and

similarity

landscape

analysis

identifies

PARP

as

a

novel

off-target

for

the

drug

Vatalanib

Background:

Searching

for

two-dimensional

structural

similarities

is

a

useful

tool

to

identify

new

active

compounds

in

drug-discovery

programs.

However,

as

similarity

measures

neglect

important

structural

and

functional

features,

similarity

by

might

be

underestimated.

In

the

present

study,

we

used

combined

and

three-dimensional

similarity

comparisons

to

reveal

possible

new

functions

and/or

side-effects

of

known

bioactive

compounds.

Results:

We

utilised

more

than

compounds

from

the

SuperTarget

database

with

known

inhibition

values

for

twelve

different

anti-cancer

targets.

We

performed

all-against-all

comparisons

resulting

in

similarity

landscapes.

Among

the

regions

with

low

similarity

scores

are

inhibitors

of

vascular

endothelial

growth

factor

receptor

(VEGFR)

and

inhibitors

of

poly

ADP-ribose

polymerase

(PARP).

To

demonstrate

that

landscape

comparison

can

identify

similarities,

which

are

untraceable

in

similarity

comparisons,

we

analysed

this

region

in

more

detail.

This

analysis

showed

the

unexpected

structural

similarity

between

inhibitors

of

VEGFR

and

inhibitors

of

PARP.

Among

the

VEGFR

inhibitors

that

show

similarities

to

PARP

inhibitors

was

Vatalanib,

an

oral

"multi-targeted"

small

molecule

protein

kinase

inhibitor

being

studied

in

phase-III

clinical

trials

in

cancer

therapy.

An

in

silico

docking

simulation

and

an

in

vitro

HT

universal

colorimetric

PARP

assay

confirmed

that

the

VEGFR

inhibitor

Vatalanib

exhibits

off-target

activity

as

a

PARP

inhibitor,

broadening

its

mode

of

action.

Conclusion:

In

contrast

to

the

search,

the

landscape

comparison

identifies

new

functions

and

side

effects

of

the

known

VEGFR

inhibitor

Vatalanib.

similarity

landscape

comparison,

as

shown

in

this

study,

has

the

potential

to

identify

new

targets

of

known

drugs.

As

a

proof

of

principle,

we

identified

Vatalanib's

additional

ability

to

target

PARP,

which

was

demonstrated

in

vitro

and

in

vivo.

Thus,

combined

and

similarity

landscape

comparison

analysis

can

identify

new

functions

and/or

side

effects

of

known

bioactive

compounds

that

are

untraceable

with

similarity

searching

alone.

fingerprints

similarity

search

methods

are

widely

used

approaches

in

the

discovery

of

novel

molecules

with

high

affinity

to

specific

targets

and,

despite

the

fact

that

molecules

are

active

in

three

dimensions,

surprisingly

powerful

In

this

study

we

used

the

freely

available

software

packages

OpenBabel

and

ChemAxon

to

analyse

the

of

about

inhibitors

against

twelve

promising

anti-cancer

targets.

Among

these

VEGFR

and

PARP

inhibitors

showed

only

little

structural

similarity,

however,

similarity

by

might

be

underestimated

Accurate

target

prediction

can

be

achieved

by

combining

different

measures

of

chemical

similarity

based

on

both

chemical

structure

and

molecular

shape

Furthermore

it

has

been

shown

recently

that

the

combination

of

a

similarity

search

and

a

shape/flexibility-based

similarity

search

led

to

an

increased

hit

rate

Therefore

of

VEGFR

and

PARP

inhibitors

was

then

analysed

by

a

proprietary

algorithm,

which

produces

reproducible

results

because

of

pre-calculated

conformers

for

every

compound.

The

method

in

combination

with

comparison

performs

quite

well

by

applying

a

quantile

threshold

(corresponding

to

an

rmsd-value

of

for

early

discovery

detection.

This

is

possible

as

our

data

follows

a

normal

distribution.

Nevertheless,

using

a

threshold

means

missing

out

on

compounds

with

larger

rmsd-values

which

could

have

the

same

inhibitory

function.

By

taking

conformers

into

account

to

simulate

the

flexibility

and

to

cover

the

conformational

space,

superpositions

of

the

two

compounds

were

calculated

and

the

best

superposition

with

a

minimal

rmsd-value

was

taken

for

the

similarity

measurement.

This

makes

the

method

robust

with

respect

to

conformation

changes.

Although

the

landscape

of

the

shows

an

overall

reduced

similarity

of

inhibitors

compared

to

the

selected

PARP

inhibitors

display

high

similarity

when

compared

to

the

VEGFR

inhibitors,

confirming

that

it

is

important

to

take

in

addition

to

similarity

into

account

to

increase

the

hit

rate.

Among

the

inhibitors

with

similarity

we

found

the

anticancer

agent

Vatalanib,

and

Compound

a

similarity

not

identified

by

different

fingerprint

algorithms.

It

might

be

that

the

algorithms

failed

to

identify

this

similarity,

because

these

fingerprints

are

based

on

atom

labels

whereas

rmsd

does

not

consider

these

labels.

Structural

comparisons

as

performed

by

the

algorithm

used

here

might

perform

better

generally,

as

they

compensate

for

fragments

of

the

molecule

and

their

connections

of

different

atom

types.

Still,

there

might

be

clusters

where

similarity

algorithms

might

be

faster

and

better..

Compound

was

identified

as

a

direct

inhibitor

in

a

yeast

screen

assay

with

an

EC

of

approximately

μM

By

using

the

HT

universal

colorimetric

PARP

assay

kit

we

calculated

an

IC

value

of

approximately

μM.

The

differences

in

the

IC

values

of

Compound

can

be

explained

by

differences

in

the

assays

used

as

well

as

by

differences

of

PARP

expressed

in

yeast

compared

to

an

isolated

PARP

enzyme

used

in

our

assay.

According

to

the

colorimetric

PARP

assay,

Vatalanib,

with

an

IC

value

of

μM

is

fifteen-fold

more

effective

than

Compound

in

inhibiting

PARP.

The

in

silico

docking

simulation

indicates

that

Vatalanib's

additional

chloride

atom,

which

is

missing

in

Compound

is

in

close

proximity

to

the

arginine

at

the

bottom

of

the

binding

site

of

PARP.

A

halogen

bond

between

the

nitrogen

of

arginine

might

be

formed,

stabilising

the

binding

position,

which

results

in

a

more

effective

drug

target

inhibition..

Both

Vatalanib

and

Compound

have

higher

LibDock

docking

scores

than

which

might

be

attributed

to

the

relative

small

molecule

size

of

Despite

better

LibDock

docking

scores,

both

Vatalanib

and

Compound

are

less

potent

than

the

prototype

PARP

inhibitor

which

has

an

IC

of

about

μM

Despite

these

comparatively

high

IC

values

of

Compound

and

Vatalanib,

μM

of

either

of

the

drugs

was

able

to

induce

foci

formation

in

human

breast

cancer

cells,

demonstrating

DNA

damage

and

PARP

inhibition.

In

regard

to

this

activity,

these

results

again

point

to

an

in

vivo

vs.

in

vitro

discrepancy

with

a

higher

bioactivity

in

cells

compared

to

the

enzyme

assay.

Due

to

the

relative

high

IC

of

Vatalanib,

Vatalanib

might

be

of

interest

as

a

new

PARP

inhibitor

or

for

drug

design.

In

addition

a

putative

positive

side

effect

of

Vatalanib

against

PARP

might

be

important

for

the

use

of

Vatalanib

as

a

chemotherapeutical.

Vatalanib

doses

up

to

mg

twice-a-day

are

well

tolerated

reaching

plasma

concentration

in

patients

in

the

μM

range.

Accordingly,

the

μM

of

Vatalanib,

able

to

induce

foci,

is

in

the

rage

of

c

max

plasma

concentrations

achieved

in

patients

Vatalanib

was

initially

described

as

a

selective

tyrosine

kinase

inhibitor

(TKI)

of

TKIs

commonly

have

additional

activity

against

other

tyrosine

kinases.

