Glioblastoma is the most common and most lethal primary brain tumor to affect adults. While current treatment options provide temporary recourse, the majority of patients experience tumor recurrence and few survive five years past their initial diagnosis. Recently, tumor microtubes (TMs) were identified in an in vivo model of glioblastoma. These membrane-bound structures formed physical connections between tumor cells, over short and long distances, and facilitated intratumoral communication, invasion, treatment resistance, and post-treatment tumor recovery. To date, this is the first instance of TMs being reported in glioblastoma. The lack of an in vitro model for these structures has delayed further characterization of how TMs form between cells, facilitate intercellular exchange, and how they can be therapeutically targeted to increase treatment susceptibility. The study presented here is the first instance of TMs characterized in an in vitro model of primary glioblastoma (PriGO) cells. These TMs recapitulated many of the structural and functional properties of those observed in vivo, making it a suitable model for further experimentation. Using this model, Rac1, a known orchestrator of cytoskeletal remodeling and motility, was shown to be integral to establishing a TM network between PriGO cells, as demonstrated by siRNA-mediated protein knockdowns. PREX1, a GEF necessary for Rac1 signaling activity, also played a role in PriGO TM formation as evidenced by CRISPR/Cas9-based knockouts. Re-introducing a PREX1 domain with Rac-GEF activity into cells lacking the protein led to a functional rescue of TM growth, thus confirming PREX1’s involvement. Characterizing a cell culture model of glioblastoma TMs is a necessary first step in the study of these structures, ultimately paving the way for future development of therapies that disrupt this network.

Chronic diseases can often be managed by constantly delivering therapeutic amounts of drug for prolonged periods. A controlled release for extended duration would replace the need for multiple and frequent dosing. Local drug release would provide added benefit as a lower dose of drug at the target site will be needed as opposed to higher doses required by whole body administration. This would provide maximum efficacy with minimum side effects. Nonetheless, a problem with the known implantable drug delivery devices is that the delivery rate cannot be controlled, which leads to drug being released in an unpredictable pattern resulting in poor therapeutic management of patients. This dissertation is the result of development of an implantable drug delivery system that is capable of long-term zero order local release of drugs. The device can be optimized to deliver any pharmaceutical agent for any time period up to several years maintaining a controlled and desired rate. Initially significant efforts were dedicated to the characterization, biocompatibility, and loading capacity of nanoporous metal surfaces for controlled release of drugs. The physical characterization of the nanoporous wafers using Scanning electron microscropy (SEM) and atomic force microscopy techniques (AFM) yielded 3.55 x 10⁴ nm³ of pore volume / μm² of wafer surface. In vitro drug release study using 2 - octyl cyanoacrylate and methyl orange as the polymer-drug matrix was conducted and after 7 days, 88.1 ± 5.0 % drug was released. However, the initial goal to achieve zero order drug release rates for long periods of time was not achieved. The search for a better delivery system led to the design of a perforated microtube. The delivery system was designed and appropriate dimensions for the device size and hole size were estimated. Polyimide microtubes in different sizes (125-1000 μm) were used. Micro holes with dimensions ranging from 20-600 μm were fabricated on these tubes using photolithography, laser drilling, or manual drilling procedures. Small molecules such as crystal violet, prednisolone, and ethinyl estradiol were successfully loaded inside the tubes in powder or solution using manual filling or capillary filling methods. A drug loading of 0.05 – 5.40 mg was achieved depending on the tube size and the drug filling method used. The delivery system in different dimensions was characterized by performing in vitro release studies in phosphate buffered saline (pH 7.1-7.4) and in vitreous humor from the rabbit’s eye at 37.0 ± 1.0°C for up to four weeks. The number of holes was varied between 1 and 3. The tubes were loaded with crystal violet (CV) and ethinyl estradiol (EE). Linear release rates with R²>0.9900 were obtained for all groups with CV and EE. Release rates of 7.8±2.5, 16.2±5.5, and 22.5±6.0 ng/day for CV and 30.1±5.8 ng/day for EE were obtained for small tubes (30 μm hole diameter; 125 μm tube diameter). For large tubes (362-542 μm hole diameter; 1000 μm tube diameter), a release rate of 10.8±4.1, 15.8±4.8 and 22.1±6.7 μg/day was observed in vitro in PBS and a release rate of 5.8±1.8 μg/day was observed ex vivo in vitreous humor. The delivery system was also evaluated for its ability to produce a biologically significant amounts in cells stably transfected with an estrogen receptor/luciferase construct (T47D-KBluc cells). These cells are engineered to produce a constant luminescent signal in proportion to drug exposure. The average luminescence of 1144.8±153.8 and 1219.9±127.7 RLU/day, (RLU = Relative Luminescence Units), yet again indicating the capability of the device for long-term zero order release. The polyimide device was characterized for biocompatibility. An automated goniometer was used to determine the contact angle for the device, which was found to be 63.7±3.7degreees indicating that it is hydrophilic and favors cell attachment. In addition, after 72 h incubation with mammalian cells (RAW 267.4), a high cell distribution was observed on the device’s surface. The polyimide tubes were also investigated for any signs of inflammation using inflammatory markers, TNF-α and IL-1β. No significant levels of either TNF-α or IL-1β were detected in polyimide device. The results indicated that polyimide tubes were biocompatible and did not produce an inflammatory response. / text

O objetivo do presente trabalho foi a fabricação de micro e nanotubos de silício através de métodos eletroquímicos, utilizando como material de partida o silício poroso. Os resultados obtidos durante o desenvolvimento deste trabalho apresentam contribuições significativas nos campo da micro e nanotecnologia, bem como no campo da eletroquímica do silício. Esses resultados mostraram que a formação dos microtubos é sensível às variações do pH, tipo e concentração dos oxidantes, tempo de corrosão, espessura das camadas porosas e tratamento prévio da superfície polida das amostras com alumínio e posterior recozimento térmico. A análise estrutural, via microscopia eletrônica de varredura (MEV), mostra que os microtubos de silício (MTS) podem ser formados em valores de pH entre 5,5 a 7,5, dependendo do tipo e concentração dos oxidantes. A análise da composição química dos MTS, realizada mediante a espectroscopia por dispersão de energia (EDS). Os resultados da espectroscopia Raman indicaram que as estruturas são tubos de Silício, cuja estequiometria é a mesma que a do cristal de Silício. Foi demonstrada a possibilidade de formação de microtubos metálicos através do controle das condições químicas e eletroquímicas da solução, utilizadas na formação das estruturas tubulares de silício. A condição da solução eletroquímica com nível de pH 6,5 permitiu formar microtubos de níquel, enquanto que estruturas tubulares de manganês foram obtidas em soluções com nível de pH 7,5. Em ambos os casos, foram utilizados agentes oxidantes originados de sais de níquel e manganês, respectivamente. Os resultados indicam que o processo de formação destas estruturas acontece por efeito do deslocamento galvânico. Os resultados apresentados neste trabalho são completamente inéditos e mostram-se vantajosos em relação àqueles reportados na literatura, já que estes últimos são obtidos mediante processos de alto custo que requerem sofisticados equipamentos, enquanto os microtubos de silício obtidos mediante técnicas eletroquímicas, aqui usadas, são de baixo custo e fácil montagem. / The aim of this work was to fabricate micro and nanotubes of silicon, through electrochemical methods and using the porous silicon as starting material. The results achieved during the development of this work present significant contributions into the micro and nanotechnology field, as well as in the silicon electrochemistry field. The obtained results have shown that the formation of the microtubes is sensitive to variations of pH, type and concentration of the oxidants, time of corrosion, thickness of the porous layers, and previous treatment of the polished silicon surface with aluminum and subsequent thermal annealing. The results of the structural analysis by scanning electron microscopy (SEM) show that the silicon microtubes can be formed at pH values ranging between 5,5 and 7,5, depending on the type and concentration of the oxidants. The chemical analysis upon the composition of these silicon tubular structures was carried out by Energy Dispersion Spectroscopy (EDS). Raman spectroscopy results indicated that the tubular structures are silicon tubes, whose stoichiometric structure is similar to the silicon crystal. The possibility of formation of metallic microtubes, just by controlling the chemical and electrochemical conditions of the etching solution used to form the silicon tubular structures was shown. The electrochemical condition of the solution with level of pH 6,5 allowed the formation of nickel microtubes structures, and the formation of manganese tubular structures was obtained in solutions with pH of 7,5. In both cases there have been used oxidants agents originated from nickel and manganese salts, respectively. The results indicate that the formation process of these metallic structures happens by a galvanic displacement process. The results presented in this work are completely unpublished and reveal advantageous in relation to those reported in literature because these last ones are obtained by using high cost processes which require sophisticated equipment, while the silicon microtubes obtained by electrochemical techniques used in the present work are of low cost and of easy assembly.

