Assez rapidement après son apparition à la fin des années 1980, la Microscopie à Force Atomique (AFM) a démontré des perspectives prometteuses d’applications biomédicales. À l’heure actuelle, elle permet l’étude d’échantillons biologiques allant de la molécule unique à la cellule vivante proche des conditions physiologiques. Bien qu’étant applicable aux cellules eucaryotes et procaryotes, elle est entravée par son faible débit. Alors qu’elle peut être fortement automatisée sur certains échantillons bien caractérisés en air, l’automatisation de l’AFM en liquide reste rare, en particulier en multi-échantillon. Lors de ce projet doctoral, une approche automatisée a été développée pour l’étude des cellules en milieu liquide. Après une introduction du système et des développements nécessaires, nous démontrons l’approche sur des bactéries fixées et vivantes, ainsi que sur des cellules épithéliales. L’utilisation d’automatisation multi-échantillon permet d’augmenter le nombre d’échantillons rassemblés tout en limitant les interactions avec l’utilisateur. Enfin, les développements ultérieurs sont discutés pour aller vers un système automatisé à plus grande échelle sur échantillons vivants. / Soon after its development in the late 1980s, atomic force microscopy (AFM) has shown promising applications in the biomedical field. It now allows investigating biological samples from single molecules to living cells under conditions close to physiological. Despite its applicability to both eukaryotic and prokaryotic cells, it is hampered by its low throughput. While heavily automated on some well-characterized samples in air, AFM automation in fluid is very scarce, especially at the multi-sample level. During this doctoral project, an automated approach was developed in fluid, on cells. After introducing the system and the developments required, we demonstrate the approach on fixed and living bacteria as well as on epithelial cells. The usage of multi-sample automation allows gathering a greater number of samples with limited user interaction. Finally, further developments are discussed to lead the path toward higher-scale AFM automation of live samples.

Coordinated movement is critical not only to sports technique and performance but to daily living and as such represents a fundamental area of research. Coordination requires being able to produce the right actions at the right time and has to incorporate perception, cognition, and forceful neuro-muscular interaction with the environment. Coordinated movements of the hands and fingers are some of the most complex activities undertaken where continuous learning and adaptation take place, but the temporal variability of the most basic movement components is still unknown. This thesis investigates the extent of temporal variability in the execution of four different simple hand and finger coordination tasks, with the purpose to find the various intrinsic temporal variability which limit the ability to coordinate the hands in space and time. Study one showed that in a synchronized bi-lateral two finger tapping test (<<1 cm movement to target) the best participant had a temporaltiming variability of 4.8 ms whereas the largest time variability could be as high as 24.8 ms. No obvious improvement was found after transfer practice, whereas the average time variability for asynchronized tapping decreased from 62.1 ms to 30.3 ms after instructed practice indicating a likely change in task grouping. Study two showed that in a unilateral thumb-index finger pinch and release test, the largest mean timing variability was 12 ms for pinching irrespective of performing the task in a slow alert manner or at a faster speed. However, the mean temporal variability for release was only 6.3 ms when the task was performed in a more alert manner and indicates that release is more accurately controlled temporally than grip. Study three suggested that in a unilateral sagittal plane throwing action of the lower arm and hand, that elbow and wrist coordination for dynamic index finger tip location was better with a radial-ulnar deviation, darts-type, throwing action than a wrist flexor-extensor type action, basketball free throw type action (the mean variability was 37.5 ms and 27.2 ms, respectively). Study four compared the variability in bi-lateral finger tapping between voluntary tapping and involuntary finger contraction tapping. Electrically stimulated neural contractions had significantly lower force onset variability than voluntary or direct magnetic stimulation of muscles (6 ms, 9.5 ms, and 10.3 ms for electrically stimulated, voluntary and Transcranial Magnetic Stimulation stimulated contraction). This work provides a comprehensive analysis of the temporal variability in various fundamental digital movement tasks that can aid with the understanding of basic human coordination in sporting, daily living and clinical areas.

