4.

7 Descrierea proiectului
<Prezintă activitățile și sub-activitățile proiectului (planurile de lucru PL), rezultatele așteptate. De asemenea este
prezentat perioada de implementare, rezultatele așteptate și resursele alocate pentru fiecare activitate și sub-
activitate în parte.>
Schimbul de cunoștințe privind cercetarea/activitățile de transfer: O seriedepachete de lucruși activitățiau fost
createîn vederea completării și asigurării succesuluiproiectuluiștiințificși în același timps-a lucrat intens la
dezvoltarea mediuluide cercetareîn cadrulTMși pregătireaUniversității pentruaconcura în mod eficientpe piața UE.
Punerea în aplicarepe termen scurtmediu și lungapoliticilorprivind elementele depracticăde cercetareși etică, cum
sunt cele folosite de cătreMMUși în conformitatecualte instituții de lideri mondiali din Europa, acestea vor fi
adaptatepentru a se potrivinevoilorinstitutuluigazdă.Politica pe termenscurtva dezvoltaprofileindividuale de
cercetare, team building, planificarea cariereipersonale pe o durată de 1-3-5anietc.Politica pe
termenmediuvainformaUniversitatea privind strategiadedezvoltare a cercetăriiși culturii, inclusiv managementul
schimbuluide cunoștințe, rețeletehnice, economiceșiadaptate nevoilor/interacțiuniinterdisciplinare colaborative.
Planificarea pe termen lungva implicaconstruireadurabilitățiitemelor de cercetareși încheierea de
parteneriateexterneșiinternaționale, inclusiv cuorganismede finanțare a cercetării, cum ar fi NIHR, MarieCurie,
Ministerul Apărării șialte inițiative ale UE. THa demonstratdejaunangajament puternicatât în ceea ce privește resursele cât
și partea financiară, pentru a permite efectuarea acestor îmbunătățiriîn asocierecu noul lorCentrude cercetare finanțatde
UE(£ 7.5.000.000-a fi finalizatîndecembrie 2015 vacoincide cuinițiereaacestui proiect în cazul în care va aveasucces) și un
al doileaCentru

Contextul proiectului științific:PCRmeste un monomer al moleculei native, PCR pentamerică.Proteina C reactivă este
una sintetizată în ficat ca răspuns la unele infecții la om. Rolul săufiziologiceste acela de ase lega lafosfocolinăcare se
găseștepesuprafața celulelormoartesau pe cale de deces, stimulândactivareasistemuluicomplement
princomplexulC1q.În contactcuceluleși țesuturiseconverteștela formamonomerică, biologic activă și, ca atarese
găseșteînregiunilede leziunitisulareunde poaterămânecronicînECMșicelulele. Formaredusă aPCRm(PCRrm) se
leagăputernic de colesterol din plutelipidicemembranare și acesta este locul pe care noidorim să o blocăm, pentru a
inhibaacțiunilePCRm.

It is important to recognise that published work over the last 5 years or so has identified a strong biological and
pathological role for mCRP in tissue-associated disease progression and prognosis that is completely distinct from
the classical and well-documented role defined for nCRP, its parent molecule. For this reason, the work has a high
novelty rating.
Este important să serecunoască faptul că,materialul publicatîn ultimii5 anisau cam așa cevaa
identificatunrolbiologicșipatologicputernic pentruPCRmîn progresia șiprognosticul boliiasociatețesutului care este
totaldiferită derolulclasicșibine documentatdefinitpentruPCRn, molecula sapărinte. Din această cauză, lucrarea are
un gradde noutate ridicat.........
Un anticorp anti PCRmeste oferit ca șiprofilacticdupă oincidențăcerebrovascularăcum ar fiun accident vascular
cerebraldeprevenire aneurodegenerării. În plus, existăîn continuareposibilitatea caun anticorpanti-PCR să
aibăunefectbeneficca tratament pe termenlung, inhibândsemnalizareaneurodegenerativăcare rezultă
dindepunereaPCRm petermenlung.Studiileclinice recenteprivindBAau încercat să eliminesubstraturilepatologicecum
ar fiplăcile de amiloidșiîncurcăturile neurofibrilare(ÎNF). Până în prezent acestea nu au avut succes.
Unmodulatordețintă propus(PCRm) oferă oabordare cu totul nouăprotejânddezvoltareaDVa(și BAasociat)după AVC.
Noi propunemo terapieunică aceea de a bloca atașamentul celulelor-plutelor lipidice de mCRPproduse în urmaECM a
regiunilorafectate dincreier. Mecanismulnostrude blocare pe bază de anticorpiar trebui să
permităclaritateaimunologicăaexcesului de proteinedinparenchimul cerebraleliminândastfel risculdeperpetuarea
bolii neurodegenerativecronicesemnalizândactivareaîn cascadășiformarea aberantă devaseși semne distinctive ale
demenței.Indiceleterapeuticideal arscădearapid de la inițiereainflamației(datorită faptului că PCRmstimuleazăîn mod
activacestproces) (de exemplu, <24 de ore- 72 de oredupă un evenimentischemic), dar înainte
demajoritatearevascularizăriiși a remodelării timpurieîn cazul în care microvascularizația
aregeneratșimaturizat(deoarece PCRmar putea aveaun efect negativ asupraacestuiprocesprin stimularea
uneiangiogenezeaberanteetc.;circa7-21zile). Prin urmare, această terapiear trebui să aibă, de fapt, un efect
pozitivasupra remodelării țesutului. Aceasta ar fiprimaîncercare a uneiterapiicare vizeazăo moleculă cheieasociată
culeziunilevasculare suferitedupăun accident vascular cerebralșistrâns legate depatologianeurodegenerativă.
Aceastaeste în primul rândostrategiede prevenire/protecție mai degrabă decât untratamentpentru cei care
dejasuferă de DVa/BA.Acest lucru esteîn acord cuexperiența anterioară avută în cazul BA, unde cele mai multe
dintreterapiiau fostîncepute mult prea târziuînprocesul degenerativ.
Leziunilevasculare, hemoragiașiinflamarea, accidentul vascular cerebral, suntfactoriicare intensifică
dezvoltareapatologicăa bolii. PCRm/PCRrmapare la câteva oredupă evenimentulvascular, rămânândpentru a
activamesajeleneurodegenerativeulterior. Vezi Figura1 de mai jospentrumecanisme.Efectul therapeutic va fi acela
de a anula aceste semnale pe termen nelimitatîn urma unui tratamentsingular care dureazăcâtevazile
dupăaccidentul vascular cerebral(când inflamațianaturalădispare), prinîndepărtareasau"curățarea"celuleiPCRrm
dincadrulregiuniiischemicecerebrale.
Beneficiul terapeuticarfi: 1) Reducerea directăa semnalizăriineuronaleneurodegenerative. 2)
Prevenireaangiogenezeiaberante, ceea ce duce lahemoragie, ischemie și inflamație, și
protejareamicrovasculaturiiexistente și nouformate. 3) Reducereainflamațieiindusă dePCRrm. 4)
Reglementareaactivării BV/BAlegat desemnalizareainsulina(de exemplu, IR, IRS-1) șifibrină/RAGE.
Indicațiisuplimentare pot includeprevenireaprogresieineurodegenerativedupăleziuni
cerebraletraumaticesauslăbiciunevascularăgenetică, cum ar fi în cazulCADASILși, de asemenea,
înaltedomeniialemedicinei, cum ar fi bolilecardiovascularesi aterosclerozaundemodularealegăturiicelularePCRrmar
putea reduceriscul de formare aplăciiinstabileșia trombozei.