O objetivo do presente trabalho foi a fabricação de micro e nanotubos de silício através de métodos eletroquímicos, utilizando como material de partida o silício poroso. Os resultados obtidos durante o desenvolvimento deste trabalho apresentam contribuições significativas nos campo da micro e nanotecnologia, bem como no campo da eletroquímica do silício. Esses resultados mostraram que a formação dos microtubos é sensível às variações do pH, tipo e concentração dos oxidantes, tempo de corrosão, espessura das camadas porosas e tratamento prévio da superfície polida das amostras com alumínio e posterior recozimento térmico. A análise estrutural, via microscopia eletrônica de varredura (MEV), mostra que os microtubos de silício (MTS) podem ser formados em valores de pH entre 5,5 a 7,5, dependendo do tipo e concentração dos oxidantes. A análise da composição química dos MTS, realizada mediante a espectroscopia por dispersão de energia (EDS). Os resultados da espectroscopia Raman indicaram que as estruturas são tubos de Silício, cuja estequiometria é a mesma que a do cristal de Silício. Foi demonstrada a possibilidade de formação de microtubos metálicos através do controle das condições químicas e eletroquímicas da solução, utilizadas na formação das estruturas tubulares de silício. A condição da solução eletroquímica com nível de pH 6,5 permitiu formar microtubos de níquel, enquanto que estruturas tubulares de manganês foram obtidas em soluções com nível de pH 7,5. Em ambos os casos, foram utilizados agentes oxidantes originados de sais de níquel e manganês, respectivamente. Os resultados indicam que o processo de formação destas estruturas acontece por efeito do deslocamento galvânico. Os resultados apresentados neste trabalho são completamente inéditos e mostram-se vantajosos em relação àqueles reportados na literatura, já que estes últimos são obtidos mediante processos de alto custo que requerem sofisticados equipamentos, enquanto os microtubos de silício obtidos mediante técnicas eletroquímicas, aqui usadas, são de baixo custo e fácil montagem. / The aim of this work was to fabricate micro and nanotubes of silicon, through electrochemical methods and using the porous silicon as starting material. The results achieved during the development of this work present significant contributions into the micro and nanotechnology field, as well as in the silicon electrochemistry field. The obtained results have shown that the formation of the microtubes is sensitive to variations of pH, type and concentration of the oxidants, time of corrosion, thickness of the porous layers, and previous treatment of the polished silicon surface with aluminum and subsequent thermal annealing. The results of the structural analysis by scanning electron microscopy (SEM) show that the silicon microtubes can be formed at pH values ranging between 5,5 and 7,5, depending on the type and concentration of the oxidants. The chemical analysis upon the composition of these silicon tubular structures was carried out by Energy Dispersion Spectroscopy (EDS). Raman spectroscopy results indicated that the tubular structures are silicon tubes, whose stoichiometric structure is similar to the silicon crystal. The possibility of formation of metallic microtubes, just by controlling the chemical and electrochemical conditions of the etching solution used to form the silicon tubular structures was shown. The electrochemical condition of the solution with level of pH 6,5 allowed the formation of nickel microtubes structures, and the formation of manganese tubular structures was obtained in solutions with pH of 7,5. In both cases there have been used oxidants agents originated from nickel and manganese salts, respectively. The results indicate that the formation process of these metallic structures happens by a galvanic displacement process. The results presented in this work are completely unpublished and reveal advantageous in relation to those reported in literature because these last ones are obtained by using high cost processes which require sophisticated equipment, while the silicon microtubes obtained by electrochemical techniques used in the present work are of low cost and of easy assembly.

Hybride Mikroschwimmer, die einen biologischen Antrieb und eine künstlich hergestellte Mikrostruktur enthalten sind ein attraktiver Ansatz um kontrollierte Bewegung auf kleinstem Maßstab zu erreichen. In dieser Dissertation wird ein neuer hybrider Mikroschwimmer vorgestellt, der aus ferromagnetischen Nanomembranen besteht, die sich zu Mikroröhrchen aufrollen und in der Lage sind, einzelne Spermien einzufangen. Dieser Mikrobioroboter nutzt die starke Antriebskraft der Spermazelle um das magnetische Mikroröhrchen fortzubewegen. Die vorliegende Arbeit beschreibt, wie dieser Mikroschwimmer seine Bewegung vollzieht und wie verschiedene Faktoren wie Temperatur, Radius der Mikroröhrchen, Eindringtiefe der Spermien in das Röhrchen und Länge der Röhrchen einen Einfluss auf sein Verhalten haben. Richtungskontrolle wird durch externe magnetische Felder realisiert und es wird dargestellt, wie dies zur Trennung der Mikrobioroboter aus einer Mischung von Spermien und Mikroröhrchen genutzt werden kann. Weiterhin werden zwei Oberflächenmodifizierungsmethoden angewandt um die Kupplungseffizienz zwischen Mikroröhrchen und Spermien zu erhöhen. In diesen Methoden wird das extrazelluläre Protein Fibronektin auf die innere Röhrchenoberfläche aufgebracht und dient als Bindungsstoff für Spermien. Schließlich wird durch den Einbau temperatursensitiver Material in die Mikroröhrchen ein ferngesteuerter Freisetzungsmechanismus für die Spermazelle vorgestellt. Dabei falten sich die Mikroröhrchen bei kleinen Temperaturerhöhungen auf und setzen die Zelle frei. Diese Arbeit diskutiert letztendlich das Potential solch eines hybriden Mikroschwimmers für die Anwendung in assistierter Reproduktion. / The search for autonomously moving, highly functional and controllable microdevices is a purpose of current micro/nanobiotechnology research, especially in the area of biomedical applications, for which reason, biocompatible solutions are in demand. In this thesis, a novel type of hybrid microswimmer is fabricated by the combination of rolled up thin nanomembranes with bovine spermatozoa. The microbiorobot presented here uses the powerful motion of the sperm flagella as a propulsion source for the magnetic microtube. This work demonstrates how the microswimmer performs its motion and how several factors such as temperature, radius of the microtube, penetration of the cell inside the microtube and length of the tube have influence on its performance. Directional control mechanisms are offered by external magnetic fields and are presented to be useful for the on-chip separation of the microbiorobots from a mixture of cells and microtubes. Two surface modification methods are presented as means to improve the coupling efficiency between the microtubes and the sperm cells. By these surface functionalizations, the extracellular matrix protein fibronectin is attached on the inner microtube walls and serves as binding agent for the spermatozoa. Finally, a remote release mechanism for the sperm cells is demonstrated by the incorporation of thermoresponsive material into the microtubes, which makes them fold and unfold upon small temperature changes. This work discusses the potential of such microswimmers for the application in assisted reproduction techniques and gives an outlook on future perspectives.