A Na, K-ATPase (NKA) é uma proteína de membrana encontrada em organismos eucariotos multicelulares cuja atividade e funções já são amplamente discutidas na literatura. Sua unidade funcional corresponde a um heterodímero formado por duas subunidades , com regiões transmembrana. Espécies multiméricas como dímeros e tetrâmeros dessa enzima também são conhecidos por exercer atividade enzimática. As interações lipídio-proteína são intrínsecas para a NKA, por tal motivo, proteolipossomos constituídos de DPPC e DPPC:DPPE foram preparados por co-solubilização. Como controle, lipossomos de mesma composição foram produzidos por extrusão e/ou sonicação. Para as imagens de AFM, as amostras foram fixadas com glutaraldeído, para proteção mecânica e contra desidratação das vesículas. Para lipossomos de DPPC as imagens topográficas de AFM das vesículas apresentaram formato oval, superfície perfeitamente lisa e diâmetro médio de 151 + 46 nm, enquanto as vesículas de composição DPPC:DPPE, apesar de lisas, tiveram cantos pontiagudos e diâmetro médio de 98 + 28 nm. Imagens de fase de ambas as composições não apresentaram qualquer indicativo de diferenças na composição química, provavelmente devido à natureza de carga neutra dos dois fosfolipídios. As imagens de fase por AFM para os proteolipossomos tanto de DPPC-NKA, quanto DPPC:DPPE-NKA, revelaram resultados inéditos na literatura, onde a inserção da NKA aparece como nítidas regiões transições de fase de composição química distinta quando comparadas com os lipossomos. No entanto, as mudanças de fase são diferentes entre as composições estudadas, aparecendo como manchas escuras circulares para DPPC-NKA e mais visíveis como interstícios brilhantes para composição de DPPC:DPPE-NKA. As vesículas de DPPC-NKA apresentaram diâmetro médio de 390 + 326 nm e, nas imagens de topografia tridimensionais, protusões de 38 a 115 nm correspondentes às regiões de mudanças de fase, que, indicaram o diâmetro dos microdomínios relacionados à proteína. Já nas imagens para DPPC:DPPE-NKA o diâmetro médio dos proteolipossomos foi de 189 + 156 nm, e as protusões apareceram entre os interstícios, variando de 20 a 66 nm. O estudo de DSC dos lipossomos revelou que a concentração de glutaraldeído nas condições das análises de AFM, em torno de 5% (v/v), afetam as características físico-químicas para as composições com DPPE. A AFM foi eficiente para confirmar a reinserção da NKA em proteolipossomos pelas imagens de fase, e, para medir o diâmetro dos microdomínios pelas imagens de topografia. / Na, K-ATPase (NKA) is a membrane protein present in eukaryotic multicellular organisms. Its functions and activity are already widely described in the literature. Its minimal functional structure is a heterodimer of two main subunits , with transmembrane domains. However, dimers and tetramers of the enzyme are also known to have enzymatic activity. Since there are intrinsic lipid-protein interactions, NKA proteoliposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by the co-solubilization method and liposomes of the same compositions were obtained by extrusion and/or sonication to be used as control. The samples to the AFM study were prepared using glutaraldehyde to protect the vesicles from mechanical shocks and dehydration. Liposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by extrusion and sonication, respectively, as control. The topographical images for DPPC liposomes showed vesicles with an oval shape and smoothed surfaces with a mean diameter of 151 + 46 nm. DPPC:DPPE vesicles also presented smoothed surfaces, but with pointed corners and mean diameter of 98 + 28 nm. Phase images for both lipid compositions showed no differences in chemical composition. For DPPC:DPPE samples, this can be explained by the neutral net charge of both lipids. The proteoliposomes observed in the AFM phase images showed darker and large circular spots in the vesicles. These spots represent delays in the phase oscillation of the AFM probe and are associated with different chemical composition. The phase changes showed the reconstitution of the NKA in the proteoliposomes. When compared with topographical images, this spots matched protrusions. The mean diameter of DPPC-NKA proteoliposomes determined by AFM was 390 + 326 nm. In the three-dimensional topographical images of composition, protrusions from 38 to 115 nm near the areas of different phases indicate the diameters of the NKA microdomains. The phase changes for DPPC:DPPE-NKA appeared as bright interstices with the protrusions of the topographical images in between them. The size of these protrusions ranged from 20 to 66 nm and the mean diameter of the proteoliposomes was 189 + 156 nm. The DSC liposomes data showed that the glutaraldehyde concentration used in the AFM analysis affect the physical chemistry properties of the samples with DPPE. AFM proved to be an efficient method to confirm the reconstitution of into proteoliposomes with phase images and to determine the diameter of the protein microdomains with the topographical images.