Likewise

Vatalanib,

which

at

higher

concentrations

also

inhibits

other

protein

tyrosine

kinases

of

the

same

family,

such

as

the

platelet-derived

growth

factor

receptor

beta

tyrosine

kinase

and

the

c-Kit

protein

tyrosine

kinase

Interestingly,

activity

across

other

classes

of

drug

targets

have

also

been

documented

for

Vatalanib.

It

has

been

shown

that

Vatalanib

significantly

inhibits

aromatase

and

thus

might

cross-inhibit

two

important

classes

of

targets

in

breast

cancer

This

"multi-targeting"

activity,

which

might

also

include

PARP

as

a

target,

could

potentially

contribute

to

the

antitumor

effect

of

Vatalanib

and

indicates

that

a

drug's

efficacy

often

might

not

only

be

based

on

the

inhibition

of

one

target

but

of

multiple

targets

First,

we

performed

an

in

silico

screening

for

a

variety

of

known

inhibitors

against

twelve

promising

anti-cancer

targets

(listed

in

Fig.

using

our

SuperTarget

database

The

overall

of

about

inhibitors

was

then

displayed

in

heat-maps

(Additional

file

Figure

where

the

values

are

coloured

according

to

the

similarity

of

the

analysed

inhibitors

as

calculated

by

the

Tanimoto

score:

high

similarity

is

displayed

in

red

and

low

similarity

in

yellow

data

points.

To

allow

for

a

better

access

to

these

values,

we

represent

these

heat-maps

as

landscapes

of

similarity.

Here,

high

similarity

is

represented

by

Fig.

similarity

landscape.

similarity

landscape

of

about

inhibitors

for

the

twelve

anti-cancer

targets.

High

similarity,

calculated

by

the

Tanimoto

score,

is

visualised

by

mountains

(white-yellow)

and

low

similarity

by

valleys

(dark

red).

We

focused

on

the

similarity

between

PARP

and

VEGFR

inhibitors

(blue

square,

upper

left

corner

mountains

and

low

similarity

by

valleys

(see

Fig.

Within

each

group

and

in

between

groups,

inhibitors

display

similarity

as

indicated

by

high

Tanimoto

scores

depicted

by

ridges

and

mountains.

In

contrast,

the

VEGFR

inhibitors

showed

only

little

structural

similarity

to

the

other

classes

of

inhibitors,

but

especially

to

the

PARP

inhibitors

(Fig.

upper

left

corner)

Because

analyses

often

neglect

important

structural

and

functional

features,

we

expanded

our

comparison

to

measured

by

the

root-meansquare

deviation

(rmsd)

based

on

the

Kabsch

algorithm.

Although

current

computers

calculate

of

compounds

relatively

fast,

it

would

still

take

months

to

compare

all

inhibitors

with

each

other.

We

therefore

focused

on

the

comparison

of

VEGFR

and

PARP

inhibitors,

which

showed

only

little

structural

similarity

in

the

analyses.

For

this

comparison

up

to

conformers

were

calculated

by

using

Accelrys

Discovery

studio

(Accelrys

Software

Inc.,

Discovery

Studio

Modeling

Environment,

Release

San

Diego:

Accelrys

Software

Inc.,

To

create

diverse

ligand

conformations,

the

'fast'

search

method

was

used

to

generate

multiple

low-energy

conformations.

The

rmsd

calculations

are

based

on

overlaying

the

anchor

points

of

both

conformers

and

to

rotate

at

single

bonds

(degrees

of

freedom)

to

minimise

the

rmsd.

We

arbitrarily

chose

a

quantile

(rmsd

of

to

evaluate

the

similarity

of

related

structures

and

analysed

only

compounds

with

both

low

rmsd

(high

similarity)

and

low

Tanimoto

scores

(low

The

resulting

overall

similarities

of

VEGFR

and

PARP

inhibitors

were

again

displayed

in

heat-maps

and

as

landscapes

of

similarity

(Fig.

The

clearly

depicts

that

the

inhibitory

compounds

of

VEGFR

and

PARP

are

similar

within

each

group,

but

that

there

is

only

little

similarity

detectable

between

both

classes

of

inhibitors.

The

results

are

visualised

in

a

similar

heat-map

and

corresponding

landscape

that

shows

overall

reduced

similarity

of

inhibitors

within

each

group.

Nevertheless,

selected

PARP

inhibitors

display

high

similarity

when

compared

to

the

VEGFR

inhibitors.

Interestingly,

there

is

a

remarkable

similarity

between

some

inhibitors

of

both

classes

(rmsd

values

between

and

depicted

by

the

ridges,

which

cannot

be

seen

in

the

landscape,

thereby

proving

the

importance

of

additional

superposition

analysis..

Among

these

similar

inhibitors

(for

a

more

detailed

list

see

Additional

file

Table

we

found

Vatalanib

(N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine;

CID

an

approved

inhibitor

for

VEGFR,

which

showed

similarity

to

a

well-known

PARP

inhibitor

sulfanylphthalazine;

CID

hereinafter

referred

to

as

Compound

(Fig.

Based

on

the

fact

that

Vatalanib

is

a

well-known

drug,

we

decided

to

further

investigate

pair

of

compounds..

The

screening

identifies

Vatalanib

as

a

potential

inhibitor

of

PARP

(rmsd:

whereas

OpenBabel

fingerprints

calculated

a

low

Tanimoto

score

of

and

therefore

failed

to

recognise

the

similarity

of

these

two

compounds.

To

analyse

in

more

detail

if

other

To

verify

Vatalanib's

function

as

an

inhibitor

of

PARP,

we

first

performed

an

in

silico

docking

simulation

to

analyse

the

binding

of

both

compounds

into

the

active

site

of

PARP

(Fig.

The

docking

was

performed

by

using

the

PDB

structure.

PARP

catalyses

the

NAD-dependent

addition

of

poly

(ADP-ribose)

(PAR)

onto

various

cytoplasmic

and

nuclear

proteins,

and

PARP

inhibitors

are

thought

to

compete

with

the

enzyme

substrate

NAD+

at

the

active

site.

The

high

spatial

similarity

is

displayed

by

overlapping

both

compounds

as

well

as

docking

them

to

PARP.

By

using

the

high-throughput

docking

algorithm

LibDock

the

best

docking

positions

of

both

compounds

were

calculated.

In

addition

the

docking

score

of

was

calculated

as

a

reference.

According

to

the

integrated

scoring

function,

the

best-ranked

poses

for

Vatalanib

and

Compound

have

comparable

LibDock

scores

of

and

respectively,

showing

that

Vatalanib,

like

Compound

fits

neatly

into

the

active

site

of

PARP.

Both

structures

have

a

higher

docking

score

than

the

reference

structure

with

a

docking

score

of

for

the

best-suited

pose..

To

confirm

our

computational

hypothesis

that

Vatalanib

also

targets

PARP,

we

next

compared

the

IC

values

of

both

compounds.

IC

values

were

determined

by

the

use

of

the

HT

universal

colorimetric

PARP

assay

kit,

which

measures

the

incorporation

of

biotinylated

poly(ADPribose)

onto

histone

proteins.

Both

compounds

inhibited

PARP

activity

in

a

concentration-dependent

manner

with

IC

values

of

about

μM

and

about

μM

for

Compound

and

Vatalanib,

respectively

(Fig.

