type Drosophila larvae was measured to be ~ 1.5 µN per pillar. We demonstrated that a microjet of air can be used to apply forces in micro-Newton range. We developed a standalone system to generate a controlled air microjet. Microjet was generated using a controlled electromagnetic actuation of a diaphragm. With a nozzle diameter of 150 µm, the microjet diameter was maintained to a maximum of 1 mm at a distance of 5 mm from the nozzle. The force generated by the microjet was measured using a commercial force sensor to determine the velocity profile of the jet. Axial flow velocities of up to 25 m/s were obtained at distances as long as 6 mm. The microjet exerted a force up to 1 µN on a poly dimethyl siloxane (PDMS) micropillar (50 µm in diameter, 157 µm in height) and 415 µN on a PDMS membrane (3 mm in diameter, 28 µm thick). We also demonstrate that from a distance of 6 mm our microjet can exert a peak pressure of 187 Pa with a total force of about 84 µN on a flat surface with 8 V operating voltage. Next, we demonstrated that the response of C. elegans worms to the impinging air microjet is similar to the response evoked using a manual gentle touch. This contactless actuation tool avoids contamination and mechanical damage to the samples. Out of the cleanroom fabrication and robust design make this system cost effective and durable. Magnetic micropillars have been used as actuators. We fabricated magnetic micropillar arrays and designed actuation mechanisms using permanent magnet and a pulsed electromagnet. Force of about 19 µN was achievable using a permanent magnet actuation. In a pulsed electromagnetic field micropillar exerted a force of about 10 µN on a commercial force sensor. These techniques have promising applications when actuation needs to be controlled from long distances.

PhDENV (Geology) / Department of Mining and Environmental Geology / The study focused on the geology, structural setting and mineralisation of copper-sulphide deposits in the Musina area, located in the Central Zone of the Limpopo Mobile Belt of South Africa. The Messina copper deposits are located in the eastern part of Limpopo Province near the border with Zimbambwe. The deposits stretch from northeastern to southwestern direction for about 15 km. Previous copper mining in the area took place at Artonvilla, Messina, Harper, Campbell and Lilly copper deposits. The current study, however, focused on two main deposits, Campbell and Artonvilla. The origin, nature and mode of formation of the Cu-sulphide deposits in the Musina area have not been established with certainty. Two principal hypotheses on the origin of the Messina copper sulphide deposits have been proposed, viz; a magmatic-hydrothermal model, and meteoric waters model. Consequently, the mode of formation and mineralisation style of the Messina Cu-sulphide deposits remain contentious. Therefore, the main objective of the study was to investigate the nature and mode of formation of Cu-sulphide deposits in the Musina area. Different research methods have been applied in the current study so as to unpack the contradictory positions on the genesis of the Messina copper deposits. This included fieldwork, remote sensing data acquisition, laboratory work, and data analysis and interpretation. Fieldwork involved soil geochemical survey as well as rock and ore sampling within the study area. A total of 295 soil samples, 33 rock specimens and 21 ore samples were collected for laboratory investigation. Laboratory work consisted of a range of methods that included; geochemical analysis, petrographic and cathodoluminescence microscopy, ore mineralogy and ore microscopy, fluid inclusion geothermometry and isotope geochemistry. The work was done in different laboratories including: Mining and Environmental Geology Laboratory, Unviersity of Venda; Department of Geology Laboratory, University of Johannesburg; MINTEK Laboratory in Johannesburg; Société Générale de Surveillance Laboratory in Johannesburg, South Africa; Department of Applied Geology, Geoscience Institute, Göttingen University, Germany and Department of Geology, University of Georgia, Athens, United States of America. Remote sensing data was acquired from Southern Mapping Company, Johannesburg, South Africa. Interpretation of Remote sensing data was done at the University of Applied Sciences, Oswestfalen-Lippe, Germany. Data analysis and interpretation of laboratory results involved the use of: Desktop ArcGIS 10.4.1 for geochemical data interpretation; ENVI 5.1 and ArcGIS 10.4.1 