Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-05-24T18:44:26Z No. of bitstreams: 2 Ana Cláudia Malagutti Corsato.pdf: 1319419 bytes, checksum: c4de080588607227096826021e800839 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-24T18:44:26Z (GMT). No. of bitstreams: 2 Ana Cláudia Malagutti Corsato.pdf: 1319419 bytes, checksum: c4de080588607227096826021e800839 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-05 / The Diatraea saccharalis is the pest responsible for a great economic loss to the cultivation of sugar cane. As well as others members of Lepidoptera order, the D. saccharalis has a fast and efficient immune innate response, characterized by primary defense barrier, cellular immune response and humoral immune response. The antimicrobial peptides (AMPs) are included in humoral immune response that show cationic and amphiphilic characteristics besides its low molecular weight, what makes them potentially therapeutic agents. In this study were analyzed proteins with low molecular weight differently expressed in the hemolymph of D. saccharalis, after the interval of six and twelve hours of humoral immune response induction with different microorganisms compared to non-challenged worms (control). The 5th instar worms were divided in groups (n= 50) and submitted to six and twelve hours of septic challenges: control (group 1), challenged by Bacillus subtilis ATCC 6623 (group 2), challenged by Escherichia coli ATCC 11229 (group 3), and challenged by Beauveria bassiana 88 strain (group 4). In each worm was inoculated a known concentration of microorganism, and after the established time, the hemolymph was collected. The low molecular weight proteins were obtained submitting each hemolymph sample to an extraction solution containing Methanol, Acetic Acid and Water. After that, the protein extracts were concentrated in columns and protein dosage was realized in 280 nm. About 500 μg of proteins were submitted to two-dimensional electrophoresis (2-DE). The spots were excised from gels, digested with Tripsin and submitted to MALDI-ToF/ToF type mass spectrometry. The identification of protein orthologs was realized using the obtained data against the data available on the MASCOT online server (Matrix science), with the database (NCBI and Swiss rot) specified for Drosophila melanogaster and an internal database of Lepidoptera. The TagIdent tool was utilized for searching proteins through their molecular weight and isoelectric point. It was possible to identify eighteen proteins, of which twelve demonstrated to be involved with immune response in D. saccharalis. The six hours septic challenge with B. subtilis was responsible for modification of expression of Peptidoglycan recognition protein (PGRP), for increase of expression of Chitin-binding protein and for induction of expression of proteins like the Attacin-A and the Serine-protease inhibitor-like. The six hours septic challenge with E. coli increased the expression of a putative defense protein. The twelve hours septic challenge using B. subtilis and E. coli were responsible for increase of expression of Lysozyme. The six hours septic challenge with B. bassiana induced of expression of a Cecropin A2 and a Drosomycin-like. On the order hand, after twelve hours of fungal challenge, there was the induction of expression of multifunctional protein Apolipophorin-3. It was concluded that the septic challenge with different microorganisms were capable of change the expression of some proteins involved in the immune response in D. saccharalis, significant for understanding this process as well as for pointing the substances with antimicrobial functions. / A Diatraea saccharalis é a praga responsável por grandes perdas econômicas à cultura da cana-de-açúcar. Assim como outros integrantes da ordem Lepidoptera, a D. saccharalis possui resposta imunológica inata rápida e eficiente, constituída por barreira primária de defesa, resposta imune celular e resposta imune humoral. Os peptídeos antimicrobianos (PAMs) são parte da resposta imune humoral que apresentam características catiônicas e anfifílicas, além de sua massa molecular baixa, o que os tornam potenciais agentes terapêuticos. Neste estudo foram analisadas proteínas com baixa massa molecular, diferencialmente expressas na hemolinfa da D. saccharalis, após os intervalos de 6 e 12 horas de indução da resposta imune humoral com diferentes microrganismos em comparação às lagartas não desafiadas (controle). As lagartas em 5º instar foram divididas em grupos (n= 50) e submetidas a desafios sépticos de 6 e 12 horas: controle (grupo 1), desafiadas por Bacillus subtilis ATCC 6623 (grupo 2), desafiadas por Escherichia coli ATCC 11229 (grupo 3) e desafiadas por Beauveria bassiana cepa 88 (grupo 4). Em cada lagarta foi inoculada uma concentração conhecida do microrganismo e, após o tempo estabelecido, a hemolinfa foi coletada. As proteínas de baixa massa molecular foram obtidas submetendo cada amostra de hemolinfa a uma solução de extração contendo Metanol, Ácido Acético e Água. Posteriormente, os extratos proteicos foram concentrados em colunas e a dosagem proteica realizada em 280 nm. Aproximadamente 500 μg de proteínas foram submetidas à eletroforese bidimensional (2-DE). Os spots foram retirados dos géis, digeridos com Tripsina, e submetidos à espectrometria de massas do tipo MALDI-ToF/ToF. A identificação das proteínas ortólogas foi realizada utilizando os dados obtidos contra os dados disponíveis no servidor online MASCOT (Matrixscience), especificando a base de dados (NCBI e Swissprot) para Drosophila melanogaster e um banco interno de Lepidoptera. A ferramenta TagIdent foi utilizada para a busca de proteínas por meio de suas massas moleculares e ponto isoelétrico. Foram identificadas dezoito proteínas, das quais doze se mostraram envolvidas com a resposta imune em D. saccharalis. O desafio séptico de 6 horas com B. subtilis foi responsável pela alteração na expressão da proteína de reconhecimento de peptideoglicano (PGRP), pelo aumento da expressão da proteína ligadora de quitina e pela indução da expressão de proteínas como a Atacina-A e a proteína inibidora de serino-protease. O desafio imunológico de 6 horas com E. coli levou ao aumento da expressão de uma provável proteína de defesa. Os desafios de 12 horas utilizando as bactérias B. subtilis e E. coli se mostraram responsáveis pelo aumento da expressão da Lisozima. O desafio de 6 horas com B. bassiana causou indução da expressão de uma Cecropina A2 e uma possível Drosomicina. Por sua vez, após o desafio fúngico de 12 horas, houve a indução da expressão da proteína multifuncional Apolipoforina-3. Concluiu-se que desafios sépticos com diferentes microrganismos foram capazes de alterar a expressão de algumas proteínas envolvidas na resposta imune em D. saccharalis, importantes para o entendimento deste processo e também para o apontamento de substâncias com funções antimicrobianas.

Em artrópodes o sistema imune inato contribui para a adaptação de animais como os escorpiões à diferentes ambientes. Esse sistema é composto por mecanismos capazes de agir contra injúrias e a ação de microrganismos e entre esses mecanismos estão os peptídeos antimicrobianos (PAMs). O objetivo deste trabalho foi identificar PAMs presentes na hemolinfa de Tityus serrulatus. Para isso sua hemolinfa foi extraída e separados os hemócitos e plasma, em seguida fracionamos em 3 concentrações de acetonitrila em TFA 0,05% (05, 40 e 80%). Estas frações foram submetidas a uma cromatografia liquida de alta eficiência (CLAE) e os picos foram avaliados quanto a sua ação antimicrobiana e hemolítica. Foram identificadas 16 frações que apresentam atividade antimicrobiana. Uma das frações com atividade antimicrobiana, presente nos hemócitos apresentou similaridade com defensina descrita em carrapatos da espécie Ixodes scapularis. Essa fração possui aproximadamente 3486 Da, não apresenta atividade hemolítica e foi denominada como Serrulina. / In arthropods, its innate immune system contributes to the adaptation of animals like scorpions to different environments. This system consists of mechanisms that act avoiding injuries and against the action of microorganisms, among these mechanisms are antimicrobial peptides (AMPs). The aim of this study was to identify AMPs, present in the hemolymph of Tityus serrulatus. The hemolymph was extracted and then we separated hemocyte and plasma. The samples were fractionated in different concentrations of acetonitrile in TFA 0.05% (05, 40 and 80%). These fractions were subjected to high-performance liquid chromatography (HPLC) and the peaks obtained were evaluated for its antimicrobial and hemolytic action. We found sixteen fractions with antimicrobial activity. One of the fractions with antimicrobial activity, present in hemocytes, is similar with a defensin described in ticks, Ixodes scapularis. This fraction has about 3486 Da, has no hemolytic activity and was named as Serrulina.

