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Table of contents
INTRODUCTION
I.1. Eukaryotic cilia and flagella
I.1.1 Structure of cilia
I.1.2. Sensory functions of cilia
I.1.3. Biogenesis of cilia.
I.1.4. Ciliopathies: pathologies related to ciliary dysfunction.
I.1.5. Important models for ciliary studies.
I.2. The Paramecium model: a powerful material for research on cilia.
I.2.1. Basal bodies and cilia of Paramecium
I.2.2. Nuclear duality in Paramecium
I.2.3. Tools available for Paramecium studies.
I.3. Thesis project: IFT57 in cilia and nuclei in Paramecium.
CHAPTER 1 IFT GENES USED IN THIS WORK
1.1. Paramecium IFT proteins used in this study.
1.2. IFT57 (synonyms: HIPPI; CHE‐13)
1.3. IFT46 (synonyms: DYF‐6; FAP32)
1.4. IFT139 (synonym: FAP60)
1.5. IFT172 (synonym: OSM‐1)
1.6. Qilin (synonyms: CLUAP1; DYF‐3; FAP22)
CHAPTER 2 IFT57 IN CILIOGENESIS
PART 2.1. LOCALIZATION STUDY OF IFT57
2.1.1. Localization of IFT57A‐GFP and IFT57C‐GFP proteins in vegetative cells
2.1.2. Localization of IFT57A‐GFP in growing cilia
2.1.3. Conclusion of Part 2.1
PART 2.2. EFFECTS OF INACTIVATION OF IFT57 GENES
2.2.1. Possibilities of co‐inactivation within the Paramecium IFT57 gene family
2.2.2. Effect on IFT57 RNAi on Paramecium growth rate
2.2.3. Effect on IFT57 RNAi on Paramecium cilia
2.2.4. Effect on IFT57 RNAi on Paramecium expressing IFT57A‐GFP
2.2.5. Conclusion of Part 2.2
PART 2.3. IFT57 WITHIN THE INTRAFLAGELLAR TRANSPORT
2.3.1. Localization of IFT46 and qilin GFP fusions.
2.3.2. Effect of the depletion of different IFT proteins.
2.3.4. Conclusion of Part 2.3.
CHAPTER 3 POTENTIAL NUCLEAR ROLE OF IFT57
PART 3.1. NUCLEAR TARGETING OF IFT57A
3.1.1. Localization of IFT57A‐GFP and IFT57C‐GFP proteins in vegetative cells
3.1.2. Localization of IFT57A‐GFP during autogamy
3.1.3. Looking for the signal that targets IFT57A to the macronucleus, compared to IFT57C.
3.1.3. Conclusion of Part 3.1.
PART 3.2. LOOKING FOR THE ROLE OF IFT57A IN THE MACRONUCLEUS
3.2.1. Attempts to induce RNAi during autogamy by expression of a hairpin RNA under the NOWA1 promoter.
3.2.2. “Regular” IFT57 RNAi during sexual events
3.2.3. Conclusion of Part 3.2.
DISCUSSION
D.1. IFT57 in the IFT system for ciliogenesis.
D.1.1. Ciliary growth and maintenance in relation to IFT recycling.
D.1.2. Cytoplasmic complexes of IFT proteins
D.2. The presence of IFT57A in the macronucleus, a still unsolved mystery
D.2.1. Nuclear targeting of IFT57A‐GFP
D.2.2. Possible nuclear roles of IFT57A deduced from its localization.
D.3. IFT57A as a possible revelator of a cross talk between cilia/basal bodies and nuclei at autogamy
MATERIALS AND METHODS
M.1. Strains and culture conditions
M.2. Physiological manipulations of Paramecium
M.2.1. Deciliation
M.2.2. Trichocyst discharge
M.2.3. India ink labeling of food vacuoles
M.3. Molecular biology methods
M.4. Vectors used
M.4.1. GFP‐fusion expression vectors
M.4.2. RNAi vectors.
M.5. Transformation of Paramecium.
M.6. Immunofluorescence microscopy.
M.7. Electron microscopy.
M.8. RNAi by the “feeding” method.
M.8.1. RNAi by feeding during vegetative growth.
M.8.2. RNAi by feeding during autogamy
M.8.3. RNAi by feeding during conjugation
BIBLIOGRAPHY
