Made available in DSpace on 2016-12-12T20:17:55Z (GMT). No. of bitstreams: 1 118436.pdf: 2819178 bytes, checksum: a3bc9e10a2c0a87089122aa8b9f6c959 (MD5) Previous issue date: 2014-07-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The habit of carrying babies in baby carriers is in a significant expansion in modern culture, however there are gaps in knowledge about these new artifacts available in the market. This study aimed to evaluate and compare the feeling of weight carried and muscle fatigue perceived by adults after carry a baby with and without the aid of baby carriers. For this research we used the Soft Structured Carrier and Wrap Sling, which are considered contemporary baby carriers. A systematic literature review presented here covers concepts of ergonomics with a focus on transporting babies, as well as cultural, physical and emotional issues associated with the artifact. The results intended to add to the existing literature, new data, contributing to the construction of scientific knowledge and identifying the need for standardization of manufacturing standards and use as well as the elaboration of a recommendation of acceptable weight limits and other relevant issues order to benefit the population that chooses this type of resource. / O hábito de transportar bebês em carregadores está em significativa expansão na cultura moderna, entretanto existem lacunas no conhecimento acerca destes novos artefatos disponíveis no mercado. A presente pesquisa teve como propósito avaliar e comparar a sensação de peso transportado e a fadiga muscular percebida pelos adultos após o transporte de um bebê com e sem o auxílio de carregadores. Para a realização desta pesquisa foram utilizados o Soft Structured Carrier e o Wrap Sling, sendo estes considerados carregadores contemporâneos. A pesquisa bibliográfica sistemática apresentada neste trabalho contempla conceitos de Ergonomia com foco no transporte de bebês, além de questões culturais, físicas e emocionais associadas ao artefato. Os resultados obtidos pretendem acrescentar a bibliografia já existente, novos dados, contribuindo para a construção do conhecimento científico e identificando a necessidade de normalização dos padrões de fabricação e uso, bem como a elaboração de uma recomendação de limites aceitáveis de peso e outras questões relevantes a fim de beneficiar a população que opta por este tipo de recurso.

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The increase in capacity demand along with the advancement in digital signal processing (DSP) have recently revived the interest in coherent optical communications and led to its commercialization. However, design and development of robust DSP algorithms for example for carrier phase recovery (CPR) becomes complex as we opt for high order modulation formats such as 16QAM and beyond. Further, electrical-domain dispersion compensation (EDC), while providing many advantages, makes the system more susceptible to laser frequency noise (FN). For instance, in coherent optical links with post-reception EDC, while the transmitter frequency noise causes only phase impairment, the local oscillator (LO) FN in these systems results in a noise enhancement in both amplitude and phase. This noise is commonly known as equalization enhanced phase noise (EEPN). It results in asymmetric requirements for transmitter laser and LO laser. Further, the system design in the presence of lasers with non-white frequency noise becomes increasingly challenging for increased capacity-distance product. The main contributions of this thesis are, firstly, an