Fig.1: Mecanismede transducție a

semnaluluiînfiziopatologianeurodegenerative:

Rețineți că, PCRminduce,de asemenea,

căiinflamatoriidirecte
prininducereadeinterleukine,MCPșisemnalizareprinNF-kB.
Înplus,datele noastrenepublicatesugerează
căPCRmpoateactivagamma-secretaze(se găsesc în principal
în concentrații mariînplutelelipidice) care ducela
fosforilareaproteineiprecursoare a amiloidului(APP), care
descompunebeta-amiloidulîn formetoxice asociate
demenței.

Antibody-targeting mechanism: Mecanism de dirijarea anticorpilor

It is important to identify patients who have suffered ischaemic, haemorrhagic or lacunar stroke as early as possible
after the first substantial event. We would estimate that as this is primarily an early-stage preventative therapy as
the time between the primary event and the treatment increases, the effectiveness would decrease. The antibody,
directed to cellular- membrane-bound rmCRP would not require brain region targeting, so long as concentrations
could be achieved which were sufficient to bind all rmCRP within ischaemic zones. If targeting was considered
necessary, it would be achievable (although not within the confines of this Proof-Of-Concept study):
Esteimportant identificarea cât mai precoce, de preferat după primul eveniment, apacienților
careau suferitaccidente vasculare cerebraleischemice, hemoragicesau lacunare.Datorită faptului
că aceastaeste în primul rândoterapie preventivă aflată într-un stadiuincipient,timpul
dintreevenimentulprimarși tratament creșteiar eficacitateaar scădea. Anticorpul,
îndreptatsprePCRrm legat de membrana celulară nuar necesitadirecționareregiunii creierului,
atâtatimp câtconcentrațiilear putea fi realizate, care au fost suficientepentru
alegatoatermCRPcadrul zonelorischemice. În cazul în care dirijarea a fost necesară, ar
firealizabil(deși nuîn limitelestudiuluiDovezii de concept):
Utilizarea principală:Principala boală pentru care acest modulator ar putea fi creat este DVa iar pacienții care au
suferit leziuni cerebrale/TBIiar target este redus/monomeric C-reactive protein (mCRP/rmCRP) which is laid down
chronically within the brain in large quantities through leakage from damaged vasculature. Patients with presence of
chronic cerebrovascular disease on advanced neuroimaging and silent strokes but without clear positive stroke index
may also benefit from this approach in future.
Prezentarea generală al obiectivului principal-1 (transferul de cunoștințeși crearea infrastructurii): Aceasta va
includedezvoltareasubstanțialăa capacitățilorindividuale din cadrulinstitutuluigazdăși al organizației urmând caîn
cele din urmăsă se formuleze unmodel pentruextindereși repetareînalte domenii. Dezvoltareaunei culturide
cercetare, ethosșia unei înțelegeri, prin care se permiteintensificareastrategicăaactivității de cercetareși
optimizareacreșterii.În plus, procesul dedezvoltareva cuprindeatâtrețeleinternaționale (de exempluindustrie,
serviciide sănătate, alei clinice, farmaceutice, IMM-urietc.) cât șicolaborări de afaceriîn primul rândîn
cadrulneuroștiințelor șimai târziu îndomeniulmai larg alștiințelor vieții.
Prezentarea generală al obiectivului principal-2 (științific): To provide proof-of-concept data for work-up to clinical
trials into blocking potential neurodegenerative consequences of monomeric-C-reactive protein build up in brain
parenchyma- goal- protection against vascular dementia attributed to stroke and traumatic brain injury. This work is
based on our therapeutic rationale/hypothesis that we could block the downstream effects of mCRP within damaged
brain tissue by preventing its cellular binding via lipid rafts and associated cell signalling soon after the primary
infarction. In this way, we should be able to significantly perturb/ nullify the neurodegenerative cascade via a dual
action of protecting neurons and preventing aberrant angiogenesis thereby abolishing or at least significantly
reducing the increased risk of development of dementia in these patients.

Furnizarea datelor privind dovada de concept privind studiile clinice în vederea blocării potențialelor consecințe
neurodegenerative ale proteinei C-reactive monomerice aflat în parenchimul cerebral având ca scop protecția -
împotriva demenței vasculare atribuite accidentului vascular cerebral si a leziunilor cerebrale traumatice. Acest lucru
se bazează pe raționamentul / ipoteza noastră terapeutică prin care am putea bloca efectele în aval al PCRm din
cadrul tesutului cerebral afectat, prin prevenirea legării celuleor prin plutele lipidice și celule asociate de semnalizare
la scurt timp după primul infarct. În acest fel, ar trebui să fim în stare să perturbăm / anulăm în mod semnificativ
cascada neurodegenerativă prin intermediul unei duble acțiune de protecție a neuronilor și prevenire a angiogenezei
aberante astfel raducând semnificativ riscul crescut de dezvoltare a demenței la acești pacienți.

