nele de clercq - ruđer bošković...
TRANSCRIPT
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Scom
of (A
easoposit dige
Arca n
onal vtion aestivenoae
Pharmace
variatand me glane Linn
Adr
B
eutical and
NELE
Keramie
tel. 09 321 21
tions metalnd of naeusriatic
Aca
Biomedical
Biological
Pro
Co-prom
E DE CL
ekstraat 80 - 9
1 10 | fax 09
in bilothio Noas, 175 Sea
ademic yea
Laboratory
Laboratory
motor: Dr.
motor: Dr. B
ERCQ
9000 Gent
220 17 26
ocheoneinh’s A
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ar 2015-201
y Technolog
y Technolog
Marijana E
rita Muysse
Fac
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Ark shom th
16
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culty Educa
C
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tion, Health
Social
Campus Ve
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work
salius
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Acknowledgement
This thesis is the result of hard work from different people. My part was only a
piece of the puzzle. I appreciate that the Ruđer Bošković Institute allowed me to do
my internship there. Because of this I met a lot of new people who were very
welcoming. I am very grateful for the work that the persons did from my university
college HoGent, i.e. the teachers from the department Biomedical Laboratory
technology for making the internship happen through a lot of effort and
communication and the administrative staff who helped me with the paper work
which was very welcome since I’m not very good at that.
I want to award some persons in particular and since the Nobel prizes aren’t
awarded yet, I want to make some suggestions.
I nominate my promotor Dr.Sc. Marijana Erk for the Nobel prize in Chemistry. I am
very glad that she accepted me to do my internship in her laboratory LBUM. I
learned a lot from her during those 4 months. I want to thank her for the effort she
put in helping me with my work and giving feedback on my thesis.
I would like to give the Nobel prize in Literature to my co-promotor Dr. Brita
Muyssen. I am very grateful for the patience that she had with me for answering all
my questions. I want to thank her for the time she spent in reading my thesis and
guide me with the writing part of my thesis.
The Nobel Peace Prize should go to the colleagues from the Laboratory for
Biological Effects of Metals (LBUM): Dr. Zrinka Dragun, Dr. Vlatka Filipović
Marijić, Dr. Dušica Ivanković and Nesrete Krasnići. They and my promotor were
very supportive and helped me where they could. They showered me with
kindness, I felt at home and also a part of the team. I am happy as a clam that they
learned me some Croatian, hvala najljepša.
The nomination for Nobel prize for Physics goes to the colleagues of IRB. This is for
a different kind of physics, i.e. football. The games of football were a perfect outlet
after a work day. Thanks, guys.
-
Th
Ser
un
Th
my
Spe
Za
pro
Me
and
rep
I a
sho
he Nobel P
rgeant for
nforgettable
he Sveriges
y family fo
ecial grati
dar for SC
ofessor at
elita Pehar
d Dr.Sc.
productive
also want
owing me
Prize in Ph
r the heal
e.
Riksbank
or supportin
tude goes
CUBA divi
University
rda Uljević
Sanja Pu
e status of s
to thank
the ropes o
ysiology o
lthy and
Prize in Ec
ng me in m
to Dr.Sc.
ing and co
y of Zadar
ć from the
ljas from
shell fish.
Dr. Sc. Sa
of field wo
or Medicin
less healt
conomic Sc
my choice t
Ivan Žup
ollecting sh
r for logisti
Institute f
the Univ
anja Gottst
ork.
ne goes to
thy diners
ciences in
to do my in
pan, assista
hell fish, to
ics and dis
for Oceano
versity of
tein and D
Steffi Lem
s. You m
Memory o
nternship i
ant profess
o Dr.Sc. Jel
ssection of
ography an
f Split for
Dr. Sc. Mir
mmens and
made the e
of Alfred N
in Zagreb.
sor at Uni
lena Čulin
f shell fish
nd Fisheri
r determi
irela Sertić
d Bronwen
experience
Nobel is for
iversity of
n, assistant
h, to Dr.Sc.
ies in Split
ination of
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Table of contents
1 Introduction ...................................................................................................... 9 1.1 Noah’s ark shell ................................................................................................ 9 1.1.1 Taxonomy .......................................................................................................... 9 1.1.2 Morphology ...................................................................................................... 9 1.1.3 Reproduction .................................................................................................. 10 1.1.4 Habitat ............................................................................................................. 11 1.1.5 Consumption .................................................................................................. 12 1.2 Pollution .......................................................................................................... 12 1.2.1 Bivalves as indicator organisms in environmental studies ...................... 13 1.3 Sampling sites ................................................................................................. 15 1.3.1 Telaščica ........................................................................................................... 16 1.3.2 Pašman kanal/channel .................................................................................. 16 1.4 Biomarkers ...................................................................................................... 17 1.4.1 Metallothionein .............................................................................................. 20 1.4.2 Proteins, carbohydrates and lipids .............................................................. 23 1.5 Biotic and abiotic factors that affect biomarker variability ...................... 24 1.6 Aim of this study ............................................................................................ 26
2 Materials and methods ................................................................................. 27 2.1 Homogenization ............................................................................................. 27 2.1.1 Materials .......................................................................................................... 27 2.1.2 Protocol ............................................................................................................ 28 2.2 Quantification of metallothioneins and cytosolic proteins ...................... 30 2.2.1 Materials .......................................................................................................... 30 2.2.2 Protocol ............................................................................................................ 31 2.3 Quantification of total tissue proteins ......................................................... 35 2.3.1 Materials .......................................................................................................... 35 2.3.2 Protocol ............................................................................................................ 36 2.4 Quantification of total tissue carbohydrates .............................................. 37 2.4.1 Materials .......................................................................................................... 37 2.4.2 Protocol ............................................................................................................ 38 2.5 Quantification of total tissue lipids ............................................................. 40 2.5.1 Materials .......................................................................................................... 40 2.5.2 Protocol ............................................................................................................ 40 2.6 Statistics ........................................................................................................... 42
3 Results ............................................................................................................. 43 3.1 Metallothionein .............................................................................................. 43 3.2 Proteins ............................................................................................................ 46 3.3 Carbohydrates ................................................................................................ 49 3.4 Lipids ............................................................................................................... 52 3.5 Correlations between measured parameters ............................................. 55 3.6 Seasonal and spatial variability of measured parameters ........................ 60 3.6.1 Metallothionein .............................................................................................. 60 3.6.2 Proteins ............................................................................................................ 62 3.6.3 Carbohydrates ................................................................................................ 63 3.6.4 Lipids ............................................................................................................... 65
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4 Discussion ...................................................................................................... 67
5 Conclusion ...................................................................................................... 74
Bibliography ..................................................................................................................... 75
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Abbreviations
MT Metallothionein
PK 1 Pašman kanal 1
PK 2 Pašman kanal 2
TE Telaščica
d.w. dry weight
w.w. wet weight
MGI Mean gonadal index
CI Condition index
ISQG Interim Marine Sediment Quality Guidelines
PEL Probable Effect Level
MCL Maximum contaminant level
n.v. no value
Glossary Periostracum = an organic, outside layer that is found on shells of a mollusk.