experimentally validated theory of coherent optical links with lasers having general non-white frequency noise spectrum and corresponding system/laser design criteria and mitigation technique. Secondly, low complexity and high phase noise tolerant CPR for high order modulation formats. The general theory propounded in this thesis elucidates the origin of the laser frequency noise induced noise enhancement in coherent optical links with different DSP configurations. The thesis establishes the existence of multiple frequency noise regimes and shows that each regime results in different set of impairments. The influence of the impairments due to some regimes can ideally be reduced by optimizing the corresponding mitigation algorithms, while other regimes cause irretrievable impairments. Experimentally validated theoretical boundaries of these regimes and corresponding criteria applicable to system/laser design are provided. Further, an EEPN mitigation method and its two possible implementations are proposed and discussed. The thesis also demonstrates an intrinsic limitation of the conventional Blind Phase Search (BPS) algorithm due to angular quantization and provides methods to overcome it. Finally, this thesis proposes and demonstrates single stage and multi-stage carrier phase recovery algorithms for compensation of phase impairments due to the two lasers for higher order circular and square modulations. The proposed methods outperform the state of art algorithms both in performance and in complexity. / <p>QC 20170516</p> / European project ICONE gr. #608099

CFTR基因突變或者功能缺失是否導致包括胃腸道在內的各種組織惡性腫瘤的發生風險增加目前仍然是一個充滿爭議的問題。同時，眾所周知，緊密連接分子在腫瘤發生和轉移的過程發揮了關鍵的作用。本論文首次發現了CFTR基因與一種緊密連接分子AF-6/afadin的在人類結直腸腫瘤中的表達水平呈高度相關，并研究了CFTR和AF-6/afadin之間潛在的相互作用及其在結直腸腫瘤轉移中的功能。 / 論文的第一部份首先用實時定量PCR和免疫組織化學的方法比較了CFTR在結直腸腫瘤和正常組織的表達情況，發現CFTR表達水平在腫瘤組織中有顯著的下降。令人感興趣的是，我們同時發現CFTR和AF-6/afadin在腫瘤組織中的表達呈高度正相關，并由此展開了後續的體外實驗，研究對CFTR與AF-6/afadin之間可能的相互聯繫。利用免疫螢光染色和免疫共沉澱的方法，我們發現了這兩種蛋白分子共表達在結直腸腫瘤細胞的接觸面，并存在相互作用。用CFTR突變蛋白的免疫共沉澱實驗進一步發現，這種相互作用需要CFTR分子在細胞膜表面的正確定位及其PDZ結構域結合位點。實驗還發現與CFTR的相互作用加強了AF-6/afadin與細胞骨架蛋白系統的結合。在結直腸腫瘤細胞中CFTR基因敲减导致了AF-6/afadin蛋白定位混亂，從細胞連接位點轉移到細胞漿內，并因此破壞了上皮細胞的緊密性。極性生長細胞的跨上皮電阻降低而滲透性增強的實驗結果證實了CFTR基因敲減導致的上皮細胞緊密性的破壞。同時，AF-6/afadin蛋白水平也隨著CFTR基因敲減而降低，但mRNA水平未發生明顯的改變。蛋白降解系統的抑製劑逆轉了CFTR基因敲減細胞中AF-6/afadin蛋白的減少，提示CFTR基因敲減增加了AF-6/afadin的蛋白降解。這些實驗結果揭示了通過與細胞連接分子AF-6/afadin的相互作用以及調節，CFTR可能在上皮細胞極性的調節以及腫瘤發展過程中起重要作用。 / 論文的第二部份研究了CFTR和AF-6/afadin在結直腸腫瘤細胞上皮細胞間充質化（EMT）和轉移過程中的功能及機制。我們之前的工作已經揭示抑制CFTR的功能可以誘導結直腸腫瘤LIM1863細胞的EMT過程。本研究在另外三株不同的結直腸腫瘤細胞（SW480，SW1116和HRT-18）中進一步證實了抑制CFTR誘導的EMT過程。細胞形態轉變，上皮細胞標誌物的下調，間充質細胞標誌物的上調以及受損的上皮細胞緊密性均證實了對CFTR的抑制可以在這三種細胞中成功誘導EMT的發生。我們發現在以上所有細胞EMT的過程中，AF-6/afadin的蛋白表達水平都發生了顯著的下調。在HRT-18細胞中過表達AF-6/afadin，可以逆轉由CFTR抑製劑誘導的上皮細胞標誌分子的下調和間充質標誌分子的上調，表明抑制CFTR誘導的EMT過程是由AF-6/afadin參與介導的。此外，CFTR基因敲減導致結直腸腫瘤細胞的惡性表型強化，包括減弱的細胞粘附性，增強的貼壁依賴性生長、侵襲和遷移。另外，CFTR基因敲減激活了ERK的磷酸化，過表達AF-6/afadin可以阻斷ERK途徑的激活。CFTR基因敲減而增強的細胞侵襲性也可以被外源性AF-6/afadin或者ERK途徑的抑製劑U0126完全逆轉，提示作為AF-6/afadin的下游靶信號，ERK介導了CFTR在腫瘤侵襲中的作用。更重要的是，我們分析了CFTR和AF-6/afadin的表達水平與結直腸癌病人腫瘤進展的關係，發現在嚴重TNM腫瘤分期或者有腫瘤遠處轉移的病人中CFTR的表達水平顯著低於輕型分期或未发生转移的病人中的水平，而且CFTR和/或AF-6/afadin低表達的病人的預後更差。這些實驗結果顯示CFTR的缺失可能通過抑制AF-6/afadin和激活ERK通路而與EMT和結直腸癌癥轉移的過程高度相關。 / 綜上所述，本研究揭示了以往未報道過的CFTR在結直腸腫瘤發病機理中的功能，提示CFTR可以用作一種新的腫瘤的潛在預後指標。 / The question whether mutation or dysfunction of CFTR increases the risk of malignancies in various tissues, including the gastrointestinal tract, remains highly controversial. Meanwhile, it is well-known that adherens junctions play critical roles in the process of cancer development and metastasis. In this thesis we found for the first time a highly correlation between expression levels of CFTR and an adherens junction molecule AF-6/afadin in human colorectal tumours, and investigated the potential interaction between CFTR and AF-6/afadin and their functional roles in the metastasis of colorectal cancer. / In the first section of this thesis, we started our studies with comparing the expression of CFTR between human colorectal tumours and normal colorectal tissues. Real time quantitative PCR and immunohistochemistry results revealed a dramatically reduced CFTR level in the cancer tissues. Intriguingly, we noticed a highly positive correlation between CFTR and AF-6/afadin expression in tumours, which prompted the further in vitro investigation of possible interaction between CFTR and AF-6/afadin. Using immunofluoresent staining and co-immunoprecipitation, we found that the two proteins were colocalized at cell-cell junctions and interacted with each other in colorectal cancer cell lines. Further Co-IP experiments performed with CFTR mutations revealed that this protein interaction requires the proper localization of CFTR in cell membrane and its PDZ-interacting domain. Moreover the interaction with CFTR strengthens the binding of AF-6/afadin to the cytoskeleton system. Knockdown of CFTR in colorectal cancer cells resulted in the disorganized localization of AF-6/afadin protein from junctional sites to the cytoplasm and impaired epithelial tightness, which was confirmed by significantly reduced transepithelial resistance and increased permeability of polarized cells. Meanwhile, the protein level of AF-6/afadin was down-regulated in CFTR-knockdown cells, while no significant changes were detected at the mRNA level. Protein degradation inhibitor reversed the repression of AF-6/afadin protein in CFTR knockdown cells, suggesting the protein degradation of AF-6/afadin was increased by CFTR knockdown. These data revealed that CFTR interacts with and regulates the cell adhesion molecular AF-6/afadin in colorectal cells, which may be important in the regulation of epithelial cell polarity and cancer development. / In the second section of this thesis, we studied the functional roles and mechanisms of CFTR and AF-6/afadin in the epithelial-mesenchymal transition (EMT) and metastasis of human colorectal cancer cells. Our previous work has revealed inhibition of CFTR can induce EMT in a colorectal cancer cell line, LIM1863. This study further confirmed the induction of EMT by inhibiting CFTR in several other colorectal cancer cell lines (SW480, SW1116 and HRT-18), which was evaluated by morphological changes, down-regulation of epithelial markers or up-regulation of mesenchymal markers, and impaired epithelial cell tightness. In all these cell lines, we found that the protein levels of AF-6/afadin were significantly reduced. Over-expression of AF-6/afadin in HRT-18 cells reversed the down-regulated epithelial markers and up-regulated mesenchymal markers induced by CFTR inhibition, indicating that the CFTR inhibition-induced EMT is mediated by AF-6/afadin. Moreover, knockdown of CFTR in HRT-18 or RKO cells resulted in enhanced malignant phenotypes, including decreased cell adhesion, increased anchorage-independent cell growth, invasion, and migration. In addition, extracellular signal-regulated kinase (ERK) phosphorylation was activated by CFTR knockdown, which was abolished by over-expression of AF-6/afadin. The enhanced invasiveness of CFTR knockdown cells was also completely inhibited by either exogenous AF-6/afadin or ERK inhibitor, U0126, suggesting that ERK, the downstream target of AF-6/afadin, is