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Stenodactilina e lanceolina: due nuove potenti “ribosome-inactivating proteins” bicatenarie. Purificazione, caratterizzazione chimico-fisica e studio dei meccanismi citotossiciBortolotti, Massimo <1978> 03 June 2008 (has links)
Two lectins, called lanceolin and stenodactylin, were purified by affinity chromatography on CL
Sepharose 6B from the caudices of the Passifloraceae Adenia lanceolata and Adenia stenodactyla,
respectively. They are glycoproteins with Mw of 61,243 (lanceolin) and 63,131 daltons
(stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties,
with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from Adenia
volkensii. These two lectins agglutinate red blood cells, inhibit protein synthesis in a cell-free
system as well as in whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A).
They are highly toxic to cells, in which they induce apoptosis and strongly inhibit protein synthesis,
and to mice, with LD50s 8.16 mg/kg (lanceolin) and 2.76 mg/kg (stenodactylin) at 48 hours after
administration.
Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosomeinactivating
proteins (RIPs).
Further experiments were conducted in order to clarify the effects of these RIPs in cells. We
investigated the cronological relationship between cytotoxic activity, indirectly evaluated as
inhibition of protein synthesis, and loss of cell viability in NB100 cell line.
The induction of apoptosis was assessed by determining caspases 3 and 7 levels, which increase
8-16 hours earlier than the beginning of protein synthesis inhibition. This suggest that the arrest of
protein synthesis is not a central event in the pathway of cell poisoning by RIPs.
The high toxicity and the induction of cell death only by apoptosis and not by necrosis in two
muscular cell lines (TE671 and RD/18) suggest that lanceolin and stenodactylin may be potential
candidates for experimental chemoablation in strabism and blepharospasm.
These results show that lanceolin and stenodactylin are amongst the most potent toxins of plant
origin.
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Studi di espressione genica in linfociti T di soggetti di diversa etàPierini, Michela <1978> 03 June 2008 (has links)
No description available.
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Metodi molecolari nello studio della variabilità genetica di Tuber borchiiBonuso, Enrico <1980> 11 April 2008 (has links)
Tuber borchii (Ascomycota, order Pezizales) is highly valued truffle sold in local
markets in Italy. Despite its economic importance, knowledge on its distribution and
population variation is scarce. The objective of this work was to investigate the
evolutionary forces shaping the genetic structure of this fungus using coalescent and
phylogenetic methods to reconstruct the evolutionary history of populations in Italy. To
assess population structure, 61 specimens were collected from 11 different Provinces of
Italy. Sampling was stratified across hosts and habitats to maximize coverage in native
oak and pine stands and both mychorrizae and fruiting bodies were collected. Samples
were identified considering anatomo-morphological characters. DNA was extracted and
both multilocus (AFLP) and single-locus (18 loci from rDNA, nDNA, and mtDNA)
approaches were used to look for polymorphisms. Screening AFLP profiles, both
Jaccard and Dice coefficients of similarity were utilized to transform binary matrix into
a distance matrix and then to desume Neighbour-Joining trees. Though these are only
preliminary examinations, phylogenetic trees were totally concordant with those
deriving from single locus analyses. Phylogenetic analyses of the nuclear loci were
performed using maximum likelihood with PAUP and a combined phylogenetic
inference, using Bayesian estimation with all nuclear gene regions, was carried out. To
reconstruct the evolutionary history, we estimated recurrent migration, migration across
the history of the sample, and estimated the mutation and approximate age of mutations
in each tree using SNAP Workbench. The combined phylogenetic tree using Bayesian
estimation suggests that there are two main haplotypes that are difficult to be
differentiated on the basis of morphology, of ecological parameters and symbiontic tree.
Between these two lineages, that occur in sympatry within T. borchii populations, there
is no evidence of recurrent migration. However, migration over the history of the
sample was asymmetrical suggesting that isolation was a result of interrupted gene flow
followed by range expansion. Low levels of divergence between the haplotypes indicate
that there are likely to be two cryptic species within the T. borchii population sampled.