A inovação tecnológica é o caminho mais viável para desenvolver uma agricultura sustentada no Brasil. Portanto, o país deve investir nos setores que possam produzir tecnologias inovadoras, adequadas as suas condições peculiares e de custo mais acessível, em substituição à dependência por produtos importados. Dentro desse contexto, este trabalho tem os objetivos de desenvolver anteparos de microaspersor com boa uniformidade de distribuição para trabalhar com sobreposição e adaptar a técnica desenvolvida por Almeida, Botrel e Smith (2008) para os microaspersores convencionais. O trabalho foi conduzido no Laboratório de Hidráulica e na área experimental do Departamento de Engenharia Rural da ESALQ/USP, onde foram desenvolvidos e avaliados os protótipos de microaspersores, e avaliado o desempenho da adaptação do microtubo aos microaspersores comerciais. Para o desenvolvimento dos protótipos de microaspersores foram testados diversos materiais e processos. Os materiais utilizados foram argila, gesso, biscuit, massa de modelar, pedra talco e os tarugos de PVC, technyl, teflon e polipropileno. Já os processos foram o torneamento mecânico, modelagem e a prototipagem rápida. O desempenho da adaptação dos microtubos aos microaspersores comerciais foi avaliado através de testes em laboratório e no campo. A relação comprimento de microtubo versus pressão de operação versus vazão foi determinada, em seguida, avaliou-se a uniformidade de distribuição de água na linha lateral. Utilizou-se os coeficientes de uniformidade de Christiansen, o da uniformidade de distribuição e o da uniformidade estatística na avaliação do desempenho dos emissores. O PVC e o tecnyl foram os materiais que apresentaram mais facilidade na construção das bailarinas, porém, o PVC foi o escolhido devido ao melhor acabamento. As melhores sobreposições ocorreram com o protótipo de 6 canaletas com 350 e duas canaletas diferenciadas. Os ajustes realizados nos protótipos iniciais que não funcionaram contribuíram significativamente para que o protótipo desenvolvido alcançasse resultados positivos. A geometria das bailarinas, ângulo e número de canaletas afetaram o desempenho do modelo, sendo que, os de maiores ângulos apresentaram uniformidade de distribuição de água mais satisfatórias. A utilização da técnica de microtubos se adapta a microaspersores convencionais, uniformizando a vazão ao longo da linha lateral. / The technology innovation is the way most feasible to develop a sustainable agriculture in Brazil. In this way, the country will have to invest in sectors which can produce innovation technologies, adequate to specific conditions and with cost more accessible, to replace the dependency from imported products. In this context, this work had to objective to develop microsprinkler spinner deflector with good distribution uniformity to work with overlap and adapt the microtubes techniques developed by Almeida, Botrel e Smith (2008) to commercial microsprinkler. Experiments in laboratory and field were carried out at Escola Superior de Agricultura Luiz de Queiroz, University of Sao Paulo, Brazil, where were developed and evaluated the microsprinkler prototypes and evaluated discharge uniformity along of lateral line with commercial micro-sprinklers and microtubes as emitter. In the development of the microsprinkler prototypes were tested a lot of materials and processes. The materials utilized were clay, biscuit, plaster, modeling mass, talc stone, PVC, technyl, teflon and polypropylene. The processes were mechanic tournament, modeling and the rapid prototyping. The performance of micro-sprinkler irrigation system with microtube emitters was evaluated both laboratory and field tests. Microtube pressure-length-discharge relationship was determinate and discharge distribution uniformity along of lateral line was evaluated. Uniformity Coefficient from Christiansen (CUC), Distribution Uniformity (DU) and Uniformity Statistic (Us) were used as indicators for evaluation of performance of emitters. Among the tested materials, the PVC and the technyl showed more facility in the deflector build, however, the PVC was choose due the better finish small channels. The rapid prototyping process provided the better finish in the deflector. In the proposed overlap, the better overlap arrangement occurred with the prototype of 6 channels with 350 and two channels differentiated. The developed prototypes which not worked and the adjustments did in them, contributed significantly to reach positive results in prototypes. The deflector geometric, angle and number of channels affected directly the performance of microsprinklers, nevertheless, the biggest angle showed water distribution uniformity more satisfactory. The utilization of microtubes techniques adapted well in conventional microsprinklers, and provided good uniformity in the discharge in lateral line.