A Na, K-ATPase (NKA) é uma proteína de membrana encontrada em organismos eucariotos multicelulares cuja atividade e funções já são amplamente discutidas na literatura. Sua unidade funcional corresponde a um heterodímero formado por duas subunidades , com regiões transmembrana. Espécies multiméricas como dímeros e tetrâmeros dessa enzima também são conhecidos por exercer atividade enzimática. As interações lipídio-proteína são intrínsecas para a NKA, por tal motivo, proteolipossomos constituídos de DPPC e DPPC:DPPE foram preparados por co-solubilização. Como controle, lipossomos de mesma composição foram produzidos por extrusão e/ou sonicação. Para as imagens de AFM, as amostras foram fixadas com glutaraldeído, para proteção mecânica e contra desidratação das vesículas. Para lipossomos de DPPC as imagens topográficas de AFM das vesículas apresentaram formato oval, superfície perfeitamente lisa e diâmetro médio de 151 + 46 nm, enquanto as vesículas de composição DPPC:DPPE, apesar de lisas, tiveram cantos pontiagudos e diâmetro médio de 98 + 28 nm. Imagens de fase de ambas as composições não apresentaram qualquer indicativo de diferenças na composição química, provavelmente devido à natureza de carga neutra dos dois fosfolipídios. As imagens de fase por AFM para os proteolipossomos tanto de DPPC-NKA, quanto DPPC:DPPE-NKA, revelaram resultados inéditos na literatura, onde a inserção da NKA aparece como nítidas regiões transições de fase de composição química distinta quando comparadas com os lipossomos. No entanto, as mudanças de fase são diferentes entre as composições estudadas, aparecendo como manchas escuras circulares para DPPC-NKA e mais visíveis como interstícios brilhantes para composição de DPPC:DPPE-NKA. As vesículas de DPPC-NKA apresentaram diâmetro médio de 390 + 326 nm e, nas imagens de topografia tridimensionais, protusões de 38 a 115 nm correspondentes às regiões de mudanças de fase, que, indicaram o diâmetro dos microdomínios relacionados à proteína. Já nas imagens para DPPC:DPPE-NKA o diâmetro médio dos proteolipossomos foi de 189 + 156 nm, e as protusões apareceram entre os interstícios, variando de 20 a 66 nm. O estudo de DSC dos lipossomos revelou que a concentração de glutaraldeído nas condições das análises de AFM, em torno de 5% (v/v), afetam as características físico-químicas para as composições com DPPE. A AFM foi eficiente para confirmar a reinserção da NKA em proteolipossomos pelas imagens de fase, e, para medir o diâmetro dos microdomínios pelas imagens de topografia. / Na, K-ATPase (NKA) is a membrane protein present in eukaryotic multicellular organisms. Its functions and activity are already widely described in the literature. Its minimal functional structure is a heterodimer of two main subunits , with transmembrane domains. However, dimers and tetramers of the enzyme are also known to have enzymatic activity. Since there are intrinsic lipid-protein interactions, NKA proteoliposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by the co-solubilization method and liposomes of the same compositions were obtained by extrusion and/or sonication to be used as control. The samples to the AFM study were prepared using glutaraldehyde to protect the vesicles from mechanical shocks and dehydration. Liposomes composed of DPPC and DPPC:DPPE (1:1 molar ratio) were prepared by extrusion and sonication, respectively, as control. The topographical images for DPPC liposomes showed vesicles with an oval shape and smoothed surfaces with a mean diameter of 151 + 46 nm. DPPC:DPPE vesicles also presented smoothed surfaces, but with pointed corners and mean diameter of 98 + 28 nm. Phase images for both lipid compositions showed no differences in chemical composition. For DPPC:DPPE samples, this can be explained by the neutral net charge of both lipids. The proteoliposomes observed in the AFM phase images showed darker and large circular spots in the vesicles. These spots represent delays in the phase oscillation of the AFM probe and are associated with different chemical composition. The phase changes showed the reconstitution of the NKA in the proteoliposomes. When compared with topographical images, this spots matched protrusions. The mean diameter of DPPC-NKA proteoliposomes determined by AFM was 390 + 326 nm. In the three-dimensional topographical images of composition, protrusions from 38 to 115 nm near the areas of different phases indicate the diameters of the NKA microdomains. The phase changes for DPPC:DPPE-NKA appeared as bright interstices with the protrusions of the topographical images in between them. The size of these protrusions ranged from 20 to 66 nm and the mean diameter of the proteoliposomes was 189 + 156 nm. The DSC liposomes data showed that the glutaraldehyde concentration used in the AFM analysis affect the physical chemistry properties of the samples with DPPE. AFM proved to be an efficient method to confirm the reconstitution of into proteoliposomes with phase images and to determine the diameter of the protein microdomains with the topographical images.