Our results suggest that isolation between populations of T. borchii could have led to
reproductive isolation between two lineages. This isolation is likely due to sympatric
speciation caused by a multiple colonization from different refugia or a recent isolation.
In attempting to determinate whether these haplotypes represent separate species or a
partition of the same species we applied Biological and Mechanistic species Concepts.
Notwithstanding, further analyses are necessary to evaluate if selection favoured premating
or post-mating isolation.
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Mezzi innovativi nella difesa delle cucurbitacee da Fusarium solani f.sp. cucurbitaeSigala, Cristian <1974> 11 April 2008 (has links)
Lo studio “Lotta biologica a Fusarium solani f.sp. cucurbitae su
zucchino” si colloca nell’ambito della difesa integrata delle colture
orticole dalle fitopatie fungine, in particolare quelle causate da patogeni ad
habitat terricolo nei confronti dei quali è sempre più frequente il ricorso a
mezzi di lotta diversi dai prodotti chimici.
Interessante e innovativa appare la prospettiva di utilizzare microrganismi
adatti a svilupparsi nel suolo, competenti per la rizosfera delle piante e con
spiccate caratteristiche d’antagonismo verso i patogeni tellurici e di
stimolazione delle difese sistemiche della pianta.
Il marciume del colletto delle cucurbitacee, causato da diversi patogeni tra
cui Fusarium solani f.sp. cucurbitae, rappresenta la principale malattia
fungina di tipo tellurica che colpisce lo zucchino ed il melone nella
Pianura Padana e che può portare a consistenti perdite produttive. Indagini
condotte dal 2004 da parte del Diproval nell’areale bolognese, hanno
evidenziato un’elevata frequenza del patogeno su zucchino coltivato
soprattutto in tunnel.
Considerata la carenza di conoscenze locali di F. solani f.sp. cucurbitae e
di mezzi chimici di lotta efficaci, la ricerca svolta ha inteso approfondire la
diagnosi della malattia e le caratteristiche biologiche degli isolati locali, e
valutare la possibilità di utilizzare metodi biologici di lotta contro questo
patogeno, nonché di studiare alcune delle possibili modalità d’azione di
microrganismi antagonisti.
Sono state pertanto prelevate, da zone diverse del Bolognese, campioni di
piante di zucchino che presentavano sintomi di marciume del colletto ed è
stato isolato il patogeno, che in base alle caratteristiche morfologiche
macro e microscopiche, alle prove di patogenicità su diversi ospiti e a
saggi biomolecolari, è stato identificato come Fusarium solani f. sp.
cucurbitae W.C. Snyder & H.N. Hansen razza 1.
Dagli isolati di campo sono state realizzate un centinaio di colture
monosporiche venti delle quali sono state utilizzate per la prosecuzione
delle prove.
I venti ceppi sono stati saggiati per la loro patogenicità inoculandoli in
terriccio sterile e con trapianto di giovani piante di zucchino. E’ risultata
un’elevata variabilità del livello di virulenza tra i ceppi, stimata da 39% a
83% riguardo la gravità della malattia e da 61 a 96% per la frequenza di
malattia. Sono state condotte prove di accrescimento miceliare che hanno
evidenziato differenze tra i ceppi e tra gli esperimenti condotti a tre diverse
temperature (17, 23 e 28°C) alla luce ed al buio. La crescita maggiore
complessivamente è stata ottenuta a 23°C. I venti ceppi hanno anche
mostrato di produrre, in vitro, vari livelli di enzimi di patogenesi quali
cellulasi, poligalatturonasi, pectin liasi e proteasi. E’ stata evidenziata
unan correlazione significativa tra attività cellulasica e pectin liasica con
frequenza e gravità della malattia dei venti ceppi del patogeno.
