USF Libraries
USF Digital Collections

Characterizing the in-vitro morphology and growth kinetics of intermediate amyloid aggregates

MISSING IMAGE

Material Information

Title:
Characterizing the in-vitro morphology and growth kinetics of intermediate amyloid aggregates
Physical Description:
Book
Language:
English
Creator:
Hill, Shannon E
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Dynamic light scattering
Atomic force microcopy
Lysozyme
Protofibril nucleation
Oligomer
Dissertations, Academic -- Physics -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The mechanisms linking deposits of insoluble fibrils of amyloid proteins to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings suggest that transiently formed intermediate aggregates, and not the prominent neuronal plaques, represent the principal toxic agent. Evaluating the neurotoxicity of intermediate aggregates, however, requires unambiguous characterization of all aggregate structures present, their relative distributions, and how they evolve in time. Hen-egg white lysozyme represents an attractive model for studying intermediate aggregate formation since it is an extensively characterized globular protein, and its human variants can lead to systemic amyloidosis. Combining in-situ dynamic light scattering (DLS) with atomic force microscopy (AFM), we have characterized the morphologies and growth kinetics of intermediate aggregates formed during lysozyme fibrillogenesis. Upon incubation at elevated temperatures, small uniform oligomers form with their numbers increasing for several hours. After a variable lag period protofibrils spontaneously nucleate. The heights and widths of protofibrils closely match those of oligomers. This match in physical dimensions, combined with the delayed onset of protofibril nucleation vs. the continuous formation of oligomers, suggest that protofibrils both nucleate and grow from oligomers. Protofibril morphologies and structures, visualized with AFM, are quite distinct from subsequently emerging mature fibrils. Overall, the evolution of aggregate morphologies during lysozyme fibrillogenesis follows a clear hierarchical pathway: amyloid monomers initially coalesce into oligomers of uniform size. Their steadily increasing numbers eventually induce nucleation and growth of protofibrils. Protofibrils, in turn, nucleate and grow via oligomer addition until they start to self-assemble into micron-sized double-stranded fibrils.
Thesis:
Thesis (M.S.)--University of South Florida, 2008.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Shannon E. Hill.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 43 pages.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 002047062
oclc - 497165355
usfldc doi - E14-SFE0002752
usfldc handle - e14.2752
System ID:
SFS0027069:00001


This item is only available as the following downloads:


Full Text

PAGE 1

n r r "# $% & r'#(rr )#" *r)#" ")#" "%( +%,)-../ ("0)&r )0n)# + )1 2-../)

PAGE 2

nn 031&340 031&&$5 4353 % 6+351"$31+ 7 66r% 7 6-8*0n(%r# / 69"0:"0;3< 6=&r:&r;4> 6-r350+"r31" 6= -6 6= --#*0&67-9"0 6?-=&r8 6/-,&r36<-7&r-6-?&r3"---/#5-995$03+""$1+ -= 96"0r n" -= 9-&r@ % r-? 99@ %"0"&r "99 9=*0A8=.# 97 9,1(34 4># 9? =1+0$1+ 9< 5&5+ =.

PAGE 3

36B% *0-< 3-B "0 %&r 9,

PAGE 4

r & 68""0 8 r% 66 & -8"&r6-& 98" &r6< & =8&r -. & ,B0 8n:*0;-,& 7B&r -< & ?B&r 96 & /8"0 9=

PAGE 5

!r"# $% &!& !$ 3> % 5 ) ) C% C)%) n )% ) %%8n % C %n ) % n :"0; :&r;)%n > n $ % ) % % 3 3 ) r %

PAGE 6

) # )% n &r)! 1%) % n ( n 3 % #) ) % 88n 8 %

PAGE 7

'n(n )')*!+,!nD#>D % n C ) ) C% % 8% % % :6)-;5% ) )C :9)=;%) C) % ) % 3 %: ;:98,;:7;) :?;):/;) :<;)""0E8% :6.; 7

PAGE 8

3> ) # ) n % %:r* ) )% )%; 3 %n :66;) n %nn )> % %) % % C ?