Sub-obiective specifice/sumar: fiecare număr reprezintă un singur plan de lucru

Activitatea privind schimbul de cunoștințe-
1. Transferul de cunoștințeșiabilitățileoamenilorcare încorporeazăcele mai eficiente strategiișirutelede impact
privind instruirea personalului, stimularea acestuiași dezvoltarea programelor, inclusiv al contextuluiacademicși
tehnic, programe de dezvoltarea carierei, înființareadecompaniispin-off și dezvoltarea de
legăturicuîntreprinderilemiciși mijlocii etc
2.Infrastructură șicercetareîn vedereapublicării, scrierii granturilor șibrevetăriicursurilor, organizarea defacilitățide
cercetareși apartamente, precum șiplatformede gestionare șiintegrareapredării și învățării în dezvoltarea cercetării
strategice.
3.Rețeleleexterneși consolidarearețelelor clinice șinon-clinice precum și al partenerilor, transferul de tehnologieși
deschidereasprepiață, managementulIPcrescând astfelîn mod semnificativcapacitatea,puterea, calitateași
concentrareacercetăriișia instituțieica unîntreg.
Activitateaștiințificăa proiectului-
4. Confirmation of our pilot data results with full characterization of the direct induction of dementia using our
murine mCRP-hippocampal injection model of Alzheimer’s (as described in our Scientific Reports paper 2015, Slevin
M et al).
Confirmarearezultatelordatelorpilotcucaracterizareacompletă ainduceriidirectede demență, folosind modelul
nostruPCRm murininjecție-hipocampice deAlzheimer(așa cum este prezentatîn Rapoartele noastre științificedin
2015,SlevinMet al).
5. Production and characterization of a monoclonal antibody that will be effective in binding to and ultimately
disposing of mCRP inhabiting the brain parenchyma after tissue injury.
Producerea șicaracterizareaunui anticorpmonoclonalcareva fi eficientîn legăturășiîn cele din urmădacă dispune
dePCRmaflate înparenchimul cerebraldupa o leziune a țesutului.
6. Testing of the antibody for effective blocking/clearing of mCRP from the brain using the murine model described
in Objective 1. Testareaanticorpuluiprivindeficiența deblocare/compensareaPCRmdincreier cu
ajutorulmodeluluimurindescrisînObiectivul 1.
7. Proof-of-concept using a canine model of stroke with measurement of the effectiveness of our mCRP antibody to
block down-stream neurodegenerative pathway signalling and removal of aggregating mCRP localised to the injury-
infarct site.
-Proof of-concept, folosind un model canin care a suferit un accident vascular cerebral cu măsurarea eficacității de
anticorpi PCRm pentru a bloca în aval caleasemnalizării neurodegenerative și îndepărtarea agregării PCRmlocalizate
la site-ul-prejudiciului infarct.
8. Project management.The project will be managed by a team consisting of scientific and technical staff together
with research knowledge exchange professionals and administrators. A rigorous schedule linked to the objectives
will be applied. Dissemination will be by conventional routes linked to EU and world-wide networking strategies.
Managementul de proiect. Proiectul va fi administrat de către o echipă formată din personal științific și tehnic,
împreună cu profesioniști în domeniul cerecetării privind schimbul de cunoștințe și de administratori. Un program
riguros, legat de obiectiveva fi aplicat. Diseminarea se va face prin căi convenționale legate de strategii de rețele la
nivel mondial și UE.
Proiecte/activități viitoare
Continuarea cercetării după terminarea proiectului: The work will be linked to a Horizon 2020 bid, as the EU
Commision demonstrates a focussed and critical push for novel solutions to the growing problems of dementia- e.g.
http://www.alzheimers.org.uk/site/scripts/news_article.php?newsID=1891
Lucrarea va fipusă spre ofertare pe Horizon 2020, deoareceComisia UE demonstrează un interes crescut fașă de
soluțiile de ultimă oră legate de problemele tot mai maricauzate de demență -de exemplu
http://www.alzheimers.org.uk/site/scripts/news_article.php?newsID=1891
Links between neurosciences and then all Life Sciences research to major bodies will be synergised throughout the
period of the work including ERANET, NIHR, UK-Ro partnerships, Marie Curie actions and the Ministry Of Defence.
Legăturile dintre neuroștiințe si apoi toate de cercetare Life Sciences la organele majore vor fi synergised pe toată
durata activității, inclusiv ERANET, NIHR, parteneriate între Marea Britanie-România, acțiunile Marie Curie și
Ministerul Apărării.
Added Value statements:Declarații cu privire la valoare adăugată:
Impactul economic:Modern medicine has led to ever-increasing life expectancies in the Western world. Larger
numbers of elderly individuals has resulted in an increased prevalence of various neurodegenerative diseases,
including vascular dementia (VD). Vascular dementia is not considered a single disease but a group of syndromes,
which result following a cardiovascular event such as stroke. Approximately 1.5% of over 65s in the major western
markets suffer from vascular dementia; this is believed to be as high as 4.5% in Japan making VD the second most
common dementia behind AD. The VD market was worth approximately $932 million in 2008 and expected to be
worth $4.2 billion by 2014, making it the third largest market in memory/cognition therapeutics. The economic
impact on TM and Ro will in part be derived from the added value this project will provide in securing new jobs,
better jobs with security and enhanced environment as well as links to international collaborators, spin-offs and
industry.
Medicina modernăa determinat casperanța de viață din lumea occidentalăsă fie tot mai mare. În cazul mai multor
oameni în vârstă s-a observat o prevalență crescută a diferitelor boli neurodegenerative, inclusiv demența vasculară
(DV). Demența vasculară nu este considerată o singură boală, ci un grup de sindroame, care rezultă ca urmare a unui
eveniment cardiovascular, cum ar fi accidentul vascular cerebral. Aproximativ 1,5% din populația de peste 65 de ani
în principalele piețe vestice suferă de demență vasculară; în Japonia 4,5% din populație suferă de această boală
astfelDV a devenit a doua cea mai frecventă formă dedemență după BA. Piața privind DVvalora aproximativ 932
milioane de dolari în 2008 și este de așteptat ca acesta să ajungă la valoarea de 4.2 miliarde de dolari până în 2014,
devenind astfel a treia cea mai mare piață privind terapeuticele de memorie / cogniție. Impactul economic asupra
TM și Ro va fi parțial derivat din valoarea adăugată deorece acest proiectva asigura noi locuri de muncă, locuri de
muncă mai securizate și cu un și mediu dezvoltat, precum și colaborări cu firme internaționaleșispin-off-uri.
Industry links: Following proof-of-concept, we expect a potential therapeutic suitable for pre-clinical/clinical trials
and expect recognition and integration within a major pharmaceutical company (for example Smith and Nephew) A
team of experienced knowledge transfer staff at MMU will be assisting the team in identifying, approaching and
negotiating with suitable pharma companies for this phase of the project.
După semnarea dovezii conceptului, ne așteptăm la o potențial terapeutic adecvat pentru studii pre-clinice / clinice
și așteaptăm recunoașterea și integrarea în cadrul unei mari companii farmaceutice (de exemplu Smith and
Nephew). O echipăcu experiență în transferul de cunoștințe,angajatal MMU va ajuta echipa în identificarea,
abordarea și negocierea cu companiile farmaceutice adecvate pentru această fază a proiectului.
Brevetarea: A patent application will be filed to protect the concept of mCRP-blocking and clearing as protection
against brain injury-associated dementia. Staff in TM will have access to a programme of development from MMU in
patent generation and development.

O cerere de brevetare va fi depusă pentru a proteja conceptul de blocare și de compensare a PCRm ca proteție
împotriva leziunilor cerebrale asociate demenței. Personalul din TM vor avea acces la un program de dezvoltare de la
MMU dezvoltarea brevetării.

Activități și PL specifice:

Knowledge transfer and developing communication skills. Activity:Identifying the most effective strategies and
routes to impact training of staff, staff incentivisation and development programmes including academic and
technical frameworks, career development programmes, establishment of spin-off companies and links with small-
medium enterprises (Year 1-overall strategic development; Year 1-4-staff incentives-produce a framework of activity
and implement; career development pathways; spin offs and SMEs.

PL-1: Transferul de cunoștințe și dezvoltarea abilităților de comunicare. Activitate: identificarea celor mai eficiente
strategii privind instruirea personalului, stimularea acestuiași dezvoltarea programelor, inclusiv al
contextuluiacademicși tehnic, programe de dezvoltarea carierei, înființareadecompaniispin-off și dezvoltarea de
legăturicuîntreprinderilemiciși mijlocii etc.(Anul 1-dezvoltări strategice generale , Anii 1-4-stimularea personalului-
dezvoltarea unui cadru de activitate și implementarea acestuia; căi de dezvoltare a carierei; spin off și IMM-uri.

Sarcina1.1- Establish and embed the foundations required to reorganise the research culture (concordat/vitae). This
will also include: staff professional development reviews for career development and specifically 1-3-5 year research
plans containing detailed strategy of how to achieve goals and empower staff.