Le prove relative al contenimento della malattia sono state condotte in
cella climatica. Sono stati considerati prodotti commerciali (Remedier,
Rootshield, Cedomon, Mycostop, Proradix, Clonotry) a base
rispettivamente dei seguenti microrganismi: Trichoderma harzianum
ICC012 + T. viride ICC080, T. harzianum T22, Pseudomonas
chlororaphis MA342, Streptomyces griseoviridis K61, P. fluorescens
proradix DSM13134 e T. harzianum + Clonostachys rosea). I prodotti
sono stati somministrati sul seme, al terreno e su seme+terreno
(esperimenti 1 e 2) e in vivaio, al trapianto e vivaio+trapianto (esperimenti
3 e 4), riproducendo situazioni di pratico impiego. L’inoculazione del
patogeno (un ceppo ad elevata patogenicità, Fs7 ed uno a bassa
patogenicità, Fs37) è stata effettuata nel terreno distribuendo uno sfarinato
secco di semi di miglio e cereali colonizzati dal patogeno. La malattia è
stata valutata come intensità e gravità. I prodotti sono stati impiegati in
situazioni di particolare stress, avendo favorito particolarmente la crescita
del patogeno. Complessivamente i risultati hanno evidenziato effetti di
contenimento maggiore della malattia nel caso del ceppo Fs37, meno
virulento. La malattia è stata ridotta quasi sempre da tutti i formulati e
quello che l’ha ridotta maggiormente è stato Cedomon. Il contenimento
della malattia somministrando i prodotti solo nel terreno di semina o di
trapianto è stato in generale quello più basso. Il contenimento più elevato è
stato ottenuto con la combinazione di due tipologie di trattamento,
seme+terreno e vivaio+trapianto. Le differenze tra i prodotti sono risultate
più evidenti nel caso del ceppo Fs7.
Per quanto riguarda lo studio di alcune delle modalità d’azione dei
microrganismi contenuti nei formulati più efficaci, è stato verificato che
tutti sono stati in grado di inibire, se pur in vario modo, la crescita del
patogeno in vitro. Gli antagonisti più efficaci sono stati S. griseoviridis
K61 (Mycostop) e P. fluorescens proradix DSM13134). I ceppi di
Trichoderma, ed in particolare T.harzianum T22 (Rootshield), sono
risultati i più attivi colonizzatori del substrato. Riguardo il fenomeno
dell’antibiosi, il batterio P. fluorescens proradix DSM13134 ha mostrato
di produrre i metaboliti non volatili più efficaci nel ridurre lo sviluppo del
patogeno. Nelle condizioni sperimentali adottate anche i due ceppi di T.
viride ICC080 e T. harzianum ICC012 hanno dimostrato di produrre
metaboliti efficaci. Tali risultati, anche se relativi a prove in vitro, possono
contribuire alla comprensione dei meccanismi dei microrganismi sul
contenimento dell’infezione relativamente al rapporto diretto sul patogeno.
E’ stato inoltre verificato che tutti i microrganismi saggiati sono dotati di
competenza rizosferica e solo i batteri di endofitismo.
Si conclude che, nonostante l’elevata pressione infettiva del patogeno che
ha certamente influito negativamente sull’efficacia dei microrganismi
studiati, i microrganismi antagonisti possono avere un ruolo nel ridurre
l’infezione di F. solani f.sp. cucurbitae razza 1.
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Enzimi, acidi organici ed altri metaboliti coinvolti nella patogenesi di Penicillium sppDonati, Irene <1977> 11 April 2008 (has links)
Blue mould caused by Penicillium expansum Link is one of the most destructive
rot of pome fruit in all growing areas (Snowdon, 1990; Jones and Aldwinckle, 1991;
Tonini,1996) In the past, Penicillium rot has been controlled by fungicide postharvest
treatment mainly by thiabendazole (TBZ) and benomyl (Hardenburg and Spalding,
1972), but their intense use produced the appearance of resistant strains with a
great reduction of their activity
The aims of the present study were to characterize the isolates of Pencillium sp
causing blue mold on pear in Italy by physiological and biochemical parameters.