Protandric = having male sexual organs in young stadium of life, and female in the later stadia.
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Abstract English Noah’s Ark shell (Arca noae) is a bivalve that is common in the Adriatic Sea, it is
exploited for culinary purposes from natural populations. The aim of this study is to
determine the concentrations of total lipids, proteins, carbohydrates and
metallothionein to differentiate between the sampling sites and their respectively
different environmental pressures and potential metal exposure. Three sampling
sites were selected: one from nature park Telaščica and two from the Pašman kanal-
the main harvesting area in the central part of Eastern Adriatic Sea in Croatia. From
each site, 36 individuals were sampled monthly in the period from March 2013 to
February 2014 . The digestive gland was used for measuring total lipids, total
proteins, total carbohydrates and metallothionein concentration, all by
spectrophotometric methods. Correlations between parameters were calculated and
two-way ANOVA was used to investigate the seasonal and spational variability
between the different sampling stations. Correlations were found for metallothionein
concentrations and reproductive cycle as well as for temperature and total lipids.
This study provided first data on metallotioneins in Arca noae. Levels of
metallothionein showed the impact that nautical tourism has on nature parks. In
summer the level of metallothionein in A. noae from Telaščica is higher than that for
the sampling sites where anthropogenic stress is expected. From the results we can
conclude that metallothionein is a possible biomarker for A. noae. More research with
organisms from sampling sites of which pollution levels are more elevated is
necessary to confirm this conclusion.
Keywords: Arca Noae, biochemical composition, seasonal variation, metallothionein, digestive gland
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Abstract Nederlands Arca noae is een tweekleppige schelp die voorkomt in de Adriatische Zee. Schelpen
uit de natuurlijke populatie worden gevangen genomen voor menselijke consumptie.
Het doel van de studie is het achterhalen of de concentratie van alle lipiden,
proteïnes, carbohydraten en metallothioneïne beïnvloed wordt door de verschillende
staalafnameplaatsen en hun eigen verschillen in ecologische druk en eventuele
metaal vervuiling. Er zijn drie staalafnameplaatsen waarvan één in het natuurpark
Telaščica en twee uit het Pašman kanal wat het grootste oogstgebied is, in het
centrale gedeelte van de Adriatische Zee van Kroatië. Voor elke plaats zijn er
maandelijks 36 individuen genomen over een periode van maart 2013 tot februari
2014. Voor de metingen van alle lipiden, alle proteïnes, alle carbohydraten en
metallothioneïne werd de spijsverteringsklier gebruikt en gemeten door
spectrofotometrische bepalingen. De correlaties tussen de parameters werden
onderzocht en two way ANOVA berekende de seizoens- en plaatsschommelingen
voor de verschillende sites. Er zijn correlaties gevonden tussen metallothioneïne
concentratie en de voortplantingscyclus alsook voor de temperatuur en de lipiden.
Het is voor het eerst dat er concentraties van metallothioneïne zijn gemeten bij de
Arca noae. De metallothioneïnewaarde in de zomer voor Telaščica ligt hoger dan de
plaatsen waar er antropogenische invloeden werden verwacht. Dit toont de invloed
van boottoerisme op natuurparken. Metallothioneïne zou een mogelijke biomarker
kunnen zijn voor A. noae maar verder onderzoek is aangewezen, aangezien de
concentraties van de verontreinigde stoffen niet hoog lagen.
Keywords: Arca Noae, biochemische samenstelling, seizoensvariatie,
metallothioneïne, spijsverteringsklier
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OCHEMICAL
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METALLOT
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gmentation (
1970s for th
s likely tha
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3 to 7 year
CONTENTS
Education
to the phy
orphia and
family Ar
ndepitte L
form; this b
at a numbe
is thick, tr
marginally,
ell is white
1 below (
(molluskase
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IN ARCA N
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ylum of the
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rcidae. Th
L., 2004).
being refle
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10 BIOCH
of 50 mm
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HEMICAL C
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-growing o
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rismatic sh
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hat males
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Reprodpic observ
while sexu
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owed by a
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g (i.e. rele
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hroditic an
to April)
roditic ind
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et al., 2006
COMPOSITIO
al., 2014).
old populat
species is
decreased f
mental alg
e rings in t
hell growth
Peharda et
dominate
as size incr
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ually mat
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February
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pawning. I
6).
ON AND ME
Therefore
tion (Pehar
s slower in
food availa
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the shell. S
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t al., 2002
small she
reases sug
ment.
om previo
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ctive cycle
xtended or
gonad reco
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to May.
gonads)
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recorded
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nd the sam
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ETALLOTH
e, local fish
rda et al., 2
n winter a
ability. Dis
s can be
Since they e
face rings
2). A stud
ll length c
ggesting tha
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les have o
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onstitution
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They are
occurs in
ly after spa
one month
only dur
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sting that h
me time wh
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IONEIN CO
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2002).
as a result
sturbance
recorded
estimate th
can cause
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categories w
at some in
s showed
orange to
olves a pe
pawning ev
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ber. Femal
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ONTENTS IN
hreaten the
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an overest
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with fema
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et al., 2006
le gametes
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August.
Male gamet
harda et al.
older part
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odites wer
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ccurs durin
N ARCA NOA
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ning seawa
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it results
he Noah’s
timation of
eharda (20
ales becom
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ads. Bival
gametogene
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6).
s are in l
une and Ju
Gametoge
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., 2006).
t of the y
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paration ta
ng this per
AE
e of
ater
ator
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riod
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Me
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OCHEMICAL
geschool Gen
hanges in th
ell weight
gnificant co
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velopment
the total
tegories of
lculation o
e active (4)
fferences b
ght be the
riability in
ain exogen
06).
