The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
|
| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | XII, 163 Bl. Ill. CD-ROM (12 cm) |
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| 100 | 1 | |a Ngwa, Che Julius |d 1983- |e Verfasser |0 (DE-588)1043607463 |4 aut | |
| 245 | 1 | 0 | |a The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions |c von Che Julius Ngwa |
| 246 | 1 | 3 | |a Die Moskitomitteldarmstadien des Malariaparasiten als Ziele für übertragungsblockierende Eingriffe |
| 264 | 1 | |c 2013 | |
| 300 | |a XII, 163 Bl. |b Ill. |e CD-ROM (12 cm) | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Würzburg, Univ., Diss., 2013 | ||
| 546 | |a Zsfassung in dt. und engl. Sprache | ||
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| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| 689 | 0 | 1 | |a Anopheles stephensi |0 (DE-588)4391075-0 |D s |
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| 912 | |a ebook | ||
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Datensatz im Suchindex
| _version_ | 1804151493034508288 |
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
AFFIDAVIT III
ACKNOWLEDGEMENTS IV
TABLE OF CONTENTS V
SUMMARY IX
ZUSAMMENFASSUNG XI
1. INTRODUCTION 1
1.1 MALARIA 1
1.2 THE P. FALCIPARUM LIFE CYCLE 3
1.3 THE MALARIA VECTOR-ANOPHELES 5
1.4 THE MALARIA LIFE CYCLE IN THE MOSQUITO MIDGUT 6
1.5 ANOPHELES MOSQUITO MIDGUT BACTERIA 8
1.6 EFFECT OF MIDGUT BACTERIA ON MALARIA INFECTION IN THE MOSQUITO 11
1.7 GENE EXPRESSION AND REGULATION IN PLASMODIUM GAMETOCYTES 12
1.8 TRANSMISSION BLOCKING STRATEGIES IN THE MOSQUITO 15
1.8.1 MALARIA TRANSMISSION BLOCKING VACCINES (TBVS) 15
1.8.2 TRANSGENESIS 18
1.8.3 PARATRANSGENESIS 18
1.8.4 TRANSMISSION BLOCKING DRUGS (TBDS) 21
1.9 OVERVIEW OF ANTIMICROBIAL MOLECULES TESTED IN STUDY 22
1.10 OBJECTIVE OF THIS STUDY 24
2. MATERIALS AND METHODS 26
2.1 MATERIAL 26
2.1.1 LABWARES 26
2.1.2 INSTRUMENTS 27
2.1.3 CHEMICALS 28
2.1.4 ANTIMICROBIAL MOLECULES TESTED IN THIS STUDY 28
2.1.5 COMMERCIALLY AVAILABLE SYSTEMS AND ENZYMES 28
2.1.6 ANTIBODIES AND ANTISERA 29
2.1.7 MICROORGANISMS AND PARASITES 30
2.1.8 BUFFERS, SOLUTIONS AND MEDIA 31
2.1.9 PLASMIDS 34
2.1.10 GENES INVESTIGATED AND OLIGONUCLEOTIDES 35
2.1.11 MISCELLANEOUS MATERIALS 39
2.2 METHODS 39
V
HTTP://D-NB.INFO/1045317012
TABLE OF CONTENTS
2.2.1 ENTOMOLOGY METHODS 39
2.2.1.1 MOSQUITO REARING AND MAINTENANCE 39
2.2.1.2 MOSQUITO PREPARATION AND FEED FOR BACTERIA DIVERSITY
DETERMINATION 40
2.2.1.3 TRANSMISSION BLOCKING ASSAYS USING P. FALCIPARUM 41
2.2.1.4 TRANSMISSION BLOCKING ASSAYS WITH P. BERGHEI 43
2.2.1.5 MOSQUITO MIDGUT DISSECTION FOR OOCYST DETERMINATION 43