No description available.

With the rapid and ubiquitous deployment of mobile communications in recent years, cochannel interference has become a critical problem because of its impact on system capacity and quality of service. The conventional approach to minimizing interference is through better cell planning and design. Digital Signal Processing COSP) based interference rejection techniques provide an alternative approach to minimize interference and improve system capacity. Single channel adaptive interference rejection techniques have long been used for enhancing digitally modulated signals. However these techniques are not well suited for analog mobile phone system (AMPS) and narrowband AMPS (NAMPS) signals because of the large spectral overlap of the signals of interest with interfering signals and because of the lack of a well defined signal structure that can be used to separate the signals. Our research has created novel interference rejection techniques based on time-dependent filtering which exploit spectral correlation characteristics exhibited by AMPS and NAMPS signals. A mathematical analysis of the cyclostationary features of AMPS and NAMPS signals is presented to help explain and analyze these techniques. Their performance is investigated using both simulated and digitized data. The impact of these new techniques on AMPS system capacity is also studied. The adaptive algorithms and structures are refined to be robust in various channel environments and to be computationally efficient. / Ph. D.

We consider the problem of designing a wide-band digital receiver for an Advanced Mobile Phone Service (AMPS) cellular system, and the associated problem of choosing an appropriate Analog-to-Digital (ADC) converter. The probability density function of the voltage across a cellular receiving antenna is shown to be dependent on various cellular parameters. These parameters include mobile transmit power, mobile distance from the base station, mobile transmit frequency, and transmitting and receiving antenna characteristics. Given a high-resolution, wideband, uniform and symmetric quantizer, optimal gain factors are computed for uniformly-, sinusoidally- and normally-distributed input signals. These gain factors maximize the quantizer's Signal-to-Quantization Noise Ratio (SQNR) in a mean-square sense. Together, these techniques can be used to implement an adaptive Automatic Gain Control for cellular communications. Results from a comprehensive AMPS base station simulation will also be discussed in detail. These results illustrate several design tradeoff's including Signal-to-Noise Ratio (SNR), Carrier-to-Noise Ratio (CNR), system loading and quantizer resolution. / Master of Science

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Background: Despite the effort of scientific research on HIV therapies and to reduce the rate of HIV infection, AIDS remains one of the major causes of death in the world and mostly in sub-Saharan Africa. To date, neither a cure nor an HIV vaccine had been found and the disease can only be managed by using High Active Antiretroviral Therapy (HAART) if detected early. The need for an effective early diagnostic and non-toxic treatment has brought about the necessity for the discovery of additional HIV diagnostic methods and treatment regimens to lower mortality rates. Antimicrobial Peptides (AMPs) are components of the first line of defense of prokaryotes and eukaryotes and have been proven to be promising therapeutic agents against HIV. Methods: With the utility of computational biology, this work proposes the use of profile search methods combined with structural modeling to identify putative AMPs with diagnostic and anti-HIV activity. Firstly, experimentally validated anti-HIV AMPs were retrieved from various publicly available AMP databases, APD, CAMP, Bactibase and UniProtKB and classified according to super-families. Hidden Markov Model (HMMER) and Gap Local Alignment of Motifs (GLAM2) profiles were built for each super-family of anti- HIV AMPs. Putative anti-HIV AMPs were identified after scanning genome sequence databases using the trained models, retrieved AMPs, and ranked based on their E-values. The 3-D structures of the 10 peptides that were ranked highest were predicted using 1-TASSER. These peptides were docked against various HIV proteins using PatchDock and putative AMPs showing the highest affinity and having the correct orientation to the HIV -1 proteins gp120 and p24 were selected for future work to establish their function in HIV therapy and diagnosis. Results: The results of the in silica analysis showed that the constructed models using the HMMER algorithm had better performances compare to that of the models built by the GLAM2 algorithm. Furthermore, the former tool has a better statistical and probability explanation compared to the latter tool. Thus only the HMMER scanning results were considered for further study. Out of 1059 species scanned by the HMMER models, 30 putative anti-HIV AMPs were identified from genome scans with the family-specific profile models after the elimination of duplicate peptides. Docking analysis of putative AMPs against HIV proteins showed that from the 10 best performing anti-HIV AMPs with the highest E-scores, molecules 1,3, 8, and 10 firmly bind the gp120 binding pocket at the VIN2 domain and the point of interaction between gp120 and T cells, with the 1st and 3rd highest scoring anti-HIV AMPs having the highest binding affinities. However, all 10 putative anti-HIV AMPs bind to the N-terminal domain of p24 with large surface interaction, rather than the C-terminal. Conclusion: The in silica approach has made it possible to construct computational models having high performances, and which enabled the identification of putative anti-HIV peptides from genome sequence scans. The in silica validation of these putative peptides through docking studies has shown that some of these AMPs may be involved in HIV/AIDS therapeutics and diagnostics. The molecular validation of these findings will be the way forward for the development of an early diagnostic tool and as a consequence initiate early treatment. This will prevent the invasion of the immune system by blocking the VIN2 domain and thus designing of a successful vaccine with broad neutralizing activity against this domain.

Venenos de aranhas podem ser boas fontes de moléculas com potencial terapêutico e biotecnológico. Sendo assim, esse estudo teve como objetivo analisar moléculas bioativas do veneno de Avicularia juruensis, com foco em PAMs e enzimas. O veneno foi extraído por estimulação elétrica e, através de CLAE-FR, ensaio de inibição do crescimento microbiano e LC-MS/MS, foram identificados e caracterizados 7 PAMs. Todos possuem o motivo nó de cistina do tipo ICK e têm similaridade com neurotoxinas de venenos de outras aranhas. O perfil eletroforético do veneno de A. juruensis indicou que ele possui moléculas com massa entre 130 e abaixo de 10 kDa. Utilizando LC-MS/MS e análise transcriptômica foi possível identificar 6 metaloproteinases. Elas possuem domínios conservados de proteínas do tipo ADAMs, MMPs e metalopeptidases astacina-like. A presença destas enzimas é, provavelmente, importante para a digestão extracorpórea, visto que esta aranha geralmente consome pequenas aves. O estudo de venenos de aranhas terafosídeas é essencial, visto que este é um grupo pouco estudado. / Spider venoms can be good sources of molecules with therapeutic and biotechnological potential. Thus, this study aimed to analyze bioactive molecules from venom of Avicularia juruensis, focusing on AMPs and enzymes. The venom was extracted by electrical stimulation. By RP-HPLC, liquid growth inhibition assay and LC-MS/MS, seven AMPs were identified and characterized. All these peptides have inhibitory cystine knot motif and they showed similarity with venom neurotoxins from others spiders. The electrophoretic profile of A. juruensis venom shows that it has molecules with molecular masses between 130 kDa and below 10 kDa. By LC-MS/MS and transcriptomic analysis yielded the identification of six metalloproteinases, which possess typical conserved domains from ADAMs, MMPs and astacin-like metallopeptidases. These metalloproteinases may be involved in a key role during preoral digestion. The study of Theraphosidae spider venom is essential, since this group is poorly studied.