To

confirm

inhibition

of

PARP

and

to

rule

out

in

vivo

vs.

in

vitro

activity

discrepancies,

i.e.

differences

of

PARP

expression

in

cells

vs.

an

isolated

PARP

enzyme,

we

analysed

accumulation

of

DNA

damage

in

a

human

breast

Because

of

PARP's

involvement

in

DNA

strand

break

repair,

its

inhibition

has

been

proposed

to

lead

to

double-strand

break

(DSB)

formation

These

DSBs

induce

phosphorylation

of

histone

on

at

sites

flanking

the

breakage

Therefore,

we

analysed

whether

treated

cells

accumulate

phosphorylated

denoted

as

which

provides

a

common

marker

for

DNA

damage

in

vitro

cells

were

treated

with

increasing

concentrations

of

Compound

and

Vatalanib,

incubated

for

h

and

stained

for

Green

immunofluorescence

(Fig.

indicates

accumulation

of

foci

after

treatment

with

concentrations

of

and

μM

of

Compound

or

Vatalanib.

Treatment

with

either

μM

of

Compound

or

μM

of

Vatalanib

is

sufficient

to

cause

accumulation

of

foci,

indicating

accumulation

of

DNA

damage

resulting

from

PARP

inhibition.

Drugs

often

not

only

interact

with

their

intended

target

but

also

with

so-called

off-targets,

thereby

causing

sideeffects

Prediction

of

side-effects

is

still

a

big

challenge

during

drug

design

and

studies

have

shown

the

potential

of

computational

methods

for

target

and

off-target

analysis

These

studies

deal

with

pathway-and

networkbased

approaches

combined

with

the

chemical

structure

of

small

molecule

compounds

regarding

their

binding

site

at

the

target

protein

To

identify

compound

similarities

it

is

important

to

take

a

detailed

look

at

structural

similarity

algorithms

were

generated

to

predict

and

create

a

drug-target

adverse

drug

reactions

(ADR)

network

These

represent

the

structure

and

properties

of

small

molecules

by

a

bit

or

integer

string.

Although

several

methods

exist

to

measure

the

similarity

between

fingerprints,

the

Tanimoto

coefficient

has

been

proven

to

be

reliable

However,

several

problems

can

occur

while

working

with

fingerprints:

size

of

compounds

as

well

as

functional

groups

or

side

chains

have

an

impact

on

the

similarity

calculations.

Hence,

functional

and

structural

features

of

compounds

can

be

neglected.

These

problems

can

be

overcome

by

using

similarity

search

methods..

Non-commercial

drug-or

target-related

databases,

which

have

been

established

in

the

last

decade,

can

be

used

for

and

comparisons.

Millions

of

compounds

can

be

found

in

databases

like

ChEMBL

or

PubChem

and

their

availability

can

be

verified

via

the

ZINC

database

We

recently

established

our

SuperTarget

database,

which

was

developed

with

the

intention

to

accentuate

drug-target

interactions

and

to

provide

references

to

other

resources

for

more

elaborate

analysis

The

SuperTarget

database

contains

a

core

dataset

of

about

drug-target

interactions,

of

which

about

interactions

have

calculated

binding

affinity

data

and

were

used

to

compare

and

structures

of

promising

anticancer

drugs..

Among

these

drugs

are

inhibitors

of

the

poly

ADP-ribose

polymerase

(PARP).

PARP

binds

to

single-strand

DNA

breaks

and

plays

a

critical

role

in

cell

recovery

from

DNA

damage.

PARP

inhibitors

show

activities

not

only

in

cancer

therapy

but

are

also

being

evaluated

for

the

treatment

of

stroke,

myocardial

infarction

and

other

diseases.

Additional

promising

anticancer

drugs,

which

can

be

found

in

the

SuperTarget

database,

are

inhibitors

of

the

vascular

endothelial

growth

factor

receptor

(VEGFR).

The

approved

VEGFR

inhibitor

Vatalanib

or

PTK/ZK)

is

currently

studied

in

several

phases

of

clinical

trials

for

different

cancer

therapies

Vatalanib

is

an

oral

"multi-targeted"

small

molecule

protein

kinase

inhibitor

that

binds

to

the

intracellular

kinase

domain

of

all

VEGF

receptor

subtypes,

thereby

inhibiting

angiogenesis

In

addition,

it

binds

to

c-KIT

and

platelet-derived

growth

factor

receptor

(PDGFR)

but

with

lower

affinity..

While

applying

a

similarity

landscape

analysis

on

inhibitors

for

different

cancer

targets

by

using

the

SuperTarget

database,

we

found

unexpected

similarities

between

PARP

and

VEGFR

inhibitors,

which

could

not

be

detected

by

similarity

searches.

As

a

proof

of

concept

of

our

similarity

landscape

analysis,

both

in

silico

and

in

vitro

assays

confirmed

Vatalanib's

off-target

activity

as

a

PARP

inhibitor.

In

this

paper

we

provide

a

combined

approach

of

and

similarity

landscapes

for

target

and

off-target

analysis,

which

can

be

applied

to

a

larger

number

of

targeted

anti-cancer

therapeutics.

was

calculated

with

the

Tanimoto

coefficient

for

pairs

of

compounds,

i.e.

inhibitors

For

many

of

these

inhibitors

the

half-maximal

inhibitory

concentration

(IC

effective

concentration

(EC

or

dissociation

constant

(Kd)

values

are

listed

in

the

SuperTarget

database.

These

known

affinities

of

the

inhibitors

were

the

basis

of

our

filter

algorithm

and,

to

avoid

unwanted

and

therefore

incomputable

off-target

effects,

only

interactions

described

by

binding

affinities

μM

(IC

EC

or

Kd)

were

considered

for

further

analysis.

Using

this

filter

method,

we

identified

nearly

inhibitors

for

the

twelve

different

anticancer

targets.

To

compare

these

inhibitors,

a

combination

of

fingerprint

and

fingerprint

were

calculated.

is

used

to

compare

small

molecules;

it

links

linear

segments

of

a

fragment

up

to

seven

atoms

to

an

index

and

considers

atoms

and

bonds

of

a

fragment

and

whether

a

complete

ring

exists.

Based

on

these

calculations,

fragments

are

assigned

to

set

bits

in

a

bit

vector.

uses

SMART

patterns

of

functional

groups

of

the

small

molecules

to

set

bits

in

a

bit

vector.

The

calculated

fingerprints

were

subsequently

compared

by

the

Tanimoto

similarity

measure

for

bit

strings

The

Tanimoto

coefficient

is

based

on

a

similarity

ratio

and

can

assume

values

between

zero

and

one,

indicating

no

similarity

or

identical

structures

respectively.

It

is

calculated

using

the

bits

of

the

binary

fingerprint

vectors

set

to

one

in

molecule

A

and

molecule

B:.

where

AB

is

the

number

of

bits

set

to

one

in

both

molecules,

A

is

the

number

of

bits

set

to

one

in

molecule

A

and

B

is

the

number

of

bits

set

to

one

in

molecule

B..

Another

method

to

calculate

the

Tanimoto

coefficient

are

the

extended-connectivity

fingerprints

(ECFP)

These

are

used

to

cover

the

calculated

by

OpenBabel

fingerprints,

which

belong

to

the

class

of

radial

fingerprints

and

are

based

on

the

Morgan

algorithm

To

calculate

the

extended-connectivity

fingerprints

the

cheminformatics

toolkit

of

ChemAxon

was

used

(JChem

compr

ChemAxon

-For

comparisons

pre-calculated

conformers

are

superimposed

using

the

Kabsch

algorithm

Based

on

the

normalised

set

of

atoms

in

a

coordinate

system

the

centres

of

mass

for

both

conformers

were

calculated

and

superimposed.

Then

the

principal

axes

of

inertia

are

estimated

and

aligned.

Thereby

the

possible

rotations

are

strongly

reduced

and

only

four

orientations

have

to

be

considered.

For

every

orientation

a

mapping

of

atom

pairs

was

performed

whereupon

atoms

were

fitted

to

each

other

with

the

smallest

possible

distance.

Because

for

atom

pair

assignment

a

maximal

distance

threshold

is

applied,

not

every

atom

is

assigned.

The

rotation

with

the

highest

amount

of

mapped

pairs

was

used

for

further

calculations.

The

normalised

variant

with

the

most

minimal

distance

is

chosen

if

more

than

one

rotation

with

the

same

amount

of

mapped

atom

pairs

exists.