1.4 Hahe shell ha
ells. In the
oatian side
ell is mor
used by te
12). The N
editerranea
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ploited (Pe
L COMPOSI
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eharda et a
ITION AND
s Vesalius
on index, e
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ampling ye
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al., 2002), se
METALLOT
expressed
good indic
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ly spawned
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THIONEIN C
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e size, sam
mperature
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ound to de
h are cha
springs o
ed in the
est Indies (
rn Adriatic
2 below.
CONTENTS
Education
o of tissue
spawning
d mean go
mber of in
stage, and
ed (Gosling
ng numeri
g: inactive
pent (1). (P
pawning p
mpled shell
e is conside
ductive cyc
rocks. It is
up to dep
epths of 60
racterized
r river inf
eastern A
(Župan et
c Sea wher
IN ARCA N
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dry weigh
since a s
onadal ind
ndividuals
d dividing
g, 2003). Q
ical values
(0), early
Peharda et
periodicity
lengths or
ered to be
cles (Peha
s found on
pths of 119
0m. The N
by lower
flows (Žup
Atlantic O
al., 2012).
re it is com
NOAE 11
alization FBT
ht and wet
tatistically
dex (MGI).
from each
the result
Qualitative
s used for
active (3),
al., 2006)
in A. noae
r temporal
one of the
rda et al.,
n rocks or
9m, at the
Noah’s ark
r salinities
pan et al.,
Ocean, the
It is most
mmercially
T
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h
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-
12 BIOCH
Figure 2. Thby black d2008).
1.1.5 It is one o
harvested
harvested
consumed
compositi
acids, esp
as such it
1.2 There are
inorganic
and gaseo
inorganic
environm
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organochl
HEMICAL C
he distributidots. Main tr
Consumof the mos
d from nat
d shells is
d by the
ion of Noa
pecially eico
represents
Pollutioe different
ions, orga
ous pollut
ions. N
mental prob
in smaller
lorine inse
COMPOSITIO
ion of Arca raditional h
mption st importa
tural popu
s sold on
local pop
ah’s Ark sh
osapentaen
s a high qu
on t kinds of
anic polluta
tants. Meta
Nitrates an
blems when
r amounts
ecticides, c
ON AND ME
noae in the harvesting ar
ant edible
ulations pr
the black
pulation. D
hell indica
noic (20:5n
uality seafo
pollutant
ants, organ
al cations,
nd phosp
n found in
. Example
carbamate
ETALLOTH
Croatian Adreas are ind
bivalves in
rimarily b
k market
Data on
ate that it i
n-3) and do
ood produc
ts that can
nometallic
, nitrates a
phates are
n large qua
es of organ
insecticid
IONEIN CO
driatic. Sighdicated by a
n the easte
by scuba d
during th
the bioch
is an excel
ocosahexae
ct (Radić e
n cause ha
compoun
and phosp
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antities unl
nic polluta
des, pheno
ONTENTS IN
ting locationrrows (Mort
ern Adriat
divers. The
he tourist
emical an
llent sourc
enoic (22:6n
et al., 2014)
arm to or
ds, radioac
phates are
xic, they
like some
ants are: h
xy herbici
N ARCA NOA
ns are indicarton & Peha
tic Sea and
e majority
season or
nd fatty a
ce of n-3 fa
n-3) acid, a
).
rganisms,
ctive isoto
examples
only ca
metals wh
hydrocarbo
ides. Most
AE
ated arda,
d is
y of
r is
acid
atty
and
e.g.
pes
s of
use
hich
ons,
t of
-
BIO
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1.2In
nat
and
org
Ra
Biv
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Som
org
OCHEMICAL
geschool Gen
ese produc
mpound. A
pan in 195
vironment
sulted in
isonous fis
oastal areas
the richest
mpact in co
heries and
dustrial de
ch an envir
llution is
ganisms an
2.1 Bi1980s it ha
tural water
d their b
ganisms sh
inbow, 199
valves ha
vironment
me of the
ganism are
wide g ecolog sessile direct filter-f ability
L COMPOSI
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cts are use
A well-kno
50s. A pa
t. Bacteria
a form th
sh (Walker
s are chara
t and most
oastal areas
d aquacult
evelopmen
ronment ca
a critical
nd also for
ivalves aas been rec
rs is the la
biological
hould be pr
93).
ave been
tal monitor
e characte
e:
geographicgically abue form of li contact wifeeder y to accumu
ITION AND
s Vesalius
ed in agric
own case ab
per factor
a in the s
hat is bioa
r et al., 200
acterized b
t fragile ec
s of the A
ure, shipp
t). Organis
an accumu
environme
humans, a
as indicacognised th
ck of corre
availabilit
referred fo
used wi
ring in ord
eristics of
cal distribundant orga
iving (attacith environ
ulate pollu
METALLOT
culture. M
bout this p
ry had rel
sediments
available t
1).
by a very h
cosystems o
Adriatic Sea
ping, touri
sms (e.g. b
ulate metal
ental issue
as their fin
ator orgahat the disa
elation betw
ty. Conse
or most env
idely as
der to detec
bivalves
ution anism ched to thenmental m
utants in th
THIONEIN C
Methylated
pollutant is
leased me
then me
to fish. 10
high biodiv
on earth. M
a are the r
sm and la
ivalves, cr
l pollutants
e, due to
nal consum
anisms inadvantage
ween the c
quently, i
vironment
sentinel
ct either na
that mak
e substratemedia
he organism
CONTENTS
Education
mercury i
s the accide
tallic merc
thylated t
00 people
versity and
Main sourc
residences
and-use pr
ustaceans
s in high co
metal toxi
mers (Walke
n environ of the con
concentrati
it has be
al monitor
or indica
atural or a
e them su
)
m
IN ARCA N
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is an organ
ent in Mini
cury in th
the mercu
died of e
d they incl
ces of anth
in the coa
ractices (a
and fish) t
oncentrati
icity for th
er et al., 20
nmental ntaminant a
ions of con
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ring tasks (
ator organ
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uitable bi
NOAE 13
alization FBT
nometallic
imata bay,
he aquatic
ury which
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lude some
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that live in
ons. Metal
he aquatic
001).
studiesanalysis of
ntaminants
uded that
(Phillips &
nisms for
enic stress.
ioindicator
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c
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14 BIOCH
Currently
Ocean is p
the protec
and asses
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monitorin
in genera
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organisms
substance
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parent an
diphenyl
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as import
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exposure
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Assessme
galloprovin
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ssment are
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ng program
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urans and
ations of th
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anean Act
ent Monit
ncialis, the
ard to the
o be a gen
ge of conta
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omposition
ondition of
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ing of con
d within the
e marine e
described
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SPAR JAM
contaminan
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ic contami
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o physiolo
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ell-type ra
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nts can dif
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r (e.g. with
999). With
indicator
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992). It is
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ONTENTS IN
the North-
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990).