2.2.1.6 MOSQUITO SALIVARY GLAND DISSECTION FOR SPOROZOITES DETERMINATION
44
2.2.2 CELL BIOLOGY METHODS 45
2.2.2.1 THAWING OF P. FALCIPARUM PARASITES 45
2.2.2.2 FREEZING OF P. FALCIPARUM PARASITES 45
2.2.2.3 PREPARATION OF BLOOD SMEARS 45
2.2.2.4 ESTIMATION OF PARASITAEMIA 46
2.2.2.5 CULTIVATION OF P. FALCIPARUM 46
2.2.2.6 SYNCHRONIZATION OF PARASITE CULTURES 47
2.2.2.7 P. BERGHEI MAINTENANCE 48
2.2.2.8 COUNTING CELLS USING A NEUBAUER COUNTING CHAMBER 48
2.2.2.9 PURIFICATION OF ASEXUAL BLOOD STAGES 49
2.2.2.10 PURIFICATION OF GAMETOCYTES BY PERCOLL 49
2.2.2.11 MALSTAT ASSAY 50
2.2.2.12 MTT CYTOTOXICITY TEST 51
2.2.2.13 GAMETOCYTE TOXICITY TEST 51
2.2.2.14 ERYTHROCYTE LYSIS TEST 51
2.2.2.15 EXFLAGELLATION INHIBITION ASSAY 52
2.2.2.16 ZYGOTE DEVELOPMENT ASSAY 52
2.2.2.17 INDIRECT IMMUNOFLUORESCENCE ASSAY 53
2.2.2.18 FLUORESCENT SIGNAL QUANTIFICATION 53
2.2.2.19 AN. STEPHENSI MIDGUT HISTOLOGY 54
2.2.2.20 ELECTRON MICROSCOPY OF E. MENINGOSEPTICA ISOLATE 54
2.2.2.21 ORGANIC EXTRACT PREPARATION FROM E. MENINGOSEPTICA ISOLATE 54
2.2.2.22 STANDARD DISK DIFFUSION ASSAY 55
2.2.3 MOLECULAR BIOLOGY METHODS 55
2.2.3.1 DNA EXTRACTION 55
2.2.3.2 POLYMERASE CHAIN REACTION 56
2.2.3.3 AGAROSE GEL ELECTROPHORESIS 58
2.2.3.4 LIGATION OF PCR PRODUCTS INTO CLONING VECTORS 59
2.2.3.5 TRANSFORMATION OF CHEMICALLY COMPETENT E. COLI CELLS 60
2.2.3.6 PLASMID DNA PURIFICATION 60
VI
TABLE OF CONTENTS
2.2.3.7 RESTRICTION DIGESTION AND 16S RRNA GENE RESTRICTION FRAGMENT
LENGTH
POLYMORPHISM (RFLP) 60
2.2.3.8 DNA SEQUENCING 61
2.2.3.9 DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) 61
2.2.3.10 CONSTRUCTION OF 16S RRNA GENE CLONE LIBRAIY 64
2.2.3.11 PHYLOGENETIC TREE CONSTRUCTION 64
2.2.3.12 CONSTRUCTION OF A SUBTRACTED CDNA LIBRARY USING THE SSH METHOD
65
2.2.3.13 COLONY PCR AND ANALYSIS OF CDNA SEQUENCE DATA FROM SSH 66
2.2.3.14 E. MENINGOSEPTICA ISOLATION AND SPECIES IDENTIFICATION 66
2.2.3.15 TOTAL RNA ISOLATION 66
2.2.3.16 REMOVAL OF GENOMIC DNA AND RNA CLEAN UP 67
2.2.3.17 CDNA SYNTHESIS AND REVERSE TRANSCRIPTASE PCR 67
2.2.3.18 QUANTITATIVE RT-PCR (QPCR) 68
2.2.4 PROTEIN BIOCHEMISTRY METHODS 69
2.2.4.1 RECOMBINANT PROTEIN EXPRESSION 69
2.2.4.2 PURIFICATION OF RECOMBINANT PROTEINS 69
3. RESULTS 73
3.1 INTERPLAY BETWEEN MIDGUT BACTERIA AND THE MALARIA PARASITE 73
3.1.1 BACTERIA DIVERSITY OF LAB-REARED ANOPHELES STEPHENSI DURING
DEVELOPMENT
AND UNDER DIFFERENT FEEDING REGIMES 73
3.1.2 ISOLATION, IDENTIFICATION AND CHARACTERISATION OF E.
MENINGOSEPTICA 80
3.1.3 ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF ORGANIC EXTRACTS FROM
E. MENINGOSEPTICA 81
3.1.4 ANTIPLASMODIAL AND GAMETOCYTOCIDAL ACTIVITY OF ORGANIC EXTRACTS OF
E. MENINGOSEPTICA 82