For

this

mapping

a

root-mean-squaredeviation

(rmsd)

was

calculated

and

further

optimised..

Every

molecule's

conformation

was

compared

with

each

conformation

of

the

second

molecule,

resulting

in

up

to

separately

calculated

rmsd

values.

Here,

only

the

smallest

rmsd

value,

i.e.

the

best

superposition

of

the

compounds,

was

stored..

Ligand

Docking

-The

docking

study

was

performed

by

using

LibDock,

a

high-throughput

docking

algorithm

for

library

design

and

library

prioritisation.

This

docking

program

was

provided

by

Accelrys

Discovery

Studio

The

algorithm

positioned

ligands

in

the

protein's

active

site

based

on

polar

and

non-polar

interaction

sites..

cell

lines

-Breast

cancer

cell

lines

were

cultured

in

medium

supplemented

with

inactivated

FBS,

U/ml

penicillin

and

mg/ml

streptomycin.

Cells

were

cultured

at

with

CO

in

a

fully

humidified

atmosphere..

IC

values

of

PARP

inhibitors

-For

the

determination

of

IC

values

of

Vatalanib

and

Compound

we

used

the

HT

universal

colorimetric

PARP

assay

kit

with

histonecoated

strip

wells

(Trevigen,

USA).

Absorbance

was

measured

in

a

Sunrise

microplate

reader

(Tecan,

Switzerland)

at

nm..

foci

analysis

-For

immunofluorescence

microscopic

analyses,

cells

were

grown

on

coverslips.

h

post

treatment

with

(control),

and

μM

Compound

or

Vatalanib,

cells

were

washed

in

PBS,

fixed

in

paraformaldehyde/PBS

min),

permeabilised

with

Triton-X

min)

and

blocked

in

fetal

bovine

serum

for

min

at

room

temperature.

After

incubation

with

anti-phospho-Histone

clone

(mouse

monoclonal

IgG

from

Millipore,

Billerica,

MA,

USA)

overnight

at

cells

were

incubated

with

Alexa

Fluor

chicken

anti-mouse

IgG

secondary

antibody

(Molecular

Probes,

The

Netherlands)

for

h

at

room

temperature

and

counterstained

with

DAPI.

Images

of

foci

and

DAPI-labelled

nuclei

were

acquired

with

a

fluorescent

microscope

Olympus,

Germany)

equipped

with

a

objective

lens

(UPlanFL;

Olympus,

Germany)

and

a

camera

(micropublisher

RTV;

QImaging,

Canada)

with

Openlab

software

(Perkin

Elmer,

Germany).

O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Mitigation of Control Channel Jamming under Node Capture Attacks Abstract-Availability of service in many wireless networks depends on the ability for network users to establish and maintain communication channels using control messages from base stations and other users. An adversary with knowledge of the underlying communication protocol can mount an efficient denial of service attack by jamming the communication channels used to exchange control messages. The use of spread spectrum techniques can deter an external adversary from such control channel jamming attacks. However, malicious colluding insiders or an adversary who captures or compromises system users is not deterred by spread spectrum, as they know the required spreading sequences. For the case of internal adversaries, we propose a framework for control channel access schemes using the random assignment of cryptographic keys to hide the location of control channels. We propose and evaluate metrics to quantify the probabilistic availability of service under control channel jamming by malicious or compromised users and show that the availability of service degrades gracefully as the number of colluding insiders or compromised users increases. We propose an algorithm called GUIDE for the identification of compromised users in the system based on the set of control channels that are jammed. We evaluate the estimation error using the GUIDE algorithm in terms of the false alarm and miss rates in the identification problem. We discuss various design trade-offs between robustness to control channel jamming and resource expenditure. E FFICIENT communication in mobile networks requires the use of multiple access protocols allowing mobile users to share the wireless medium by separating user data in any combination of time, frequency, signal space, and physical space. The entire class of multiple access can thus be described by the unifying framework of orthogonal frequency division multiple access (OFDMA) [2] . Allocation of access and resources to mobile users must be periodically updated in order to maintain the efficiency of the multiple access protocol when base station group membership, user demands, and wireless channel conditions are dynamic. Hence, there is a necessary overhead involved in the multiple access protocol to handle the resource allocation to users. This overhead often takes the form of control messages exchanged between mobile users and base stations.. In many systems, dedicated channels are established for the exchange of control messages. These control channels can be used for a wide variety of functions, from topological information propagation for network routing to access control in subscription services. In a cellular system such as GSM [3] , [4] , [5] , for example, base stations and mobile users must coordinate over a variety of control channels in order to perform access control, traffic channel allocation, and intercell user handoff. Control channels thus serve as a platform on which higher-level protocol functionality is supported and, hence, as critical points of failure that can be targeted by a malicious adversary in a denial-of-service (DoS) attack [6] .. An adversary with knowledge of the underlying channel access protocol can perform a DoS attack against individual users or local neighborhoods in the mobile network by jamming the communication channels. Moreover, if the access protocol uses a fixed predetermined schedule for data and control messages, allowing the adversary to distinguish between channels for data and control messages, a control channel jamming attack focusing only on the control channels can be mounted with energy savings of several orders of magnitude less than that required to jam all communication channels [7] . The use of jamming-resistant communication protocols such as Direct Sequence Spread Spectrum (DSSS) or Frequency Hopping Spread Spectrum (FHSS) [2] , [4] introduce pseudorandomness into the access schedule by keeping the spreading or hopping sequences, respectively, unknown to the adversary. It was noted in [8] that the effect of DSSS and FHSS may be further improved by using cryptographic primitives. Alternative antijamming techniques include the use of random channel surfing [9] to randomly hop away from jammed channels and resynchronize on available channels and the use of wormholes [10] to create a channel for reports or alarms from a jammed region.. The above-mentioned antijamming techniques consider jamming attacks by an external adversary and are not intended to mitigate jamming by valid network insiders. A set of malicious colluding users or an adversary who captures or subverts network users in a node capture attack [11] , potentially inserting replicated or fabricated devices into the system [12] , is able to bypass the antijamming techniques above by assuming the collective roles of the compromised users in the network. For example, a set of malicious colluding users can use the available DSSS or FHSS sequences to perform an efficient jamming attack that follows the corresponding pseudorandom sequence as though it is a fixed schedule. An access protocol, which gives the same information to all network users, is thus ineffective against DoS attacks by internal adversaries, as a malicious insider has the ability to perform any task of a valid user. Hence, solutions to prevent or mitigate control channel jamming attacks by malicious insiders must make use of the following properties. First, multiple distinct pseudorandom sequences must exist and be held by different users. Second, the set of distinct sequences should exhibit a degree of cover-freeness [13] in that at least one of the sequences of each user should be different from the union of the set of sequences held by malicious colluding users with a non-negligible probability to ensure collusion resistance. Finally, the total number of pseudorandom sequences should scale favorably as the number of users increases, suggesting that there exist tradeoffs between the cover-free property and the resource efficiency of the protocol.. We thus approach the problem of designing control channel access schemes which allow for efficient reception of control messages while maintaining a degree of independence between the hopping sequences held by different users. In this work, we focus our attention on designing schemes which are robust to control channel jamming attacks by malicious colluding insiders or compromised users. In this paper, we addressed the mitigation of control channel jamming by malicious colluding insiders and compromised system users as well as the identification of compromised users without prior knowledge of the number of compromised users in the system. We mapped the problem of control channel access that is robust to jamming by compromised users to the problem of secure key establishment under node capture attacks. Based on the mapping, we proposed a framework for control channel access schemes using random key assignment. We proposed and evaluated metrics for resilience and delay which quantify the availability of control messages under control channel jamming attacks, and demonstrated that the use of random key assignment provides graceful degradation in availability as the number of compromised users increases. We formulated the identification of compromised users in the system as a maximum likelihood estimation problem and proposed the GUIDE algorithms using greedy heuristics for jammer identification. We provided an analytical approximation to evaluate the false alarm and miss rates in the identification of compromised users resulting from the GUIDE algorithms. We discussed design trade-offs in the key assignment parameters and the identification interval used by the TA. In future work, we will investigate modifications to the adversary's jamming strategy and the effect on the availability of control messages and the ability to identify compromised users.