Stabilirea și încorporarea bazelor necesare pentru a reorganiza cultura de cercetare (concordat / vitae).
Aceasta va include, de asemenea: analizadezvoltării profesionale alpersonalului pentru dezvoltarea carierei și, în
special planuri de cercetare pe 1-3-5 ani conținând descrierea detailată a strategiei de atingere a obiectivelor și
împuternicirea personalului.
Perioada: 1-12 luni
-Initiate a whole range of developmental and assisting mechanisms to promote World class research including post-
doctoral fellowship groups and group leader structures, studentships, research groups dedicated to primary research
and centre management, laboratory management protocols and technical support integration.
Timeline: Months 1-48

Sarcina1.2Inițierea unei serii întregi de mecanisme de dezvoltare și de asistență pentru promovarea cercetării de
talie mondială, inclusiv grupuri de burse post-doctorale și structuri lider de grup, studenți, grupuri de cercetare
dedicate cercetării primare și managementului centrelor, protocoale de management de laborator și integrarea
suportului tehnic.
Perioada: 1-48 luni
Task 1.3-Embedding sustainability of resources, a pay and reward structure for research, technical and research
active academic staff (as well as lecturers who are not research active) and orientation of developmental pathways
between teaching-research and technical avenues.
Timeline: Months 13-48

Sarcina 1.3-Integrarea durabilității resurselor, a unei structuri de remunerare și recompensare pentru personalul de
cercetare, cel tehnic și cea de cercetare academică activă (lectori care nu activează încă în domeniul cercetării) și
eficientizarea căilor de dezvoltare între predare-cercetare și modalități tehnice.
Perioada: 13-48 luni

Task 1.4- Knowledge transfer within groups and research themes at TM, for example creation of networks around
neuroscience and cardiovascular/vascular disease or dementia. Transfer beyond TM, via media coverage, web-site
management, use of Scopus and SciValtoform a database for comparison with other institutions, formation of official
partner institutions and small medium enterprises to create research network consortiums etc.
Timeline: Months 13-48
Sarcina 1.4- Transferul de cunoștințe și de teme de cercetare în cadrul grupurilor de la TM, de exemplu crearea de
rețele privindneuroștiințași bolile cardiovasculare / vasculare sau demența. Transferul de dincolo de TM, prin
intermediul mass-media, managementul paginii de internet, utilizarea bazelor de date Scopus și SciValtoform pentru
comparație cu alte instituții, formarea de instituții partenere oficiale și întreprinderi mici și mijlocii pentru a crea
consorții sau rețele de cercetare, etc.
Perioada: 13-48 luni
Results: This programme of activity is expected to result in provision of the building blocks for stable research
growth and development within the centre and optimised research activity from each sub-discipline based around
new suite structuring and groups. In relation to the scientific project, this will enable specific elements of the work to
be effectively directed within the new research centre at TarguMures, via new incorporation of dedicated sections in
tissue culture cell and molecular biology and analytical pharmaceuticals.
Rezultate:
Acest program de activitate este de așteptat să conducă la furnizarea blocurilor de construcție pentru o creștere
stabilă de cercetare și dezvoltare în cadrul centrului și activitate de cercetare optimizată din fiecare sub-disciplină în
parte bazată pe o structurare nouă, proprie și de grup. În ceea ce privește proiectul științific, aceasta va permite ca
unele elemente specifice ale activității să fie direcționate în mod eficient noului centru de cercetare de la Targu
Mures, prin încorporarea de noisecții dedicate culturii de țesutși biologie molecularăși farmaceutice analitice.
Resources: mini academies, seminar series and intense management sessions such as research leader academies.
Costs will be incurred for staff travel from MMU to TM and vice versa as well as payment in some instances for
services (expected 15 working days/annum x 3 senior staff).
Resurse: mini academii, seminarii și sesiuni intense de management, cum ar fi academiilororganizate pentru liderul
în cercetare. Costurile suportate vor fi cele pentru călătoria personalul din MMU la TM și invers, precum și plata în
unele cazuri pentru servicii (de așteptat 15 zile lucrătoare / an x 3 personal de conducere).
Total £60000

WP.2: Analysis and modification of infrastructureand culture. Infrastructure research culture to invest in publication,
grant writing and patenting academies, to organise research facilities and suites. Establish management platforms
and integrate teaching and learning with research strategic development (including set up and manage
infrastructure assessment panels; initiate and manage research-linked academies and academic networks; research
organisation and facility overview and planning.) Overall strategic research developmental planning.)
PL.2: Analiza si modificarea culturii și infrastructurii. Dezvoltarea infrastructurii de cercetare, în vedereapublicării,
scrierii granturilor șibrevetăriicursurilor, organizarea defacilitățide cercetare. Stabilirea platforme de gestionare și
platformede gestionare șiintegrareapredării și învățării în dezvoltarea de cercetare strategică(inclusiv constituirea și
administrarea panourilor de evaluare a infrastructurii; inițierea și gestionarea academiilorlegate de cercetare și a
rețelelor academice;organizarea de cercetare și privire de ansamblu facilitatile si planificare) Planificarea generală de
dezvoltare a strategicii de cercetare).

Task 2.1- Formulation of sustainable core research groups with access to all facilities and a research management
system to allow appropriate personnel access to the grant academies, publication academies, patenting and IP
sessions and sabbatical opportunities which will be instilled into the culture here.
Timeline: Months 1-24
Sarcina 2.1- Crearea de grupuri de cercetare de bază durabile cu acces la toate facilitățile și un sistem de
management de cercetare pentru a permite personalului adecvat accesul la academiile de grant, de publicare,
sesiuni de brevetare și IP și oportunități sabatice care vor fi însuflate în cultura de aici.
Perioada: 1-24 luni
Task 2.2- Organisation of research facilities and suites in order to optimise research output. This means identifying,
setting up and promoting organisation of research structures within the institute such as genomics, proteomics, cell
culture, animal facilities, microscopy suites, imaging suites etc. Also links to human resources knowledge exchange
support, IT/technical etc.
Timeline: Months 13-48
Sarcina 2.2- Organizarea de facilități de cercetare și spații în scopul optimizăriicapacității de cercetare. Aceasta
înseamnă identificarea, crearea și promovarea organizăriiunor structuri de cercetare în cadrul Institutului, cum ar fi
genomică, proteomică, culturi de celule, facilități pentru animale, spații pentru microscoape, spațiipentru imagistică
etc. De asemenea link-uri către resurse umane care oferă sprijin cu privire la schimbul de cunoștințe, IT / tehnic etc.
Perioada: 13-48 luni

Task 2.3- Integration of teaching and learning with research strategic development. It is critical that research informs
teaching at all levels and student satisfaction is partly achieved by integration of application of current and future
research ideas with basic teaching elements and curriculum. We will consult with teachers and researchers to
maximise these opportunities
Timeline: Months 13-24
Sarcina 2.3-Integrarea predării și învățăriiîn strategia de dezvoltare a cercetării. Este esențial ca cercetarea
informeaza predare la toate nivelurile și satisfacția studenților se realizează în parte prin integrarea aplicării ideilor
actuale și viitoare de cercetare cu elemente de predare de bază și curriculum. Ne vom consulta cu profesorii și
cercetătorii pentru a maximiza aceste oportunități.
Perioada: 13-24luni

Task 2.4-Senior research management training and development to enable a coherent approach to research
strategy. Training the trainers sessions and cross disciplinary and cross sectional meetings between accounting,
human resources, finance, technical, research and teaching.
Timeline: Months 3-48

Sarcina 2.4- Dezvoltarea și formarea managementului principal de cercetare pentru a permite o abordare coerentă
a strategiei de cercetare. Instruirea formatorilor și sesiunile cruce disciplinare și cruce întâlniri sectionale între
contabilitate, resurse umane, finanțe, tehnic, de cercetare și de predare.
Adăugat: Luni 3-48
Results: Here, we will have produced a system for progressing project ideas from the bench to the bedside with
assisted support mechanisms on how to maximise the impact of publications, to link this to funding applications and
to produce case studies showing impact within healthcare science policy and treatment.