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2007).
ve epitheli
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mpling site
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L COMPOSI
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atural env
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THIONEIN C
vironment
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Croatia witharvesting are
CONTENTS
Education
tal stresso
EA) metho
edentary a
aminant bu
polluted w
vailability
Sea in C
h indicated sea and TE is
IN ARCA N
BLT - specia
ors in th
odology the
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NOAE 15
alization FBT
he mussel
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sediment.
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T
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-
16 BIOCH
1.3.1 The samp
m) located
impact. H
vessels in
daily visit
because o
(Favro &
indeed be
Nickel an
compared
µg/g dry
weight. T
other unp
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in the cen
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and orga
different
municipal
industrial
transport
Two samp
northwest
Sampling
while site
potential
waters) is
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Telaščicpling site w
d on the is
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ts and excu
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Gržetić, 2
e used as a
nd chrom
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weight w
The levels o
polluted are
Pašmanman channe
ntral Adria
represents
However,
anisms livi
sources. P
l waste fro
l waste of
of chemica
pling sites
t–southeas
site PK 1
e PK 2 (at
influence
to be expe
COMPOSITIO
ca was selecte
sland Dugi
he nature p
During the
ursions. Th
from the
2008). So w
reference
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ON AND ME
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i otok, and
park is a p
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his leads to
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site.
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lead and
čić et al., 2
hannel one of the
e 3). It will
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oth PK 1 an
ETALLOTH
ature park
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2010).
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ONTENTS IN
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ower than
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depth of 10
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AE
0-14
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anges at th
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netics cau
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data avail
iomarkerare bioch
or effects
tal scientis
were alread
seful as m
icators of s
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he level of c
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evel expose
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used by
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s Vesalius
able of me
rs emical, ph
s of xenob
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s at individ
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METALLOT
easured con
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THIONEIN C
ncentration
al, or his
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Walker et
n toxicolog
xposure to
yndromes
ation and
or below,
st level of
gure 4).
of ecosystem
stress, i.e.
exchange i
cause chr
CONTENTS
Education
ns of any p
tological i
omarkers
al., 2001)
gy, in whic
o specific c
(Hill, 1965
communit
i.e. cell an
f environm
m health ca
cell and ti
is a bioma
romosoma
IN ARCA N
BLT - specia
pollutants i
indicators
were first
). Before
ch they ha
chemicals o
5; Forbes et
ty are con
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mental stre
aused by env
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arker for c
al aberrat
NOAE 17
alization FBT
in Pašman
of either
t used by
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ve proven
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sidered as
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structural
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tions e.g.
T
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l
n
.
-
18 BIOCH
dichlorop
organism,
the organ
heavy me
Nowaday
effects of e
In order
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ind
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pes
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and
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environme
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minolaevuli
etylcholine
sticides an
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mong differ
hemical spe
act differen
hibit ALAD
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should be c
a reaction
her criteria
s importan
t only in th
the limita
ns (Hugge
COMPOSITIO
alker et al
ad to an in
r, e.g. avoi
ker et al., 20
kers are oft
ental pollu
ate candid
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ker respon
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inic acid d
esterase ge
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rent species
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ntly to ch
D which ca
otective m
checked if
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a are appli
nt to know
he laborato
ations of a
ett, 1989).
ON AND ME
., 2001). Th
ncrease of d
idance beh
001).
ten used as
utants.
date bioma
ds to a va
useful in
ehydratase
ets inhibite
ex mixture
a criterio
o endpoin
biomarker
must be k
s (Huggett
s also diffe
hemicals (H
an be mor
mechanism
the bioma
re of pollut
icability to
w if the biom
ory.
a method
ETALLOTH
he second
diseases o
haviour ha
s monitors
arkers’ str
ariety of di
monitorin
e (ALAD)
ed by orga
e of polluta
on which
nts such as
rs (Hugget
kept in m
t, 1989).
erent for ev
Huggett, 1
rtal while l
(Walker et
arker is a tr
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level of t
r death. C
as been us
s for quanti
rengths an
ifferent ch
ng (Hugge
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looks at
s lethality
tt, 1989).
mind since
very bioma
1989), e.g.
lead also in
t al., 2001).
ransient or
ggett, 1989)
ditions and
useful for f
e consider
ONTENTS IN
the respon
hanges in
sed as a b
ifying expo
nd weakn
hemicals, th
ett, 1989).
hibited by
horous and
er et al., 20
the sensi
or growth
biomarke
arker, som
pollution
nduces me
r permanen
).
d validation
field measu
red before
N ARCA NOA
nse affects
behaviour
biomarker
osure and
nesses seve
hus a gene
The enzy
lead wher
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001).
itivity of
h impairm
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me biomark
of lead w
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nt occurre
n in the fie
urements a
e making a
AE
the
r of
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mate
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ment
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kers
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If b
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biomarker
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tablished:
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induce
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health
L COMPOSI
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rs are mea
changes a
the organi
withstand
hout havin
on or situa
energy exp
environme
ctuation in
lution) (Hu
hemical po
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ed stress.
asic physio
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the increa
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ITION AND
s Vesalius
asured in a
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stress. If
ng negativ
ation that
penditure,
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n the seawa
uggett, 198
ollution th
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METALLOT
a dose- or
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89).
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uggett, 198
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time-depe
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rocarbon r
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ett, 1989).
rker the fo
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of the bio
een natura
ganism sho
nimized.
ker should
anges in bio
netic adap
iomarker s
89).