3.2 CHANGES IN THE TRANSCRIPTOME DURING THE INITIAL PHASE OF MALARIA
PARASITE
TRANSMISSION FROM HUMAN TO MOSQUITO 84
3.2.1 DETERMINATION OF TRANSCRIPTOME CHANGES BY SUPPRESSIVE SUBTRACTIVE
HYBRIDIZATION (SSH) 84
3.2.2 COMPARISON OF GENES IDENTIFIED BY SSH WITH PLASMODIUM PROTEOME
DATA.... 85
3.2.3 GENES WITH TRANSCRIPT CHANGES REGULATED BY DOZI AND CITH 99
3.2.4 QUANTIFICATION OF TRANSCRIPT LEVELS BY QRT-PCR 101
3.2.5 CHANGES IN PROTEIN EXPRESSION OF SSH SELECTED GENES 103
3.3 ROLE OF ANTIMICROBIAL MOLECULES AS TRANSMISSION BLOCKING AGENTS 108
3.3.1 TRANSMISSION BLOCKING EFFECT OF HARMONINE 108
3.3.1.1 ANTIPLASMODIAL EFFECT OF HARMONINE 108
VII
TABLE OF CONTENTS
3.3.1.2 EFFECT OF HARMONINE ON GAMETOCYTES DEVELOPMENT AND MOSQUITO
MIDGUT STAGES 109
3.3.1.3 IN VIVO TRANSMISSION BLOCKING EFFECT OF HARMONINE 110
3.3.1.4 CYTOTOXIC EFFECT OF HARMONINE 110
3.3.2 TRANSMISSION BLOCKING EFFECT OF RECOMBINANTLY PRODUCED PEPTIDES
111
3.3.2.1 TRANSMISSION BLOCKING EFFECT OF SMI ILL
3.3.2.2 TRANSMISSION BLOCKING EFFECT OF PCFK DERIVATIVES 113
4. DISCUSSION 115
4.1 INTERPLAY BETWEEN MIDGUT BACTERIA AND THE MALARIA PARASITE 115
4.1.1 BACTERIA DIVERSITY IN AN. STEPHENSI DURING DEVELOPMENT AND
DIFFERENT
FEEDING REGIMES 115
4.1.2 DOMINANCE OF E. MENINGOSEPTICA IN AN. STEPHENSI MIDGUT 116
4.1.3 TRANSSTADIAL AND VERTICAL TRANSMISSION E. MENINGOSEPTICA 118
4.1.4 E. MENINGOSEPTICA AS A POTENTIAL PARATRANSGENIC WEAPON 119
4.2 CHANGES IN THE TRANSCRIPTOME DURING MALARIA PARASITE TRANSMISSION
FROM
HUMAN TO MOSQUITO 120
4.2.1 CHANGES IN GENE EXPRESSION BY SSH FOLLOWING GAMETOCYTE ACTIVATION
120
4.2.2 CHANGES IN TRANSCRIPT LEVELS BY QRT-PCR 122
4.2.2.1 GENES WITH HIGH TRANSCRIPT LEVELS IN ACTIVATED GAMETOCYTES AS
COMPARED TO OTHER STAGES 122
4.2.2.2 GENES WITH HIGH TRANSCRIPT LEVELS IN GAMETOCYTES COMPARED TO
ASEXUAL STAGES 123
4.2.2.3 GENES WITH HIGH TRANSCRIPT LEVELS IN ASEXUAL BLOOD STAGES 125
4.2.3 CHANGES IN PROTEIN EXPRESSION OF SSH GENE 128
4.3 ROLE OF ANTIMICROBIAL MOLECULES AS TRANSMISSION BLOCKING AGENTS 129
4.3.1 HARMONINE 130
4.3.2 RECOMBINANT PRODUCED PEPTIDES 131
5. CONCLUSIONS AND FUTURE PERSPECTIVES 133
6. REFERENCES 135
7. APPENDIX 158
7.1 ABBREVIATIONS 158
7.2 16S RRNA GENE AND SSH SEQUENCES 159
PUBLICATIONS 160
VIII
|
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| spelling | Ngwa, Che Julius 1983- Verfasser (DE-588)1043607463 aut The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions von Che Julius Ngwa Die Moskitomitteldarmstadien des Malariaparasiten als Ziele für übertragungsblockierende Eingriffe 2013 XII, 163 Bl. Ill. CD-ROM (12 cm) txt rdacontent n rdamedia nc rdacarrier Würzburg, Univ., Diss., 2013 Zsfassung in dt. und engl. Sprache