Resources: creation of senior management Boards for overall strategic planning, infrastructure assessment panels
and academies will incur costs, salaries,travel and subsistence, space and office consumables. (2-3 people from
Manchester at specified intervals to assist in running the meetings.
Total=£45.000

WP-3: Goal- External networking and industry enhancement of both clinical and non-clinical networks and partners,
technology transfer and drive towards market, IP management thereby increasing significantly the capacity, power,
quality and focus of the research and of the institution as a whole. (Year 1-Identification and procurement of clinical
and none clinical external networks; Year-2-Technology transfer and interdisciplinary research academies; Year-3-IP
and marketing and patenting development and Year-4-pathways to impact academies and training.
Task 3.1 Identify and establish a robust framework for developing external links with other research stakeholders
within and outside of Romania. This strategy to include creating and cementing links with industrial partners and to
involve clinical and non-clinical networks and partners.
Timeline: Months 3-36
Task 3.2 Utilise the expertise and knowledge of our external expert and international collaborators to establish
technology transfer and interdisciplinary research academies and embed these schemes in custom and practice
within the University.
Timeline: Months 7-48
Task 3.3 We will utilise the same expertise as in 3.1 to introduce and embed academies of Intellectual Property,
marketing and patenting development in order to strengthen research and build capacity within the institution.
Timeline: Months 7-48
Task 3.4 Pathways to impact and training linked to power and case study generation. This will include project
development based around potential impact within the field of healthcare science. Impact case study generation is a
critical element of European ’ethos-impact’, being any effects on policy, practice, culture or therapeutics. A coherent
strategy at the heart of the project is neccessary in order to maximise future potential benefits which would impact
on IP, funding generation and industry/ NHS links.
Timeline: Months 7-48

Expected results: Forming working parnerships with both local and international halthservices and industry as well
as research networks will be an ongoing long-term strategy initiated here. The reputation of the University and
research institute should benefit significantly from this strategic directive.

Resources: Will be required for meetings between appropriate staff from the collaborating universities to run
throughout the course of this project, e.g. for travel, subsistance and salary as well as space for workshops and
meetings.
Total: £55.000