O O B I I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Unequal error protection for data partitioned H.264/AVC video broadcasting Abstract Application Layer Forward Error Correction (AL-FEC) is becoming a popular addition to protocols for real-time video delivery over IP-based wireless networks. In particular, rateless codes are identified as suitable solution for AL-FEC due to their flexibility and capacity-approaching performance. Since each part of video data is not equally important for video reconstruction, it is beneficial to group it based on its importance, and then provide different degree of protection using Unequal Error Protection (UEP). Data partitioning (DP) is one such low-cost feature in H.264/AVC enabling partitioning of video data based on its importance. We propose schemes for the DP H.264/AVC video transmission using Raptor and Random Linear Codes (RLC) and investigate their performance as AL-FEC solutions in Digital Video Broadcasting. We provide comparisons between optimized Non-Overlapping Window RLC and Expanding Window (EW) RLC, which are two effective UEP RLC strategies. The results using realistic channel traces show viability of the EW RLC as a promising solution for multimedia broadcast applications. Multimedia broadcasting over wireless networks is rapidly increasing. Since wireless channels are prone to errors and packet losses, error mitigation measures must be incorporated. Forward Error Correction (FEC) is the favored approach as retransmissions in broadcasting applications are usually counter-productive [16, 27] . Indeed, FEC codes are successfully applied in Digital Video Broadcast (DVB) networks. For example, DVB-Handheld (DVB-H) specifies a Multi-Protocol Encapsulation -FEC (MPE-FEC) solution at the link layer designed for real-time services [10] , which applies adaptive punctured Reed-Solomon (RS) codes against packet losses. The DVB-H standard also provides a possibility of using Application Layer (AL) FEC solution for Internet Protocol (IP) datacasting services using a well-known class of rateless codes called Digital Fountain (DF) Raptor codes [16, 25] . Though DF Raptor codes are currently used only for non real-time services, they have been investigated for multi-burst protection and compared to MPE-FEC in terms of performance and delay for real-time services over DVB-H [11] and DVB-NGH (Next Generation Handheld).. Apart from DF Raptor codes, a class of rateless codes that have been gaining increased popularity recently for applications in wireless networks are Random Linear Codes (RLC) [14, 17] . RLC show near-optimal performance even for very low codeword lengths, but suffer from high decoding complexity of the Gaussian Elimination (GE) decoder as the codeword length increases. However, for real-time video transmission, RLC is usually applied over short chunks of video data (called generations) thus working in the domain of practically implementable codeword lengths [24, 30] . In addition, as multihop and cooperative communications are becoming increasingly popular in emerging wireless network architectures, introduction of RLC may serve as a step forward towards exploring the benefits of network coding [17] . In the context of future DVB networks, this may be important for emerging concepts such as hybrid broadcast/cellular networks (with clients equipped with multiple wireless broadband interfaces) and device-to-device communications [4] .. The focus of this study is to analyse the use of the data partitioning (DP) error resilient feature of H.264/AVC [34] in combination with Raptor codes and RLC as an AL-FEC solution for video broadcasting. Furthermore, the benefits of unequal error protection (UEP) RLC, tailored with the DP feature of H.264/AVC is investigated in detail. The DP feature of H.264/AVC is chosen because it effectively prioritizes video stream by partitioning it into classes of decreasing importance to video reconstruction with a very small decrease in overall performance. This enables simple rate adaptation crucial in wireless broadcasting [10, 21] .. UEP using H.264/AVC with DP is suggested in [33] . DP H.264/AVC and UEP are later used for wireless video delivery in [26] using rate compatible punctured convolutional (RCPC) codes, in [3] with hierarchical quadrature amplitude modulation, in [1] with Raptor codes for IPTV, and in [20] with growth codes. In [5] , design of a Raptor generator matrix based on frame dependencies within a group of picture (GOP) has been proposed. The redundancy allocation process is tied to the knowledge of the channel loss which may not be suitable for real-time applications and video broadcasting. The proposed scheme also needs a mechanism to transport the generator matrix coefficients to decoder. DP H.264/AVC with UEP is used in [6] where only two most important partitions are protected using rateless codes, while the third partition is sent unprotected.. Expanding Window Fountain (EWF) codes [32] are LT-based UEP layered scheme where the protection of lesser important layers also includes the more important layers. In [32] , EWF codes are optimized for scalable video delivery. In [13] , a layer-aware FEC mechanism has been proposed, using UEP low-density parity-check (LDPC) codes and Raptor codes for protection of H.264 Scalable Video Coding (SVC) video. The work also explores the suitability of the proposed codes to physical and application layer FEC protection for the latest video communication standards including DVB-H. Extending the UEP design methods of [32] from DF to RLC, UEP-based RLC strategies for scalable code delivery have been investigated in [31] .. While [31] is more focused on performance analysis of UEP RLCs schemes over random erasure channel, this paper addresses design challenges of UEP RLCs schemes with DP H.264/AVC for real-world implementation for broadcasting and compares their performance to standard DF Raptor codes. While both DF Raptor codes and RLC have been individually well studied in literature, interestingly, no systematic performance/complexity comparison has been conducted yet. The results reported here are based on DVB-H and on DVB-terrestrial 2 (DVB-T2) [29] configured according to the DVB-T2 Lite profile, which is a profile intended to allow simpler receiver implementations for very low capacity applications such as mobile broadcasting, although it may also be received by conventional stationary receivers. Thus, the goal of this paper is to evaluate performance of the proposed schemes in a real-world environment and provide several optimized design recommendations.. The rest of the paper is structured as follows. Section 2 briefly covers the necessary background. The proposed UEP RLC schemes are described in Sections 3 and 4. Simulation results are given in Section 5. Finally, Section 6 concludes the paper. The study has extensively compared the performance of RLC and Raptor codes by assigning different degree of protection to the data partitions of H.264/AVC video under different packet loss scenarios at different data rates. RLC are seen to perform very close to or somewhat better than Raptor codes. Within RLC configurations, it is seen that the scheme with less number of symbols results in better performance. This is promising result for the use of RLC for multimedia broadcast applications, as the decoding complexity for such configurations is manageable.. The average PSNR of UEP schemes with NOW configuration is not as good as that of single-layer AVC at high data rates. However, it is interesting to note that at lower data rates UEP schemes provide good results with smaller number of decoding failures. The performance of RLC codes using EW-RLC has been analyzed and compared to the case of single-layer coding. EW2 and EW3 both perform up to 5dB better compared to the singlelayer case and show robustness to channel fluctuations. The EW3 configuration provides better degree of control to match a particular data rate and erasure channel characteristics, while EW2 overall provides slightly better performance. The performance of EW-RLC schemes is significantly better than corresponding NOW-RLC schemes. The number of symbols per codeword used was 68, which is a very promising result for the use of RLC for multimedia broadcast applications, as the decoding complexity for such configurations is manageable. In this section we briefly review H.264/AVC and its DP feature, Raptor codes and RLC, and parts of DVB-H/DVB-T2 protocol stack relevant to this work.

