Roles of Msb2p and other putative sensors in environmental responses of Candida albicans [Elektronische Ressource] / presented by Fabien Cottier

Beratung

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2008
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Roles of Msb2p and other putative sensors in environmental
responses of Candida albicans







Inaugural - Dissertation





Presented by

Fabien COTTIER
From Lyon, France









Submitted to attain the doctoral degree of the
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf





DÜSSELDORF
December 2007


































Referee: Prof. Dr. J.F. Ernst

Co-referee: Prof. Dr. K. Jäger

müdlichen Prüfung: 18.01.2008


i
1. Introduction ............................................................................................................................ 1
1.1 Candida albicans: friend and foe...................................................................................... 1
1.2 Dimorphism: from yeast to hyphae.................................................................................. 2
1.2.1 MAPK cascade pathways.......................................................................................... 2
1.2.2 cAMP-PKA pathway................................................................................................. 3
1.2.3 Other pathways.......................................................................................................... 5
1.3 Sensing the environment .................................................................................................. 5
1.3.1 Pheromones ............................................................................................................... 6
1.3.2 Glucose...................................................................................................................... 6
1.3.3 N-Acetylglucosamine................................................................................................ 7
1.3.4 Amino acids...............................................................................................................7
1.3.5 Ammonium................................................................................................................ 7
1.3.6 Gases ......................................................................................................................... 8
1.3.7 Thigmotropism and galvanotropism ......................................................................... 8
1.3.8 Stress ......................................................................................................................... 9
1.4 Msb2p............................................................................................................................... 9
1.5 Aims of this work........................................................................................................... 14
2. Material and methods........................................................................................................... 15
2.1 Chemical products and enzymes .................................................................................... 15
2.2 Strains and media. 15
2.2.1 Bacterial strain......................................................................................................... 15
2.2.2 Media and growth of E. coli 15
2.2.3 Yeast strains ............................................................................................................ 15
2.2.4 Media and growth conditions for yeast................................................................... 17
2.2.5 C. albicans hyphal induction in liquid media ......................................................... 17
2.2.6 hyphal induction on solid media .......................................................... 18
2.2.7 Thigmotropism........................................................................................................ 18
2.3 Plasmids and primers ..................................................................................................... 18
2.3.1 Reference plasmids ................................................................................................. 18
2.3.2 Constructed plasmids .............................................................................................. 19
2.3.3 Primers .................................................................................................................... 19
2.3.4 Plasmid construction ............................................................................................... 20
2.4 Transformation............................................................................................................... 21
2.4.1 Transformation of E. coli ........................................................................................ 21
2.4.1.1 Electrocompetent cells ......................................................................................... 21
2.4.1.2 Transformation 21
2.4.2 Transformation of S. cerevisiae .............................................................................. 22
2.4.3 TransformaC. albicans ................................................................................ 22
2.5 Preparation of nucleic acids ........................................................................................... 22
2.5.1 Extraction of plasmids from E. coli ........................................................................ 22
2.5.2 Extraction of plasmid from S. cerevisiae ................................................................ 22
2.5.3 Extraction of genomic DNA from C. albicans ....................................................... 22
2.5.4 Extraction of total RNA from .............................................................. 23
2.6 Methods of molecular biology ....................................................................................... 23
2.6.1 Restriction enzyme.................................................................................................. 23
2.6.2 Formation of blunt-ended DNA .............................................................................. 23
2.6.3 Phosphatase reaction ............................................................................................... 24
2.6.4. Ligation .................................................................................................................. 24
2.6.5 Size markers for DNA fragment ............................................................................. 24
2.6.6 Purification of DNA from agarose gels................................................................... 24 ii
2.6.7 Quantification of nucleic acids by photometry ....................................................... 24
2.6.8 Southern blot ........................................................................................................... 24
2.6.8.1 DNA transfer on Nylon membrane ...................................................................... 24
2.6.8.2 Labelling of probes............................................................................................... 25
2.6.8.3 Staining of blots ................................................................................................... 25
2.6.9 Polymerase Chain Reaction (PCR) ......................................................................... 25
2.6.10 Quantitative real-time RT-PCR (qRT-PCR)......................................................... 26
2.6.10.1 DNAase I treatment............................................................................................ 26
2.6.10.2 RNA purification................................................................................................ 26
2.6.10.3 Reverse transcription.......................................................................................... 26
2.6.10.4 Polymerase Chain Reaction ............................................................................... 26
2.6.11 DNA-microarrays.................................................................................................. 27
2.6.11.1 cDNA synthesis 27
2.6.11.2 Hybridization and washing of the DNA-microarray.......................................... 28
2.6.11.3 DNA-microarray scanning ................................................................................. 28
2.6.11.4 Normalization and statistical analysis................................................................ 28
2.7 Methods of biochemistry 29
2.7.1 Protein analysis ....................................................................................................... 29
2.7.1.1 Protein extraction ................................................................................................. 29
2.7.2 Cell wall composition.............................................................................................. 29
2.7.2.1 Isolation of cells walls.......................................................................................... 29
2.7.2.2 Determination of mannoproteins.......................................................................... 29
2.7.2.3 Determination of chitin ..............................................................