PAGE 9

)'-./ $ &0*!1 *%% n) n:*0;* 6=)9//F 6-<)*0D % C%n G= % )%*0 :69;n> %8 ) nA*% n )*0 %% %n 8 :6=; /

PAGE 10

)'2( &3(4 "0! n 4C) % %) 5% %) r % : ; 8 3 4 % % 4n ) 3 n %( <

PAGE 11

38 n % C %8 n ) 6:H; ( 3 C n% ( 6:H;GIC:8"!-H; !%%3 %%%( !G:=JFK;:LF-; %C )K% % )L &) )") % >8! ) 5G>43F7JM" >44n)3 )M% 6. 6 ; : 6

PAGE 12

36..% n% 3n 3 n )) &)r%Nn +)! .7 7 ,) n nr ) n :9.; 66

PAGE 13

)'5&$ 34+,!3 3&r %3 ) % %3 >%N ,r % ) %) %:96; 6-

PAGE 14

&r%: ; :8;) >' n >3 %> n D &)8 %! 3! % %&) %D C % 69

PAGE 15

-'n(.( -')r %)C > n! :)! )&r)3 r); % %F ::6,;&C) % n:67;"0 %n! 8%%) %)% n 8 >:6?;3 %"0 % 5 &r)) % ) 8%> n % nn 8 :&r; 6=

PAGE 16

4n &r % n % !n % &r n %"0% 8n 1 % % %% 6,

PAGE 17

-'-1%./& !6$ n:6?F0;G-. 6?,r+ %nn C6(6+F ) # C)n =,O % <),.. ,) % --.-.n -.6+0n $P KG-/.:Q-/.G-7=R6R6;:-?;0n =8,,.O &>8 ) :; 67

PAGE 18

-'2( &3(4,$ "0 Nn+ :r% 0)$;9*B+ :KG799; 8 % : ,;) %6. 7. %n ST %Nn+ #n %% 6?

PAGE 19

-'5&$34, 5 rY" :r>r+97F+ +###8&r58,.; 6.?% 3%%7. ?.>n.,n! 6.-=C6.-=C 5 6/

PAGE 20

-'76"r!!$ &r, :44 )U,;#88:6V; % 6.)n 3 6(-.n 6 )n) &9)* '&r ,2 "n 3n &r:; *& )*% 5:-/; 6<

PAGE 21

*% % C %:&=4; &:-/;)* 5 ) % %3) 5 3 8% 5:&=;5 C ,5 #$ %&n'#( : ;&r ,: ;% *:; : ;35% ) -.

PAGE 22

-'8&%& !$ "0 ) 6.W0! >"0 % &r 6..86?,r+FG-. ?,W0 %, ) n $"0) n,.O :;)& r -6

PAGE 23

-'9%&$%( 3 :&9;)8 r > %3 &r 3 >) 8 &r ) :-/; --

PAGE 24

-':1!%. !! &!%&1 (&$$ ) n C&r #D :6/;C& 'C C) % :-<; &)$* +*, ) *-.$,/+0+./ ) C3 0 )% &)./-+*/,+./ +1+*/,+./.*,0 ) *.-+12r34,.0 -9

PAGE 25

2'n((n 2')($,&%($6, !#$ &,% 6:H; n G-.3G,. )6:H;8C% 6. )6:H; % 3 3) % n :#";)r r3#" :&,4; )n >8 3 > n n G=,) n% :6/;% ,.X)#"% %) > ) 6
PAGE 26

,7 n $ 5678 567, 34 3% 6:H; *0 G-.3G,. 6. % 34 8n :# ";% 6:H;)#"): C;>&C 6.> #" 34 08 > > ) n: ( ;08% 3% % -,

PAGE 27

&:; % ) 5 -,:&,; >:&,4; :&,";) 8 :&,";) % -89:&,;& )n > > CC % ) % "0 > :+G6-; -7

PAGE 28

2'-&!;,* $$% &r% "0 ) C >:&,4; %% "0Z Z n Z % &! %) n&r :&9;)&r &=:)r r; n 3)8 "0 &r)n "0! &r% -?

