RIASSUNTI DEI PROGETTI DI RICERCA DEL CORSO DI … · 1.Beltrame Monica: Deciphering the molecular...

RIASSUNTI DEI PROGETTI DI RICERCA DEL CORSO DI

DOTTORATO IN BIOLOGIA MOLECOLARE E CELLULARE

A.A. 2014-15 (XXX CICLO)

1.Beltrame Monica: Deciphering the molecular network involving the transcription factor Sox18 in blood

vascular and lymphatic development

2. Berruti Giovanna: Zebrafish as animal model to study in vivo the ESCRT-DUB USP8

3.Bertoni Giovanni Detailing of small RNA-based regulatory networks by parallel transcriptomic-proteomic profiling 4.Bolognesi Martino: Structural-Based Drug Discovery against RNA-based VIRUSES

5. Cappelletti Graziella, Francesco Demartin (Co-Tutor): Dissecting microtubule dynamics at the synapse 6.Caretti Giuseppina: Role of the SMYD3 methylase in embryonic stem cells differentiation and zebrafish development

7.Cattaneo Elena: Stem cells in human neurodegenerative disorders

8.Colombo Lucia: Network controlling seed size and quality

9. Fornara Fabio: Molecular control of flowering in rice

10. Gissi Carmela: Evolutionary dynamics of nuclear genes involved in replication and repair of the mitochondrial genome in fast-evolving chordates

11.Gnesutta Nerina: NF-Y partners in the regulation of cell cycle promoters

12.Guerrini Luisa: Analysis of the molecular mechanisms at the basis of thalidomide-induced limb defects in zebra fish embryos

13. Kater Martin: Yield Increase in Rice by Altering Inflorescence Architecture

14.Landini Paolo: Identification of RNA thermosensors in the opportunistic pathogens Pseudomonas

aeruginosa and Escherichia coli.

15.Lazzaro Federico: Role of Translesion Synthesis (TLS) polymerases in rNMPs incorporation during DNA replication

16. Mantovani Roberto: NF-YA isoforms in ES cells and cancer

17. Marini Federica: DAXX protein and the human DNA damage response: chromatin remodeling, gene expression, genome stability and tumorigenesis

18.Messina Graziella: Role of the anthocyanins in slowing down the progression of Muscular Dystrophies

19.Moroni Anna: HCN channel dysfunction in Alzheimer's disease

20.Muzi Falconi Marco: Function and regulation of nuclease activities in the maintenance of genome

stability

21.Nardini Marco: Structural analysis of transcription factor/DNA complexes

22. Pellicioli Achille: Targeting Slx4-Esc4 complex to prevent adaptation to DNA damage, genetic instability

and cancer

23. Petroni Katia: Role of anthocyanin-enriched diet on cardioprotection

24.Pesaresi Paolo: FRUIT of the Plastid: a new strategy to uncover genes involved in fruit development and

ripening

25.Ricagno Stefano: Amyloid Light-chain amyloidosis: uncovering the molecular bases of cardiotoxicity

26.Riva Paola: Molecular mechanisms involved in phenotype expression variability of Neurofibromatosis

type 1

27.Soldà Giulia: Identification of genetics and molecular bases of inherited sensorineural hearing loss by whole-exome sequencing

28.Tonelli Chiara/Conti Lucio: Site of ABA action in flowering

29.Zuccato Chiara: A molecular study of the impact of synaptic dysfunction in Huntington’s Disease

Project leader: Monica Beltrame ([email protected])

Department of Biosciences, University of Milan

RESEARCH PROJECT SUMMARY

Deciphering the molecular network involving the transcription factor Sox18 in blood vascular

and lymphatic development

Our group is interested in gene expression regulation during embryonic development in vertebrates.

We are studying a family of transcription factors, the Sox (Sry-related HMG box) proteins, which are found

throughout the animal kingdom and play key roles in embryonic development. Mutations in several Sox

genes have been shown to result in developmental anomalies, from fly to mammals. The emerging picture

of SOX transcription factors is one of tissue-specific switches that induce changes in gene expression

required for cell-type specification or differentiation.

Our interest is currently centered on SOX18, which is mutated in patients affected by the

Hypotrichosis-Lymphedema-Telangiectasia syndrome. SOX18 is transiently expressed in the endothelial

component of nascent blood and lymphatic vessels during embryonic development and in adult life, when

neovascularization occurs.

Despite its relevance, relatively little is known about upstream factors and downstream targets

involving SOX18 in endothelial cell differentiation and vascular development. We are addressing these

questions using the zebrafish model system, as it provides several advantages over other vertebrate model

organisms for the in vivo imaging of the vascular system and for the genetic or experimental manipulation

of vascular development. We have shown that Sox18 and the closely related Sox7 protein (both belonging

to the SoxF group) play redundant roles in arterio-venous differentiation of endothelial cells in zebrafish,

while only Sox18 plays a conserved role in lymphatic development (Cermenati et al., 2008, Blood;

Cermenati et al. 2013, ATVB). Gene expression profiling, at key developmental stages, under conditions of

perturbed SoxF protein expression will serve as a basis to shed light on the molecular networks controlling

blood vascular and lymphatic development. Loss-of-function and gain-of-function approaches in specific

transgenic lines will be used to assess the functional relevance of interesting genes, whose expression is

modified when SoxF proteins are perturbed. Given the pathological relevance of

angiogenesis/lymphangiogenesis and lymphatic dysfunction in humans, the identification of new players

might open up new therapeutic perspectives.