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


A novel method to assess collagen architecture in skin Background: Texture within biological specimens may reveal critical insights, while being very difficult to quantify. This is a particular problem in histological analysis. For example, cross-polar images of picrosirius stained skin reveal exquisite structure, allowing changes in the basketweave conformation of healthy collagen to be assessed. Existing techniques measure gross pathological changes, such as fibrosis, but are not sufficiently sensitive to detect more subtle and progressive pathological changes in the dermis, such as those seen in ageing. Moreover, screening methods for cutaneous therapeutics require accurate, unsupervised and high-throughput image analysis techniques. Results: By analyzing spectra of images post Gabor filtering and Fast Fourier Transform, we were able to measure subtle changes in collagen fibre orientation intractable to existing techniques. We detected the progressive loss of collagen basketweave structure in a series of chronologically aged skin samples, as well as in skin derived from a model of type 2 diabetes mellitus.We describe a novel bioimaging approach with implications for the evaluation of pathology in a broader range of biological situations. Our improved measurement of texture and divergence from regular structure in multiple planes provides superior measurement of collagen orientation in skin and thus is widely applicable to dermatological research. In addition, the combination of the Gabor filter and FFT is likely to have utility beyond the quantification of texture in skin and biological imaging as the fundamental principle of measuring divergence from a regular shape is of wider utility across scientific and mathematical disciplines. Image analysis techniques that exploit the frequency domain are attractive as they generate spectra informed by texture. We have developed a methodology that can quantify the organised structure or texture within images. Due to the highly-organised basketweave conformation of healthy mammalian dermis being lost or at least compromised in pathological conditions, or with chronological age, the investigation of these pathological states should be tractable to image analysis. Indeed the loss of both fibrillar collagens and the well-organised collagen structure with increasing age has long been known [8, 9, 30] , and loss of collagen organization in gross pathological states such as fibrosis (as assessed by FFT methods) were previously documented [6, 13, 19, 31] . However, these are untested with respect to more subtle changes in structure (which still have many pathological sequelae). Moreover, they are also untested in mouse tissue, in which the finer collagen structure is considerably more difficult to discern than in humans. FFT-based quantification of multi-photon microscopic images of mouse dermis has been reported, but this was not designed to specifically assess basketweave, and this technology is expensive and beyond the reach of many laboratories [9] . A wide range of therapeutic and cosmetic interventions target skin structure, texture and function, and hence tools to determine incremental changes in the integrity of the collagen basketweave are needed. These analytical tools would be particularly attractive if they could extract information from images generated by routine or inexpensive laboratory equipment, rather than electron micrographs or confocal imaging.. Simple H&E stained images are informative (particularly if eosin auto-fluorescence is exploited), but they do not reveal collagen basketweave. Cross-polar images of picrosirius-stained histological skin sections are able to reveal collagen architecture, and relevant optics are inexpensive and retro-fit to many standard microscopes. We found that by applying a FFT to cross-polar photomicrographs, we were able to quantify a shift in collagen organisation in extreme age (i.e. in skin from 20mth old mice). However, incipient collagen changes went undetected in samples from one year old mice. To improve sensitivity, we decided to deploy a Gabor filter to improve edge detection prior to application of a FFT. This yields a more complex spectrum, but by quantifying pixel distributions in four planes we were able to create a sensitive collagen orientation index. In this way, we were able to detect subtle changes in collagen that were not revealed by FFT alone. Further testing revealed that 5°rotations of the images used in this study still facilitated discrimination between biological groups, although larger rotations that take the basketweave out of phase with the Gabor filter (i.e. 20-30°) result in a loss of sensitivity (not shown). The skin is composed of three principle layers; an outer protective epidermal barrier comprised largely of keratinocytes; a deeper layer of connective tissue dermis that confers strength and elasticity; and an underlying energy store, the sub-cuticular fat. The dermis is primarily composed of extracellular matrix (ECM) proteins, assembled into a meshwork of primarily collagen fibres [1] . To date, 28 different types of collagen are known, which are categorized in eight subfamilies based on function, assembly and domain homology [2, 3] . Fibrillar collagens are the major ECM component, with collagen I and IV being the predominant dermal constituents [4] . The dermis may be further subdivided into two discrete reticular and papillary layers. The reticular dermis consists of large, mature, well-organised collagen fibres in the lower layer of the dermis, interfacing with the subcutaneous fat. The papillary dermis is adjacent to the basement membrane, with thinner collagen fibres and a distinct collagen organisation [5] .. In a healthy state, dermal collagen forms a 'basketweave' structure, with perpendicular collagen fibres intersecting at approximately 90°angles [6, 7] . This basketweave structure provides textural information regarding the skin and as a loss of collagen organisation characterises many different physiological situations, including ageing [8, 9] , diabetes [10] , scarring [11] [12] [13] , fibrosis [14] and more specific diseases of connective tissue, such as Ehlers-Danlos syndrome [15] , thus providing methods to assess texture in biological images has direct applications within dermatological research.. The ability to quantify collagen conformation in health and disease has important consequences for basic research, drug development and clinical diagnosis. For example, techniques facilitating accurate measurement of photo-damage, scarring, wound repair or other agerelated damage are of value in determining the effectiveness of collagen repair therapies. Histological stains, such as picrosirius, effectively identify collagen in tissue specimens, and one may make qualitative assessments of ECM integrity from photomicrographs. However, unbiased image analysis methods are preferable. Segmentation-based methods have been described to assess collagen bundle thickness and orientation, although a degree of user intervention is required [16] . More sophisticated unbiased methods exploit frequency domain transformation methods, or power spectral analysis tools.. Power spectral analysis estimates the power/energy variation of an image in different frequency sub-ranges, and is directly related to the autocorrelation of an image in that it describes how closely related two points in an image are as a function of their distance and orientation. The Fourier Transform, in particular the Fast Fourier Transform (FFT), has been used to estimate the power spectrum of images, and this approach was reported by several groups in measurements of bundle thickness and spacing, as well as collagen fibre orientation [9, 11, 12, 14, [17] [18] [19] .. One particular variation of this approach, the Fourier zeroth-order maximum analysis, has been used to measure the orientation of collagen fibres [14] . This method was extensively applied to a variety of different clinical situations, including identifying fibrosis in scleroderma [14] , evaluating new treatments for hypertrophic scars and keloids [11, 12] , and assessing the effectiveness of dermal substitutes in clinical trials [18] . Initial attempts to utilise first-order maximum Fourier analysis required substantial observer input [14] but this approach was refined so that the user simply selected the area of interest for analysis and a measure of collagen orientation was calculated by determining stretch or elongation of the FFT spectrum [19] . In this way, differences in bundle thickness, spacing and orientation in scar tissue compared to normal skin were measured.. Although the methods described above identify gross collagen changes associated with pathological states, they are not sufficiently sensitive to measure incremental changes in architecture seen in, for example, the progressive loss of basketweave with chronological age. A method to facilitate the quantification of textural information is, therefore, required. Gabor filters are known for their similarity to human visual system (HVS) models in interpreting image texture as they provide a multi-channel bank of filters capable of analysing images at different narrow spatial frequencies and orientations. The link between HVS and Gabor filters was established by the pioneering work of Daugman on image analysis/compression and iris recognition [20, 21] . It has since been successfully used for texture representation, segmentation and discrimination. Therefore, we sought to build on these published methods by combining Gabor filter and Fourier transform techniques to measure collagen fibre orientation in a series of images derived from picrosirius-stained mouse skin. Polarised light microscopy clearly reveals the basketweave structure of the dermis [22] and by initially applying a Gabor filter in eight angles to our images before creating a Fourier spectrum, we can generate a metric for collagen structure, indicating the integrity of the collagen basketweave. This provides increased sensitivity and decreased user input which is prone to human error and bias. To test the analysis platform we have measured the effect of chronological ageing in wild-type (WT) mice and assessed dermal integrity in a mouse model of type 2 diabetes. The improved performance of the Gabor analysis in mouse skin, which is notoriously difficult to analyze compared to human skin, confirms the superior nature of this platform for dermal structure analysis.