PAGE 29

&rn C :9.[.-; :9/[./;! :&7\36;* n % --79% -6-9n :6<;3 8n)%) :-.; ,.) C n n :&74;& %>) 3&r%) ) ]]>"0 %%C r 4 )% /6 n) 3 "0:&,; &r :&7; ) % C 38 )6, 8 3 n36 -/

PAGE 30

,8 $8r # G-.3G,. 34 4:3G-.;)&r *0 : ;&r*0 9 ,.X ) : ;&r % 6.: ( ;&r-, : ;&r C6.. )> 3 &r:84; 67:8;6-, 3) ) % &r n36 6) 57 % &r% % *0 -<

PAGE 31

3&r%% &) % ) % >C :6=; %)C : 36&?;3! % :-6; 3 > r &r 3 ) %) &r 8 % 0:&,"; %) 3 %)&r"0 8 9.

PAGE 32

,9 n%9"$ r : ;&r *09 .X: ;&r *0 % 46-, % C8 & ) :&7"; % "0:&,;&r)%) C) % % -..3 8 8&7 -89 )%) 8 ) "0 C 96

PAGE 33

9.. =..) 8 8 6, 4 % 8% :&7;3 n %) %&r A8=. r :&6:--;; 9-

PAGE 34

2'2;,*&$%("( )% n 3 : ; :-9;) n % !n 8 ) &) )> n ) :&/; &r)) :&74; 3 C3! ]>]"0 % &r3 n # %n 6.. "0 -.89. 99

PAGE 35

3 ) % &r ) % "0 ,: 9 : ;3#"S >T >) 6<-=3 8n:&=4;: ; 6:H;8C 8C 6/89/ 8&r *8 C : ;) % &r%C 9=

PAGE 36

*Cn ) &r:&9;3 ) :&7;) C 6-,[-/7 $#E : rr:-=;;) ) 3n n)) 8> :&r; r % :"0;"% ) &r > "0 3 n ) &r 6# 5& :;r n:.;) % :9; :6.,; "0:9 ;#n ) &r 9,

PAGE 37

2'5./&$<5=1! 0n> > nA8 =.&r] ]n n A) %'C n:9)=; n >8%8) >A8=. "0:&6:7;; n>:& ,4;&)%n n 8 :&7; &rA8 =.:--; 3 ) ) % 8 )8 ) :6;) n "08&r > 3 97

PAGE 38

2'7'&$0,!+$%1%6 r" 1 38 % n *n ) "0&r % n 3n )%) ]]:9)=; :7; 1 %% % &)! % )C) ) "0&r ) %) 8 ) 9?

PAGE 39

&) ) 3 % &r) % ) %3 :-,; A8 :6=)-7;&r C 8 A8 r ) 8 "+C3) 8 C #) )C > :&7; 9/

PAGE 40

5'nn ) )n n3 % # ) 8 9<

PAGE 41

n 6)^*:6<<7;% % % #n:9rnr;r 8> 6686? -$%>)P+)3)))^5 )\&>)0:6<<<;# # r r 7> 6..686.69")+)r)r))*4)4 >)^)0) :r :-..-; 1&8# + P <;r# -99> 97.=7897.,9 =)5)))3)^0)r) 3r)r) :r :-..9; 1 r # 2==> =/78=/< ,)5)))n)&))3) )* :r :-..?;& ) n n=n -> 6/6686/66 7_)*)0>))>n)r")3 )"4))8 :r :-..-; 8> 8 "= ??> 6,.86,= =.