The dynamics of rigid body motion are dependent on the inertial properties of the body - that is, the mass and moment of inertia. For complex systems, it may be necessary to derive these results empirically. Such is the case for manual wheelchairs, which can be modeled as a rigid body frame connected to four wheels. While 3D modeling software is capable of estimating inertial parameters, modeling inaccuracies and ill-defined material properties may introduce significant errors in this estimation technique and necessitate experimental measurements. To that end, this thesis discusses the design of a device called the iMachine that empirically determines the mass, location of the center of mass, and moment of inertia about the vertical (yaw) axis passing through the center of mass of the wheelchair. The iMachine is a spring-loaded rotating platform that freely oscillates about an axis passing through its center due to an initial angular velocity. The mass and location of the center of mass can be determined using a static analysis of a triangular configuration of load cells. An optical encoder records the dynamic angular displacement of the platform, and the natural frequency of free vibration is calculated using several techniques. Finally, the moment of inertia is determined from the natural frequency of the system. In this thesis, test results are presented for the calibration of the load cells and spring rate. In addition, objects with known mass properties were tested and comparisons are made between the analytical and empirical inertia results. In general, the mass measurement of the test object had greater than 99% accuracy. The average relative error for the x and y-coordinates of the center of mass was 0.891% and 1.99%, respectively. For the moment of inertia, a relationship was established between relative error and the ratio of the test object inertia to the inertia of the system. The results suggest that 95% accuracy can be achieved if the test object accounts for at least 25% of the total inertia of the system. Finally, the moment of inertia of a manual wheelchair is determined using the device (I = 1.213 kg-m²), and conclusions are made regarding the reliability and validity of results. The results of this project will feed into energy calculations for the Anatomical Model Propulsion System (AMPS), a wheelchair-propelling robot used to measure the mechanical efficiency of manual wheelchairs.

Hintergrund: Schlaf ist ein streng regulierter Zustand körperlicher Ruhe und reduzierten Bewusstseins, der evolutionär im ganzen Tierreich konserviert ist. Schlafmangel ist in der modernen Gesellschaft weit verbreitet und betrifft 10 – 30 % der Erwachsenen. Dies stellt ein ernstes gesundheitliches Problem dar, da Schlafmangel mit vielen Krankheiten assoziiert ist, darunter Depressionen, Krebs und Herz-Kreislauf-Erkrankungen. Umgekehrt beeinflussen auch Krankheiten und das Immunsystem das Schlafverhalten. Trotz der fundamentalen Rolle dieser Wechselbeziehung sind grundlegende molekulare Mechanismen, die Funktionen des Immunsystems und Schlafkontrolle verbinden, bisher kaum verstanden. Da die Schlafregulation in Säugetieren sehr komplex ist, ist es sinnvoll konservierte Mechanismen zuerst in einfacheren Modellorganismen zu untersuchen. Der Rundwurm C. elegans ist ein solcher etablierter, simpler und vielseitiger Modellorganismus für die Schlafforschung. Er schläft sowohl im Rhythmus seiner Larvenentwicklung immer jeweils während des Lethargus kurz vor der Häutung, als auch nach besonderem Stress, wie zum Beispiel Hunger oder Hitze. C. elegans besitzt ein invariantes Nervensystem, in dem eine rapide Depolarisation des einzelnen RIS-Interneurons genügt, um Schlaf zu induzieren. Eine Mutation des AP2 Transkriptionsfaktors APTF-1 verhindert die Expression von FLP-11, dem schlafinduzierenden Neuropeptid von RIS. Dies führt praktisch zu völliger Schlaflosigkeit, die in C. elegans in der Regel nicht tödlich ist, und deshalb ein nützliches Modell für genetisch-chronischen Schlafmangel darstellt. Unser Labor fand heraus, dass eine Gain-of-function-Mutation in der Kollagenase NAS-38 über Signalwege der angeborenen Immunität und RIS-Aktivierung zu vermehrtem Schlaf während des Lethargus führt. Gleichzeitig wird dabei die Expression einer ganzen Familie antimikrobieller Peptide (AMP) hochreguliert. Derselbe Signalweg, einschließlich der AMP, sowie das Schlafverhalten werden auch durch Verletzungen induziert. Interessanterweise sterben nicht-schlafende Würmer nach einer Verletzung häufiger. Insgesamt deutet dies darauf hin, dass AMP als Signalmoleküle fungieren könnten, die Schlaf als Teil einer globalen Schutzreaktion vom peripheren Gewebe zum Nervensystem signalisieren. Für diese Hypothese fehlten bisher jedoch die Beweise. Fragestellungen und Hypothesen: Mein Ziel war es, den molekularen Mechanismus zu entschlüsseln, durch den verschiedene Reize der angeborenen Immunität, das heißt NAS-38 sowie epidermale Verletzungen, Schlaf induzieren. Zwei Fragen habe ich hierbei im Speziellen adressiert: Welche Domänen des NAS 38-Proteins sind an der Schlafregulation beteiligt? Da die Astacin-Domäne als aktive Proteasedomäne von NAS-38 angesehen wird, erwartete ich eine Schlüsselrolle dieser Domäne auch in der Schlafinduktion. Zweitens, welche Rolle spielen AMP bei der Signalisierung von immunitätsinduziertem Schlaf? Da gezeigt wurde, dass AMP während des NAS-38 Schlafes und auch nach Verwundung hochreguliert sind, erwartete ich, dass AMP an der Signalisierung von Schlaf von der Epidermis zum Nervensystem beteiligt sind. In einem zweiten Schritt untersuchte ich die molekularen Mechanismen, die den Vorteilen von Schlaf für das Überleben von Verletzungen zugrunde liegen. Auch hier habe ich speziell zwei Fragestellungen untersucht: Verändert genetischer Schlafentzug die transkriptionelle Reaktion auf epidermale Verletzungen? Da Schlaf für viele fundamentale Prozesse wichtig ist und Schlaflosigkeit die Sterblichkeit nach Verletzungen erhöht, vermutete ich, dass genetischer Schlafentzug die transkriptionelle Reaktion auf Verletzungen beeinträchtigt. Zweitens, ist Schlaf wichtig für die Entwicklung von Robustheit, um im Falle einer Verletzung weniger Schaden zu nehmen? Während der Larvenentwicklung fällt die Cuticula-Synthese mit Schlaf zeitlich zusammen. Daher stellte ich die Hypothese auf, dass Schlafentzug die korrekte Bildung einer Cuticula beeinträchtigt. Methoden: Zur Analyse der Signalmechanismen, durch die sowohl NAS-38 als auch Verletzungen Schlaf induzieren, filmte ich das Schlafverhalten von C. elegans mittels Langzeit-Bildgebung in Agarose-Mikrokammern. So führte ich eine Struktur-Funktions-Analyse mit verschiedenen nas-38 Mutanten durch, in denen jeweils eine andere NAS-38 Domäne deletiert war. Darüber hinaus testete ich verschiedene Suppressoren für immunvermittelten Schlaf, der durch NAS 38 oder Verletzungen induziert war. Die Redundanz des Suppressionseffektes der verschiedenen Mitglieder der AMP-Familie auf immunvermittelten Schlaf testete ich, indem ich den Suppressionsphänotyp einer CRISPR/Cas9-editierten Multi-Knockout-Mutante analysierte, in der insgesamt 19 AMP deletiert waren. Um