involved in mediating the effect of CFTR in cancer invasion. More importantly, we analyzed the association of CFTR and AF-6/afadin expression levels with tumour progression of patients with colorectal cancer, and revealed that CFTR expression was significantly lower in patients with more severe TNM stage or with metastasis to distant organs than those with milder stage or with no metastasis. The prognosis was poorer in patients with lower expression of CFTR and/or AF-6/afadin than those with higher expressions. These data showed that dysfunction of CFTR is highly associated with EMT and colorectal cancer metastasis, probably via repression of AF-6/afadin and activation of ERK pathways. / In summary, the present study has revealed a previously undefined role of CFTR in the pathogenesis of colorectal cancer and indicated its potential as a new prognostic indicator. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Tingting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 113-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 中文摘要 --- p.iv / Publications --- p.vi / Conference Abstract --- p.vii / Declaration --- p.viii / Acknowledgements --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Colorectal Cancer --- p.1 / Chapter 1.1.1. --- Structure of Human Normal Colon and Rectum Epithelium --- p.1 / Chapter 1.1.2. --- Staging of Colorectal Cancer --- p.3 / Chapter 1.1.3. --- Metastasis of Colorectal Cancer --- p.3 / Chapter 1.1.4. --- K-Ras mutation and It Downstream Pathways in Colorectal Cancer Metastasis --- p.11 / Chapter 1.1.5. --- Prognosis of Colorectal Cancer --- p.14 / Chapter 1.2. --- Epithelial Cell Junctional Complexes --- p.14 / Chapter 1.2.1. --- Junctional Complexes and Epithelial Cell Polarity --- p.15 / Chapter 1.2.2. --- Classic Cadherin-catenin Complex --- p.17 / Chapter 1.2.3. --- Novel Nectin-afadin Complex --- p.19 / Chapter 1.2.4. --- Cell Polarity and Cancer Progression --- p.23 / Chapter 1.3. --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.24 / Chapter 1.3.1. --- Structure of CFTR --- p.24 / Chapter 1.3.2. --- Mutations of CFTR --- p.24 / Chapter 1.3.3. --- Functions of CFTR --- p.26 / Chapter 1.3.4. --- Cancer Risk of CF Patients --- p.33 / Chapter 1.4. --- Hypothesis and Aims --- p.34 / Chapter Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1. --- Materials --- p.35 / Chapter 2.1.1. --- Reagents and Chemicals --- p.35 / Chapter 2.1.2. --- Antibodies --- p.35 / Chapter 2.1.3. --- Primers --- p.35 / Chapter 2.1.4. --- Solutions and Buffers --- p.35 / Chapter 2.1.5. --- Human Specimens --- p.36 / Chapter 2.2. --- Methods --- p.36 / Chapter 2.2.1. --- Cell Culture --- p.36 / Chapter 2.2.2. --- Transfection --- p.36 / Chapter 2.2.3. --- Selection of Stable Clones --- p.40 / Chapter 2.2.4. --- RNA Extraction and RT-PCR --- p.40 / Chapter 2.2.5. --- Quantitative Real Time PCR --- p.41 / Chapter 2.2.6. --- Protein Extraction and Western Blotting --- p.42 / Chapter 2.2.7. --- Immunostaining --- p.45 / Chapter 2.2.8. --- In vitro Cell Functional Assays --- p.46 / Chapter 2.2.9. --- Epithelial Tightness Measurement --- p.48 / Chapter 2.2.10. --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Interaction of CFTR with AF-6/afadin and Its Importance in Maintaining Colorectal Epithelial Cell Polarity --- p.50 / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Objectives --- p.53 / Chapter 3.3. --- Experimental plan --- p.54 / Chapter 3.4. --- Results --- p.55 / Chapter 3.4.1. --- The expression of CFTR and AF-6/afadin is decreased and positively correlated in human colorectal cancer --- p.55 / Chapter 