O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O B O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O B O O O O O O O O B O O O O B O O O O O O O O B O O B O O O B O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O B O O O B O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Social debt in software engineering: insights from industry Social debt is analogous to technical debt in many ways: it represents the state of software development organisations as the result of "accumulated" decisions. In the case of social debt, decisions are about people and their interactions. Our objective was to study the causality around social debt in practice. In so doing, we conducted exploratory qualitative research in a large software company. We found many forces together causing social debt; we represented them in a framework, and captured anti-patterns that led to the debt in the first place. Finally, we elicited best practices that technicians adopted to pay back some of the accumulated debt. We learned that social debt is strongly correlated with technical debt and both forces should be reckoned with together during the software process. Software engineering success is increasingly dependent on the well-being of development communities [1] . In some of our previous work [2] [3] [4] , we found many decisions influencing community well-being. For example, changing the organisational structure [5] of the development community (e.g., through outsourcing), changing the development process (e.g., by adopting agile methods), leveraging on global collaboration (e.g., by striking a balance between formal and informal communication across global sites) are all socio-technical decisions, i.e. social and technical at the same time, that influence the state and welfare of developing communities and their members [5] . The social connotation of these decisions, changes the way people work and interact with others -i.e., their organisational and social structure [4] . The technical connotation of these decisions, changes the way in which development tasks are worked out. In agile methods, for example using Kanban boards, a "pull" task-allocation is often used, as opposed to classic "push".. Some socio-technical decisions eventually cause additional cost (e.g., through delays) on software projects and the development community around them. This cost, is *Correspondence: damianandrew.tamburri@polimi.it 1 VU University Amsterdam, Amsterdam, The Netherlands Full list of author information is available at the end of the article not immediately visible and its resolution is often postponed. Also, if the same decisions remain into place, project costs may increase. For example, organisations often employ strict information-filtering protocols to protect industrial secrets when they embark in open-source [6] . This decision, however, might slow down development interactions, causing delays or even inciting fear. This extra cost is conceptually similar to technical debt [7] , i.e., the additional project cost caused by sub-optimal technical decisions. However this extra cost is not necessarily related to code and it is actually "social" in nature, i.e. connected to people and their development organisation. Paraphrasing Cunningham [8] who first introduced technical debt, social debt can be thought as: "not quite right development community -which we postpone making right". In Layman's terms, by social debt, we indicate the additional cost occurring when strained social and organisational interactions get in the way of smooth software development and operation.. While technical debt has received increased attention over the last 10 years, this other form of debt, namely "social debt", has remained latent and relatively unexplored. For instance, software engineering practitioners still lack a way to formalise socio-technical decisions and measure the connected debt.. This article takes one small step forward towards defining and studying social debt by means of an industrial exploratory case study. The case study was driven by the following research questions: "What are the factors at play around social debt during the software lifecycle? Are there patterns in said factors? Can they be mitigated?". From our study we learned that, indeed, there is a strong correlation between social debt and sub-optimal characteristics in organisational-social structures behind software development communities. Also, social debt is inextricably related to technical debt in many ways, e.g., uninformed socio-technical decisions generate both social and technical debt in a compounding manner that cannot be trivially "payed back". In addition, our findings suggest the presence of community "smells", i.e., precursors to the emergence of social debt, much like code smells may lead to technical debt, e.g. as shown in [9] by Zazworka et al. Finally, our findings uncovered some "mitigation strategies", i.e., ways in which some community "smells" may be averted and avoid the debt, even if partially.. In summary, this article offers four contributions: (1) a framework to define and interpret social debt; (2) a list of community "smells", intended as socio-technical antipatterns that may appear normal but in fact reflect unlikeable community characteristics, such as anti-social organisational behaviour across the community, e.g., developers that refuse or delay information sharing; (3) a list of mitigations to said community "smells", as emerging from our case study; (4) a list of lessons learned as part of our case study that further discuss social debt and its implications.. The rest of the article is structured as follows: Section 2 outlines previous work related to the study of social debt. Section 3 outlines our study design, discussing our research methods and materials. Section 4 presents our contribution while Section 5 discusses their implications, pointing out to relevant threats to validity and ways in which they were tackled. Finally, Section 6 concludes the article. In this paper we discuss and elaborate on the notion of social debt that was originally introduced in [3] . To investigate social debt in action we conducted a case study in industry. To answer our primary research questions, namely, "What are the factors at play around social debt during the software lifecycle? Are there patterns in said factors? Can they be mitigated?", this article offers four key contributions.. First, a framework to define and interpret social debt. This can be used in practice to identify the variables that govern social debt, e.g., for more informed decisionmaking.. Second, a list of community "smells". These are sociotechnical anti-patterns that may apparently look normal but reflect unlikeable community characteristics, such as anti-social organisational behaviour across the community, e.g., developers that refuse or delay information sharing. These patterns can be used in practice to "sense" the precursors of social debt in much the same way code smells fathom technical debt.. Third, a list of mitigation strategies to community "smells", as emerging from our case study. These can be used in practice to reduce the negative effects connected to some "smells".. Fourth, a list of lessons learned as part of our case study that further discuss social debt and its implications. These can be used by researchers interested in pursuing further social debt research.. From our study we learned that social debt is a force to be reckoned with, hand in hand with technical debt. This force is connected to sub-optimal characteristics in software development communities. Further research is needed to generate a more systematic approach. For example, the use of community detection and characterisation mechanisms via social networks analysis might be used to understand if social debt can somehow be measured automatically. Also, investigating further the relation between software architectures and "organisational architectures" might reveal patterns that lead to social and technical debt.. In the future, we plan to devise mechanisms to visualise and study the social community structure of development communities, by putting together their socio-technical properties and observable characteristics. In so doing, we hope to discover ways to mine data from software development communities that can lead to the discovery of their sub-optimal characteristics, and, possibly, any connected "smells". Also, we plan to elaborate further on the notion of community "smells" perhaps starting from analysing literature in software engineering (e.g., studying software failure stories). More in particular, we plan to answer the following research questions:. "What software engineering artefacts are affected by community smells?" Much information and data in our dataset concerning software artefacts (e.g., software architecture, requirements, integration requests, etc.) in our investigation scenario remains to be analysed -perhaps this data can be analysed in continuation with the study in this paper, e.g., to understand if community smells have a direct or indirect impact on software itself.. "How can social debt be measured?" Social debt clearly entangles a number of software and lifecycle artefacts, finding measurements for the negative characteristics connected to social debt in said artefacts is the starting point to measure social debt in the first place. This research venue might benefit from research in Mining Software Repositories (MSR) since it involves mining software products to establish causality. However, an exploratory study is needed to establish what should be measured and how.. "Are there community smells previously reported in literature?" Software engineering literature offers empirical research in software failure that could potentially contain a number of community smells. Mapping this portion of software engineering literature could be a valuable tool for practitioners embarking on complex software engineering endeavours involving more than one team or organisation. This section discusses our findings and their implication. Also, we report a number of lessons learned from the study. These are general insights that might benefit further research into social debt as well as practitioners at hands with it. Finally, we discuss a few threats to validity we identified for this study.

B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O B I O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O


Object Segmentation and Labeling by Learning from Examples Abstract-We propose a system that employs low-level image segmentation followed by color and two-dimensional (2-D) shape matching to automatically group those low-level segments into objects based on their similarity to a set of example object templates presented by the user. A hierarchical content tree data structure is used for each database image to store matching combinations of low-level regions as objects. The system automatically initializes the content tree with only "elementary nodes" representing homogeneous low-level regions. The "learning" phase refers to labeling of combinations of low-level regions that have resulted in successful color and/or 2-D shape matches with the example template(s). These combinations are labeled as "object nodes" in the hierarchical content tree. Once learning is performed, the speed of second-time retrieval of learned objects in the database increases significantly. The learning step can be performed off-line provided that example objects are given in the form of user interest profiles. Experimental results are presented to demonstrate the effectiveness of the proposed system with hierarchical content tree representation and learning by color and 2-D shape matching on collections of car and face images. The primary benefits of the proposed learning by example method are: 1) ability to produce a semantic level of segmentation without user intervention and 2) capability to significantly improve the speed of subsequent searches as a consequence of capturing semantic object information in composite nodes. These are demonstrated in the tables and figures presented in the results section.. Some concluding observations are in order: 1) The quality of resulting semantic object segmentation depends on the granularity of the initial low-level segmentation. An initial segmentation with perfectly uniform low-level regions will yield a more accurate potential semantic object boundary; hence, improved matching performance. However, there is a tradeoff between the number of regions in the initial segmentation and the time required to perform the initial search.. 2) The proposed learning by example concept can employ any object matching method. The 2-D shape matching technique employed in this work has been shown to handle translation, rotations in the image plane, and isometric scaling, but not general affine or perspective transformations. Inaccuracies in low-level matching affect the performance of learning (labeling) as quantified by TP and Precision measures. Also limited by the object matching methods, the current system does not handle partially occluded objects.. 3) The initial search is generally expensive for real life images as can be seen from Tables I and II . However, given a user search profile, e.g., a collection of example objects similar to those in Figs. 3 and 4, this initial search can be done offline. We are working on improving the speed of initial search through hierarchical low-level segmentation and partial match guided search procedures. IMAGE PROCESSING (1994 , and the Journal of Multidimensional Systems and Signal Processing (1994 Processing ( -1999 . He was an Area Editor for Graphical Models and Image Processing (1995) (1996) (1997) (1998)