PAGE 42

?)^")*))0)r)\0 )#3:6<; 5> 66<86-, /)\):-..6;#) C %b =r=nr 5> /,<8/7. < )5) 8N))#)r)0) ^)+)0 :r :-..9; r&4 r $&r ;r 669,866== 6.0)r#)4)))4) ))&)5 :r :6< 7==/87=,9 66+ )r))5)r))\) :-..?;r 3 1n &n#" 6.9668 6.9-=6-#)r4)>)#+)4)"5)P )"r)3) : r :6<<9; n =n 28-> ,,98,,? 69 %)0+\P)5:-..,;3 & *0n ;r ,6,8,-7 6= )r\4>)&:6< 6-986,< =6

PAGE 43

6,4))&)))r)\ 3)":-..,;+ C 8 )-> //8<, 67>)3\n)"r:6<<<; nE8 (+ 8 "=' ?8> 97//897<9 6?4)*:6<<9; ? :1C$%#)+_>; 6/#)))#)\r )r :-..?; 80n "+ 0n ;# )-?> --=8-9= 6<)"r\)+:6<7<;r 0n )87> =,=8=7, -." )4)>)+)\4>)4:6 ,6,/8,67= -6*)"r)0>))4>)4) )rr)\3)"4 :6< --9798--9?--))0 )#3^)\)^* :6<<<;( "=' ?8> <
PAGE 44

-94)3)r>))_))>)3) ) :r :-..7; 5&1%& 997??8997/9 -=#)&:6<97;r %4E 5 3 6866-,"r)r0\")P:-..=; -,&%(# =' )=)> --<98--) :r :-..,; 8`a8> =n 527> ??98??/ -? )^)5))) "+)\P):6<7-; # 0nn 66.?8666-/3')"^)@ ))0)r5)\ 4%):6<<<;@ ( r&r%r 55> 96-89-7 -;),<8/99.r% &@ABA2+ $ 96*)# :-..?; n %:" : r :#+);)68<= =9


xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam 2200385Ka 4500
controlfield tag 001 002047062
005 20100112140330.0
007 cr mnu|||uuuuu
008 100112s2008 flu s 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0002752
035
(OCoLC)497165355
040
FHM
c FHM
049
FHMM
090
QC21.2 (Online)
1 100
Hill, Shannon E.
0 245
Characterizing the in-vitro morphology and growth kinetics of intermediate amyloid aggregates
h [electronic resource] /
by Shannon E. Hill.
260
[Tampa, Fla] :
b University of South Florida,
2008.
500
Title from PDF of title page.
Document formatted into pages; contains 43 pages.
502
Thesis (M.S.)--University of South Florida, 2008.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
3 520
ABSTRACT: The mechanisms linking deposits of insoluble fibrils of amyloid proteins to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings suggest that transiently formed intermediate aggregates, and not the prominent neuronal plaques, represent the principal toxic agent. Evaluating the neurotoxicity of intermediate aggregates, however, requires unambiguous characterization of all aggregate structures present, their relative distributions, and how they evolve in time. Hen-egg white lysozyme represents an attractive model for studying intermediate aggregate formation since it is an extensively characterized globular protein, and its human variants can lead to systemic amyloidosis. Combining in-situ dynamic light scattering (DLS) with atomic force microscopy (AFM), we have characterized the morphologies and growth kinetics of intermediate aggregates formed during lysozyme fibrillogenesis. Upon incubation at elevated temperatures, small uniform oligomers form with their numbers increasing for several hours. After a variable lag period protofibrils spontaneously nucleate. The heights and widths of protofibrils closely match those of oligomers. This match in physical dimensions, combined with the delayed onset of protofibril nucleation vs. the continuous formation of oligomers, suggest that protofibrils both nucleate and grow from oligomers. Protofibril morphologies and structures, visualized with AFM, are quite distinct from subsequently emerging mature fibrils. Overall, the evolution of aggregate morphologies during lysozyme fibrillogenesis follows a clear hierarchical pathway: amyloid monomers initially coalesce into oligomers of uniform size. Their steadily increasing numbers eventually induce nucleation and growth of protofibrils. Protofibrils, in turn, nucleate and grow via oligomer addition until they start to self-assemble into micron-sized double-stranded fibrils.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
590
Advisor: Martin Muschol, Ph.D.
653
Dynamic light scattering
Atomic force microcopy
Lysozyme
Protofibril nucleation
Oligomer
690
Dissertations, Academic
z USF
x Physics
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.2752