CONTENTS
Education
endent ma
allow for
m can be de
ot endure
sm is not h
o mobilize
er tempera
fluctuatio
ature) and
n between
1989), e
receptor (W
ost immedi
on with bio
ollowing p
sensitive,
omarker s
al variabilit
ould be kn
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omarker co
tion.
should be
IN ARCA N
BLT - specia
anner, it is
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healthy. A
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on inside th
d anthropo
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.g. the b
Walker et
iate, and f
omarkers b
properties
reliable an
should be
ty and con
nown so th
n.
oncentratio
correlated
NOAE 19
alization FBT
s easier to
essment of
he residual
d stressful
stressor is
urces and
essors that
he estuary,
genic (e.g.
cant and a
inding of
al., 2001).
faster than
before any
should be
nd easy.
known in
ntaminant-
hat sources
on are due
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T
o
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a
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-
20 BIOCH
1.4.1 Metalloth
cysteine-r
serve as li
variety of
bismuth, a
essential
contain ca
MTs from
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and inver
MTs are
metabolis
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Figure 5. Mbeads are m
HEMICAL C
Metallohioneins (M
rich, metal
igands for
f metal io
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of metals
ent with t
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Metallothionemetal atoms (
COMPOSITIO
othioneinMT) consti
l binding
metal che
ons, such
. Native M
nc and co
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crustacean
is a differ
ompared to
o be invol
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to metallo
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thesis seem
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ON AND ME
n itute a fam
proteins a
lation. The
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zinc (Hug
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rence betw
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ting tissue
exercise, an
ms to be me
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ucture. Modnd small yell
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mily of lo
and oligop
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um, coppe
ontain mix
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ggett, 1989)
of two 9-
ween MTs f
al mamma
nterrelated
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MTs supp
nthesis, an
es. MT als
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sects and c
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ow molecu
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s of MT can
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cysteine d
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processes
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nding sites ore sulfur atom
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The cyste
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mercury, c
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mium the
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mammalia
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zinc and
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nd possibly
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role in the
icals. The
ticoid horm
s (Huggett,
of metallothims (Branisla
N ARCA NOA
ht (6-7 kD
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ced by a w
cobalt, nick
ntaining b
protein m
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an vertebra
d with me
copper, a
investigat
ly copper,
processes
e response
stress rela
mones. Thi
, 1989).
ionein. Red av et al., 2013
AE
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ues
wide
kel,
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dmium re
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ethod of de
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ethod whic
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ethods for
ere at least
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ecific reag
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s can be inf
gestive gla
an in the w
L COMPOSI
nt – Campus
been sho
n normal c
vered in b
Ts are dif
may be met
bound. W
emains bo
on of MT.
al metallot
cies and di
eterminatio
used for
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nts depend
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and than in
whole organ
ITION AND
s Vesalius
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cellular me
blue crabs
fferent pro
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When MT b
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thionein co
ifferent tiss
on.
metallothi
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reference
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n the whol
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METALLOT
an effectiv
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the one use
ković et a
metallothion
nitude lowe
he particu
standards
udies are c
the values
le organism
mple.g. in M
THIONEIN C
ve free-rad
and in m
es sapidus)
ch observ
s of biodeg
es copper a
he steady
ons are exp
differentia
antification
ed in this s
al. (2003) w
neins spec
er in comp
ular samp
applied.
ompared.
s of MT co
m, while th
M. edulis.
CONTENTS
Education
dical scave
odifying r
) that cad
ations sug
gradation o
and zinc w
y state of
pressed in
al pulse pol
n in Table
study, i.e.
who comp
trophotom
parison wi
le treatme
Therefore,
oncentratio
he values i
IN ARCA N
BLT - specia
enger, whi
radiation s
dmium- an
ggest that
of MT are d
will be lost.
f biosynth
d.w. or w.
larography
I is a vol
spectroph
pared two
metric mea
ith the vol
ent as we
, caution
ons are hig
in the gills
NOAE 21
alization FBT
ich can be
sensitivity.
nd copper
MT gene
dependent
. However
hesis and
w. for
y as the
ltammetric
hotometric.
SH-based
surements
ltammetric
ell as two
should be
gher in the
are lower
T
e
.
r
e
t
r
d
c
.
d
s
c
o
e
e
r
-
22 BIOCH
Table I. Babivalves fro
In the stu
gland for
from 520–
and autum
these resu
interspeci
HEMICAL C
sal metallotom sites con
udy perform
r M. gallop
–870, 360–7
mn (0.52-0
ults could
ies differen
COMPOSITIO
thionein connsidered to b
med by (Iv
provincialis
760, 225–6
0.87, 0.36-0
be comp
nces would
ON AND ME
ncentrations e uncontami
vanković e
determin
15 and 180
0.76, 0.225-
ared to th
d be expect
ETALLOTH
determinedinated (Ami
et al., 2005
ned by spe
0–860 µg/
-0.615 and
he data ob
ted - as lon
IONEIN CO
d by Differeniard et al., 20
5) MT conc
ectrophoto
g d.w. in w
0.18-0.86
btained in
ng as you c
ONTENTS IN
ntial Pulse P006).
centrations
ometric me
winter, spr
mg/g d.w
this thesi
convert w.w
N ARCA NOA
Polarograph
s of digest
ethod rang
ring, summ
w.). Therefo
is – althou
w. to d.w..
AE
y in
tive
ged
mer
ore,
ugh
-
BIO
Ho
1.4Ch
cau
bec
ma
Wh
req
ene
str
is n
(H
Gly
tox
fro
gla
ene
the
For
sto
198
wh
are
al.,
Lip
inv
sou
sea
of l
dar
by
as
OCHEMICAL
geschool Gen
4.2 Prhanges in
used by ph
cause of l
anifested u
hen the e
quirements
ergy reserv
ess or repr
not the ma
uggett, 198
ycogen is
xicant-indu
om the gon
abrata). Gen
ergy dema
erefore obs
r vertebrat
orage organ
89). Glyco
hen lipids a
e released.
, 2014).
pids are
vertebrates
urce. Altho
asons, spec
lipids is va
rk muscle
toxicant-in
well follow
L COMPOSI
nt – Campus
roteins, cthe concen
hysical and
long-term,
under acute
energy int
s of the or
ves are mo
roduction.
ain role be
89).
a storage
uced stress
nadal regio
nerally, th
and. Glyco
served alte
tes the pri
ns are the
gen reserv
are not ava
This proc
the most
s, fish and
ough it can
cies or stad
ariable in a
than in th
nduced str
wed by an
ITION AND
s Vesalius
carbohyntration o
d chemical
, sublethal
e exposure
take excee
ganism th
obilized w
Proteins c
ecause thei
form of g
s. Ansaldo
on can be u
ere will be
ogen storag
erations in
mary stora
hepatopan
ves are gen
ailable, sin
ess is follo
importan
birds. For
n tend tow
dia in life c
an organism
he white m
ress but a
increase in
METALLOT
ydrates aof stored e
stressors.