O O O O O B I I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Preamble-based Channel Estimation in OFDM/OQAM Systems: A Review Filter bank-based multicarrier communications (FBMC) have recently attracted increased interest in both wired (e.g., xDSL, PLC) and wireless (e.g., cognitive radio) applications, due to their enhanced flexibility, higher spectral efficiency, and better spectral containment compared to conventional OFDM. A particular type of FBMC, the so-called FBMC/OQAM or OFDM/OQAM system, consisting of pulse shaped OFDM carrying offset QAM (OQAM) symbols, has received increasing attention due to, among other features, its higher spectral efficiency and implementation simplicity. It suffers, however, from an imaginary inter-carrier/inter-symbol interference that complicates signal processing tasks such as channel estimation. This paper focuses on channel estimation for OFDM/OQAM systems based on a known preamble. A review of the existing preamble structures and associated channel estimation methods is given, for both single-(SISO) and multiple-antenna (MIMO) systems. The various preambles are compared via simulations in both mildly and highly frequency selective channels. Orthogonal frequency division multiplexing (OFDM) has become quite popular in both wired and wireless communications [7, 40, 86] , mainly because of its immunity to the frequency selectivity of the channel, which allows a significant increase in the transmission rate [83, 69] . Using the cyclic prefix (CP) as a guard interval against intersymbol interference (ISI), OFDM manages to turn a frequency selective channel into a set of parallel flat channels with independent noises. This greatly simplifies both the estimation of the channel and the recovery of the transmitted data at the receiver. However, these advantages come at the cost of an increased sensitivity to frequency offset and Doppler spread. This is due to the fact that, although the subcarrier functions are perfectly time limited, they suffer from spectral leakage in the frequency domain and hence inter-carrier interference (ICI) results under nonideal conditions. Moreover, the inclusion of the CP entails a waste in transmitted power as well as in spectral efficiency, which, in practical systems, can go up to 25% [7] .. An alternative to CP-OFDM, that can mitigate these drawbacks, is given by filter bank-based multicarrier (FBMC) systems, which have recently attracted increased interest [76] for both wired (e.g., power line comms (PLC) [10, 15, 1] ) and wireless (e.g., cognitive radio [30, 89, 54] and DVB-T [4] ) applications. For an excellent presentation of FBMC systems, including a review of recent application areas and related standardization activities, the reader could refer to [31] . FBMC enjoys enhanced flexibility, higher spectral efficiency, and better spectral containment compared to CP-OFDM [2, 51] . Special attention has been recently paid to an OFDM filter bank-based variant employing offset quadrature amplitude modulation (OQAM), known as OFDM/OQAM [58, 31, 46, 47] . This was, in its principle, first proposed back in the 60's [20, 78] , but the interest in this scheme was only recently revived due to its desirable features including the potential for maximum spectral efficiency as well as implementation simplicity over alternative FBMC schemes [12, 13, 79] . It consists of pulse shaping, implemented via an IFFT/FFT-based efficient filter bank, and the subcarriers are loaded with staggered OQAM symbols, i.e., real symbols at twice the symbol rate of OFDM/QAM [39, 80] . This allows the pulses to be well localized in both the time and the frequency domains [18] , thus increasing the system's robustness to frequency offsets [9, 77] and Doppler effects [16, 74, 27, 56] and providing better spectral containment in bandwidth sensitive applications [3, 6, 81] . Furthermore, although the OQAM-based OFDM scheme can be seen to exhibit similar peak-to-average power ratio (PAPR) performance with classical OFDM/QAM [55] , the presence of spectral leakage in the latter may ultimately generate higher peak power values [85] . Moreover, because of the good time localization, a CP does not have to be included in the OFDM/OQAM transmission, 1 which leads to still higher transmission rates [80, 5, 74] . However, the subcarrier functions are now only orthogonal in the real field, which means that there is always an intrinsic imaginary interference among (neighboring) subcarriers and symbols [45] . This makes the channel estimation task for OFDM/OQAM systems more challenging. A number of training schemes and associated estimation methods have recently appeared in the literature, including both preamble-based [48, 62, 65, 41, 42, 25, 52, 53] and scat-tered pilots-based [45, 72, 48, 63, 43, 44, 88, 64, 8, 37, 57, 59] ones. These can be roughly characterized as aiming at cancelling/avoiding the undesired interference or constructively exploiting it to improve the estimation performance. The focus of this paper is on OFDM/OQAM channel estimation based on a known preamble. Existing preamble structures and their associated estimation methods are reviewed, for both the single-antenna (SISO) and multiple-antenna (MIMO) scenarios. The problem of preamble optimization to achieve minimum estimation error is also shortly discussed, based on recently reported results. The estimation performances of the principal methods are compared via simulations for both mildly and highly frequency selective channels.. The rest of the paper is organized as follows: In Section 2, the discretetime baseband equivalent model for the OFDM/OQAM system is described. Section 3 reviews the main approaches proposed in the literature for structuring the preamble and exploiting it for SISO channel estimation. The MIMO case is addressed in Section 4. Comparative simulation results are reported in Section 5. Section 6 concludes the paper. Notation. The complex conjugate of a complex number z is denoted by z ⋆ . Also,  = √ −1. ℜ(z) or z R denotes the real part of z, while its imaginary part is written as ℑ(z) or z I . The set of real numbers is denoted by R. Boldface lower and upper case letters are used for vectors and matrices, respectively. The nth-order identity matrix is denoted by I n . · is the Frobenius norm. The expectation is denoted by E(·). ⊗ is the (left) Kronecker product. The problem of preamble-based channel estimation in OFDM/OQAM systems was revisited in this paper, and a review of the preamble structures and associated estimation methods was given, for both SISO and MIMO systems. Results on MSE-optimal design of preambles with zero guard symbols were also reviewed for SISO systems. Analogous results for the MIMO scenario are still to be derived. The estimation performances of the principal methods (including IAM variants and the iterative method of [41] ) were demonstrated via results of simulation in a realistic (including interference from data) and fair scenario and for both mildly and highly frequency-selective channels. A recently developed method of the IAM type, E-IAM-C, was seen to outperform the rest under such conditions and at all considered SNR values. Further investigation is required to assess the loss in performance from tail truncation in the IAM preambles and mitigate the high PAPR problem associated with their periodic structure. Moreover, improved IAM designs as well as optimality results are needed for MIMO-OFDM/OQAM. latter is known to equal zero for all g's that can be used as prototype filters in such filter banks [28] . Hence, g m,n m±2,n = 0 2) q = −1, p ∈ {−2, −1, 0, 1, 2}. For p = ±1:. while for p = ±2:. 3) q = 1, p ∈ {−2, −1, 0, 1, 2}. Substituting q = 1 in (26) yields [42] ; (b) [48] ; (c) [88] , and avoidance: (d) [41, 25] . M = 8. OQPSK modulation is assumed.

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