3.4.2. --- CFTR colocalizes and interacts with AF-6/afadin in human colorectal cancer cells --- p.58 / Chapter 3.4.3. --- PDZ binding motif and membrane localization of CFTR are necessary for the interaction between CFTR and AF-6/afadin --- p.64 / Chapter 3.4.4. --- Knockdown of CFTR interferes with cell junction formation in colorectal cancer cells --- p.66 / Chapter 3.5. --- Discussion --- p.71 / Chapter Chapter 4 --- CFTR as a Suppressor and Prognosis Indicator of Metastasis in Human Colorectal Cancer --- p.77 / Chapter 4.1. --- Introduction --- p.77 / Chapter 4.2. --- Objectives --- p.80 / Chapter 4.3. --- Experimental plan --- p.81 / Chapter 4.4. --- Results --- p.82 / Chapter 4.4.1. --- CFTR inhibition-induced EMT in colorectal cancer cells involves AF-6/afadin --- p.82 / Chapter 4.4.2. --- Knockdown of CFTR aggravates malignant phenotype of colorectal cancer cells --- p.86 / Chapter 4.4.3. --- AF-6/afadin mediates the effect of CFTR on cell invasion in colon cancer through ERK --- p.91 / Chapter 4.4.4. --- CFTR and AF-6/afadin expression is correlated with the prognosis of colorectal cancer --- p.97 / Chapter 4.5. --- Discussion --- p.100 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.105 / Chapter 5.1. --- The diversified roles of CFTR in epithelial cells --- p.105 / Chapter 5.2. --- The unfolding relationship between CFTR and cancer development --- p.107 / Chapter 5.3. --- Future studies --- p.109 / Chapter 5.4. --- Conclusions --- p.112 / Reference List --- p.113 / Chapter Appendix A --- Reagents and Chemicals --- p.128 / Chapter Appendix B --- Antibody List --- p.131 / Chapter Appendix C --- Primer List --- p.132 / Chapter Appendix D --- Solution Recipe --- p.133

Lead halide perovskites have shown remarkable device performance in both solar cells and LEDs. Whilst the research efforts so far have been mainly focussed on device optimisation, little is known about the photophysical properties. For example, the nature of the bandgap is still debated and an indirect bandgap due to a Rashba splitting has been suggested. In this thesis, we study the early-time carrier relaxation and its impact on photoluminescence emission. We first study ultrafast carrier thermalization processes using 2D electronic spectroscopy and extract characteristic carrier thermalization times from below 10 fs to 85 fs. We then investigate the early-time photoluminescence emission during carrier cooling. We observe that the luminescence signal shows a rise over 2 picoseconds in CH3NH3PbI3 while carriers cool to the band edge. This shows that luminescence of hot carriers is slower than that of cold carriers, as is found in direct gap semiconductors. We conclude that electrons and holes show strong overlap in momentum space, despite the potential presence of a small band offset arising from a Rashba effect. Recombination and device performance of perovskites are thus better described within a direct bandgap model. We finally study carrier recombination in perovskites and the impact of photon recycling. We show that, for an internal photoluminescence quantum yield of 70%, we measure external yields as low as 15% in planar films, where light out-coupling is inefficient, but observe values as high as 57% in films on textured substrates that enhance out-coupling. We study the photo-excited carrier dynamics and use a rate equation to relate radiative and non-radiative recombination events to measured photoluminescence efficiencies. We conclude that the use of textured active layers has the ability to improve power conversion efficiencies for both LEDs and solar cells.