Softwares for remote sensing data; and Triplot version 4.1.2 software for ternary plot for compositional variation of rocks. Soil geochemical survey revealed geochemical anomalies for Pb, Zn, Cu, As and Ni over the known copper deposits in the area as well as over six other areas that have not been associated with any sulphide mineralisation. Such new anomalous areas have been identified as target areas for future exploration of sulphide ore mineralisation. Petrographic studies of the rocks confirmed the host rocks to be amphibolite-quartz granulite, biotite-garnet-quartz granulite, amphibolite, quartzite, hornblende gneiss, quartzo-feldspathic gneiss, potassium-feldspathic gneiss and cal-silicate gneiss. These rocks were subjected to hydrothermal alteration during ore mineralisation within the area. It was further noted that epidote alteration was quite intensive in ore samples, while in unmineralised rock samples it was less intensive. Remote sensing data interpretation revealed spatial distribution and intensity of epidote alteration within the study area and in places coincided either with the known copper deposits or structural features, thus led to the identification of target areas for future mineral exploration in the Musina area. The current study established that the process of ore mineralisation in the Messina copper deposits took place in two distinct phases: first the formation of garnet, graphite, magnetite and hematite during regional metamorphism of the Limpopo Mobile Belt; and secondly, sulphide ore mineralisation resulting in the formation of copper ore comprising, veined, disseminated and brecciated ores. Sulphide ore mineralisation consisted mainly of pyrite, chalcopyrite, sphalerite, bornite, chalcocite and minor pyrrhotite and galena as well as traces of pentlandite, tennantite, mollybdenite, cobaltite and tetrahedrite. This confirms that the Messina copper deposits had complex sulphide ore mineralisation that is typical of hydrothermal mode of ore mineralisation from a magmatic source. The study further establishes the paragenitic sequence of ore mineralisation, comprising four stages: Stage I (Garnet- graphite – Fe oxides); stage II (Quartz- pyrite); stage III (Pyrite- sphalerite - chalcopyrite); and stage IV (Carbonates). Stage III represented the main stage of sulphide ore mineralisation in the area, while Stage IV comprising calcite, dolomite and ankarite marked the final stage of hydrothermal ore mineralisation. Paragenetic sequence identified three generations of quartz; first generation being associated with garnet, graphite, magnetite and hematite, second generation with pyrite and third generation with pyrite, sphalerite and chalcopyrite. Previous studies, however, indicated that there was only one generation of quartz that formed at the temperature between 210o to 150°C, but the current study established that the entrapment temperature of first generation quartz ranges from 315o to 200°C; second generation quartz from 235o to 135°C and third generation quartz from 240o to 115°C. At the same time, sulphur isotope investigation of chalcopyrite-pyrite pair from Campbell deposit registered a temperature of 359°C. The study therefore concluded that the temperature of ore formation within the Messina copper deposits ranged between 359°C and 115°C. The presence of halite and calcite as daughter minerals within the fluid inclusions was noted and this apparently is indicative of high salinity of fluid inclusions, which is considered as a product of direct exolution of crystalizing magma. Raman spectroscopy revealed the composition of gases in the fluid inclusions to be CH4 and N2 with 80% and 20% composition respectively, however, some inclusions were gas-poor. The presence of gases in the fluid inclusions is an indication that there was boiling at the time of entrapment. A narrow range of 34S values of -0.5 to 0.5‰ obtained in this study further confirms the magmatic source of Sulphur as Sulphur from the host rock was found to have high 𝛿34S value of 8.2‰. A genetic model for copper ore mineralisation within Musina area is proposed. The deposits are of polymetallic vein type that are genetically associated with porphyry copper deposits. According to this model, copper ore bodies were formed from hydrothermal fluids originating from magma and were epigenetic in nature. Geological structures in the area acted as conduits for hydrothermal fluids that resulted in the alteration of the host rocks and mineralisation of copper sulphide ore. Thus, the