Effektoren zu identifizieren, die den AMP nachgeschaltet sind, induzierte ich Schlaf durch Überexpression des AMP NLP 29 unter der Kontrolle eines Hitzeschock-Promotors und analysierte die Sschlafsuppression durch verschiedene Knockout-Mutanten. Im zweiten Projekt beschäftigte ich mich mit der Frage, wie genau Schlaf das Überleben nach Verletzungen unterstützt. Ich verglich die Expression von literaturbekannten Reportern für verschiedene Aspekte der Verwundungsreaktion mittels Langzeit-Fluoreszenzmikroskopie im Wildtyp sowie dem Modell für chronisch-genetischen Schlafmangel. Darüber hinaus habe ich die Transkriptome zwischen jeweils adulten verwundeten und unverwundeten Wildtypen und schlaflosen Mutanten verglichen. Um die Struktur der Cuticula des Wildtyps und der schlaflosen Mutante zu vergleichen, analysierte ich außerdem rasterelektronen-mikroskopische Aufnahmen. Ergebnisse: Im ersten Projekt konnte ich zeigen, dass NAS-38 Schlaf durch seine Astacin-Domäne verlängert. Dieser Prozess wird moderiert durch die TSP-1-Domäne. Weiterhin konnte ich zeigen, dass viele AMP redundant wirken um immunvermittelten Schlaf, verursacht durch NAS-38 oder Verletzungen, zu signalisieren. Ich konnte zeigen, dass das AMP NLP-29 über den Neuropeptidrezeptor NPR-12 wirkt. Dieser kann NLP-29-induzierten Schlaf vermitteln, wenn er in einem neuronalen Netzwerk exprimiert wird, welches nachweislich RIS aktiviert. Interessanterweise fand ich außerdem heraus, dass für NLP-29-vermittelten Schlaf der EGFR Signalweg notwendig ist. Im zweiten Projekt entdeckte ich, dass Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht dramatisch verändert. Allerdings ist das Transkriptionsprofil bereits in der unverletzten schlaflosen Mutante verändert. Dies betraf unter anderem eine Gruppe oszillierender Gene, die Cuticula-assoziierte Proteine codieren, und deren Expression normalerweise ihren Höhepunkt gegen Ende des Lethargus erreicht. Da angenommen wird, dass der Zeitpunkt der Kollagenexpression entscheidend für eine fehlerfreie Cuticula-Bildung ist, analysierte ich die Cuticula der schlaflosen Mutante. Ich konnte zeigen, dass die Cuticula des adulten Tieres tatsächlich einen strukturellen Defekt aufweist. Dieser betrifft speziell Furchen in der Region nahe den Alae und könnte möglicherweise die Strapazierfähigkeit der Cuticula gegenüber bestimmten Belastungen verringern. Daher könnte Schlaf erforderlich sein, Robustheit in Form einer strukturierten Cuticula zu fördern. Schlussfolgerungen: In diesem Dissertationsprojekt vollendete ich die Charakterisierung eines neuentdeckten Mechanismus in C. elegans, durch den Verwundungen Schlaf als Teil der Immunantwort aus der Peripherie zum Nervensystem signalisieren. Ich konnte zeigen, dass AMP gewebeübergreifend Signale von der Epidermis an ein neuronales Netz vermitteln, welches wiederum RIS aktiviert und dadurch Schlaf induziert. Da Komponenten dieses Signalweges konserviert sind, könnten AMP auch in anderen Tieren, einschließlich des Menschen, Schlaf zur Genesung fördern. Darüber hinaus habe ich die Grundlagen für die Analyse molekularer Mechanismen geschaffen, die den essentiellen Funktionen des Schlafes für Heilung und Überleben zugrunde liegen. Obwohl Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht drastisch zu verändern scheint, deuten meine Ergebnisse auf eine Rolle des Schlafes bei der richtigen Cuticula-Bildung und möglicherweise sogar auf eine vielfältigere Rolle bei der zeitlichen Regulierung der Genexpression hin.:Summary I Zusammenfassung IV Contents VII List of Figures XII List of Tables XIV Abbreviations XV 1. Introduction 1 1.1. Sleep is fascinating 1 1.1.1. The origin and basic features of sleep 1 1.1.2. Regulation of sleep in higher animals 3 1.1.2.1. Neuronal control of sleep 3 1.1.2.2. Molecular control of sleep 5 1.1.3. The functions of sleep 6 1.2. The immune system and its relationship to sleep 7 1.3. Wound healing and its relationship to sleep 10 1.4. Caenorhabditis elegans is a well-studied model organism 12 1.4.1. Sleep in C. elegans 15 1.4.2. The C. elegans cuticle 18 1.4.3. Immunity in C. elegans 19 1.4.4. Wound healing response in C. elegans 22 2. Previous results 25 2.1. A strong gain-of-function mutation in the astacin metallo-proteinase NAS 38 increases lethargus duration and movement quiescence in C. elegans 25 2.2. NAS-38 increases sleep mostly through the RIS neuron 25 2.3. NAS-38 is expressed in the epidermis and oscillates with the developmental rhythm 25 2.4. nas-38(ok3407) acts via innate immunity pathways to increase lethargus duration and AMP expression 27 2.5. Overexpression of AMPs induces RIS dependent quiescence 30 2.6. Epidermal wounding induces RIS-dependent sleep, which is beneficial for survival 31 3. Thesis Aims 34 3.1. Aim 1 – Characterizing the molecular mechanism through which NAS-38, innate immunity, and wounding induce sleep 34 3.2. Aim 2 – Analyzing how sleep promotes survival after wounding 35 4. Materials and Methods 36 4.1. C. elegans maintenance 36 4.2. C. elegans crossing and genotyping 41 4.3. Creation of transgenic animals 45 4.3.1. Creating the npr-12 rescue in nmr-1 expressing neurons 45 4.3.2. Microparticle bombardment 45 4.3.3. CRISPR/Cas9 system 46 4.4. Synchronizing worm cultures by hypochlorite treatment 48 4.5. Imaging 49 4.5.1. Imaging setups 49 4.5.2. DIC Imaging of worm development, lethargus, and sleep behavior 50 4.5.2.1. Imaging of heterozygous mutants 50 4.5.3. DIC imaging in the temperature control device 51 4.5.4. Fluorescent imaging experiments 51 4.5.4.1. nas-38p::d1GFP and nlp-29p::GFP during L1 development 51 4.5.4.2. nlp-29p::GFP in L4 larvae 52 4.5.4.3. nlp-29p::GFP after heat shock-induced lin-3 overexpression 52 4.5.4.4. Imaging fluorescent markers in (wounded) young adults 52 4.5.4.5. Functional Ca2+ imaging in young adults 52 4.5.4.6. Fluorescence imaging across the whole developmental time 54 4.5.4.7. Nuclear decompaction assays 55 4.5.4.8. Transcription factor localization with spinning disc confocal microscopy 55 4.5.4.9. Imaging DPY-13::mKate2 in young adults 56 4.6. Image analysis 56 4.6.1. Assessment of developmental time and lethargus detection 56 4.6.2. Sleep detection in DIC mode 56 4.6.3. Analyzing functional Ca2+ images 57 4.6.4. Fluorescent reporter analysis during long-term imaging 57 4.7. RNAi-by-feeding 58 4.8. Transcriptome analysis 59 4.8.1. Analysis of the nas-38(ok3407) transcriptome 59 4.8.2. Analysis of the wounding transcriptome 59 4.9. Epidermal wounding 62 4.9.1. Laser wounding 62 4.9.2. Needle wounding 62 4.9.3. Survival assay 63 4.10. Scanning Electron Microscopy (SEM) 63 4.11. Histamine-inducible hyperpolarization of RIS 64 4.12. Cuticle integrity test with Sodium hypochlorite 64 4.13. NPR-12 receptor modeling 64 4.14. Quantification and statistical analysis 65 5. Results 66 5.1. Aim 1 – Characterizing the pathway through which NAS 38, wounding and innate immunity induce sleep 66 5.1.1. The loss of function mutation nas-38(tm2655) shows the opposite phenotype to the gain of function mutation nas-38(ok3407) 66 5.1.2. nas-38 gain-of-function mutants act through their astacin protease domain and are semi-dominant 66 5.1.3. Transcriptome analysis of