Die vorliegende Arbeit ist der Verbesserung der Carrier-Envelope Phasenstabilisierung von ultrakurzen Laserimpulsen gewidmet. Zur Realisierung von Fortschritten auf diesem Gebiet werden die grundlegenden Rauschquellen identifiziert, die das erzielbare Restphasenrauschen limitieren, und geeignete Maßnahmen zu deren Verringerung vorgeschlagen. Es wird gezeigt, dass sowohl die Messung der Carrier-Envelope Phase (CEP) als auch deren Kontrolle durch verschiedene Rauschbeiträge beeinträchtigt wird. Der Detektionsprozess ist dabei einerseits durch technische Rauschquellen beeinflusst, die vor allem in den verwendeten nichtlinearen Interferometern auftreten. Andererseits repräsentiert das Detektionsrauschen während der elektro-optischen Wandlung eine fundamentale Limitierung, da das optische Schrotrauschen sowie das Rauschen des Lichtdetektors die Messung der CEP unausweichlich beeinträchtigen. Es wird demonstriert, wie solche Beschränkungen durch geeignete Wahl der Interferometertopologie, bzw. durch Optimierung des spektralen Verbreiterungsmechanismus verringert werden können. Experimentell gelingt es dadurch den Signal-Rauschabstand der Phasenmessung um 20 Dezibel zu steigern. Hinsichtlich der CEP Kontrolle von Oszillatoren wird in dieser Arbeit ein neuartiges Doppelstabilisierungskonzept vorgestellt, welches eine feed-forward Stabilisierung, die auf einem akustooptischen Frequenzschieber beruht, mit einer klassischen Feedback Regelung kombinert. Mit diesem Konzept gelingt eine Reduzierung des Phasenrestrauschen auf beispiellose 20 Milliradian. Darüber hinaus werden weitere neue Stabilisierungskonzepte vorgestellt, die ohne Feedback zu dem Laseroszillator auskommen. Bei einem dieser Konzepte, handelt es sich um eine gepulste feed-forward Stabilisierung, die speziell für das Zusammenwirken mit einer Verstärkerstufe konzipiert ist. Erste experimentelle Ergebnisse zeigen, dass Phasenrestrauschen von weniger als 100 Milliradian auch für Verstärkersysteme erreichbar sind. / The present thesis is dedicated to improvements of the carrier-envelope phase stabilization of ultrashort laser pulses. In order to realize such improvements, the fundamental noise sources are identified, and suitable measures for their reduction are proposed. It is shown that both, the measurement of the carrier-envelope phase (CEP) as well as its control are corrupted by different noise contributions. On the one hand, the detection process is influenced by technical noise sources, which arise especially in the used nonlinear interferometers. On the other hand, the detection noise in the electro-optic conversion represents a fundamental limitation, since the optical shot noise as well as the noise induced by the light detector inevitably influence the measurement of the CEP. It is demonstrated how such limitations can be minimized by a suitable choice of the interferometer topology and by an optimization of the spectral broadening process in a micro-structured fiber. This way an enormous improvement of the signal-to-noise ratio by 20 dB is obtained experimentally, which significantly reduces the limitation of detection noise. For controlling the CEP of mode-locked oscillators, a novel double stabilization scheme is introduced in this thesis, which combines a feed-forward stabilization based on an acousto-optic frequency shifter, with a classical feedback loop. This method enables a reduction of the residual phase jitter to an unprecedented value of 20 milliradian. Beyond that, several further concepts are introduced that are capable of stabilizing the CEP without any feedback to the laser oscillator. One of these concepts, represents a pulsed feed-forward stabilization, which is specifically designed for the use in combination with a subsequent amplification stage. First experimental results indicate that residual phase jitters of less than 100 milliradian are within reach also for amplified laser systems.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