Messina coper deposits are of magmatic hydrothermal origin although the apparent location of a batholith is still unknown and the study recommends further viii research work on the location of the batholith that is presumed to have been the magmatic source. The study further recommend dating of later rocks as well as orebody s it is essential for understanding the process of ore formation in this area. For further exploration, areas that have undergone “moderate” to “high” degree of epidote alteration and lie in close proximity to geological structures such as faults and thrust folds that could have acted as conduits for hydrothermal fluids and resulted in sulphide ore mineralisation and registered high geochemical anomalies for Pb, Zn, As and Ni should be targeted. In support of further mineral exploration within the study area, the study recommend a detailed geostatistical application for the purpose of delineating homogeneous areas based on the combination of lineaments, interpolated soil geochemical maps and thematic maps. / NRF

Η παρούσα εργασία αφορά στη μελέτη της ανταγωνιστικής αλληλεπίδρασης των Α1/D1 υποδοχέων στο επίπεδο έκφρασης του πρώιμου γονιδίου zif/268 (δείκτης νευρωνικής δραστηριότητας) και της in vivo μεταγωγής σήματος των Α1 και Α2Α υποδοχέων αδενοσίνης κάτω από τη ντοπαμινεργική απονεύρωση στο μυ weaver. Ο μυς weaver αποτελεί ένα γενετικό μοντέλο ντοπαμινεργικής απονεύρωσης, η οποία συμβαίνει σταδιακά, έτσι ώστε το μοντέλο αυτό να προσομοιάζει τη Νόσο Πάρκινσον (ΝΠ) στον άνθρωπο. Στο πρώτο στάδιο της μελέτης προέκυψε το ενδιαφέρον αποτέλεσμα ότι με την ταυτόχρονη ενεργοποίηση των Α1 και D1 υποδοχέων παρατηρήθηκε η αναμενόμενη ανταγωνιστική αλληλεπίδραση (ενδεχομένως μέσω σχηματισμού του ετεροδιμερούς), ενώ με την ενεργοποίηση μόνο των Α1 υποδοχέων στους weaver μύες παρατηρήθηκε αυξημένη ενεργοποίηση των νευρώνων του ραβδωτού σώματος και συγκεκριμένων περιοχών του εγκεφαλικού φλοιού. Η ενεργοποίηση αυτή ήταν μη αναμενόμενη, δεδομένου ότι οι Α1 υποδοχείς (A1Rs) είναι συζευγμένοι με Gi πρωτεΐνες και καταστέλλουν τη μεταγωγή σήματος που οδηγεί στην επαγωγή του zif/268 μέσω του D1R/Gs/cAMP/PKA/pDARPP-32/pCREB μονοπατιού. Η ακόλουθη διερεύνηση του μηχανισμού έδειξε ότι η Α1R-επαγόμενη ενεργοποίηση του zif/268 καταστέλλεται από τον ειδικό ανταγωνιστή των Α2Α υποδοχέων αδενοσίνης (A2ARs) ZM241385 και από τον ειδικό αγωνιστή των D2 υποδοχέων ντοπαμίνης Quinpirole, υποδεικνύοντας την ενεργοποίηση των Α2ΑRs και άρα ενεργοποίηση της έμμεσης οδού. Το αποτέλεσμα αυτό επιβεβαιώθηκε, δεδομένου ότι η διέγερση των Α1Rs προκάλεσε αύξηση της έκφρασης του mRNA της εγκεφαλίνης, αλλά όχι της δυνορφίνης, που αποτελούν δείκτες ενεργοποίησης της έμμεσης και της άμεσης οδού, αντίστοιχα. Το γεγονός ότι ο αγωνιστής των Α1Rs δεν προκαλεί στα φυσιολογικά ζώα ενεργοποίηση του zif/268 mRNA υποδεικνύει την υπερευαίσθητη απόκριση των Α2ARs. Η μελέτη της υπερευαίσθητης αυτής απόκρισης στο μυ weaver έγινε με τη διερεύνηση της μεταγωγής σήματος μετά από in vivo ενεργοποίηση των Α2ΑRs: α) του καθιερωμένου μονοπατιού Α2ΑRs/Gs/AC/cAMP/PKA/pDARPP-32/pCREB, το οποίο οδηγεί στη επαγωγή του zif/268 και β) του μονοπατιού των ΜΑΡΚ. Τα αποτελέσματα έδειξαν αυξημένα βασικά επίπεδα φωσφορυλίωσης της DARPP-32 στη θέση Thr-34. Τα αυξημένα επίπεδα της φωσφορυλιωμένης DARPP-32 πολλαπλασιάζουν τη δράση της ΡΚΑ και άρα διευκολύνουν τη μεταγωγή σήματος μέσω Α2ΑRs/Gs/AC/cAMP/PKA/pDARPP-32/pCREB μονοπατιού. Επομένως, η υπερευαίσθητη απόκριση των Α2ΑRs κάτω από την έλλειψη ντοπαμίνης στο μυ weaver φαίνεται να οφείλεται στα αυξημένα ενδογενή επίπεδα της φωσφορυλιωμένης DARPP-32. Ενδιαφέρον παρουσιάζει το γεγονός ότι τα βασικά επίπεδα φωσφορυλίωσης των πρωτεϊνών ERK1/2(MAPK44/42) είναι αυξημένα στον μυ weaver, αλλά μειώνονται σημαντικά μετά από την ενεργοποίηση των Α2ΑRs. Δεν γνωρίζουμε το μηχανισμό μέσω του οποίου αυξάνονται τα ενδογενή επίπεδα των ERK1/2 και πρέπει να διερευνηθεί περαιτέρω. Το συμπέρασμα όμως που εξάγεται είναι ότι κάτω από τη ντοπαμινεργική απονεύρωση η μεταβίβαση σήματος μέσω των Α2ΑRs δεν ενεργοποιεί την οδό των MAP κινασών. Στην παρούσα in vivo μελέτη αναδεικνύεται ο ρόλος των Α1 και Α2Α υποδοχέων στην λειτουργία των βασικών γαγγλίων κάτω από τη ντοπαμινεργική απονεύρωση. Τα αποτελέσματα αυτά έχουν ιδιαίτερη σημασία δεδομένου ότι εμφανίζονται σε ένα γενετικό μοντέλο παρκινσονισμού, στο οποίο η εκφύλιση των ντοπαμινεργικών νευρώνων είναι σταδιακή και προσομοιάζει τη ΝΠ, και όχι οξεία, όπως σε άλλα τοξικά μοντέλα. Επιπλέον, τα αποτελέσματα αυτά παρουσιάζουν ενδεχομένως κλινικό