nas-38(ok3407) reveals upregulation of genes associated with secretion, innate immunity and cuticle formation 69 5.1.4. nas-38(knu568) increased movement quiescence can be suppressed by mutations of innate immunity pathways 72 5.1.5. Multiple NLPs and CNCs act in parallel to mediate nas-38(ok3407) induced sleep 75 5.1.6. Wounding-induced sleep requires RIS, ALA, EGFR and immune signaling 77 5.1.7. NLP-29 signals via the NPR-12 receptor in neurons upstream of RIS 80 5.1.8. NLP-29 requires neuronal EGFR signaling to induce sleep 81 5.1.9. Simple in silico models suggest that many different NLPs can bind to NPR-12 83 5.1.10. AMPs contribute to the survival after wounding 85 5.2. Aim 2 – Identifying the advantages sleep provides that help to survive harmful conditions 87 5.2.1. Wounding decreases the lifespan in the wild type and the aptf 1(gk794) mutant 87 5.2.2. Histamine-inducible RIS hyperpolarization suppresses wounding sleep 87 5.2.3. Genetic sleep deprivation decreases translocation of DAF-16 into the nucleus immediately after wounding 89 5.2.4. Genetic sleep deprivation hardly changes the transcriptional wounding response 95 5.2.5. Genetic sleep deprivation and wounding increase nuclear PHA 4 101 5.2.6. Oscillating genes and genes associated with the cuticle and the unfolded protein response are upregulated in young adult aptf 1(gk794) mutants 106 5.2.7. Genetic sleep deprivation leads to a malformation of cuticular furrows 109 5.2.8. Genetic sleep deprivation leads to an increased transcription of lethargus specific oscillating genes in young adults 114 5.2.9. Genetic sleep deprivation does not significantly affect development time or body size 120 5.2.10. Expression of fluorescent reporters of oscillating genes is not phase-shifted in the aptf-1(gk794) mutant 122 6. Discussion and Outlook 128 6.1. NAS-38 acts through its astacin domain to increase sleep via innate immunity pathways 128 6.2. NAS-38 during larval lethargus and epidermal wounding in the adult signal sleep via many AMPs as part of a peripheral immune response 130 6.3. Epidermal AMPs activate a neuronal circuit to induce sleep 131 6.4. Genetically sleep deprived worms can mount a proper wounding response in many ways, except for DAF-16/FOXO regulation 132 6.5. Genetic sleep deprivation alters cuticle formation 135 6.6. The role of PHA-4/FOXA in genetically sleep-deprived animals 137 6.7. Conclusion 139 7. References 140 8. Acknowledgements 163 9. Appendix 166 9.1. Standard reagents 166 9.2. Sequence summary of PHX3754 167 9.3. MATLAB script to analyze the intensity of fluorescent reporters over time 171 9.4. Permissions to reprint figures 174 9.5. Experimental author contributions 175 9.6. Predicted interactions between the NPR-12 receptor and peptides of the nlp and cnc families 176 9.7. Overlap of the adult wounding transcriptome with other data sets 179 9.8. Curriculum Vitae – Marina Patricia Sinner 181 / Background: Sleep is a tightly regulated state of behavioral quiescence and reduced consciousness, which is conserved throughout the animal kingdom. In modern societies 10 – 30 % of the adult population suffer from insufficient sleep, which poses a serious health problem as sleep deprivation is associated with a variety of diseases including depression, cancer, and cardiovascular diseases. Conversely, sickness and the immune system also influence sleep patterns. Despite the important role of this interrelationship between sleep and immunity, basic molecular mechanisms that link both vital functions are only poorly understood yet. As sleep regulation is complex in mammals and is thus difficult to address experimentally, it is reasonable to investigate its basic conserved mechanisms in simpler models first. The nematode C. elegans is such a well-established, simple, and powerful model organism for sleep research. It displays stress-induced sleep, for example upon starvation or heat shock, but also developmentally-timed sleep during lethargus prior to each larval molt. C. elegans possesses an invariant nervous system in which rapid depolarization of the single RIS interneuron is sufficient to induce sleep. Mutation of the AP2 transcription factor APTF 1 deprives RIS of its sleep-inducing neuropeptide FLP-11 and thus virtually abolishes sleep. This is not per se lethal in C. elegans, thereby presenting a powerful model for genetic sleep deprivation. Our lab found that a gain-of-function mutation in the collagenase NAS-38 strongly increases RIS-dependent sleep during lethargus with a concomitant upregulation of a large family of antimicrobial peptides (AMPs) via immunity pathways. Epidermal wounding also triggers AMP expression via immune signaling and induces sleep in the adult worm. Moreover, genetic sleep deprivation increases mortality upon epidermal injury. Together, this suggests AMPs to act as somnogens from peripheral tissues to the nervous system as part of a protective response. This hypothesis, however, was hitherto lacking final evidence and pathway components. Research questions and hypotheses: I aimed to characterize the molecular mechanism by which separate triggers of innate immunity, i. e. NAS-38 and wounding, induce sleep. I specifically addressed two questions: Firstly, which domains of the NAS-38 protein are involved in sleep regulation? As the astacin domain is predicted to be the active protease domain of NAS-38, I expected a role for it also in sleep induction by NAS-38. Secondly, what is the role of AMPs in signaling immunity-induced sleep? As they have been shown to be upregulated during times of increased sleep in the nas-38 mutant and after wounding, I expected AMPs to be involved in signaling sleep from the epidermis to the nervous system. In a second step, I investigated the molecular mechanisms underlying the benefits of sleep for surviving injury. Again, I addressed two questions: Firstly, does genetic sleep deprivation alter the transcriptional wounding response? As sleep has a role in many fundamental processes and sleeplessness increases mortality upon wounding, I hypothesized that genetic sleep deprivation impairs wounding-induced changes of transcriptional activity. Secondly, does sleep help building robustness before encountering injury? During larval development the synthesis of a new cuticle coincides with sleep. Thus, I hypothesized that genetic sleep deprivation impairs proper cuticle formation. Methods: To dissect the signaling mechanisms by which NAS-38 and wounding induced sleep, I followed sleep behavior of C. elegans by long-term imaging in agarose microchambers. I performed a structure-function analysis with different nas-38 mutants, each carrying a deletion of a different domain. Moreover, I screened for suppressors of sleep induced by NAS 38 or wounding. To test for redundancy of the AMP family, I investigated the suppression-phenotype of a CRISPR/Cas9 edited multi-knockout mutant lacking 19 AMPs. To identify downstream effectors of the AMP NLP 29, I induced sleep by overexpressing NLP 29 from a heat-shock promoter and analyzed the suppression-phenotype of different knockout mutants. For the second project, I addressed the question how sleep aids recovery from injury. I