Association between human rhinovirus C and severity of acute asthma in children

303 Pages·2011·7.09 MB·English

The Immunogenetics of Asthma Exacerbations
in Children

Joelene Ann Bizzintino B.Sc. (Hons)

This thesis is presented for the degree of
Doctor of Philosophy
at The University of Western Australia,
School of Paediatrics and Child Health.

2011

”Apply your heart to instruction
and your ears to words of knowledge”

Proverbs 23:12

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DEDICATION

This work is dedicated to

Theodore Box

a truly great man who has always inspired me and imparted countless words of wisdom.
He has taught me many things that I will never forget,
the most important of which is

“always think for yourself”

and whilst I have drawn from the support and expertise of supervisors and colleagues,
this thesis is definitely written proof of that.

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DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR
WORK PREPARED FOR PUBLICATION

This thesis contains published work and/or work prepared for publication, some of
which has been co-authored. The bibliographical details of the work and where it
appears in the thesis are outlined below.

1) Bizzintino J and Lee W-M, Laing IA, Vang F, Pappas T, Zhang G, Martin AC,
Khoo S-K, Cox DW, Geelhoed GC, McMinn PC, Goldblatt J, Gern JE, Le Souëf
PN
Association between human rhinovirus C and severity of acute asthma in
children. Published in: European Respiratory Journal. 2011;37:1037-42.
This constitutes Chapter 2 of this thesis. J Bizzintino was involved in the
recruitment of patients, sample collection, data collection, detection, and typing
assays and was responsible for data analysis and drafting the article. W-M Lee
developed and was responsible for the molecular detection and typing assays,
which was assisted by F Vang, T Pappas and S-K Khoo. IA Laing was involved
in the study design, initiation and management of the study and revision of the
manuscript. G Zhang supported the data analysis. AC Martin was primarily
responsible for the recruitment of patients, sample and data collection, and was
assisted by DW Cox. GC Geelhoed and PC McMinn contributed to study
design, as did JE Gern who also manages the team that performed the detection
and typing assays on the nasal aspirates. J Goldblatt and PN LeSouëf initiated
the study, contributed to its design, data analysis and drafting of the article.

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2) JA Bizzintino, C McLean, G Zhang, S-K Khoo, L Subrata, AC Martin, K
Rueter, CM Hayden, A Sharafi, GC Geelhoed, J Goldblatt, PG Holt, IA Laing
and PN LeSouëf
Polymorphisms in Toll-Like Receptor (TLR) genes influence susceptibility
and severity of acute asthma in children
This constitutes Chapter 3 of this thesis and the manuscript has been prepared
for publication. J Bizzintino was involved in study design, the recruitment of
patients, sample collection, data collection, DNA extraction, genotyping and was
responsible for data analysis and writing the article. C McLean was partially
responsible for genotyping and contributed to the study design. G Zhang
assisted the statistical analysis, as did IA Laing who was involved in
experimental design and manuscript revision. S-K Khoo assisted the genotyping
and data collection. L Subrata conducted micro-array and qRT-PCR analyses.
AC Martin, K Rueter and A Sharafi also conducted patient recruitment and
assessments. GC Geelhoed, J Goldblatt, PG Holt, CM Hayden and PN LeSouëf
contributed to the study design.

3) JA Bizzintino, G Zhang, C McLean, S-K Khoo, L Subrata, AC Martin, K
Rueter, CM Hayden, A Sharafi, GC Geelhoed, J Goldblatt, PG Holt, PN
LeSouëf and IA Laing
Polymorphisms in RIG-I-like Receptor Pathway Genes Involved in the
Innate Immune Response to Rhinovirus Affect Susceptibility and Severity
of Acute Asthma in Children.
This constitutes chapter 4 of this thesis and manuscript has been prepared for
publication. J Bizzintino was primarily responsible for study design, performed
all statistical analyses, wrote the manuscript, was involved in patient
recruitment, and performed DNA extraction and genotyping. G Zhang supported
the statistical analysis. C McLean and S-K Khoo performed some of the
genotyping. L Subrata was responsible for micro-array and qRT-PCR analyses.
AC Martin, K Rueter and A Sharafi were all involved with patient recruitment
and assessments. GC Geelhoed, J Goldblatt, PG Holt and PN LeSouëf initiated
the study, and assisted revision of the article. IA Laing supported data analysis,
was involved in initiation of the study, experimental design, and manuscript
revision.

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4) JA Bizzintino, LS Subrata, G Zhang, S-K Khoo, J Goldblatt, PG Holt, GC
Geelhoed, PN LeSouëf and IA Laing.
Genotype-driven expression of novel genes involved in childhood acute
asthma, identified by micro-array.
This constitutes Chapter 5 of this thesis and manuscript has been prepared for
publication. J Bizzintino was responsible for data analysis and writing the
article, was involved in study design, the recruitment of patients, sample
collection, data collection, DNA extraction and genotyping. L Subrata
performed micro-array and qRT-PCR analyses. G Zhang supported statistical
analysis. S-K Khoo assisted genotyping and data collection. J Goldblatt, PG
Holt, GC Geelhoed and PN LeSouëf contributed to study initiation, design and
manuscript revision. IA Laing was involved in study initiation, experimental
design, manuscript revision and supported statistical analysis.