Malariamücke (DE-588)4168696-2 gnd rswk-swf Malaria (DE-588)4037197-9 gnd rswk-swf Krankheitsübertragung (DE-588)4384937-4 gnd rswk-swf Anopheles stephensi (DE-588)4391075-0 gnd rswk-swf Mitteldarm (DE-588)4448343-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Malariamücke (DE-588)4168696-2 s Anopheles stephensi (DE-588)4391075-0 s Mitteldarm (DE-588)4448343-0 s Krankheitsübertragung (DE-588)4384937-4 s Malaria (DE-588)4037197-9 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-83594 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-83594 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026840040&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ngwa, Che Julius 1983- The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions Malariamücke (DE-588)4168696-2 gnd Malaria (DE-588)4037197-9 gnd Krankheitsübertragung (DE-588)4384937-4 gnd Anopheles stephensi (DE-588)4391075-0 gnd Mitteldarm (DE-588)4448343-0 gnd |
| subject_GND | (DE-588)4168696-2 (DE-588)4037197-9 (DE-588)4384937-4 (DE-588)4391075-0 (DE-588)4448343-0 (DE-588)4113937-9 |
| title | The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions |
| title_alt | Die Moskitomitteldarmstadien des Malariaparasiten als Ziele für übertragungsblockierende Eingriffe |
| title_auth | The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions |
| title_exact_search | The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions |
| title_full | The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions von Che Julius Ngwa |
| title_fullStr | The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions von Che Julius Ngwa |
| title_full_unstemmed | The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions von Che Julius Ngwa |
| title_short | The mosquito midgut-specific stages of the malaria parasite as targets for transmission blocking interventions |
| title_sort | the mosquito midgut specific stages of the malaria parasite as targets for transmission blocking interventions |
| topic | Malariamücke (DE-588)4168696-2 gnd Malaria (DE-588)4037197-9 gnd Krankheitsübertragung (DE-588)4384937-4 gnd Anopheles stephensi (DE-588)4391075-0 gnd Mitteldarm (DE-588)4448343-0 gnd |
| topic_facet | Malariamücke Malaria Krankheitsübertragung Anopheles stephensi Mitteldarm Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-83594 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026840040&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT ngwachejulius themosquitomidgutspecificstagesofthemalariaparasiteastargetsfortransmissionblockinginterventions AT ngwachejulius diemoskitomitteldarmstadiendesmalariaparasitenalszielefurubertragungsblockierendeeingriffe |