l exposure
e condition
eds the m
he energy w
when there
can also be
ir role is t
glucose. D
o et al. (20
used as a g
e a depleti
ge and mo
n glycogen
age locatio
ncreas (dig
nerally us
nce the rese
owed by th
nt, readily
r most of th
wards a se
cycle or re
m. For inst
muscle of a
decrease i
n specific c
THIONEIN C
nd lipidsenergy res
Changes in
e to a str
ns (Hugget
maintenanc
will be sto
is an incre
e used as an
that of a s
Depletion o
06) proved
general bio
ion of glyc
obilization
concentra
on is the li
gestive gla
sed during
erves of lip
he gametog
y available
he species
econdary e
productive
tance there
fish. A de
in total lip
component
CONTENTS
Education
s erves in in
n stored en
ressor and
t, 1989).
ce, growth
red as gly
eased ener
n energy s
structural u
of glycoge
d in their
marker for
cogen beca
is restricte
ations are o
iver. For m
and) and th
g the game
pids deplet
genetic dev
e energy
lipids are
energy sou
e cycle. Th
e are more
ecrease in l
id concent
ts of the lip
IN ARCA N
BLT - specia
invertebrat
nergy rese
d are nor
h and rep
ycogen or l
rgy deman
source, alth
unit of an
en can occ
study that
r snails (Bi
ause of an
ed to certa
often tissu
mussels th
he mantle
etogenetic
tes when t
evelopmen
source fo
e the prima
urce among
he tissue di
lipids pre
lipids can
trations m
pid constit
NOAE 23
alization FBT
tes can be
rves occur
mally not
productive
lipids. The
nd such as
hough that
n organism
cur due to
t glycogen
iomphalaria
increased
ain tissues,
ue specific.
he primary
(Huggett,
processes
he gonads
t (Radić et
or aquatic
ary energy
g different
istribution
sent in the
be caused
might occur
tuents, e.g.
T
e
r
t
e
e
s
t
m
o
n
a
d
,
.
y
,
s
s
t
c
y
t
n
e
d
r
.
-
24 BIOCH
an increas
term stres
occur dur
humans to
Structural
an energy
mobilize p
but depen
circumsta
used as an
the last so
(Huggett,
nutritiona
1.5 In genera
and seaso
such as th
decussate (
and chron
an increa
1989). Sim
Ruditapes
noae is ne
hydrate v
by Radić e
coinciding
reserves i
demand (
HEMICAL C
se of trigly
ss, althoug
ring acute
o detect pr
l proteins a
y source. H
proteins as
nding on
ances, prot
n individu
ource of en
1989). To
al stress, ho
Biotic aal the glyco
on (Hugge
he mussel M
(Radić et a
nic exposu
ased energ
milar result
decussatus
egatively a
value was d
et al. (2014
g with high
is strongly
Radić et al
COMPOSITIO
ycerids. Li
gh it is po
e stress (H
rostate can
are abunda
However
s an energ
the seaso
teins can b
ual biomark
nergy used
otal protei
owever for
and abiotogen conte
ett, 1989).
M. gallopro
al., 2014). T
ure to meta
gy demand
ts have bee
(Hamza-C
affected by
detected in
4), a second
h phytopla
y influence
l., 2014).
ON AND ME
ipids are m
ossible that
Huggett, 1
ncer (Zhou
ant in an o
under con
y source. N
n, reprodu
be a quite
ker, becau
d and there
in concent
r chemical
tic factorent can be
This is de
ovincialis an
The glycoge
als. A decr
d combine
en found f
Chaffai et a
y seawater
n December
d carbohyd
ankton abu
ed by food
ETALLOTH
most usefu
t changes
1989). Lipi
et al., 2012
organism. T
nditions of
Normally l
uctive stat
important
use of the fa
efore will n
tration in
pollutants
rs that a influenced
emonstrate
nd the clam
en content
rease in gly
ed with ch
for snails (A
al., 2003).
r temperat
r (Radić et
drate peak
undance. T
d availabi
IONEIN CO
ul as suble
in specific
ids are us
2).
These prote
f severe st
lipids and
tus and th
t energy s
act that ge
not be a ve
the plasm
s it is not se
ffect biod by diet,
ed in vario
ms Venerup
t of fish tis
ycogen us
hemical-in
Ansaldo et
Carbohydr
ture, there
t al., 2014).
k in A. noae
The season
ility, in ad
ONTENTS IN
thal indica
c lipid com
sed as a b
eins are no
tress the o
glycogen
he length
ource. Pro
enerally pr
ery sensitiv
ma of a fis
ensitive (H
marker vreproduct
ous specie
pis philippi
ssue is affe
ually occu
duced stre
t al., 2006)
rate concen
fore the h
In the stud
e was obser
al variation
ddition to
N ARCA NOA
ators of lo
mponents
biomarker
ot intended
organism
are used f
of the str
oteins are
roteins will
ive biomar
sh can det
Heath, 1995
variabilittive condit
es of bival
inarum and
ected by ac
urs because
ess (Hugg
and the sh
ntration in
highest car
dy perform
rved in Ap
n in glycog
reproduct
AE
ng-
can
r in
d as
can
first
ress
not
l be
rker
tect
5).
ty tion
lves
d V.
cute
e of
gett,
hell
n A.
rbo-
med
pril,
gen
tive
-
BIO
Ho
In
rea
in t
Pro
pri
aft
biv
and
Lip
gro
gen
dec
car
gly
201
Th
tem
in
sig
rec
gon
Au
For
mu
sea
(Iv
For
gro
me
exp
wit
OCHEMICAL
geschool Gen
a study p
ached the m
the concen
otein is a m
ior to spaw
er spawni
valves. In m
d constitut
pid conten
owth, repr
neral, lipid
creases. In
rbohydrate
ycogen to
14).
here is no
mperature
mean go
gnificant c
corded in t
nad index
ugust) (Peh
r the mus
ussel repro
ason of sa
vanković et
r oysters i
owth and t
easuremen
posure to
thout the i
L COMPOSI
nt – Campus
performed
maximum
ntration of
major organ
wning (Jun
ing (from
marine biv
te a major c
nt of variou
roductive
d content i
n A. noae, li
e content.
lipids bios
o proof o
for Arca na
nad index
orrelation
the period
x during t
harda et al.
ssel M. ga
oductive st
ampling, c
t al., 2005).
it is the rep
the molt c
nt of MT
metals (H
influence o
ITION AND
s Vesalius
by Radić
value in J
proteins an
nic compon
e) may sup
July to
valves, lipi
componen
us species
condition
increases b
ipid variati
This relati
synthesize
of a signi
aoe in Mali
x is follow
between
from July
the period
., 2006).