The shift from analogue to digital data has been a trend the last couple of decades. One reason for such a switch is the ease and effectiveness with which it can be stored and spread. Among the most common methods people use to spread their data today is by e-mail and therefore, the goal should be to decrease the cognitive load when utilizing e-mail services. The main hypothesis of this thesis is that a prototype of an e-mail application designed to induce lower levels of cognitive load to its users is more time effective and less mentally demanding than one of its market counterparts; Gmail. Male and female participants with the age group 18-40 (N=21) performed five different tasks on both the prototype and the Gmail application. These tasks were accompanied by a dual-task, which was to tap their finger(s) rhythmically with their non-dominant hand on a metal plate to induce further cognitive load. The result showed that the tasks were more mentally demanding and frustrating when using the Gmail application compared to the prototype. The results showed that the execution time for five out of five tasks where longer when using the Gmail application compared to when using the prototype, but only three out of five had a statistically significant difference in time. / Växlingen från analogdata till digitaldata har varit en trend de senaste två decennierna. En anledning till en sådan förändring är fördelarna i att mer lätthanterligt kunna spara och sprida digitaldata. Bland de vanligaste metoderna människor använder idag för dataspridning är e-mejl och därför bör vi sträva att minska den kognitiva belastningen vid användandet av e-mejl tjänster. Hypotesen av denna uppsats är att en e-mejls applikations prototyp designad för att ge mindre nivåer av kognitiv belastning är mer tidseffektiv och är mindre mentalt belastande än en av dess marknadsmotparter; Gmail. Manliga och kvinnliga deltagare i åldersgruppen 18–40 (N=21) fick utföra fem olika uppdrag utförda på både prototypen och Gmailapplikationen. Uppdragen var kompletterade med ett samtida sidouppdrag vilket var att under uppdragens gång slå deras finger rytmiskt mot en metallplatta med deras icke-dominanta hand för att öka kognitiv belastning. Resultaten visade att uppdragen krävde mer mentala resurser och att deltagarna hade högre frustrationnivåer under användningen av Gmailapplikationen än på prototypen medan 3 utav 5 uppdrag visade statistisk signifikant skillnad i tid.