ενδιαφέρον, δεδομένου ότι η ενεργοποίηση της έμμεσης οδού μέσω Α1Rs από την ενδογενή αδενοσίνη θα επιδείνωνε περαιτέρω τις κινητικές δυσλειτουργίες της ΝΠ. Η πληροφορία αυτή, καθώς και η γνώση για την ενισχυμένη μεταγωγή σήματος μέσω των Α2Α υποδοχέων ενισχύουν την πρόταση για χρήση των Α2Α ανταγωνιστών ως αντιπαρκινσονικά φάρμακα. Δεδομένου ότι σήμερα το ενδιαφέρον είναι στραμμένο στη δημιουργία διμερών προσδεμάτων (bivalent ligands) που μπορούν να δρουν ταυτόχρονα σε δύο υποδοχείς, η συγκεκριμένη πληροφορία θα μπορούσε να χρησιμοποιηθεί για μελλοντική δημιουργία φαρμακευτικού σχήματος που να δρα ταυτόχρονα ως αγωνιστής των D2 υποδοχέων ντοπαμίνης και ως ανταγωνιστής των Α2Α υποδοχέων αδενοσίνης. / The present work studied the antagonistic interaction of A1/D1 receptors at the level of mRNA expression of the immediate early gene zif/268 (used as a marker of neuronal function). In parallel we studied the in vivo signal transduction of A1 and A2A adenosine receptors under dopamine deficiency in weaver mutant. The weaver mutant represents the only genetic animal model of gradual nigrostriatal neuron degeneration, which can be characterized as a pathophysiological phenocopy of Parkinson’s Disease. In the first part of the study, the co-activation of A1 and D1 receptors revealed the well-known antagonistic interaction of these receptors (possibly through the formation of A1/D1 heterodimer) in weaver mutant. An interesting result was that the activation of A1 receptors alone did induce zif/268 mRNA expression in stiatal and specific cortical neurons in weaver mutant. This induction was not expected, since A1 receptors are Gi-coupled and suppress the signal transduction pathway that leads to zif/268 induction through AC/PKA/p-DARPP-32/pCREB cascade. Further study, revealed that the A1 receptor-induced zif/268 mRNA expression is counteracted by the A2A receptor selective antagonist ZM241385 and by the D2 receptor selective agonist Quinpirole, suggesting the activation of A2A receptors and thus the activation of the “indirect pathway”. Moreover, A1 receptors activation induced the expression of enkephalin mRNA, but not of dynorphin, which are considered as marker of neuronal activation of the “indirect” and the “direct” pathway, respectively. The fact that the A1 receptor agonist did not induced zif/268 mRNA expression in +/+ animals indicates that under dopamine deficiency the A2A receptors react with a supersensitive response. This response was analyzed in weaver mouse after in vivo A2A receptor activation: a) by examining the classical signal transduction pathway of A2A receptors/AC/PKA/p-DARPP-32/pCREB, which leads to zif/268 expression and b) by studying the MAPK cascade. Results showed increased basal phosphorylation levels of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, MW 32kDa) of Thr-34 in weaver compared to control mice. Increased phosphoThr34-DARPP-32 would amplify the effects of the PKA and thus facilitating the signal transduction through A2A receptors/AC/PKA/p-DARPP-32/pCREB. Therefore, the A2A receptors supersensitive response under dopamine deficiency in weaver mutant seems to be due to elevated endogenous phosphorylation levels of DARPP-32. Interestingly, while the basal phosphorylation levels of ERK1/2 (MAPK44/42) are elevated in weaver mutant, they are significantly reduced after A2A receptor activation. Although we do not know the mechanism through which the endogenous ERK1/2 levels are elevated, the conclusion is that, under dopamine deficiency, A2A receptors do not activate MAPK cascade. The present in vivo study demonstrates the role of A1 and A2A adenosine receptors in the function of basal ganglia under dopamine deficiency. Our results are significant since the expreriments were performed in a genetic parkinsonian model, in which the dopaminergic neurons are gradually degenerated and thus simulate the human PD, and not in an acute toxic model. Moreover, these results could be of possible clinical relevance, since the activation of A1 receptors by endogenous adenosine would exaggerate the motor dysfunctions of PD. Furthermore, the enhanced signal transduction pathway through A2A receptors supports the suggestion that the A2A receptor antagonists as antiparkinsonian agents. Given the well-known A2A/D2 antagonistic interaction, new therapeutical prospectives would involve the development of pharmacological bivalent ligands, which can interact with the A2A/D2 receptors and act simultaneously as A2A receptor antagonists and as D2 receptor agonists.