WP-4: Production of characterised effective monoclonal antibodies to monomeric C-reactive protein (mCRP).
Activity:-this schedule of activity is required to produce and optimise a tailored antibody fit-for-purpose, specific for
targeting and binding to mCRP and characterizing the biological inhibitory activity. (Year 1-production; Year 2-
optimization and biological activity).
Antibody discovery/development strategy: Proposedstrategy/task cascade to:
Task 4.1 Generate and characterize novel mouse antibodies to human target antigen.
Task 4.2 Humanize and characterize existing antibodies to the target, to generate an antibody that blocks rmCRP
functional activity.
Recombinant mCRP for immunization can be generated using protein containing the C36S and C97S mutations to
expose the cholesterol-binding site and potentially expose novel epitopes. We will generate a panel of monoclonal
antibodies which will be screened for binding to human, mouse and cynomCRP. A number of commercial anti-human
and mouse CRP are available and will be purchased for benchmarking purposes and development of cell based
assays. In addition mCRP for human, mouse, cynomologus and dog will be made in house using an E. coli expression
system. The protocol is used currently within our group. Recombinant mCRP has been supplied purified from our
collaborator and project associate Dr Larry Potempa (Roosevelt, US).
Monoclonal against human mCRP will also need to cross react with mCRP in cynomologus monkeys in order to
conduct PK tests and toxicology. Downstream of the project the efficacy of a lead antibody will be tested in a canine
model of stroke.
For the purposes of this project we will generate a panel of Abs to human mCRP and check cross reactivity with
mouse, and dog cynomCRP will be retained (percentage identity between species is moderately high, with 70%
homology between mouse and human and 91% homology between human and cynomologus monkey and 60%
between human and dogs).
Task 4-1
Recombinant mCRP will be supplied to an experienced Research Associate for hybridoma generation. Hybridoma
supernatants will be screened by ELISA for binding to human mouse and cyno-mCRP. As a counter screen, we will
use an ELISA and SPR analysis for human nCRP.
Preparation phase
• Sourcing of commercially available Abs to CRP
• In-house generation of human, mouse, dog and cynomCRP
• Transfer/establish cells assay system and test
• Establish ELISAs to detect binding to human, mouse and cynomCRP
• Determine affinity of mCRP for the ECM and develop mCRP membrane disassociation assay
Time line: Months 0-6 months
Hybridoma Generation (to run in parallel with preparation phase)
• Via Research Associate generate panel of hybridomas and assay for binding to human,
• Mouse, cynomCRP and counter screen against normal and mutant mCRP plus nCRP.
• Initial functional screening – cell based signaling assay
• Select panel of MAbs for purification.
• Rank Abs for binding affinities via Biacore
Timeline: Months 0-6 months
Functional characterization
Confirmation of functional blocking activity of the anti-human mCRP Abs by screening for functional activity in a
variety of assays including the cholesterol-binding assay, cell-binding assay, cell activation/signalling assays using
endothelial cell lines and primary neurons-see below for assay details.
Timeline: Months 7-12
Assays: Assays which will be used to determine the antibody potency-ability to block mCRP function are already in
use:
Cholesterol binding assay: Monolayer insertion-Langmuir Trough: mCRP would be injected into a sub-phase of a
monolayer that has the right amount of cholesterol on the surface. Lateral pressure differences would be identified
when the protein inserts into the monolayer. The experiment will be repeated using the mAb at different ratios to
antigen in the fluid phase (i.e. inject the complexes in the sub-phase). In this way, we will prove/show that the
antibody prevents mCRP from inserting under optimal conditions (Ji SR et al, 2009).
Inflammation activity assay: In this assay, the ability of mCRP to stimulate monocyte (U-937)/endothelial cell
expression of inflammatory markers including IL-1-beta, IL-8, IL-6, MCP-1 will be measured by standard ELISA and
signaling molecules NF-kappa-B and p38-MAP Kinase phosphorylation will be measured by Western blotting.
Receptor binding assays: Fc Gamma R1 in monocytes and Insulin Receptor in endothelial cells-presumed as IRS-1 is
phosphorylated (neuronal receptor not known at the present time). Standard protocols used here.
mCRP Monocyte adhesion to HbMEC:-is a standard in vitro assay and will be measured under static as well as under
laminar flow conditions.
mCRP Platelet aggregation assay: Again, a standard assay using light aggregometry and analysis by flow cytometry
Cell based assays using our fully characterized human brain microvessel endothelial cell line (HbMEC) and primary
rat cortical neurons available from Lonza Pharmaceuticals: Tau/ERK/IRS-1 phosphorylation assays will be used to
show level of signal transduction activation for neurons and endothelial cells.
Task 4.2Generation of lead Antibodies (humanization)
Design, build and generate chimeric antibody (IgG1, kappa) as control.
Design (using molecular modelling and sequence analysis) humanised versions of
candidate Abs. Generate and test for optimal versions using ELISA and SPR.
Timeline: Months 13-18
TAk 4.3 Structural characterization of Fab; Antigen complex
Generate Fab fragment of candidate Ab (recombinant chimeric) in sufficient quantities for NMR and/or
crystallography.
Generate recombinant human mCRP and Fab fragment and supply to structural group
Timeline: Months 18-21
Task 4.4 Characterization of Lead Antibodies
Determine potency using the Biacore assay
In vitro functional validation: using cell based assays outlined above.
Selectivity: Test tissue microarrays (commercially available) to determine tissue cross-reactivity with relevant
antibodies. Measure biophysical properties: aggregation, solubility, thermostability, non-specific serum binding,
affinity.
Timeline for biophysical analysis plus in vitro validation: Months 21-24
Optimization of Lead Antibodies
(If required – dependent on the binding affinity and performance of the humanized Antibody in the cell based assay).
Task 4.5 Affinity maturation:
Reconfigure humanized mAb as a scFv and build two scFv libraries based on a random mutagenesis and focused
mutagenesis approaches to the CDRs – if available use structural information. Screening and selection of scFv will be
based on affinity and biophysical properties.
Candidate mAbs will be re-made as whole IgG4 mAbs and re-tested for affinity, solubility, aggregation and
nonspecific serum binding.
Timeline: Months 21-24
Expected results: Generation of mAbs with the desired characteristics is key and is likely to take 6-8 months,
including characterization. Ideally the lead candidate should be specific for mCRP rather than the pentameric but
there is a risk that may not be possible to retain functional activity and cross species reactivity. Species-specific
ortholog antibodies may have to be developed although there is significant shared sequence identity between. Once
a candidate mAb has been selected this project should progress rapidly as a straightforward humanization program
(~12-18 months), assuming that the affinity of the murine antibody is considered sufficient when tested. If an affinity
maturation step is required this will extend the timelines for an additional 6-9 months and this can be incorporated
within the first 2 year time frame.
Allocated resources for WP4:
Preparation Phase (4 months):
Transfer reagents, source commercially available Abs, establishing ELISA and assay protocols
(1 FTE BioT)
Production mCRP-1 (0.5 FTE)
Cell based assay development: (0.5 FTE Bio)
Hybridoma Generation (to run in parallel with preparation phase)
Timeline: Months 6-8
Outsource Hybridoma production.
Characterisation of Mabs generated (4 months, 1 FTE BioT, 1 FTE Biology)
Generation of lead Abs (3 months)
Design, build express and test humanized versions (1 FTE BioT)
Structural characterization of Fab:antigen complex (to run in parallel with generation of lead Abs)
Generation Fab fragments, 2-3 months 0.5 FTE
Generation mCRP and structural studies 6 months 0.5 FTE
Timeline: Months 3-6
Characterization of Lead Abs (3-6 months)
Biochemical and biophysical potency 1 FTE 3 months
In vitro function validation and selectivity testing 1 FTE 6 months
Measurement of Biophysical properties 1 FTE 3 months
Full in vitro characterization of cell activation effects on endothelial cells, neurons and glia combined with
immunohistological confirmations of protein/phosphorylated protein expression within the brain.
Costing: Materials for Western blotting, antibodies against specific proteins, Kinexus focused Western array analysis,
tissue culture materials and rat neuronal/glia. Histological materials, kits and antibodies for immuno-
histochemistry/fluorescence-additional £12,000.
Antibody blocking testing in vitro-cell based assays, Western blotting and ELISA for optimization and kinetic studies
£10,000.
Total person time=FTE 36 months
Total material costs=£
WP.5: Full characterization of the murine model for measuring mCRP-induced dementia. Our pilot-published data
demonstrated the murine model using stereotactic hippocampal injection allowed us to identify cognitive,
behavioral and pathobiological changes linking mCRP deposition within the cortex to onset of dementia. Activity:-
We will optimise and detail the technique, fully characterizing cognitive, behavioural and memory defects over time
and identifying key neuro-inflammatory and neurodegenerative markers, cellular localization and binding
characteristics of mCRP thus enabling us to measure accurately the competency of the antibody. (Year 1-
experimental work; Year 2-assessement of pathobiological changes to the brain).
In vivo validation of AD model:
Task 5.1 -Characterization of effects on cognitive function, memory and behavior (1-12 months)
Animals:
mCRP, will be delivered into the CA1 region of the mouse hippocampus by stereotactic surgery procedures. Four-
month old mice will be anesthetized with 10 mg/kg xylacine (Rompun 2%, Bayer, Leverkusen, Germany) i.p. and 80
mg/kg ketamine (Ketolar 50 mg/ml, Pfizer, Alcobendas, Madrid, Spain) i.p. and placed in a stereotactic apparatus
(David Kopf Instruments, Tujunga, CA). Bilateral infusions of either an experimental agent solution or artificial CSF
(NaCl 148 mmol/l, KCl 3 mmol/l, CaCl2 1 mmol/l, MgCl2 0.8 mmol/l, Na2HPO4 0.8 mmol/l, NaH2PO4 0.2 mmol/l) will
be performed into the CA1 area of the hippocampus. Solutions of mCRP contained a final content of 50µg. Injections
will be performed at a rate of 1μl/min at coordinates relative to Bregma of -2.0 mm A/P, ±1.2 mm M/L, -1.5 mm V/D.
One microliter of the testing solutions will be delivered to the application point with a 25-gauge stainless steel
cannula (Small Parts Inc., Miami, FL) connected to a Hamilton syringe through a Teflon tube. The syringe will be
attached to a micro-infusion pump (Bioanalytical systems Inc., West Lafayette, IN). The cannula will be left in position
for 5 min after delivery to prevent the solution from surging back.
Testing points and end-points (euthanasia) at 1 month 3 months and 6 months after injection
C57BL6 mice per test
Sham controls x 6 (n=18)
mCRP injected x 12 (n=36)
3 x Tg positive control x 6 (n=18)
Total animals=72