followed fluorescent reporters of previously described wounding response pathways by fluorescent long-term imaging in wild-type and genetically sleep-deprived animals. Moreover, I compared the transcriptomes of adult wild-type and genetically sleep-deprived worms both wounded and unwounded. To investigate the structure of the cuticle, I analyzed scanning electron microscopy images. Results: In the first project, I could show that NAS-38 indeed increases sleep via its astacin domain in a process that is modulated by the TSP-1 domain. Moreover, I could show that many AMPs act redundantly in mediating immunity-induced sleep downstream of NAS-38 and after wounding. I demonstrated that the AMP NLP-29 signals sleep via the neuropeptide receptor NPR 12. This receptor can mediate sleep when it is specifically expressed in command interneurons of a circuit that has been shown to activate RIS. Interestingly, I also found that EGFR signaling is required to mediate NLP-29-induced sleep. In the second project, I found that sleeplessness does not dramatically alter the transcriptional wounding response. However, I could show that transcription is altered already in the unwounded non-sleeping mutant. This affects, among others, a specific subset of oscillating collagen-coding genes, whose expression usually peaks around the end of lethargus. As the timing of expression of collagens is thought to be highly important for proper cuticle formation, I characterized the cuticle of the aptf-1(gk794) mutant. I could show that young adult aptf 1(gk794) worms indeed have a structural defect affecting cuticular furrows in the region adjacent to the alae, which could potentially decrease specific aspects of resilience of the cuticle. Thus, sleep might be required to build robustness in the form of a properly structured cuticle. Conclusion: In this PhD project, I completed the characterization of a novel mechanism by which wounding signals sleep from the periphery to the nervous system as part of the immune response in C. elegans. I could show that AMPs act as cross-tissue signals from the epidermis to a neuronal RIS-controlling circuit that ultimately leads to sleep induction. As components of this molecular pathway are highly conserved, AMPs might also induce sleep to promote recovery from injury in other organisms, including humans. Moreover, I laid the foundations for dissecting the molecular mechanisms behind the functions of sleep for healing and survival. Even though the disability to sleep did not seem to drastically change the transcriptional response to wounding, my results indicate a role for sleep in proper cuticle formation in C. elegans and potentially even a broader role in the regulation of precise gene expression timing.:Summary I Zusammenfassung IV Contents VII List of Figures XII List of Tables XIV Abbreviations XV 1. Introduction 1 1.1. Sleep is fascinating 1 1.1.1. The origin and basic features of sleep 1 1.1.2. Regulation of sleep in higher animals 3 1.1.2.1. Neuronal control of sleep 3 1.1.2.2. Molecular control of sleep 5 1.1.3. The functions of sleep 6 1.2. The immune system and its relationship to sleep 7 1.3. Wound healing and its relationship to sleep 10 1.4. Caenorhabditis elegans is a well-studied model organism 12 1.4.1. Sleep in C. elegans 15 1.4.2. The C. elegans cuticle 18 1.4.3. Immunity in C. elegans 19 1.4.4. Wound healing response in C. elegans 22 2. Previous results 25 2.1. A strong gain-of-function mutation in the astacin metallo-proteinase NAS 38 increases lethargus duration and movement quiescence in C. elegans 25 2.2. NAS-38 increases sleep mostly through the RIS neuron 25 2.3. NAS-38 is expressed in the epidermis and oscillates with the developmental rhythm 25 2.4. nas-38(ok3407) acts via innate immunity pathways to increase lethargus duration and AMP expression 27 2.5. Overexpression of AMPs induces RIS dependent quiescence 30 2.6. Epidermal wounding induces RIS-dependent sleep, which is beneficial for survival 31 3. Thesis Aims 34 3.1. Aim 1 – Characterizing the molecular mechanism through which NAS-38, innate immunity, and wounding induce sleep 34 3.2. Aim 2 – Analyzing how sleep promotes survival after wounding 35 4. Materials and Methods 36 4.1. C. elegans maintenance 36 4.2. C. elegans crossing and genotyping 41 4.3. Creation of transgenic animals 45 4.3.1. Creating the npr-12 rescue in nmr-1 expressing neurons 45 4.3.2. Microparticle bombardment 45 4.3.3. CRISPR/Cas9 system 46 4.4. Synchronizing worm cultures by hypochlorite treatment 48 4.5. Imaging 49 4.5.1. Imaging setups 49 4.5.2. DIC Imaging of worm development, lethargus, and sleep behavior 50 4.5.2.1. Imaging of heterozygous mutants 50 4.5.3. DIC imaging in the temperature control device 51 4.5.4. Fluorescent imaging experiments 51 4.5.4.1. nas-38p::d1GFP and nlp-29p::GFP during L1 development 51 4.5.4.2. nlp-29p::GFP in L4 larvae 52 4.5.4.3. nlp-29p::GFP after heat shock-induced lin-3 overexpression 52 4.5.4.4. Imaging fluorescent markers in (wounded) young adults 52 4.5.4.5. Functional Ca2+ imaging in young adults 52 4.5.4.6. Fluorescence imaging across the whole developmental time 54 4.5.4.7. Nuclear decompaction assays 55 4.5.4.8. Transcription factor localization with spinning disc confocal microscopy 55 4.5.4.9. Imaging DPY-13::mKate2 in young adults 56 4.6. Image analysis 56 4.6.1. Assessment of developmental time and lethargus detection 56 4.6.2. Sleep detection in DIC mode 56 4.6.3. Analyzing functional Ca2+ images 57 4.6.4. Fluorescent reporter analysis during long-term imaging 57 4.7. RNAi-by-feeding 58 4.8. Transcriptome analysis 59 4.8.1. Analysis of the nas-38(ok3407) transcriptome 59 4.8.2. Analysis of the wounding transcriptome 59 4.9. Epidermal wounding 62 4.9.1. Laser wounding 62 4.9.2. Needle wounding 62 4.9.3. Survival assay 63 4.10. Scanning Electron Microscopy (SEM) 63 4.11. Histamine-inducible hyperpolarization of RIS 64 4.12. Cuticle integrity test with Sodium hypochlorite 64 4.13. NPR-12 receptor modeling 64 4.14. Quantification and statistical analysis 65 5. Results 66 5.1. Aim 1 – Characterizing the pathway through which NAS 38, wounding and innate immunity induce sleep 66 5.1.1. The loss of function mutation nas-38(tm2655) shows the opposite phenotype to the gain of function mutation nas-38(ok3407) 66 5.1.2. nas-38 gain-of-function mutants act through their astacin protease domain and are semi-dominant 66 5.1.3. Transcriptome analysis of nas-38(ok3407) reveals upregulation of genes associated with secretion, innate immunity and cuticle formation 69 5.1.4. nas-38(knu568) increased movement quiescence can be suppressed by mutations of innate immunity pathways 72 5.1.5. Multiple NLPs and CNCs act in parallel to mediate nas-38(ok3407) induced sleep 75 5.1.6. Wounding-induced sleep requires RIS, ALA, EGFR and immune signaling 77 5.1.7. NLP-29 signals via the NPR-12 receptor in neurons upstream of RIS 80 5.1.8. NLP-29 requires neuronal EGFR signaling to induce sleep 81 5.1.9. Simple in silico models suggest that many different NLPs can bind to NPR-12 83 5.1.10. AMPs contribute to the survival after wounding 85 5.2. Aim 2 – Identifying the advantages sleep provides that help to