lloprovincia
tate, age an
contribute
productive
ycle that c
in organi
Huggett, 19
of the repro
METALLOT
et al. (20
June, just b
nd lipids.
nent of biv
pport this h
September
ids play a
nt of reprod
of bivalve
n, salinity
before mas
ions from
ionship is
ed during
ificant cor
i Ston Bay
wed by a
these two
to Decemb
d when tem
alis differe
nd sex, sea
to variat
e cycle/sea
can affect M
isms does
989). For
oductive cy
THIONEIN C
14), protei
before spaw
valve oocyt
hypothesis
r) was sim
crucial rol
ductive ma
es (Radić e
and tem
ss spawnin
February t
usually a
the forma
rrelation b
. However
an increase
o variable
ber when s
mperature
ent biotic
a temperatu
tions in th
ason of th
MT concen
not nece
detecting
ycle on the
CONTENTS
Education
in and lip
wning, foll
tes. Theref
s. The decr
milar to m
le in matur
aterial (Rad
et al., 2014)
perature
ng occurs,
to June are
ttributed t
ation of ga
between g
r, most of t
e in temp
s is proba
spawning c
e was still
and abiot
ure and sa
he MT ce
e year and
ntration. It
essarily re
exposure
e level of M
IN ARCA N
BLT - specia
pid levels
lowed by
fore, protei
rease in pr
many othe
ring gonad
dić et al., 2
4) is affecte
(Huggett,
and then
e inversely
to the con
ametes (Ra
gonadal in
the times a
perature. L
ably due
caused a d
increasin
tic factors
alinity, as w
ellular con
d for blue
t is thus ev
eflect the
in the env
MT, a meth
NOAE 25
alization FBT
in A. noae
a decrease
in maxima
otein level
er marine
dal tissues
2014).
ed by diet,
1989). In
markedly
y related to
version of
adić et al.,
ndex and
an increase
Lack of a
to values
decrease in
ng (July to
including
well as the
ncentration
crabs it is
vident that
degree of
vironment
hod is used
T
e
e
a
l
e
s
,
n
y
o
f
,
d
e
a
s
n
o
g
e
n
s
t
f
t
d
-
26 BIOCH
where the
The conce
between
(Pellerin &
Therefore
differentia
example
example
caused by
of biomar
induced b
1.6 This thesi
much larg
determina
seawater
metals in
was not th
The aim o
and carbo
differentia
environm
to provid
on the ob
bioindicat
environm
the levels
Peharda U
the gonad
HEMICAL C
ey transpo
entrations
the two s
& Amiard,
e, when u
ate betwee
gonadal
temperatu
y the anthr
rker respo
by anthrop
Aim of is focused
ger resear
ation of m
and the s
the soft ti
he main aim
of this stud
ohydrates i
ate betwe
mental pres
e informat
btained res
tor species
mental stres
s of metall
Uljević and
ds.
COMPOSITIO
ort species
are meas
sites to co
2009)
sing biom
en the phy
developm
ure and sa
opogenic s
onse may i
pogenic sou
this stud on analys
rch that w
metal conce
sediments.
issue. Accu
m of that r
dy was to
in the tissu
een the
sures. The
tion about
ults of this
s and dige
ss that coul
lothionein
d Dr. Sc. Sa
ON AND ME
from an u
ured at bo
onclude if
markers in
ysiological
ent and
alinity) an
stressor stu
interfere w
urces.
dy ses of the
was done o
entrations
. The unp
umulation
research.
determine
ue of diges
sampling
e levels of
t potential
s study we
estive glan
ld be cause
are measu
anja Puljas
ETALLOTH
unpolluted
oth sites.
there is a
environm
l changes
food abun
nd the co
udied. In o
with the es
digestive g
on this spe
on the wh
published
was only
e the conce
stive gland
sites an
metallothi
metal exp
e will eval
nd as the t
ed by meta
ured for th
s were in ch
IONEIN CO
area to th
Then they
any expos
mental stud
caused by
ndance),
ntribution
other word
stimation o
gland of A
ecies. The
hole soft ti
results sh
measured
entrations
d of Noah’
nd their
ionein will
posure in t
luate the p
target orga
al pollution
he species
harge of st
ONTENTS IN
he area of i
y compare
ure in sit
dies it is
y biotic in
abiotic inf
to bioma
s, the natu
of the leve
A. noae as
other par
issue, as w
howed accu
d in March
of total pro
s Ark shel
respective
l be determ
the environ
potential o
an in the a
n. It is the f
A. noae. D
tudying the
N ARCA NOA
investigati
e the chan
te of inter
important
nfluences (
nfluences (
arker chan
ural variatio
el of chan
one part o
rt focused
well as in
umulation
h 2013 sinc
roteins, lipi
lls in order
ely differ
mined aim
nment. Ba
of A. noae a
assessment
first time t
Dr. Sc. Me
e histology
AE
ion.
nges
rest.
t to
(for
(for
nge
ons
nges
of a
on
the
n of
ce it
ids,
r to
rent
ming
sed
as a
t of
that
elita
y of
-
BIO
Ho
2 Th
Ma
tra
the
usi
sam
con
Ou
site
so
nec
pro
to
bio
sam
80°
2.1
2.1In
Tab
Pro
β-
Leu
PM
Tri
Tri
Ab
OCHEMICAL
geschool Gen
Mhe Noah’s
arch 2013 u
ansported i
ey were im
ing stainle
mples wer
ntained dig
ur samples
e there are
for one s
cessary wh
oteins and
be homo
ochemical c
mples for a
°C for later
1 Ho
1.1 MTable II th
ble II. Produ
oduct/soluti
mercaptoe
upeptine
MSF (pheny
is- HCl
is- base
bsolute eth
L COMPOSI
nt – Campus
aterials Ark shell
until Febru
in dark an
mmediately
ess steel e
re collected
gestive gla
consist of
e four diffe
ampling s
hen measu
the level o
ogenized.
compositio
analysis of
r use.
omogen
Materials he products
cts used for h
ion
ethanol
ylmethylsu
anol
ITION AND
s Vesalius
and metls from na
uary 2014 b
nd cool co
y washed w
equipment
d for each
ands dissec
tissue of t
erent pools
site there a
uring bioch
of metallot
There is
on and MT
f biochemi
ization
s needed fo
homogenizat
ulfonyl fluo
METALLOT
hods atural pop
by SCUBA
ontainers t
with seawa
t to extrac
h sampling
cted from 9
the digestiv
s; A, B, C
are 36 ind
hemical com
thionein (M
a differen
T measurem
ical compo
or homoge
tion.