In particular differencing also the behavior of isolates to relationship with sensitivity
or resistance to TBZ treatments.
We have examined the early stage of infection in relation to enzyme activity, local
modulation of pH, production of organic acids, and to secondary metabolism of
pathogen.
The results described here confirm that the majority of P. expansum isolates from
pears packing houses are resistant to TBZ,
Among the TBZ-resistant isolates scored in this work, different isolates (RR) showed
higher percentage of conidial germination on TBZ-amended medium compared to non
amended medium. This may indicate a stimulatory effect of TBZ on conidial
germination. Therefore TBZ treatments are not only ineffective for controlling P.
expansum, but they may also increase the severity of blue mould on fruits.
In the absence of fungicide, isolates showed a significant difference for infection
severity, R and RR isolates are characterized by higher pathogenic fitness on fruits,
producing larger lesions than S isolates. These data are supported by the study with
laboratory-induced resistant isolates, which shows the lack of correlation between
TBZ resistance and osmotic sensitivity, and highlights the association between TBZ
resistance and infection severity (Baraldi et al 2003).
Enzymatic screening gave a positive reaction to esterase, urease, pectinase activity,
in addition, the pathogen is able to synthesize a complex enzyme act to degrade the
main components of the cell wall especially pectin and cellulose.
Isolated sensitive and resistant are characterized by a good activity of pectinase,
especially from poligactoronase, which, as already reported by several studies
(D'hallewin et al, 2004; Prusky et al, 2004), are the basis of degradative process of
cell wall. Also, although the measure was minor also highlighted some activities of
cellulase, but even note in the production of this kind of cellulase and hemicellulase
P. Expansum were not targeted, studies have found no other source of information in
this regard.
Twenty isolates of Penicillium expansum, were tested in vitro ad in vivo for acid
production ability and pH drop.
We have found that modulation of pH and the organic acids extrusion were influence
to various parameter:
Initial pH: in general, the greatest reduction of pH was observed in isolates
grown at pH 7, except for four isolates that maintained the pH of the medium
close to 7, the others significantly decreased the pH, ranging from 5.5 to 4.1..
In extreme acid condition (pH 3,0) growth and modulation of pH is most lower
respect optimal condition (pH 5,0). Also isolates R and RR have showed a
greater adaptation to environmental condition more than isolates S.
Time: although the acidification continues for some days, PH modulation is
strongest in early hours (48-72 hours)of inoculation process. Time also affects
the quality of organic acids, for example in vitro results showed an initial
abundant production of succinc acid, followed to important production of
galacturoinc acid.
Substrates: there are many differences for the type of acids produced in vitro
and in vivo. Results showed in vivo an abundant production of galacturonic,
malic, and citric acids and some unknown organic acids in smaller
concentrations.
Secondary metabolite analysis revealed intra-specific differences, and patulin was
found in all isolates, but most significant reduction was observed between in vitro and
in vivo samples.
There was no correlation between the concentration of patulin, and the percentage of
infected fruits, but sample with a lower infection severity of rotten area than the
others, showed a significantly lower mycotoxin concentration than samples with a
higher lesion diameter of rotten area. Beyond of patulin was detected the presence
of another secondary metabolite, penitrem A.