survive harmful conditions 87 5.2.1. Wounding decreases the lifespan in the wild type and the aptf 1(gk794) mutant 87 5.2.2. Histamine-inducible RIS hyperpolarization suppresses wounding sleep 87 5.2.3. Genetic sleep deprivation decreases translocation of DAF-16 into the nucleus immediately after wounding 89 5.2.4. Genetic sleep deprivation hardly changes the transcriptional wounding response 95 5.2.5. Genetic sleep deprivation and wounding increase nuclear PHA 4 101 5.2.6. Oscillating genes and genes associated with the cuticle and the unfolded protein response are upregulated in young adult aptf 1(gk794) mutants 106 5.2.7. Genetic sleep deprivation leads to a malformation of cuticular furrows 109 5.2.8. Genetic sleep deprivation leads to an increased transcription of lethargus specific oscillating genes in young adults 114 5.2.9. Genetic sleep deprivation does not significantly affect development time or body size 120 5.2.10. Expression of fluorescent reporters of oscillating genes is not phase-shifted in the aptf-1(gk794) mutant 122 6. Discussion and Outlook 128 6.1. NAS-38 acts through its astacin domain to increase sleep via innate immunity pathways 128 6.2. NAS-38 during larval lethargus and epidermal wounding in the adult signal sleep via many AMPs as part of a peripheral immune response 130 6.3. Epidermal AMPs activate a neuronal circuit to induce sleep 131 6.4. Genetically sleep deprived worms can mount a proper wounding response in many ways, except for DAF-16/FOXO regulation 132 6.5. Genetic sleep deprivation alters cuticle formation 135 6.6. The role of PHA-4/FOXA in genetically sleep-deprived animals 137 6.7. Conclusion 139 7. References 140 8. Acknowledgements 163 9. Appendix 166 9.1. Standard reagents 166 9.2. Sequence summary of PHX3754 167 9.3. MATLAB script to analyze the intensity of fluorescent reporters over time 171 9.4. Permissions to reprint figures 174 9.5. Experimental author contributions 175 9.6. Predicted interactions between the NPR-12 receptor and peptides of the nlp and cnc families 176 9.7. Overlap of the adult wounding transcriptome with other data sets 179 9.8. Curriculum Vitae – Marina Patricia Sinner 181

從無線語音通訊(Voice)的日漸普及，以及日益下降的語音ARPU(Average Revenue Per User)的趨勢看來，所有的行動電信營運商(Telecom Operator)都開始在鑽研如何去推動數據的收入(Data Revenue)，以及所謂的加值型服務的利潤，借以廣增收入及營運利潤的提昇。所以如何建立一個正確的『營利模式』成為全世界所有行動電信營運商最關切的焦點之一。 台灣在行動電信數據服務以及加值型服務方面，其實發展得很早，曾經有過兩次相當大而且相當慘痛的失敗經驗。一次是在2000年和信電訊股份有限公司曾經有過引進日本NTT DoCoMo i-mode的慘痛失敗經驗；另外一件是在2003年亞太行動寬頻電信股份有限公司也曾經歷過引進韓國SK Telecom的SKVM數據服務平台技術移轉的失敗經驗。這兩起的案例由於個人都剛好身歷其境，所以印象特別深刻，檢討起來失敗的原因固然很多，但是其中如何去正確建立一個適合台灣的主客觀環境的『營運模式』卻是一個相當值得探討的問題。 手機一直是行動電話產業裡最熱門的話題，從以前的所謂的『大哥大』(AMPS類比式)手機時代，走入今天的第三代行動通訊所謂的3G『智慧型手機』的時代。手機應該算得上是繼PC電腦以來，發展最快，對人類的生活文明最具影響力的產品之一。PC是坐著那邊使用的工具，而手機卻是人們天天帶在身邊，帶著到處跑的『不可或缺的一部分』。有的人每天也許可以沒有去用電腦，但是很多人每天如果沒有手機在身邊的話，那會是一件非常嚴重的事情。手機發展的很快，也愈做愈像一台能夠隨身帶著走的『智囊包』了。不但包辦所有個人週邊的服務，尤其是所謂的『多媒體影音及下載』功能(有人稱之為:「多媒體手機」)，『網際網路』上網及網路週邊的服務功能，甚至所謂的『人對機』、『機對人』、以及『機對機』等未來無比的想像空間的一些功能及發展。所有設計出來的『營運模式』都要先透過手機去呈現在消費者面前，電信營運商也要先去做許多的驗證實驗(Initial Service Launch)以後，再去正式的推到市面上去。然後再從消費者的滿意度指標(KPI)裡去確認這些被建立的『營運模式』是正確的。 本論文就是站在是一個行動電信營運商(Telecom Operator)的立場，從『智慧型手機』的發展角度來探討未來應該如何去正確建立一個『營運模式』來。主要論述點是從國外一些成功的行動電信營運商們的『營運模式』做切入點，再探討一些台灣的第三代行動通訊上所謂的『加值型服務』(Data Service數據服務)一直發展不起來的原因；另外再從『智慧型手機』以及『手機上網』的角度去探索出一些建議，以做為正確建立一個『營運模式』（Business Model）的基礎。本研究著眼於在行動電話傳統以來就以「語音(Voice)」為主要獲利的「營運模式」，在經過「第三代行動通訊」從「建置期」到「營運期」動態競爭市場愈演愈烈的情勢下，行動電話營運商從一個完全「主導的角色」逐漸轉變成一個「中介者的角色」，其「營運模式」的選擇固然會因為各家電信營運商的「財力、物力」；及「可動用的資源」有所差異，但是在「策略」及「營運模式」的釐訂上也有相當大的學問，有時候看人家(國外)的成功，及複製人家的「成功關鍵因素」並不見得就會「一定成功」的。重要的是要能在「不同的競爭環境」下，以及「艱困險惡的競爭態勢裡」，要能「看得到別人沒看到的市場」商機，要能「把握到每一個轉折的時間點」，然後將「有效的資源集中的投下去」，勝利成功是必然的。 本研究主要的結論包括： 1. 第三代行動通訊數據服務及加值型應用服務之營運模式(Business Model)與傳統的電信服務以voice語音為主的營運模式有顯然的不同點，無法以現有的營運模式來經營數據服務及加值行應用服務。但是採行「老二主義」(也就是所謂的「跟隨主義」)在第三代行動通訊市場動態競爭環境下是可行的策略之一。（本研究案例--日本KDDI及亞太行動即為其例） 2. 傳統以「語音」為主的「營運模式」與「數據服務及加值型應用服務」為主的「營運模式」差異點為以語音為主的營運模式其主要在於對語音服務的『資費價格』(Rate price)做調制，就會對市場及競爭者產生影響力，而且這些調制的主控權是可以完全操控於營運商的掌控中的。對數據及數據加值型應用服務營運模式來說，電信營運商必須退居於「中介者」的角色，以撮合各個價值鏈的「供應商」們一起來，創建「最高的客戶價值」，並從這些撮合的過程中分得應有的利潤。(本研究案例--日本NTT DoCoMo i-mode) 3. 本研究案例是以國內行動電話，一個已完全競爭的市場裡，以一個後進者(本研究案例公司—亞太行動）開始進入市場，在幾經仔細觀察競爭者的動態後，終於發現了一個「競爭者忽略的市場空間」；以及「競爭者沒有發現的市場空間」，然後再以「更便宜的產品」切入市場，終於勝出於市場。 4. Apple繼iPOD及iTune成功之後，以一個在Music Download世界裡，完全以提供消費者Content內容服務的角度。另外，Google也以一個提供消費者全方位Internet網際網路服務的角度，雙雙切入「智慧型手機」市場，甚至於「整個的行動電話市場」，確實帶給整個手機市場無比的震撼。本研究發現iPhone也好，Google Phone也好，都是從消費者使用行動電話的角度，利用「開放型營運模式」去切入市場。這與傳統行動電話營運商，採完全「封閉型營運模式」有顯著的不同。到底「開放型營運模式」與「封閉型營運模式」最後誰會勝出呢？ 5. 數據產品的營運模式與傳統以語音為主的營運模式不同，語音產品受「價格調制」的影響很大，而價格的調制是完全掌控在行動電話營運商手中的；但是，數據產品的使用量必須經過「學習」、「認知」、「確認」及「喜好」等過程才能逐漸增加它的使用量，而這些過程中的因素變動並非完全掌控於行動電話營運商手中，因為行動電話營運商在數據產品市場中扮演的角色是「中介者」撮合的角色。本研究發現數據產品的初期投資較高，但是同質性高，容易在上述過程中被市場淘汰，但是一旦其中有一項產品能成功的在競爭的市場裡存活下來，其他同質產品的失敗都會因為這項成功的產品而能於短期內回收回來。所以數據產品必須要採「多路徑投入」方式之營運模式。 成功的數據產品，其營運模式所營造出來的利潤，都是在所謂的「長尾期」。正如同「i-mode」的母親：松永真理小姐所說的：「追求wants而非追求needs」，「我們想做一個連自己母親都能夠輕易操作的服務」等等，這些原則或信念，看似無甚高論，但卻是市場、行銷、消費的原理。而松永真理之於「i-mode」的可貴之處正在於，她去除了高科技產業的「不可親」性格，她把高科技產品拉回到人性面、市場面來處理。她看到了連結於高科技產品與市場間的「niche」。 最後歸根結底，一個數據產品的「營運模式」被成功的碩造出來，其實是來自於「不懂科技」、來自於「原創性」、來自於「生活化」、來自於「人性」、「自然」及「生活中不斷的那種來自於心靈深處的創新思維」。 / From wireless voice communications growing popularity, as well as declining voice ARPU (Average Revenue Per User) of the trends, all of the operations of telecommunications operators have begun to study how to promote the income data (Data Revenue), as well as the so-called value-added services to profit, in order to widely by income and operating profit improved. So how to create a correct 『Profitable Business Model』 as the world's telecom operators all actions focus of most concern. Taiwan in the operation of telecommunications data services, as well as value-added services, in fact, the development of very early, there have been two very large and very painful experience of failures. One was in 2000 and the KG Telecom Co., Ltd. had introduced Japan NTT DoCoMo i-mode failure of the painful experience; another in 2003 Asia Pacific Broadband Wireless Co., Ltd. has experienced through the introduction of SK Telecom of South Korea SKVM data services platform technology transfer failures. The two cases are just for personal involved, so was particularly impressed to review with a lot of reasons for the failure of course, but how to correct for the establishment of a right 『Business Model』to fit Taiwan's subjective and objective environment is a very worth exploring problems. Mobile Handset has been the mobile phone industry's hottest topics, from the previous so-called 『Big Brother』AMPS analog phone era into today's third generation mobile communications (3G) so-called 『Smart Phone』3G era. Mobile phones should be considered following the PC computer has been the fastest growing of civilized human life the most influential one of the products. PC is sitting side-use tools, and mobile phones are necessarily carried it every day, with around an integral part of you. Some people did not go a day may be able to use computers, but many people every day if there is no Mobile phone in the side, it would be a very serious matter. Mobile phone development soon, but also more like to do more one can carry away 『a think, smart-tank pack』. Not only do all individuals of the