Thesis (M.Sc.), Faculty of Engineering (Electrical Engineering), University of the Witwatersrand, 1994

As operações de usinagem compreendem uma parcela significativa do universo da manufatura sendo que atualmente algumas tendências bem definidas, como a usinagem de peças já endurecidas, vêm ganhando espaço no campo da fabricação de moldes e matrizes, em especial, mas também na indústria automotiva e de construção de máquinas. O torneamento e o fresamento de materiais, nesta difícil condição de usinagem, já estão estabelecidos na indústria. Assim, a conversão do processo de produção atual de materiais em estado mole para endurecido só será possível com todas as operações de usinagem incluídas. Devido a isso, a demanda por furação e roscamento de peças endurecidas está em constante crescimento. Essas operações apresentam dificuldades muito maiores do que as de torneamento e fresamento, daí a importância de ferramentas especificamente projetadas para elas. Dessa forma, este trabalho teve como objetivo estudar o processo de desgaste sofrido pelas ferramentas durante a furação e roscamento de aços AISI D2 e AISI H13 com a utilização de diferentes velocidades de corte. As forças de corte e o torque também serão medidos durante o processo para avaliar seus comportamentos com o aumento da velocidade. Com os resultados obtidos verificou-se que na furação e roscamento de aços endurecidos de baixa usinabilidade, como no caso do aço AISI D2, o desgaste da ferramenta é muito acentuado, podendo tornar o processo inviável economicamente caso não sejam empregadas velocidades de corte extremamente reduzidas. De uma forma geral, a furação e o roscamento do AISI H13 pode ser viável, pois o número de furos/roscas obtidos dentro dos critérios especificados mostrou-se muito superior ao esperado. Os principais mecanismos de desgastes que atuaram nos machos de corte durante o processo de roscamento de ambos os aços (AISI D2 e AISI H13) foram abrasão nas superfícies de folga e adesão nas superfícies de saída de forma acentuada nos três primeiros filetes. / The cutting processes compose a huge part of the manufacturing universe and, nowadays, some well-defined trends, like cutting hardened materials, are increasing not only in die molding production but also in automotive and machine industries. The turning and milling processes of materials, in this difficult condition of cutting, are already applied in the mechanical industries. Therefore, the total conversion of the present production process of regular materials into hardened ones can only be reached when all the cutting processes are included. Because of that, the demands of drilling and tapping hardened materials have increased constantly. These operations require tools specifically designed for them, because they have proven themselves to be much more difficult than the turning and milling processes. In that way, this work aimed to study the wear process of drills and taps used to machining the AISI D2 and AISI H13 hardened steels with different cutting speeds. The cutting forces and the torque generated were also acquired during the cutting process to evaluate its behavior with the speed increase. After analyzing the results, a very aggressive tool wear was confirmed in the drilling and tapping process of hardened steels with bad machinability, like the AISI D2 steel, and this can make the process economically unfeasible if an extremely reduced cutting speed is not used. Generally, the drilling and tapping process of the AISI H13 can become economically viable, because the number of holes/threads achieved in accordance with the specified criteria was superior to the expected ones. In the cutting process of both materials (AISI D2 and H13), the main wear mechanism observed on the taps were abrasion, on the clearance surface, and adhesion, on the rake surface. This was severe for the first three threads of the tap.

As operações de usinagem compreendem uma parcela significativa do universo da manufatura sendo que atualmente algumas tendências bem definidas, como a usinagem de peças já endurecidas, vêm ganhando espaço no campo da fabricação de moldes e matrizes, em especial, mas também na indústria automotiva e de construção de máquinas. O torneamento e o fresamento de materiais, nesta difícil condição de usinagem, já estão estabelecidos na indústria. Assim, a conversão do processo de produção atual de materiais em estado mole para endurecido só será possível com todas as operações de usinagem incluídas. Devido a isso, a demanda por furação e roscamento de peças endurecidas está em constante crescimento. Essas operações apresentam dificuldades muito maiores do que as de torneamento e fresamento, daí a importância de ferramentas especificamente projetadas para elas. Dessa forma, este trabalho teve como objetivo estudar o processo de desgaste sofrido pelas ferramentas durante a furação e roscamento de aços AISI D2 e AISI H13 com a utilização de diferentes velocidades de corte. As forças de corte e o torque também serão medidos durante o processo para avaliar seus comportamentos com o aumento da velocidade. Com os resultados obtidos verificou-se que na furação e roscamento de aços endurecidos de baixa usinabilidade, como no caso do aço AISI D2, o desgaste da ferramenta é muito acentuado, podendo tornar o processo inviável economicamente caso não sejam empregadas velocidades de corte extremamente reduzidas. De uma forma geral, a furação e o roscamento do AISI H13 pode ser viável, pois o número de furos/roscas obtidos dentro dos critérios especificados mostrou-se