臺灣博碩士論文加值系統

Public Health is a multidisciplinary field aiming to promote populations health trough organized community efforts. Through such efforts, many infectious diseases that were huge burdens are nowadays under control. The Primary Amoebic Meningoencephalitis (PAM) is, for the most of communities, an unknown thus neglected infectious disease caused by the brain eating amoeba: Naegleria fowleri. Like the disease it causes, this single cell free-living amoeba is not well known; especially in ecological and biological fields.
Herein, we (1) investigated the nutrient-related and temperature-related effects on N. fowleri. The growth profiles of N. fowleri using liquid media with different ingredient compositions at different range of temperature were established. Secondarily, we (2) investigated the presence of essential non-neuronal cholinergic system genes and the ability of N. fowleri to produce acetylcholine. Subsequently, we evaluated the role of acetylcholine in N. fowleri trophozoites migration, proliferation and morphology.
From the first part of this thesis, we demonstrated that supplementation with peptone promotes the parasites growth at 25, 37 and 43°C. Besides, yeast extract had an inhibitory effect and was able to swamp the growth promoting effect of peptone.
In the second part of the thesis, we found that N. fowleri LDL possesses and expressed mAChR, AChE and CLT genes. However, no significant nucleotides sequence of ChAT was discovered. Furthermore, we demonstrated that the parasite was producing ACh with extracellular accumulation. Thus, we noted that ACh was (a) involved in the parasite migration, (b) keeping the trophozoites alive in non-growth promoting medium, and (c) involved in the transformation of trophozoites in cysts.
Overall, we believe that populations should avoid diving in waters especially if they contain organic debris like peptone promoting the parasite growth. Moreover, we suggest that a better prevention or treatment of PAM requires the development of anticholinergic drugs.

DEDICATION i
ACKNOWLEDGEMENTS ii
ABSTRACT iii
TABLE OF CONTENTS iv
List of Tables viii
List of Figures ix
List of abbreviations xii
1. INTRODUCTION 1
2. LITERATURE REVIEW 7
2.1. Morphology and life cycle 7
2.2. Cultivation and pathogenicity 8
2.3. Primary amoebic meningoencephalitis (PAM) 9
2.3.1. Clinical features. 9
2.3.2. Pathophysiology 10
2.3.3. Risk factors 11
2.4. Neurotropic Preferences of Naegleria fowleri 13
2.5. Epidemiology 13
2.6. Diagnostic 14
2.6.1. Microscopic identification of amoeba. 15
2.6.2. Molecular detection of Naegleria fowleri 16
2.7. Treatment 16
2.8. Prevention and control 17
2.9. Non-neuronal cholinergic system 17
3. MATERIAL AND METHODS 21
3.1. Study 1: Optimization of Naegleria fowleri axenic culture 21
3.1.1. Naegleria fowleri LDL strain and culture media 21
3.1.2. Growth curves 22
3.1.3. Giemsa staining 23
3.2. Study 2: Non-neuronal Cholinergic system 23
3.2.1. Measurement of ACh concentration 23
3.2.2. Bioinformatics search 24
3.2.3. Naegleria fowleri LDL strain DNA and RNA extraction 25
3.2.4. Cholinergic system genes amplification. 26
3.2.5. Annotation of signal peptide, cleavage sites and domains. 26
3.2.6. Sequence Alignments 27
3.2.7. Phylogenic tree 27
3.2.8. Quantitative Reverse Transcription PCR (qRT-PCR) 27
3.2.9. Trough assay (chemotaxis under agar assay) 28
3.2.10. Naegleria fowleri growth in Nelson medium supplemented with serum or without serum 28
4. RESULTS 30
4.1. Study 1: Optimization of Naegleria fowleri axenic culture 30
4.1.1. The growth profiles of Naegleria fowleri in the Nelson and the modified PYNFH media at 25oC, 37oC and 43oC 30
4.1.2. Effects of medium components on the growth of Naegleria fowleri 31
4.2. Study 2: Non-neuronal cholinergic system in Naegleria fowleri 36
4.2.1. Acetylcholine measurement in different media 36
4.2.2. Bioinformatics result 39
4.2.3. Amplification of the cholinergic system genes 41
4.2.4. Alignments and phylogenic trees. 43
4.2.5. Quantitative Reverse Transcription PCR (qRT-PCR) results 51
4.2.6. Role of ACh on Naegleria fowleri migration 53
5. DISCUSSION 57
5.1. From study 1 57
5.1.1. Naegleria fowleri growth is temperature-dependent. 57
5.1.2. Naegleria fowleri growth and temperature effects are medium-dependent. 57
5.2. From study 2 59
5.2.1. Naegleria fowleri LDL possesses a non-neuronal cholinergic system 59
5.2.2. Acetylcholine extracellular accumulation in N. fowleri culture is medium dependent 59
5.2.3. Acetylcholine effects on Naegleria fowleri. 60
6. CONCLUSION AND PERSPECTIVES 62
7. REFERENCE 64
8. APPENDICES 79
A. Naegleria fowleri ATCC 30863 Strain and Naegleria gruberi 79
A.1. Muscarinic receptor 79
A.2. Acetylcholinesterase 80
A.3. Choline transporter-like protein 82
A.4. Choline acetyltransferase 83
B. Naegleria fowleri LDL Strain 84
B.1. Muscarinic receptor 84
B.2. Acetylcholinesterase 86
B.3. Choline transporter-like protein 87
C. RT-PCR sequencing results 88
C.1. Muscarinic receptor 89
C.2. Acetylcholinesterase 89
C.3. Choline transporter-like protein 89
C.4. Melting curves 90
D. Naegleria fowleri LDL amino acids Alignment file 97
D.1. Muscarinic receptor 97
D.2. Acetylcholinesterase 98
D.3. Choline transporter-like protein 100
D.4. Choline acetyltransferase 101

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