B
S
S
oride) S
S
S
K
THIONEIN C
pulations w
A diving d
o the labo
ater and di
ct the dige
g site and
9 individua
ve gland o
and D. On
dividuals. P
mpositions
MT). As a f
nce in the
ments. Afte
osition are
enization o
Brand/firm
Sigma
Sigma
Sigma
Sigma
Sigma
Kemika
CONTENTS
Education
were sam
one by Dr
oratory wit
issected by
estive glan
time. Each
als.
of Arca noae
ne pool con
Preparatio
s such as li
first step, a
e homoge
er the hom
stored in
of the samp
IN ARCA N
BLT - specia
mpled mon
r.Sc. Ivan Ž
thin 6 hou
y Dr.Sc. Jel
nd. Four
h composi
e. For each
ntains 9 in
on of the s
ipids, carbo
all the sam
enization b
mogenizatio
several al
ples are list
NOAE 27
alization FBT
nthly from
Župan and
urs, where
lena Čulin
composite
ite sample
h sampling
ndividuals,
samples is
ohydrates,
mples need
buffer for
on process
iquots at -
ted.
T
m
d
e
n
e
e
g
,
s
,
d
r
s
-
-
28 BIOCH
PreparatBiochemic
100
MT homo
20
+ P
hom
+ l
3µl
+ β
add
2.1.2 ‐ Tra
‐ Sw
‐ Tur
‐ Lab
ice
‐ We
‐ Tak
‐ We
‐ We
sto
Procedure
We use t
Dondero,
Add 3 m
homogeni
HEMICAL C
tion of bucal compos
0 mM Tris-
ogenization
mM Tris-H
PMSF diss
mogenizat
leupeptin
l/ml homo
β- mercap
dition 1 µl/
Protocoansfer the t
witch on the
rn on the c
bel the sam
.
eigh the tis
ke 1 g of th
eigh about
eigh the re
ore them at
e
the metho
& Fabbri,
ml homog
ization tub
COMPOSITIO
uffers sition hom
-HCl pH =
n buffer (ad
HCl pH = 8
solved in
tion buffer
dissolved
ogenization
ptoethanol
/ml homo
ol tissues of t
e ice maker
centrifuge
mple tubes
ssue and cu
he tissue fo
0.4 g for b
emaining t
t -20 °C. Th
for
od accord
1997)
enization
be.
ON AND ME
mogenizatio
8.6 (at 5 °C
dd fresh ad
8.6 (at 5 °C
ethanol (s
.
in 20 mM
n buffer.
dissolved
genization
the digestiv
r.
to cool the
and put th
ut with scis
or MT mea
biochemica
issue and
hese will be
samples
ing to Vi
buffer pe
ETALLOTH
on buffer:
C)
dditives ev
C)
stock solut
M Tris-HCl
d in MQ-
n buffer.
ve gland fr
e rotor (4-9
he tubes an
ssors until
asurement.
al composit
put in a cr
e used for
s
iarengo et
er 1 g of
IONEIN CO
very day):
tion 58 mg
(stock sol
H2O(stock
rom -80 °C
°C).
nd the hom
you get a
tion measu
ryogenic v
lyophiliza
to
t al (1997)
tissue an
ONTENTS IN
g/ml) - ad
lution 1 m
k solution
C to -20 °C.
mogenizati
pulp.
urement.
vial of 1.5 o
tion .
measure
). (Viareng
nd transfe
N ARCA NOA
dd 1.5 µl/
mg/ml) – a
n 100 µl/m
ion buffers
or 1.8 ml a
M
go, Ponza
er to a gl
AE
/ml
add
ml)-
s on
and
MT:
ano,
lass
-
BIO
Ho
Pro
OCHEMICAL
geschool Gen
‐ Homo
‐ Rinse
sampl
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centrif
‐ Before
balanc
sampl
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‐ Separa
pipetti
fat, cen
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Make
defros
(SN) f
3 subs
furthe
‐ Store t
ocedure fo
‐ Add 1
homog
ml buf
‐ Homo
PTFE
‐ Rinse
sampl
L COMPOSI
nt – Campus
ogenize on
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ifuge for 12
ate the su
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sted and f
or determi
samples of
er be referr
the aliquot
or samples
10 volume
genization
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ogenize the
pestle.
the pestl
les.
ITION AND
s Vesalius
ice using a
le and ot
mogenates
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20 min at 5
upernatant
n top) and
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ination of c
30 µl for c
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e samples
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METALLOT
a motor-dr
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tor. A diffe
50000 ×g (t
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pellet (at t
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nd transfer
lyses neede
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cytosolic p
cytosolic pr
50.
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re biochem
ogenization
e the ratio
s on ice us
ther equip
THIONEIN C
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pment use
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Pasteur-p
the bottom
min.
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proteins. W
roteins. Th
C.
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for digest
sing a mo
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CONTENTS
Education
ogenizer w
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heck their
s than 0.5
ivalent to 1
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lled Eppen
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We collected
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position:
er volume
tive gland
otor-driven
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IN ARCA N
BLT - specia
with PTFE p
De-H2O in
and keep o
weight in
g between
1 h at 10000
king care
is a large
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never be r
parts of su
d 2x250 µl f
rnatant sam
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is 0.4 g tis
n homogen
De-H2O in
NOAE 29
alization FBT
pestle.
n between
on ice until
n order to
n opposite
00 ×g).
to avoid
fraction of
es (on ice);
repeatedly
upernatant
for MT and
mples will
to a glass
ssue and 4
nizer with
n between
T
n
l
o
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f
;
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t
d
l
s
4
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-
30 BIOCH
‐ Tra
cov
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tub
for
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2.2
2.2.1 The matecytosolic pTable III. P
Product/so
Chloroph
Ethanol, 9
NaCl
EDTA
HCl
DTNB acid)
GSH (glut
KH2PO4
NaOH
BSA (bovi
H3PO4
G- Cooma
HEMICAL C
ansfer hom
ver with p
mples.
rtex befor
bes, use the
r determina
termination
ansfer the r
ore the aliq
Quantif
Materiaerials that proteins arroducts used
olution
horm
95%
(5,5-dithi
tathion)
ine serum
assie brillia
COMPOSITIO
mogenates
arafilm an
re you ma
e blue pipet
ation of lip