Isoelectric trapping (IET) has become an accepted preparative-scale electrophoretic separation technique. However, there are still a number of shortcomings that limit its utility. The performance of the current preparative-scale IET systems is limited by the serial arrangement of the separation compartments, the difficulties in the selection of the appropriate buffering membranes, the effect of Joule heating that may alter separation selectivity and a lack of methods for the determination of the true, operational pH value inside the buffering membranes. In order to bolster the current membrane pH determination methods which rely on the separation of complex ampholytic mixtures, a fluorescent carrier ampholyte mixture was synthesized. The use of a fluorescent mixture allows for a reduced load of carrier ampholytes, thereby reducing a possible source of error in the pH determinations. A mixture of carrier ampholytes tagged with an alkoxypyrenetrisulfonate fluorophore was shown to have suitable fluorescence and ampholytic properties and used to accurately determine the pH of high pH buffering membranes under actual IET conditions. In a more elegant solution to the difficulties associated with pH determinations, a method utilizing commercial UV-transparent carrier ampholytes as the ampholyte mixture to be separated was developed. By using commercial carrier ampholytes and eliminating the need to synthesize, purify, and blend fluorescently tagged ampholytes, the new method greatly simplified the determination of the operational pH value of the buffering membranes. In order to address the remaining limitations, a new system has been developed that relies on (i) parallel arrangement of the electrodes and the collection compartments, (ii) a directionally-controlled convection system for the delivery of analytes, (iii) short anode-to-cathode distances, (iv) short intermembrane distances, and (v) an external cooling system. This system has been tested in four operational modes and used for the separation of small molecule ampholytic mixtures, for the separation of protein isoforms, and direct purification of a target pI marker from a crude reaction mixture.

This thesis entitled ‘ Unusual Acylation Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Proteins’ describes the discovery of self-acylation and malonyl transferase activity in acyl carrier proteins involved in type II fatty acid biosynthesis and assigns a physiological role to these processes inside the cellular milieu. Acyl carrier protein (ACP) is one of the most abundant proteins present inside the cell and almost 4% enzymes require it as a cofactor. Acyl carrier proteins can exist either as discrete proteins or as domains of large functional proteins. They function in a variety of synthases as central molecules to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final product. A prototypical bacterial ACP is composed of 70-80 amino acids and is generally expressed in the apo form. It is post-translationally modified to active holo form by the addition of 4'-phosphopantetheine moiety to an absolutely conserved serine residue in a reaction catalyzed by holo-ACP synthase or 4'-phosphopantetheine transferase. Chapter 1 surveys literature related to carrier proteins inside the cell and describes the thesis objective. It also presents an overview of the acyl carrier proteins and their involvement in various metabolic pathways inside the cell. The chapter details the structural organization of acyl carrier proteins from various sources revealing the conservation in their structure and also details the molecular basis of interaction of ACP with other enzymes inside the cell. The discovery of unusual self-acylation property in acyl carrier proteins involved in polyketide biosynthesis and its absence in acyl carrier proteins involved in fatty acid biosynthesis prompted me to investigate the reasons for this selective behavior. Discovery of self-acylation property in acyl carrier proteins Plasmodium falciparum and chloroplast targeted Brassica napus acyl carrier proteins involved in type II fatty acid biosynthesis and the mechanism of this reaction forms the basis of Chapter 2. In this chapter it has been shown that self-acylation property is intrinsic to a given acyl carrier protein and is not dependent on the pathway in which it is involved. Based on primary sequence analysis and site directed mutagenesis studies presence of an aspartate/glutamate has been identified to be critical for the self-acylation event. Furthermore, it has also been shown that the self-acylation event in type II fatty acid biosynthesis acyl carrier proteins is highly specific in nature employing only dicarboxylic acid –CoAs as substrates unlike the polyketide biosynthesis acyl carrier