Analysis:
Animals will be tested for changes of non-cognitive and cognitive behavior at three weeks, 11 weeks and 22 weeks
after hippocampal injections. A battery of tests will be applied on fourteen daily consecutive sessions.
Sensorimotor responses: Visual reflex and posterior legs extension reflex will be measured by holding the animal by
its tail and slowly lowering it towards a black surface. Motor coordination and equilibrium will be assessed by the
distance covered and the latency to fall off a horizontal wooden rod and a metal wire rod. Prehensility and motor
coordination will be measured as the distance covered on the wire hang test, which consisted of allowing the animal
to cling from the middle of a horizontal wire (2 mm diameter x 40 cm length) with its forepaws for two trials of 5 s
and a third 60 s trial.
Corner test: Neophobia to a new home-cage will be assessed by introducing the animal into the center of a standard
square cage (Macrolon, 35 x 35 x 25 cm) with fresh bedding and counting the number of corners visited and rearings
during a period of 30 s. The latency of the first rearing will be recorded.
Open field test: Mice will be placed in the center of the apparatus (home-made, wooden, white, 55 x 55 x 25 cm
high) and observed for 5 min. Patterns of horizontal locomotor activity (distance covered and thigmotaxis) and
vertical movement (rearings) will be analyzed throughout the test. Initial freezing, self-grooming behavior and the
number of urine spots and defecation boli will be also recorded.
Dark and light box test: Anxiety-like behavior will be measured in a dark-light box. The apparatus consists of two
compartments (black: 27 x 18 x 27 cm with a red light; white: 27 x 27 x 27 cm with white lighting intensity of 600 lux)
connected by an opening (7 x 7 cm). The mice will be introduced into the black compartment and observed for 5
min. The latency to enter the lit compartment, the time spent in the lit compartment and the number of rearings will
be recorded.
Boissier’s four hole-board test: Exploratory behavior will be measured as the number of head-dips and time spent
head-dipping on each of the four holes (3 cm diameter) equally spaced in the floor of the hole-board (woodwork
white box of 32 x 32 x 32 cm). The latencies of movement, first dipping and four hole dipping will be recorded.
Tail suspension test: Mice will be suspended by the tail to assess depression-like behavior. The mice will be hanged
30 cm above the surface. The tail will be fixed with adhesive tape at 1 cm from its tip. The duration of immobility
(defined as the absence of all movement except for those required for respiration) will be scored during 6 min.
Object recognition test: Animals will be placed in the middle of a black maze with two arms angled 90º, each
measuring 25 cm x 5 cm. The 20 cm high walls can be lifted off for easy cleaning. The lighting intensity will be 30 lux.
The objects to be discriminated will be made of wood (5-6 cm high, brightly colored). After two previous days of
habituation, the animals will be submitted to a 10 min acquisition trial (first trial) during which the mouse will be
placed in the maze in the presence of two identical novel objects (A+A’) placed at the end of each arm. A 10 min
retention trial (second trial) occurred 2 h later, replacing object A’ in the maze by object B. Another 10 min retention
trial (third trial) occurrs 24 h later, replacing object A in the maze by object C. The time that the animal explors the
new object and the old object will be recorded. In order to avoid object preference biases, the sequence of
presentation of the different objects will be counter-balanced in each experimental group. The maze and the objects
will be cleaned with 96° ethanol between different animals, to eliminate olfactory cues.
Morris water maze test: Animals will be tested for spatial learning and memory in the Morris water maze (MWM),
consisting of one day of cue learning and six days of place task learning for spatial reference memory, followed by
one probe trial. To test the spatial learning acquisition, mice will be trained to locate a hidden platform, 10 cm in
diameter, located 20 cm from the wall and 0.5 cm below the water surface. This will be placed in a circular pool 100
cm in diameter, 40 cm height, with 25ºC opaque water, surrounded by black curtains. The animals will learn to find
the platform using distinctive landmarks as visual cues (four trial sessions of 60 s per day). On day seven, after one
trial of place learning, the platform will be removed and the mice perform a probe trial of 60 s to test the retention
of learning. A computerized tracking system (SMART, Panlab S.A., Barcelona, Spain) will be used to measure the
distance covered during the learning tasks, along with the time spent in each quadrant of the pool after the removal
of the platform in the probe test. For statistical analysis, 2-way ANOVA will be used and significance will be defined
as p<0.05*.
Time line 1-12 months

Task 5.2Histological Analysis:– Characterization of brain pathobiological changes linked to dementia (13-24 months)

After completion of the behavioral tests, at 1/3/8 months after injection, mice will be anesthetized as described
above and transcardially perfused with 100 mM phosphate buffer (PB, pH 7.4) containing 0.1 mg/ml heparin (Mayne
Pharma, Spain) followed by 4% paraformaldehyde in PB. Brains will be removed and post-fixed overnight in cold
paraformaldehyde, rinsed with cold PB and then dehydrated in a graded ethanol series, cleared in xylene and
embedded in paraffin. Serial sections will be cut throughout the brain at 5μ, and IHC will be carried out at 1mm
intervals throughout the brain-details described below in the section on immunohistochemistry (animals n=6 per
group) in order to determine expression and localization of mCRP, p-Tau, p-IRS-1 and Aβ.
Immunohistochemistry:
Double immunofluorescence and/or immunohistochemistry will be used to assess the distribution of mCRP (mouse
anti-human mCRP-specific antibodies 8C10 and p-IRS-1-Y1179) and activated microvessels (CD105/endoglin rabbit
polyclonal antibody) as well as the presence of β-amyloid and p-tau (rabbit polyclonal antibodies). After incubation
with primary antibodies for 1h at room temperature (1:100), sections will be washed and then incubated with the
appropriate secondary antibodies (1:50) – peroxidase (HRP), fluorescein isothiocyanate-conjugated sheep anti-
mouse IgG (Jackson) or tetramethylrhodamineisothiocyanate-conjugated rabbit anti-goat (Jackson). Images will be
captured with Nikon 80i Digital Microscope using Nis Elements 3.21 software with multichannel capture option.
Negative control slides will be included where the primary antibody will be replaced with PBS. Vecor ABC kits will be
used for all IHC and the Vector mouse on mouse (M.O.M) will be used when applying mouse primary CRP antibodies
to the murine brain sections with mouse secondary.
Time line-13-24 months

Results: We will have produced a fully characterized proof-of-concept model to be able to test the therapeutic
effects of ‘modulation’ of mCRP aggregation within the brain after injury.
Whilst we will use this to test for therapeutic removal of mCRP, this model could also be used in combination with
stroke, inflammatory conditions or traumatic brain injury since CRP production is NOT a major part of the murine
response to acute inflammation (unlike in humans and other higher mammals) and so the additional or confounding
effects of mCRP on these processes within the brain can be managed by examining the model in the presence versus
the absence of administered CRP/mCRP.
Allocated resources for WP-5
Post-doctoral researcher x 2 for 2 years : £142,579
Characterization of localization, morphological changes and effects of mCRP on cognition and behavior using a
murine model of dementia.
Costing: Animal purchase, housing, food C57BL/6 (total n=72)-£75,000
Immunohistochemical study-AbCam kits for double immunohistochemistry/ fluorescence, other materials for
histology £58,500.
General Lab Consumables: £5,000

Total resources=

WP.6:Testing of our antibodies using the murine model to optimise effective protection aginst mCRP-induced
dementia. In order to obtain initial proof-of-concept and mechanistic data to support the antibody-protective effects
of the therapeutic. Activity:- Using the murine model (now fully characterized) and various injection protocols and
establishing changes to cognition, behaviour, memory and brain pathobiology compard with mCRP-injected animals
without protective antibody. (Year 3).
Task 6.1- Characterization of effects on cognitive function, memory and behavior (25-36 months)
Task 6.2- Characterization of brain pathobiological changes linked to dementia (37-48 months)
Here, we will examine blocking efficiency of the newly generated rmCRP-antibodies and the ability to protect against
neuronal degeneration and plaque formation.
End-points at 1 month and 3 and 6 months after injection
C57BL6 mice per test
Sham controls x 6 (n=18)
mCRP injected x 12 (n=36)
mCRP + antibody x 12 (n=36)
Antibody alone x 6 (n=18)
3 x Tgcontrol x 6 (n=18)
Total animals=126
Using the same techniques described above, mice will be injected stereotactically with mCRP with or without the
blocking antibody and n=6 sham-operated controls; n=6 antibody only controls; n=6 3 x Tg positive controls for each
time point. Total =126 mice) during the same surgery (dose of mCRP-50µg).
And the same strategy as described above in WP4.5- will be used to assess the impact on mouse cognition, behavior
and neurodegenerative decline.
Timeline of activity:
The work in WP-6 and activity timeline will follow the exact format as for WP.5
WP.6.1 – mimicking 5.1-Months 25-36
WP.6-2 – mimicking 5.2-Months 37-48
Results: Proof-of-concept that our therapeutic antibody injection will abrogate the neuropathological effects of
aggregated mCRP within the brain. Here we will be able to assess the level of effectiveness and define if any further
optimization or modifications will be required prior to the canine model pilot study (below) and clinical trial
organization.
Allocated resources for WP.6