5) JA Bizzintino, LS Subrata, G Zhang, S-K Khoo, J Goldblatt, PG Holt, PN
LeSouëf and IA Laing
Genotypes within steroid responsive genes identified by micro-array are
associated with childhood acute asthma susceptibility, severity and response
to treatment.
This constitutes chapter 6 of this thesis and manuscript has been prepared for
publication. J Bizzintino performed all statistical analyses, wrote the article, was
involved in the recruitment of patients, sample and, data collection, DNA
extraction and genotyping. L Subrata performed micro-array and qRT-PCR
analyses. G Zhang supported the data analysis. S-K Khoo supported genotyping
and data collection. J Goldblatt, PG Holt and PN LeSouëf contributed to the
study design and revision of the paper. IA Laing supported data analysis and
manuscript revision, was involved in initiation of the study and was primarily
responsible for experimental design.

Each author has given permission for the work arising in the above
manuscripts to be presented in this thesis.

______________________ ____________________________
Mrs Joelene Bizzintino Professor Peter Le Souef
PhD Candidate Principal Supervisor
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STATEMENT OF CANDIDATE CONTRIBUTION

The work presented in this thesis was performed at the Institute for Child Health
Research and the School of Paediatrics and Child Health, University of Western
Australia, Princess Margaret Hospital, Subiaco, Perth, Western Australia, under the
supervision of Professor Peter LeSouef, Dr Ingrid Laing and Professor Jack Goldblatt.

With the exception of acknowledged contributions by others, the author performed all
experimental and statistical work presented herein. This thesis is my own account of my
research and has not previously been submitted for a degree at this or any other
University.

Joelene Bizzintino
February 2011

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ACKNOWLEDGMENTS

I would like to firstly thank my supervisors, Prof. Peter LeSouef, Dr Ingrid Laing and
Prof. Jack Goldblatt. Peter, your constant support and encouragement, challenging
discussions, fantastic view of the bigger picture and passion for respiratory research
have made you an absolute joy to work with. I have shared some great times overseas
whilst attending international conferences and I thankyou for your willingness to
introduce me to numerous collaborators and help out last-minute when posters don’t
make the trip. I have also immensely appreciated your contagious enthusiasm for
adventure and your great sense of humour that has me laughing out loud (especially in
emails during the early hours of the morning). Ingrid, your understanding and ability to
interpret scientific data will always be a true inspiration for me and I am extremely
fortunate to have learnt a great deal from you during my candidature. I am also grateful
for your willingness to meet up with me to discuss my project, provide feedback on my
work, and set-up meetings or discussions for me with contacts you have made. Jack,
your ability to provide great feedback in a very short time frame or dissolve arguments
with your words of knowledge has always been very impressive and much appreciated.

I’d like to thank the people from our asthma genetics team that I have loved working
with over the years: Kim for your tremendous support, team-player spirit, amazing
generosity, your problem-solving abilities and your true friendship - you have been
absolutely amazing to work with; En nee for your honesty and your faith, Holly for your
constant support and shared love of Edward; Pierre for your humour and valued
friendship; Guicheng for your great statistical support; Catherine for all your good
advice; Carryn for your joy; Des for your passion and humility, Ash for your
enthusiasm; Selma for your kindness and helpfulness; May for your wit, Andrew Martin
for your encouragement and care-free nature; Gareth for being lots of fun to be around;
Jasminka for sharing my stress; Shah for your support that I miss; and Lauren, Olga,
Alicia, Philippa, Michelle and Giovanni for your friendship.

A huge thank you to all those at/affiliated with the School of Paediatrics and Child
Health who have supported and encouraged me and shared some great times during my
candidature. These people include Ruth, Angela, Karla, Clara, Julie, Jen, Sam,
Sunalene, Anthony, Tony, Andrew Currie, Eva, Lea-Ann, Lisa, Gina, Dianne, Steve
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Stick, Erika, Peter Franklin, Michael, Mel, Salina, Catherine Gangell, Jan, Karen and
Bill. In particular, I wish to truly thank Tony for actively emphasising the word
“support” in IT support. And I especially thank my shared-office champs (adopted
sisters, also my adopted brother Patrick) for great friendship and for showing me some
of the best times in my life. Ange, OMG, your help formatting the thesis has been
“gigantic”, and Julie, thanks for your help and attention to detail. Ruth, there aren’t
words. How do you thank someone who goes above and beyond when you need it
most, who constantly encourages you to “just keep swimming” when you are crazy-
overtired or frustrated, and without whom you would not meet an impossible deadline?

I wish to thank my colleagues over the years at the Telethon Institute for Child Health
Research, especially Prof. Wayne Thomas, Belinda, Michael, Jamie, Claire, Lee,
Jacquie, Tracey, Serena, Tatjana, Clint, Paula, Wendy, and Jua, for their individual
contributions and helpful advice.

Many thanks to Dr Wai-Ming Lee, Prof Jim Gern, Tressa, Fue and the team from the
University of Wisconsin-Madison, School of Medicine and Public Health for their
advice and technical assistance regarding the RMA and typing assay. Thank you also
for your friendship, for sharing your laboratory with me and for teaching me new
molecular techniques.

Thankyou Katie Lindsay and Tony Keil for your assistance retrieving samples and
specimen-related data, your constant support is much appreciated.

To my family (Mum, Dad, Poppa, Carrie, Trav, Greg, Kel, Rizzi) and friends (Sonya,
Joe, Dylan, Kelsey, Elina, Fiona, Candace) who loved me, called to encourage me,
prayed for me, helped me, cooked for me, and were very patient with me during my
writing-up stage.

Saving the best for last, I want to thank my awesome husband Carmelo for being very
supportive and loving, for infinite understanding, for his willingness to help wherever
he can and for the best hugs ever. I thank God for you.
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