proteins which utilize both dicarboxylic acid and β-keto acid thiol ester -CoAs as substrates. The detailed kinetics of these reactions has also been worked out. Combining all the results a plausible mechanism for the self-acylation reaction has been proposed. Chapter 3 describes the discovery of a novel malonyl transferase behavior in acyl carrier proteins involved in type II fatty acid biosynthesis. Malonyl transferase property in ACPs of type II FAS from a bacterium (Escherichia coli), a plant (Brassica napus) and a parasitic protozoon (Plasmodium falciparum) were investigated to present a unifying paradigm for the mechanism of malonyl transferase behavior in ACPs. Identification of malonyl transferase property in Plasmodium falciparum ACP and Escherichia coli ACP (EcACP) and the absence of this property in Brassica napus ACP has been described in this chapter. Detailed investigations demonstrated that presence of an arginine or a lysine in loop II and an arginine or glutamine at the start of helix III as the residues that are critical for the transferase activity. In order to assign a physiologic function to these unusual acylation properties, fabD(Ts) mutant strain of Escherichia coli was utilized for heterologous complementation by the various wild type and mutant ACPs that are able to catalyze either or both of the activities. Growth of the mutant strain at non-permissive temperature, when complemented with ACPs catalyzing both the reactions confirmed that these properties have a physiologic relevance. Extensive mutagenesis experiments in conjunction with complementation studies allowed me to propose a plausible mechanism on how the self-malonylation and malonyl transferase properties operate in tandem. Chapter 4 describes the thermodynamic characterization of self-acylation process using Isothermal Titration Calorimetry. Isothermal Titration Calorimetric studies on the binding of malonyl, succinyl, butyryl and methylmalonyl –CoA to Plasmodium falciparum and Brassica napus acyl carrier proteins were performed to investigate the role of thermodynamic parameters in the specificity of self-acylation reaction. Calculation of the parameters showed that the thermodynamics does not control the self-acylation reaction. The evolution of self-acylation property in various acyl carrier proteins and its possible significance in the evolution of various metabolic events is described in Chapter 5. Extensive bioinformatics search was performed and phylogenetic analysis on acyl carrier proteins from 60 different taxa was done using the MEGA4 program. Analysis showed that this property was first found in cyanobacterium. Later, during the course of evolution this property was lost in most acyl carrier proteins, and was retained either in acyl carrier proteins that are targeted to organelles of cyanobaterial orgin viz. apicoplast in apicomplexans and chlorplasts in plants or in acyl carrier proteins involved in secondary metabolic events such as polyketide biosynthesis. Chapter 6 summarizes the findings of the thesis. Acyl carrier protein from Plasmodium falciparum, Brassica napus and Escherichia coli were characterized for their self-acylation and malonyl transferase properties and a combined mechanism for these two properties is proposed. The work done also provides an in vivo rationale to these in vitro processes. Furthermore, the evolutionary significance of the self-acylation behavior is also discussed in the thesis. The thesis also probes into the thermodynamics of the self-acylation reaction in Plasmodium falciparum and Brassica napus acyl carrier proteins. Thus, the thesis adds a new dimension to the much unexplored ACP biology and paves the way to study in vivo roles of these processes in detail. Appendix I describes the Isothermal Titration calorimetric characterization of binding of various acyl-PO4 molecules to Escherichia coli PlsX (Acyl-phosphate acyltransferase). PlsX, the first enzyme of phosphatidic acid biosynthesis pathway catalyzes the conversion of acyl-ACP into acyl-PO4, which is further used by other enzymes leading to the formation of phosphatidic acid. ITC results presented in this section show that longer chain length acyl-PO4 molecules show better binding to PlsX, as compared to the smaller ones demonstrating that long chain acyl molecules serve as better substrates for phosphatidic acid synthesis.