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Risposte di resistenza a batteri fitopatogeni di importanti specie coltivate indotte da molecole segnale di diversa naturaBiondi, Enrico <1977> 11 April 2008 (has links)
A modern management of crop protection should be based on integrated control programmes, including the use of environmentally safe products. Antagonistic/beneficial bacteria and resistance inducers may have a great potential in the prophylaxis of diseases caused by common and quarantine pathogens. This work was carried out to confirm the ability of the known strain IPV-BO G19 (Pseudomonas fluorescens) against fire blight (Erwinia amylovora), as well as to evaluate their efficacy against southern bacterial wilt of tomato (Ralstonia solanacearum) and grapevine crown gall (Agrobacterium vitis). A virulent strain of R. solanacearum race 3 was inhibited by the antagonist on plate. When the pathogen was inoculated 48 h after their application to the root apparatus of tomato plants grown in a climatic chamber, bacterial wilt progression rate was clearly reduced. Moreover the defence response evoked by IPV-BO G19 was studied in tomato plants by monitoring the transcription of genes codifying for three PRs as PR-1a, PR-4, PR-5 and for an intracellular chitinase using multiplex RT-PCR and Real Time RT-PCR. In two field trials during 2005 and 2006, the strain IPV-BO G19 was compared with biofungicides and some abiotic elicitors to protect actively growing shoots of pear scions against fire blight. In both trials, IPV-BO G19 plus Na-alginate gave a high level of protection, three weeks after wound inoculation with E. amylovora. In pear leaf tissues treated with the antagonistic strain IPV-BO G19, catalase, superoxyde dismutase and peroxidise activity was evaluated as markers of induced resistance. The IPV-BO G19 strain was compared with other bioagents and resistance inducers to prevent grapevine crown gall under glasshouse and vineyard conditions.
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Induzione di resistenza agli agenti della fusariosi della spiga del frumento duro attraverso applicazioni di elicitori chimiciTonti, Stefano <1978> 27 April 2009 (has links)
No description available.
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The Integrated European Project "GEHA - GEnetics of Healthy Aging": recruitment, health status assessment and survival of the Italian 90+ sibpairsCevenini, Elisa <1979> 08 June 2009 (has links)
The present study is part of the EU Integrated Project “GEHA – Genetics of Healthy Aging” (Franceschi C et al., Ann N Y Acad Sci. 1100: 21-45, 2007), whose aim is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced age in good cognitive and physical function and in the absence of major age-related diseases.
Aims
The major aims of this thesis were the following:
1. to outline the recruitment procedure of 90+ Italian siblings performed by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS).
The procedures related to the following items necessary to perform the study were described and commented: identification of the eligible area for recruitment, demographic aspects related to the need of getting census lists of 90+siblings, mail and phone contact with 90+ subjects and their families, bioethics aspects of the whole procedure, standardization of the recruitment methodology and set-up of a detailed flow chart to be followed by the European recruitment centres (obtainment of the informed consent form, anonimization of data by using a special code, how to perform the interview, how to collect the blood, how to enter data in the GEHA Phenotypic Data Base hosted at Odense).
2. to provide an overview of the phenotypic characteristics of 90+ Italian siblings recruited by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The following items were addressed: socio-demographic characteristics, health status, cognitive assessment, physical conditions (handgrip strength test, chair-stand test, physical ability including ADL, vision and hearing ability, movement ability and doing light housework), life-style information (smoking and drinking habits) and subjective well-being (attitude towards life). Moreover, haematological parameters collected in the 90+ sibpairs as optional parameters by the Bologna and Rome recruiting units were used for a more comprehensive evaluation of the results obtained using the above mentioned phenotypic characteristics reported in the GEHA questionnaire.
3. to assess 90+ Italian siblings as far as their health/functional status is concerned on the basis of three classification methods proposed in previous studies on centenarians, which are based on:
• actual functional capabilities (ADL, SMMSE, visual and hearing abilities) (Gondo et al., J Gerontol. 61A (3): 305-310, 2006);
• actual functional capabilities and morbidity (ADL, ability to walk, SMMSE, presence of cancer, ictus, renal failure, anaemia, and liver diseases) (Franceschi et al., Aging Clin Exp Res, 12:77-84, 2000);
• retrospectively collected data about past history of morbidity and age of disease onset (hypertension, heart disease, diabetes, stroke, cancer, osteopororis, neurological diseases, chronic obstructive pulmonary disease and ocular diseases) (Evert et al., J Gerontol A Biol Sci Med Sci. 58A (3): 232-237, 2003).