Project leader: Giovanna Berruti ([email protected])



Zebrafish as animal model to study in vivo the ESCRT-DUB USP8

Mouse USP8, originally termed UBPy (1), is an Ub-specific protease highly expressed in the nervous system

and male germ cells (1-3). It works as a regulator of the down-regulation of tyrosine kinase receptors at the

endosomal compartment so that it is known also as ESCRT-DUB (4). mUSP8 knock-out/USP8 conditional

mutants are lethal (5) so that no in vivo functional study founded on the targeted disruption of the Usp8

gene has been till now possible. Interestingly, in the wobbler mouse, a spontaneous mutant whose

phenotype is due to a point mutation in the Vps54 gene (6) and used as animal model to study ALS (3) and

globozoospermia (7), the cross-talk Vps54-USP8 is hampered. Usp8 gene is conserved in human,

chimpanzee, dog, cow, mouse, rat, chicken, frog, zebrafish, fruit fly and C.elegans. The embryo lethality of

USP8-KO in the mouse, nothwithstading this expresses more than 90 DUBs, may indicate that USP8 has an

unrecoverable role in the development. The zebrafish (Danio rerio) system with its morpholino (MO)

technology and/or KO techniques is suited for easily studying the loss-of-function phenotype in vivo. It

could represent the model of election for studying gene products as USP8. In our Department at the

University of Milan, we can dispose of the Zebrafish Laboratory, directed by Prof. F. Cotelli, with the

relative facility and researchers of excellent expertise. With the present project, we propose to investigate

about the role of USP8 in vivo by exploiting zebrafish as animal model.

References

1. Gnesutta N, Ceriani M, Innocenti M. et al. (2001). Cloning and Characterization of Mouse UBPy, a

Deubiquitinating Enzyme That Interacts with the Ras Guanine Nucleotide Exchange Factor CDC25Mm/Ras-

GRF1. J. Biol. Chem. 276, 39448-39454.

2. Berruti G, Ripolone M, Ceriani M. (2010). USP8, a regulator of endosomal sorting, is involved in mouse

acrosome biogenesis through interaction with the spermatid ESCRT-0 complex and microtubules. Biol.

Reprod. 82, 930-939.

3. Paiardi C, Pasini M, Amadeo A, Gioria M and Berruti G. (2013). The ESCRT-deubiquitinating enzyme USP8

in the cervical spinal cord of wild-type and Vps54-recessive (wobbler) mutant mice. Histochem Cell Biol

141, 57-73.

4. Wright MH, Berlin I, Nash PD (2011) Regulation of endocytic sorting by ESCRT-DUB-mediated

deubiquitination. Cell Biochem Biophys 60, 39–46

5. Niendorf S, Oksche A, Kisser A, et al (2007) Essential role of USP8 for receptor tyrosine kinase stability and endocytic trafficking in vivo. Mol Cell Biol 27: 5029–39 6. Schmitt-John T, Drepper C, Mussmann A, et al (2005) Mutation of Vps54 causes motor neuron disease

and defective spermiogenesis in wobbler mouse. Nat Genet 37, 1213–1215

7. Paiardi C, Pasini M, Berruti G (2011) Failure of acrosome formation and globozoospermia in the wobbler mouse, a Vps54 spontaneous recessive mutant. Spermatogenesis 1, 52-62.

Project leader: Giovanni Bertoni ([email protected])



Detailing of small RNA-based regulatory networks by parallel transcriptomic-proteomic profiling

Pseudomonas aeruginosa is an important opportunistic pathogen in immune-compromised and cystic fibrosis patients responsible for numerous acute and chronic infections. Crucial traits contributing to pathogenicity of P. aeruginosa are the production of a large assortment of virulence factors, biofilm formation and the ability to rapidly develop resistance to multiple classes of antibiotics. Expression of these traits is fine-tuned by a dynamic and intricate regulatory network [1], in which more than 50 regulatory proteins play key roles as transcription regulators. On the contrary, the involvement in this context of small RNAs (sRNAs), important regulatory molecules acting post-transcriptionally on target mRNAs and/or via interactions with proteins, has been studied to a lesser extent.

Preliminary results suggest that four novel P. aeruginosa sRNAs, which were identified in the

proponent lab [2], are involved in the regulation of virulence traits in response to infection-relevant stimuli.