Post-doctoral researcher x 2 for 2 years : £142,579

Characterization of localization, morphological changes and effects of mCRP on cognition and behavior using a
murine model of dementia.
Costing: Animal purchase, housing, food C57BL/6 (total n=126)-£125,000
Immunohistochemical study-AbCam kits for double immunohistochemistry/ fluorescence, other materials for
histology £58,500.
General Lab Consumables: £10,000
Total resources=

WP.7: Pre-clinical further proof-of-concept using the canine model of stroke-induced dementia and our proposed
mCRP blocking/clearing mechanism to prevent cognitive decline. This large mammal pilot study will pave the way to
clinical trial possibilities, since CRP production is a feature of stroke associated neuro-inflammation in dogs, we will
assess the ability of our mCRP-antibody therapeutic to remove lesion associated mCRP from the brain parenchyma.
Activity:- We will perform temporary middle cerebral artery perfusion on aged canine animals and carry out a
preliminary assessement to characterise the potential clearance of mCRP using our therapeutic antibody.
WP.7: Progression beyond initial in vivo validation (year 4; months 37-48)
Pilot study to examine mCRP production, blocking/clearance in aged canine model-after MCAO. Consideration of
the ideal models for mCRP physiology representing the human acute phase response, the canine model seems the
most appropriate since it also represents one of the best stroke models mimicking human pathophysiology with
similar CRP acute phase response (e.g. Teck-Kang et al, 2007), and is also useful for inclusion of MRI-neurobehavioral
status indications (2009 same author).
Aged animals can be used and dogs are susceptible to development of cognitive decline and dementia with similar
pathobiological changes to the human within the brain. Currently there is no published scientific work on combined
models of stroke-VD/AD in larger mammals.
In this section of the work we will be carrying out experiments to measure the ability of the antibody to ‘clear’ or
remove mCRP following creation of anmCRP-Ab complex opsonizing-uptake by macrophages/glia and phagocytosis.
Six healthy aged (10 years) female beagle dogs (mean body weight, 11.7 kg; range, 9.0 ∼14.5 kg) will be used for this
study. The dogs will be monitored for at least 2 weeks before the surgical procedure. Results of the physical and
neurological examinations during this period should be consistently normal.
The animals will be screened for metabolic diseases by complete blood count (CBC) and serum chemistry analysis,
and also screened for infectious pathogens such as canine parvovirus and canine distemper virus. Each dog will be
housed in an individual cage and fed commercial dry food (Limited Ingredient Diets; Natural Balance Pet Foods, USA)
twice daily.
The end points of the study will be 1 week following induction of ischemic stroke via permanent MCAO (with or
without concomitant injection of mCRP therapeutic antibodies) n=3 per group.
All dogs will be euthanized at these end points.The surgical and experimental protocols, including euthanasia, will be
approved by the institutional animal care and use committee.
Protocols:
MCAO
Task 7.1-Temporary MCAO will be induced via placement of microvascular clips at the proximal MCA trunk and the
orbitofrontal branch following craniectomy in anaesthetized animals. The clips will be removed after 6 hours to allow
reperfusion. This method will result in production of small sub-cortical infarcts in the animals (Young AR et al 1997).
Three of the animals will also be concomitantly treated with our mCRP therapeutic antibody (months 37-40).
Task 7.2-Injection of antibody
A single injection of our therapeutic antibody (dose/titre to be determined from the studies above) will be injected
distal to the clamping points at the time of release in three of the animals (months 41-42).
Task 7.3-Measurement of Neurological status
Standard neurological scoring will be used to test motor function, responsiveness vision, head turning; and memory
and whole body MR imaging used to identify the lesion at 6h 24h and 7 days (Zu QQ et al 2013-Lab Invest) (months
43-48).
Task 7.4-Histology and tissue analysis
After gross examination, coronal sections will be cut through the cortex and analyzed by 1) staining with TCC to
identify the infarcted regions and b) paraffin processed for histological analysis and IHC as described above (months
45-48).
Time line of activity: Months 37-48
Results: The data generated from this final part of the study will enable us to characterize-in pilot form – the ability
of our therapeutic injected antibody at binding and clearing and/or blocking the biological activity of aggregated
mCRP. In this case originating from the animals own inflammatory response to stroke and hence it will act as a pilot
proof-of-concept for consideration of phase-1 clinical trials.
Allocated resources for WP.7
Post-doctoral researcher x 2 for 1years: £71,290
Characterization of localization, morphological changes and effects of mCRP on cognition and behavior using a
murine model of dementia.
Costing: Animal purchase, housing, food (total n=6)-£155,000
Immunohistochemical study-AbCam kits for double immunohistochemistry/ fluorescence, other materials for
histology £58,500.
General Lab Consumables: £12,000
Total resources=

WP.8-Project Management Establish a robust management structure that will ensure that the project is delivered on
time and within budget. A Project Management Steering Committee, consisting of the PI, and applicants DrPetiOlah,
PETI PLEASE COMPLETE THE NAMES OF OTHERS HERE will be set upto specify the schedules of work, monitor
budgets and define the roles of team members. The Management Steering Committee will also be responsible for
monitoring and reporting on progress, plus dealing with/solving any problems that may arise. The Committee will
meet on a quarterly basis (in Romania or Manchester) but full use will be made of video conferencing facilities at
each institution in order that weekly contact is maintained. Risk analysis – although the impact to the project was
categorized as low, risk register will be established as part of the management process and ongoing progress will be
monitored against this. In this manner, any problems that arise can be responded to in a timely manner. The
management team will review progress at their quarterly meetings with one of their key tasks being the overcoming
of any problems that might cause delay to the research. Should any major problems occur between the quarterly
meetings members of the management team can make contact with others via Skype or other video conferencing
facilities to ensure a timely resolution to such incidents. In this manner the smooth running of the project is
guaranteed.
Task 8.1 Interview for Project Manager who will be responsible for the day-to-day management of the project, and
researchers required to carry out the project. The Project Manager will be responsible for creating and maintaining
the project website, to be hosted by TarguMures. (Month 1).
Task 8.2 Kick-off meeting at TarguMures involving all members of the Project Management Steering Committee to
establish systems for monitoring the project.Set up an electronic Risk Register using a project website, to which all
the Management Steering Committee have access. (Month 1).

Task 8.3 Employment of the Project Manager and researchers is to begin in Month 2. The Project Manager, in
collaboration with the Project Management Steering Committee, will be responsible for setting up (book rooms,
equipment, marketing and advertising, etc.) the first of a series of academies on project management, grant writing
and other areas of applying for research funding that will inform, strengthen and empower researchers at
TarguMures and beyond in order that research capacity is increased.
Task 8.4 The Project Manager will explore and identify other avenues of research funding that will enable successful
capacity building within the institution. Our planned strategy is that we will target, in particular, other FP7 and
Horizon 2020 projects in similar areas, using the expertise of our PI and international collaborators (month 3
onwards).

Tasks 8.5-8.6 Project scientific management via weekly local meetings, monthly Skype and tri-monthly face-to-face
meetings to ensure continuity and progression in all elements (month 1 onwards).

Assimilation of data: This will take place at the end of each specific WP-sub activity and a general and final
assimilation of results will take place over the final two months of the project.

Ethics:The study has been approved by the local animal experimentation ethics committees (e.g. Ref: DAAM-6991,
CEEA, UB). All procedures will be carried out in accordance with approved local/European guidelines/legislation
concerning the protection of animals used for experimental and other scientific purposes and the European
Commission Council Directive 86/609/EEC on this subject. All experimental protocols have been approved by the
above authorities.