Firstly these available models to define the health status of long-living subjects were applied to the sample and, since the classifications by Gondo and Franceschi are both based on the present functional status, they were compared in order to better recognize the healthy aging phenotype and to identify the best group of 90+ subjects out of the entire studied population.
4. to investigate the concordance of health and functional status among 90+ siblings in order to divide sibpairs in three categories: the best (both sibs are in good shape), the worst (both sibs are in bad shape) and an intermediate group (one sib is in good shape and the other is in bad shape). Moreover, the evaluation wanted to discover which variables are concordant among siblings; thus, concordant variables could be considered as familiar variables (determined by the environment or by genetics).
5. to perform a survival analysis by using mortality data at 1st January 2009 from the follow-up as the main outcome and selected functional and clinical parameters as explanatory variables.
Methods
A total of 765 90+ Italian subjects recruited by UNIBO (549 90+ siblings, belonging to 258 families) and ISS (216 90+ siblings, belonging to 106 families) recruiting units are included in the analysis. Each subject was interviewed according to a standardized questionnaire, comprising extensively utilized questions that have been validated in previous European studies on elderly subjects and covering demographic information, life style, living conditions, cognitive status (SMMSE), mood, health status and anthropometric measurements. Moreover, subjects were asked to perform some physical tests (Hand Grip Strength test and Chair Standing test) and a sample of about 24 mL of blood was collected and then processed according to a common protocol for the preparation and storage of DNA aliquots.
Results
From the analysis the main findings are the following:
- a standardized protocol to assess cognitive status, physical performances and health status of European nonagenarian subjects was set up, in respect to ethical requirements, and it is available as a reference for other studies in this field;
- GEHA families are enriched in long-living members and extreme survival, and represent an appropriate model for the identification of genes involved in healthy aging and longevity;
- two simplified sets of criteria to classify 90+ sibling according to their health status were proposed, as operational tools for distinguishing healthy from non healthy subjects;
- cognitive and functional parameters have a major role in categorizing 90+ siblings for the health status;
- parameters such as education and good physical abilities (500 metres walking ability, going up and down the stairs ability, high scores at hand grip and chair stand tests) are associated with a good health status (defined as “cognitive unimpairment and absence of disability”);
- male nonagenarians show a more homogeneous phenotype than females, and, though far fewer in number, tend to be healthier than females;
- in males the good health status is not protective for survival, confirming the male-female health survival paradox;
- survival after age 90 was dependent mainly on intact cognitive status and absence of functional disabilities;
- haemoglobin and creatinine levels are both associated with longevity;
- the most concordant items among 90+ siblings are related to the functional status, indicating that they contain a familiar component. It is still to be investigated at what level this familiar component is determined by genetics or by environment or by the interaction between genetics, environment and chance (and at what level).
Conclusions
In conclusion, we could state that this study, in accordance with the main objectives of the whole GEHA project, represents one of the first attempt to identify the biological and non biological determinants of successful/unsuccessful aging and longevity. Here, the analysis was performed on 90+ siblings recruited in Northern and Central Italy and it could be used as a reference for others studies in this field on Italian population. Moreover, it contributed to the definition of “successful” and “unsuccessful” aging and categorising a very large cohort of our most elderly subjects into “successful” and “unsuccessful” groups provided an unrivalled opportunity to detect some of the basic genetic/molecular mechanisms which underpin good health as opposed to chronic disability. Discoveries in the topic of the biological determinants of healthy aging represent a real possibility to identify new markers to be utilized for the identification of subgroups of old European citizens having a higher risk to develop age-related diseases and disabilities and to direct major preventive medicine strategies for the new epidemic of chronic disease in the 21st century.