In this project we studied the effect of different parameters on heat transfer in two-dimensional microchannels, microtubes and also tube-in-block heat exchangers for various engineering applications. These included the use of flow restrictions to enhance local heat or mass transfer rate, enhancement of conjugate heat transfer with discrete heating and magnetic coolers (or heaters) associated with magnetic refrigeration system. The results of this research will help in designing the heating or cooling systems and selection of their appropriate geometrical dimensions and materials for specific applications. Types of problems studied in this project are: steady state analysis of forced convection around a restricted flow passage in a micro channel, effect of protrusions in a microtube or microchannel for conjugate heat transfer with uniform or discrete heating and transient analysis of heat transfer in trapezoidal microchannels under time varying heat source. For each of these problems a numerical simulation model was developed. The mass, momentum, and energy conservation equations were solved in the fluid region and energy conservation in the sold region to arrive at the velocity and the temperature distributions. Detailed parametric study was carried out for each problem. The parameters were shape and size of the restriction/protrusion, number of restrictions/protrusions, wall thickness, Reynolds number, solid materials and working fluids. The results showed that in microtubes the local value of Nusselt number increases at the restriction/protrusion and the global value for the whole tube is somewhat lower. For a two-dimensional microchannel, both the local Nusselt number at the restriction/protrusion as well as the global Nusselt number for the entire channel is higher. In the trapezoidal channels the results showed that with the increase in Reynolds number, the outlet temperature decreased and the average heat transfer coefficient increased. With an increase in magnetic field there was an increase in the solid fluid interface temperature and in turn the average heat transfer coefficient increased. With a decrease in the channel height and width there was an increase in the average Nusselt number in the channel.

A inovação tecnológica é o caminho mais viável para desenvolver uma agricultura sustentada no Brasil. Portanto, o país deve investir nos setores que possam produzir tecnologias inovadoras, adequadas as suas condições peculiares e de custo mais acessível, em substituição à dependência por produtos importados. Dentro desse contexto, este trabalho tem os objetivos de desenvolver anteparos de microaspersor com boa uniformidade de distribuição para trabalhar com sobreposição e adaptar a técnica desenvolvida por Almeida, Botrel e Smith (2008) para os microaspersores convencionais. O trabalho foi conduzido no Laboratório de Hidráulica e na área experimental do Departamento de Engenharia Rural da ESALQ/USP, onde foram desenvolvidos e avaliados os protótipos de microaspersores, e avaliado o desempenho da adaptação do microtubo aos microaspersores comerciais. Para o desenvolvimento dos protótipos de microaspersores foram testados diversos materiais e processos. Os materiais utilizados foram argila, gesso, biscuit, massa de modelar, pedra talco e os tarugos de PVC, technyl, teflon e polipropileno. Já os processos foram o torneamento mecânico, modelagem e a prototipagem rápida. O desempenho da adaptação dos microtubos aos microaspersores comerciais foi avaliado através de testes em laboratório e no campo. A relação comprimento de microtubo versus pressão de operação versus vazão foi determinada, em seguida, avaliou-se a uniformidade de distribuição de água na linha lateral. Utilizou-se os coeficientes de uniformidade de Christiansen, o da uniformidade de distribuição e o da uniformidade estatística na avaliação do desempenho dos emissores. O PVC e o tecnyl foram os materiais que apresentaram mais facilidade na construção das bailarinas, porém, o PVC foi o escolhido devido ao melhor acabamento. As melhores sobreposições ocorreram com o protótipo de 6 canaletas com 350 e duas canaletas diferenciadas. Os ajustes realizados nos protótipos iniciais que não funcionaram contribuíram significativamente para que o protótipo desenvolvido alcançasse resultados positivos. A geometria das bailarinas, ângulo e número de canaletas afetaram o desempenho do modelo, sendo que, os de maiores ângulos apresentaram uniformidade de distribuição de água mais satisfatórias. A utilização da técnica de microtubos se adapta a microaspersores convencionais, uniformizando a vazão ao longo da linha lateral. / The technology innovation is the way most feasible to develop a sustainable agriculture