surrounding services, especially the so-called audio-visual and multimedia 『download function (some call it: 『Multimedia Mobile phone』，Internet surrounding、the Internet and network service functions, and even the so-called 『People to Machine』, 『Machine to People』, as well as 『Machine to Machine』 『next great imagination, such as some of the features of space and development. All designed 『Business Model』 must first go through a cell phone in front of consumers, telecom operators have also done a lot of verification experiment (Initial Service Launch) after the formal go to the market to go. And then from the consumer's satisfaction indicators (KPI) where these have been established to confirm the 『Business Model』 is correct. In this paper, is standing on is a mobile telecommunication Mobile Operators position, from 『Smart phone』 development perspective to explore how the future should be the correct 『Business Model』 of the establishment. The main discussion points from a number of successful operations abroad carrier's 『Business Model』 make an entry point, and then explore some of Taiwan's third-generation mobile communication; so-called 『Value-added Services』(Data Services) has been developed with not that much successful by now the reasons; additional from 『Smart phone』and 『Mobile Internet』 perspective to explore a number of recommendations to serve as the correct Business Model of the establishment basis. This study focused on the mobile phone which traditionally has been to 『Voice』 as the main profit "Business Model", after "third-generation mobile communications" from the "build phase" to "the service and operational period" become increasingly dynamic and competitive markets situation, mobile phone operators from a completely "leading role" gradually transformed into an "intermediary role", And its "business model" option of course, because each carrier's "financial and material resources"; and "available resources" be different, but in the "strategy" and "operating mode" setting also has considerable big science, and sometimes they look (outside) the success of, and copying other people's "critical success factors" and will not "necessarily successful". Is important to be in the "different competitive environment" and "difficult competitive situation in the sinister", to be able to "see other people do not see the market" business opportunities, to be able to "grasp the turning point of each point in time "and then" effective concentration of the resources go to vote, "the victory of success for granted. The main conclusions of this study include: 1. Third-generation mobile data services and value-added application services business model and traditional voice telecommunications services to voice-based mode of operation have clearly different points, not to the existing mode of operation to run data services and value-added application services firms. However, adopting the "second doctrine" (that is, the so-called "follow the doctrine") in the third generation mobile communications market and competitive environment is feasible strategies. (In this case study - Japan's KDDI and the Asia-Pacific Broadband Wireless operations shall Example) 2. The traditional "voice"-based "business model" and "data services and value-added application services," mainly "business model" point of difference to a voice-based mode of operation lies in its main voice services 『 tariff price 』(Rate price) make modulation, will be on the market and competitors influence and ownership of these modulators can be completely controlled in the control of operators. Data and data value-added application services business model for telecom operators must take a back seat in the "intermediary" role to bring together the various value chain "suppliers" are one up, to create "the highest customer value," and From these match the process of due share of profits. (In this case study - Japan's NTT DoCoMo i-mode shall Example) 3. This case study is based on the domestic mobile phone, a perfectly competitive market, with a backward person (in this case study the company - the Asia-Pacific Broadband Wireless), began to enter the market, after careful observation in the dynamics of competitors finally found a "competition who neglected market space "; and" did not find a competitor's market space, "and then" cheaper products "into the market, finally winning in the market. 4. Apple following the iPOD and iTunes success, to a Music Download world, completely in order to provide the consumer point of view Content Services. In addition, Google also provides consumers with a full-service Internet point of view, both into the "smart phone" market, and even "the whole mobile phone market", indeed to the entire mobile phone market unparalleled shock. The study found that iPhone, or, Google Phone, or are using a mobile phone from a consumer point of view, by using the "open mode of operation" to cut into the market. This is the traditional mobile phone operators, mining completely "closed mode of operation" has significantly different. In the end "open business model" and the "closed mode of operation," Who will win the final then? 5. Data products mode of operation with traditional voice-based mode of operation is different from voice products are "price modulation" a great impact, while the price is in complete control of the modulation in the hands of mobile phone operators; However, the use of data products must go through "learning," "cognitive," "confirmed" and "preferences" and other process to be a gradual increase in its usage, and these factors in the process of change is not complete control over the mobile phone operators hands, because mobile phone operators in the data market's role as "intermediary" role comes together. The study found data products a higher initial investment, but the homogeneity of the high, easy to in the above-mentioned process were eliminated, but once one has a product can be successful in a competitive market survived the failure of other homogeneous products because it will of successful products that can come back in a short period of recovery. Therefore, data mining product must be "multi-path input" mode of the mode of operation. The success of data products, and its profits created out from business model are in the so-called "long tail period." As "i-mode" mother: Miss Matsunaga Mari said: "the pursuit of wants rather than the pursuit of needs", "A Service we want to do, even their own mothers can easily operate the service," and so on, These principles or beliefs, seemingly high theory, but it is the market, marketing, consumption principle. Matsunaga Mari in the "i-mode" is a valuable point to, apart from her to go high-tech industries "not nice" character, her high-tech products back to human face, face to deal with the market. She saw the link in the high-tech products with the market between "niche". Finally, after all, a data product "business model" has been successful created made out, in fact, come from "do not understand technology" from the "original" from the "life" and from "human", "natural "and" the kind of life continued from the soul of innovative thinking. "