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Studies on biotic and abiotic elicitors inducing defense responses in tomatoLancioni, Pietro <1980> 27 April 2009 (has links)
Tomato (Lycopersicon esculentum Mill., Solanum lycopersicon L.) is one of the most popular vegetable throughout the world, and the importance of its cultivation
is threatened by a wide array of pathogens. In the last twenty years this plant has been successfully used as a model plant to investigate the induction of defense
pathways after exposure to fungal, bacterial and abiotic molecules, showing triggering of different mechanisms of resistance. Understanding these mechanisms in order to improve crop protection is a main goal for Plant Pathology.
The aim of this study was to search for general or race-specific molecules able to determine in Solanum lycopersicon immune responses attributable to the main
systems of plant defense: non-host, host-specific and induced resistance. Exopolysaccharides extracted by three fungal species (Aureobasidium pullulans, Cryphonectria parasitica and Epicoccum purpurascens), were able to
induce transcription of pathogenesis-related (PR) proteins and accumulation of enzymes related to defense in tomato plants cv Money Maker,using the chemical inducer Bion® as a positive control. During the thesis, several Pseudomonas spp. strains were also isolated and tested for their antimicrobial activity and ability to produce antibiotics. Using as a positive control jasmonic acid, one of the selected strain was shown to induce a form of systemic resistance in tomato. Transcription of PRs and reduction of
disease severity against the leaf pathogen Pseduomonas syringae pv. tomato was determined in tomato plants cv Money Maker and cv Perfect Peel, ensuring no direct contact between the selected rhizobacteria and the aerial part of the plant. To conclude this work, race-specific resistance of tomato against the leaf mold Cladosporium fulvum is also deepened, describing the project followed at the Phytopathology Laboratory of Wageningen (NL) in 2007, dealing with localization of a specific R-Avr interaction in transfected tomato protoplast cultures through fluorescence microscopy.
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Preclinical development of cancer vaccinesPalladini, Arianna <1978> 05 May 2009 (has links)
Triplex cell vaccine is a cancer immunopreventive cell vaccine that can prevent almost completely mammary tumor onset in HER-2/neu transgenic mice. A future translation of cancer immunoprevention from preclinical to clinical studies should take into account several aspects. The work reported in this thesis deals with the study of three of these aspects: vaccine schedule, activity in a therapeutic set-up and second-generation DNA vaccines. An important element in determining human acceptance and compliance of a
treatment protocol is the number of vaccinations. In order to improve the vaccination schedule a minimal protocol was searched, i.e. a schedule consisting of a lower number of administrations than standard protocol but with a similar efficacy. A candidate optimal protocol was identified by the use of an in silico model, SimTriplex simulator. The in vivo test of this schedule in HER-2/neu transgenic mice only partially confirmed in silico predictions. This result shows that in silico models have the potential ability to aid in searching of optimal treatment protocols, provided that they will be further tuned on experimental data. As a further result this preclinical study highlighted that kinetic of antibody response plays a major role in determining cancer prevention, leading to the hypothesis of a threshold that must be reached rapidly and maintained lifetime. Early clinical trials would be performed in a therapeutic, rather than preventive, setting. Thus, the activity of Triplex vaccine was investigated against experimental lung metastases in HER-2/neu transgenic mice in order to evaluate if the immunopreventive Triplex vaccine could be effective also against a pre-existing tumor mass. This preclinical model of aggressive metastatic development showed that the vaccine was an efficient treatment also 4 for the cure of micrometastases. However the immune mechanisms activated against tumor mass were not antibody dependent, i.e. different from those preventing the onset of primary mammary carcinoma. DNA vaccines could be more easily used than cellular ones. A second generation of Triplex vaccine based on DNA plasmids was evaluated in an
aggressive preclinical model (BALBp53neu female mice) and compared with the preventive ability of cellular Triplex vaccine. It was observed that Triplex DNA vaccine was as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.
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