in Brazil. In this way, the country will have to invest in sectors which can produce innovation technologies, adequate to specific conditions and with cost more accessible, to replace the dependency from imported products. In this context, this work had to objective to develop microsprinkler spinner deflector with good distribution uniformity to work with overlap and adapt the microtubes techniques developed by Almeida, Botrel e Smith (2008) to commercial microsprinkler. Experiments in laboratory and field were carried out at Escola Superior de Agricultura Luiz de Queiroz, University of Sao Paulo, Brazil, where were developed and evaluated the microsprinkler prototypes and evaluated discharge uniformity along of lateral line with commercial micro-sprinklers and microtubes as emitter. In the development of the microsprinkler prototypes were tested a lot of materials and processes. The materials utilized were clay, biscuit, plaster, modeling mass, talc stone, PVC, technyl, teflon and polypropylene. The processes were mechanic tournament, modeling and the rapid prototyping. The performance of micro-sprinkler irrigation system with microtube emitters was evaluated both laboratory and field tests. Microtube pressure-length-discharge relationship was determinate and discharge distribution uniformity along of lateral line was evaluated. Uniformity Coefficient from Christiansen (CUC), Distribution Uniformity (DU) and Uniformity Statistic (Us) were used as indicators for evaluation of performance of emitters. Among the tested materials, the PVC and the technyl showed more facility in the deflector build, however, the PVC was choose due the better finish small channels. The rapid prototyping process provided the better finish in the deflector. In the proposed overlap, the better overlap arrangement occurred with the prototype of 6 channels with 350 and two channels differentiated. The developed prototypes which not worked and the adjustments did in them, contributed significantly to reach positive results in prototypes. The deflector geometric, angle and number of channels affected directly the performance of microsprinklers, nevertheless, the biggest angle showed water distribution uniformity more satisfactory. The utilization of microtubes techniques adapted well in conventional microsprinklers, and provided good uniformity in the discharge in lateral line.

Hybride Mikroschwimmer, die einen biologischen Antrieb und eine künstlich hergestellte Mikrostruktur enthalten sind ein attraktiver Ansatz um kontrollierte Bewegung auf kleinstem Maßstab zu erreichen. In dieser Dissertation wird ein neuer hybrider Mikroschwimmer vorgestellt, der aus ferromagnetischen Nanomembranen besteht, die sich zu Mikroröhrchen aufrollen und in der Lage sind, einzelne Spermien einzufangen. Dieser Mikrobioroboter nutzt die starke Antriebskraft der Spermazelle um das magnetische Mikroröhrchen fortzubewegen. Die vorliegende Arbeit beschreibt, wie dieser Mikroschwimmer seine Bewegung vollzieht und wie verschiedene Faktoren wie Temperatur, Radius der Mikroröhrchen, Eindringtiefe der Spermien in das Röhrchen und Länge der Röhrchen einen Einfluss auf sein Verhalten haben. Richtungskontrolle wird durch externe magnetische Felder realisiert und es wird dargestellt, wie dies zur Trennung der Mikrobioroboter aus einer Mischung von Spermien und Mikroröhrchen genutzt werden kann. Weiterhin werden zwei Oberflächenmodifizierungsmethoden angewandt um die Kupplungseffizienz zwischen Mikroröhrchen und Spermien zu erhöhen. In diesen Methoden wird das extrazelluläre Protein Fibronektin auf die innere Röhrchenoberfläche aufgebracht und dient als Bindungsstoff für Spermien. Schließlich wird durch den Einbau temperatursensitiver Material in die Mikroröhrchen ein ferngesteuerter Freisetzungsmechanismus für die Spermazelle vorgestellt. Dabei falten sich die Mikroröhrchen bei kleinen Temperaturerhöhungen auf und setzen die Zelle frei. Diese Arbeit diskutiert letztendlich das Potential solch eines hybriden Mikroschwimmers für die Anwendung in assistierter Reproduktion.:TABLE OF CONTENTS SELBSTSTÄNDIGKEITSERKLÄRUNG 0 ABSTRACT 1 TABLE OF CONTENTS 3 1 MOTIVATION AND GOALS 5 1.1 MINIATURIZATION: “THERE IS PLENTY OF ROOM AT THE BOTTOM…” 5 1.2 SPERMBOTS: POTENTIAL IMPACT 7 2 BACKGROUND AND STATE-OF-THE-ART 11 2.1 MICROBIOROBOTICS 11 2.2 SPERM MORPHOLOGY AND THEIR JOURNEY TO THE EGG 15 2.3 INFERTILITY AND ASSISTED REPRODUCTION TECHNIQUES 19 2.4 SINGLE CELL RELEASE 22 2.5 STIMULI-RESPONSIVE MATERIALS 25 3 MATERIAL AND METHODS 29 3.1 ROLLED UP TECHNOLOGY 29 3.2 TREATMENT OF BOVINE SPERMATOZOA 32 3.2.1 Preparation of Spermbots 32 3.2.2 Speed Measurements 33 3.2.3 Separation On Chip 33 3.3 SURFACE MODIFICATION OF MICROTUBES 