muito superior ao esperado. Os principais mecanismos de desgastes que atuaram nos machos de corte durante o processo de roscamento de ambos os aços (AISI D2 e AISI H13) foram abrasão nas superfícies de folga e adesão nas superfícies de saída de forma acentuada nos três primeiros filetes. / The cutting processes compose a huge part of the manufacturing universe and, nowadays, some well-defined trends, like cutting hardened materials, are increasing not only in die molding production but also in automotive and machine industries. The turning and milling processes of materials, in this difficult condition of cutting, are already applied in the mechanical industries. Therefore, the total conversion of the present production process of regular materials into hardened ones can only be reached when all the cutting processes are included. Because of that, the demands of drilling and tapping hardened materials have increased constantly. These operations require tools specifically designed for them, because they have proven themselves to be much more difficult than the turning and milling processes. In that way, this work aimed to study the wear process of drills and taps used to machining the AISI D2 and AISI H13 hardened steels with different cutting speeds. The cutting forces and the torque generated were also acquired during the cutting process to evaluate its behavior with the speed increase. After analyzing the results, a very aggressive tool wear was confirmed in the drilling and tapping process of hardened steels with bad machinability, like the AISI D2 steel, and this can make the process economically unfeasible if an extremely reduced cutting speed is not used. Generally, the drilling and tapping process of the AISI H13 can become economically viable, because the number of holes/threads achieved in accordance with the specified criteria was superior to the expected ones. In the cutting process of both materials (AISI D2 and H13), the main wear mechanism observed on the taps were abrasion, on the clearance surface, and adhesion, on the rake surface. This was severe for the first three threads of the tap.

Now a day, it is no longer optional to have security solutions even inevitable for every kind of organizations and individuals. There are number of generic tools which are common for organizations as well as for individual users to provide security which includes; Anti-Spam, Anti-Virus etc., and network security have become essential issue in MANET. Security is one of the main issues in the MANET especially with respect to size and complexity of the network. The aim of the thesis is to discuss different aspects of security in MANET (e.g. multi-layer intrusion detection technique in multi hop network of MANET, security problems relates between multihop network and mobile nodes in MANET etc) and also implement some of the solutions (e.g. comparative study of different routing protocol (AODV, DSR and TORA) security threats within MANET network like intruder behavior, tapping and integrity, MANET link layer and network layer operations with respect to information security etc) with respect to MANET network. This report also discusses different number of scenarios of MANET network which we implement in our simulation. In our simulation we use to implement different routing protocols and also did comparative study that which one is better with respect to different aspects. We also use to implements mechanisms of intruder behavior, tapping and integrity, and MANET link layer and network layer operations with respect to information security.

Timber-concrete composite (TCC) floor systems consist of a bottom layer of wood that is connected to a top layer of concrete using shear connectors. The shear connectors resist slip between the layers, thus allowing wood and concrete develop composite action when subjected to flexure. The objective of this study is to determine the flexural strength and behavior of TCC floor systems that consist of a cross laminated timber wood layer connected to a concrete top layer using hexagonally headed self-tapping screw shear connectors. To accomplish the objective, coupon specimens and full-scale TCC floor panels were tested, and a finite element modelling approach was developed. The coupon tests were used to determine the stiffness of the shear connection and to determine the effect of the screw configuration. The results from the coupon tests indicated that the inclined screw configuration provided the largest shear strength compared to the normal, crossed, and nested screw configurations. Based on the results from the coupon tests, bending and vibration (heel drop) tests were conducted on full-scale panel specimens with an inclined screw configuration and with a strong-axis panel orientation. The results from the full-scale panel tests showed that the flexural stiffness and strength of the TCC floor system was consistent and that the composite floor panels have adequate stiffness to minimize transient floor vibrations that are caused by walking for typical span lengths and typical loading. A finite element model of TCC floor systems was developed to simulate TCC floor systems and calibrated with the test data. The simulated response matched the test response fairly well for partially composite single-span and double-span panels. Additional refinement of the model is needed to better match fully composite panels. The research demonstrated that hexagonal-headed self-tapping screws may be effectively used to connect wood and concrete layers in TCC floor systems.