34 3.3.1 Surface Chemistry 35 3.3.2 Microcontact printing 39 3.4 POLYMER TUBE FABRICATION 44 3.4.1 Synthesis of photosensitive monomer 4-Acryloylbenzophenone 44 3.4.2 Synthesis of poly (N-isopropylacrylamide-co-Acryloylbenzophenone) 46 3.4.3 Photolithography of polymeric films 48 3.5 VIABILITY TESTS 51 4 RESULTS AND DISCUSSION 53 4.1 CHARACTERIZATION OF SPERMBOTS 55 4.2 TEMPERATURE INFLUENCE 60 4.3 MAGNETIC CONTROL 62 4.4 SEPARATION ON CHIP 68 4.5 EFFECT OF DECREASED MICROTUBE LENGTH 72 4.6 COUPLING EFFICIENCY 74 4.7 THERMORESPONSIVE POLYMERIC MICROTUBES FOR CELL RELEASE 80 4.8 SPERM VIABILITY TESTS 94 5 SUMMARY AND CONCLUSIONS 97 6 OUTLOOK 101 7 LIST OF FIGURES 107 8 LIST OF TABLES 113 9 ABBREVIATIONS 115 10 CURRICULUM VITAE 117 11 LIST OF PUBLICATIONS 119 JOURNAL ARTICLES 119 CONTRIBUTIONS TO COLLECTED EDITIONS/PROCEEDINGS 121 12 ACKNOWLEDGEMENTS 123 13 REFERENCES 125 / The search for autonomously moving, highly functional and controllable microdevices is a purpose of current micro/nanobiotechnology research, especially in the area of biomedical applications, for which reason, biocompatible solutions are in demand. In this thesis, a novel type of hybrid microswimmer is fabricated by the combination of rolled up thin nanomembranes with bovine spermatozoa. The microbiorobot presented here uses the powerful motion of the sperm flagella as a propulsion source for the magnetic microtube. This work demonstrates how the microswimmer performs its motion and how several factors such as temperature, radius of the microtube, penetration of the cell inside the microtube and length of the tube have influence on its performance. Directional control mechanisms are offered by external magnetic fields and are presented to be useful for the on-chip separation of the microbiorobots from a mixture of cells and microtubes. Two surface modification methods are presented as means to improve the coupling efficiency between the microtubes and the sperm cells. By these surface functionalizations, the extracellular matrix protein fibronectin is attached on the inner microtube walls and serves as binding agent for the spermatozoa. Finally, a remote release mechanism for the sperm cells is demonstrated by the incorporation of thermoresponsive material into the microtubes, which makes them fold and unfold upon small temperature changes. This work discusses the potential of such microswimmers for the application in assisted reproduction techniques and gives an outlook on future perspectives.:TABLE OF CONTENTS SELBSTSTÄNDIGKEITSERKLÄRUNG 0 ABSTRACT 1 TABLE OF CONTENTS 3 1 MOTIVATION AND GOALS 5 1.1 MINIATURIZATION: “THERE IS PLENTY OF ROOM AT THE BOTTOM…” 5 1.2 SPERMBOTS: POTENTIAL IMPACT 7 2 BACKGROUND AND STATE-OF-THE-ART 11 2.1 MICROBIOROBOTICS 11 2.2 SPERM MORPHOLOGY AND THEIR JOURNEY TO THE EGG 15 2.3 INFERTILITY AND ASSISTED REPRODUCTION TECHNIQUES 19 2.4 SINGLE CELL RELEASE 22 2.5 STIMULI-RESPONSIVE MATERIALS 25 3 MATERIAL AND METHODS 29 3.1 ROLLED UP TECHNOLOGY 29 3.2 TREATMENT OF BOVINE SPERMATOZOA 32 3.2.1 Preparation of Spermbots 32 3.2.2 Speed Measurements 33 3.2.3 Separation On Chip 33 3.3 SURFACE MODIFICATION OF MICROTUBES 34 3.3.1 Surface Chemistry 35 3.3.2 Microcontact printing 39 3.4 POLYMER TUBE FABRICATION 44 3.4.1 Synthesis of photosensitive monomer 4-Acryloylbenzophenone 44 3.4.2 Synthesis of poly (N-isopropylacrylamide-co-Acryloylbenzophenone) 46 3.4.3 Photolithography of polymeric films 48 3.5 VIABILITY TESTS 51 4 RESULTS AND DISCUSSION 53 4.1 CHARACTERIZATION OF SPERMBOTS 55 4.2 TEMPERATURE INFLUENCE 60 4.3 MAGNETIC CONTROL 62 4.4 SEPARATION ON CHIP 68 4.5 EFFECT OF DECREASED MICROTUBE LENGTH 72 4.6 COUPLING EFFICIENCY 74 4.7 THERMORESPONSIVE POLYMERIC MICROTUBES FOR CELL RELEASE 80 4.8 SPERM VIABILITY TESTS 94 5 SUMMARY AND CONCLUSIONS 97 6 OUTLOOK 101 7 LIST OF FIGURES 107 8 LIST OF TABLES 113 9 ABBREVIATIONS 115 10 CURRICULUM VITAE 117 11 LIST OF PUBLICATIONS 119 JOURNAL ARTICLES 119 CONTRIBUTIONS TO COLLECTED EDITIONS/PROCEEDINGS 121 12 ACKNOWLEDGEMENTS 123 13 REFERENCES 125

The work focused on addressing four main objectives. The first objective was to quantify protein and amino acid substitution reactions. Michael addition reactions were used to modify the amino acids and protein. Amino acids alanine, cysteine, and lysine, and protein ovalbumin (OA) were substituted with different concentrations of ethyl vinyl sulfone (EVS). The substituted products were analyzed using Raman spectroscopy and UV-spectroscopy based ninhydrin assay. In case of alanine, Raman and UV results correlated with each other. With cysteine at lower EVS substitutions amine on the main chain was the preferred site while the substitution shifted to thiols at higher substitutions. This could only be discerned using Raman spectroscopy. Lysine has amines on the main chain and side chain while main chain amine was the most reactive site at lower concentrations of EVS while at higher concentrations side chain amines were also substituted. This information could be discerned using Raman spectroscopy only and not UV spectroscopy. In case of protein as observed by Raman and UV spectroscopy the reaction continued at higher concentrations of EVS indicating the participation of glutamine and asparagines at higher substitutions. However, the reaction considerably slowed down at higher EVS substitutions. The second objective of the study was to decrease the glass transition temperature (Tg) of OA through internal plasticization and also study the effects of the substituents on the thermal stability of OA. The hypothesis was by covalently attaching substituents to OA, number of hydrogen bonds can be reduced while increasing the free volume and this would reduce Tg. EVS, acrylic acid (AA), butadiene sulfone (BS) and maleimide (MA) were the four groups used. EVS was the most efficient plasticizer of all the four substituents. The Tg decreased with the increasing concentration of EVS until all of the reactive of groups on OA were used up. Tg decreased slightly with AA and BS while no change was observed with MA. However, the substituents showed exact opposite trend in thermal stability as measured using thermogravimetric analysis (TGA). The thermal stability of MA substituted OA was the highest and that of EVS substituted OA was least. FT-IR spectroscopy results indicated that all four substituents caused structural changes in OA. This implied that there were intermolecular interactions between substituted protein chains in case of AA, BS, and MA. This caused an increase in the thermal stability. EVS on the other hand is a linear chain monomer with a hydrophobic end group and hence could not participate in the intermolecular interactions and hence caused a decrease in Tg. As mentioned above the limitation to this technique is the number of available reactive groups on the protein. However, we successfully demonstrated the feasibility of this method in decreasing Tg of protein. The third objective was to create hydrogels by crosslinking OA with divinyl sulfone (DVS). Protein hydrogels due to their biocompatible nature find applications in drug delivery and tissue engineering. For tissue engineering applications the hydrogels need to be mechanically stable. In this study the protein was substituted with EVS or AA and then crosslinked with DVS. The swelling ratio was measured as a function of pH. All the hydrogels showed the same trend and swelled the least at pH 4.5 which is the isoelectric point of the protein. At basic pH conditions EVS substituted hydrogels swelled the most while AA substituted hydrogels showed least swelling. The static and dynamic moduli of the hydrogels were determined using tensile tester and rheometer respectively. The static modulus values were three times the dynamic modulus. The modulus of the control which is crosslinked OA was least and that of AA substituted OA was highest. The stress relaxation test also showed similar results in which AA substituted OA relaxed the most and the control relaxed the least. FT-IR of the dry hydrogels showed that the amount of hydrogen bonding increased with AA substitution. The hydrophilic AA end groups interacted with each other forming hydrogen bonds. These hydrogen bonds served as additional crosslinks there by increasing the modulus of the hydrogels. EVS on the other hand was incapable of interactions due to the lack of hydrophilic end groups. We were successfully able to create protein hydrogels and control the swelling and mechanical properties by varying the amount of substituted group. The final objective of the study was to create and characterize microstructures from substituted alanine and lysine. Alanine and lysine were substituted with different concentrations of EVS. Bars and fibers were observed for alanine at moderate substitutions while at higher concentrations random structures were observed using scanning electron microscopy (SEM). Lysine formed tubes at moderate EVS substitutions and rosettes at high concentrations of EVS as evidenced by SEM. FT-IR results suggested that instead of carbonyl one of sulfonyl bonded to the available amine in modified amino acids. And only in this case fibers, tubes and rosettes were observed. X-ray diffraction (XRD) results supported this observation. Using these results we hypothesized that the self assembled structures very much depended on the amount of EVS present in the substituted product and sulfonyl forming β-sheet analogs with amine. / Ph. D.