In actual fact, overexpression of these sRNAs was shown to influence the expression of P. aeruginosa

virulence descriptors such as motility, biofilm formation, secretion of proteases, toxic secondary

metabolites and siderophores. In addition, it was observed that the expression of these sRNAs can be

responsive to temperature shift from room to body temperature, oxygen availability, iron limitation,

envelope stressors and quorum sensing, a system of stimulus and response correlated to bacterial cell

density that coordinate the expression of several virulence genes. Focusing on these four sRNAs, the main

aim of this research project is to unravel the cognate regulons, i.e. the set of genes which can be both

direct and indirect targets of the sRNA-mediated regulation. This aim will be accomplished combining

quantitative proteomics with transcriptomics. For virulence-relevant direct targets, the sRNA/target mRNA

interaction will be characterized.

References

1. Balasubramanian D, Schneper L, Kumari H & Mathee K (2013) A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res, 41:1-20.

2. Ferrara S, Brugnoli M, De Bonis A, Righetti F, Delvillani F, Dehò G, Horner D, Briani F & Bertoni G (2012)

Comparative profiling of Pseudomonas aeruginosa strains reveals differential expression of novel unique and conserved small RNAs. PLoS One, 7:e36553.

Project leader: Martino Bolognesi ([email protected])



Structural-Based Drug Discovery against RNA-based VIRUSES

The number of fatalities that occur worldwide as the result of infections by RNA viruses is in the order of many millions annually. Over the past 3 decades, many RNA virus threats were identified or “re-discovered”, including a variety of pathogenic flaviviruses. The flavivirus group includes several pathogens of global medical importance, i.e. dengue viruses (DENV). Infections by these neurotropic viruses may result in life-threatening aseptic encephalitis, with high risk of life-long debilitating neurologic sequels. There is an increasing number of cases (about 50-100 million every year) as the disease spreads to new areas, causing severe outbreaks. The threat of a possible outbreak of dengue fever now exists in Europe and local transmission of dengue, in addition to several imported cases, was reported for the first time in 2010. Vaccine development has proven particularly challenging because of the existence of 4 dengue serotypes and antibody dependent enhancement of infection. Hence neither vaccines nor antiviral therapy (or prophylaxis) are available today. We urgently need advanced levels of preparedness with which to confront and ultimately control these viral pathogens.

The infective enveloped flavivirus contains a (+) single-stranded RNA genome encoding 7 non-structural proteins involved in viral replication. The non-structural proteins NS3 and NS5 play key roles in the replication cycle, therefore are ideal antiviral targets. For discovering small-molecule inhibitors of viral replication, our approach relies on the study of the three-dimensional structure of target proteins via X-ray crystallography and other biochemical/biophysical techniques (“structure-based drug discovery”). Practical lab activities, which will be the basis for the targeted studies, but also the training grounds for the candidate are: recombinant protein production and purification, biophysical studies (e.g. dynamic light scattering, spectroscopies, …), biochemical assays (e.g. enzyme kinetics, activity tests), protein crystal growth, X-ray diffraction and crystallographic analyses, in silico simulation studies (molecular dynamics, docking of small molecules). The key outcomes of the project will be the discovery and optimization of novel inhibitors (i.e. drug hits or drug leads) that bind viral replicative enzyme domains and/or viral replication complexes. Inhibitors active against dengue may possibly also be active against other flaviviruses, including West Nile virus, Yellow fever and Japanese encephalitis virus.

This provides a unique opportunity to fully address the "Societal challenges" issue of the Horizon 2020 program, with a clear emphasis on world population health and well-being.

Project leader: Graziella Cappelletti ([email protected])


Co-Tutor: Francesco Demartin

Dipartimento di Chimica, Via Venezian 21, Milano


Dissecting microtubule dynamics at the synapse

Microtubules (MTs) are highly dynamic polymers that control many aspects of neuronal function: they

provide a scaffold to sustain axonal and dendritic architecture and supply the railway for axonal transport.

Far from being mere structural elements, MTs are emerging as key determinants of neuronal polarity. In

spite of the fact that the regulation of MT organization and dynamics has been extensively studied during

axon and dendrite formation and maintenance, much less is known about the regulation of MT dynamics at

synaptic terminals. Recently, we have unravelled the role of a synaptic protein, namely -synuclein, in

regulating MTs.

The goal of the present project is to investigate the role of -synuclein in controlling MT behaviour at the

synapse by focusing on MT nucleation that determines where, when and how polymerization of new MTs is

initiated. In the first part of the work, the PhD fellow will perform light and electron microscopy analyses on

primary neuronal cultures obtained from mice embryos. In these cultures, -synuclein will be

overexpressed or knocked-down. Next, taking advantage of the very high resolution afforded by 3D EM

tomography, a detailed analysis of the structure of MTs nucleated at the synapse will be carried out. The

labelling with immuno-gold particle will allow localizing -synuclein into the 3D structures. For image

analysis will be used a software developed by the Department of Chemistry (University of Milan). A better

understanding of MT regulation at the synapse and novel insights into the physiological role of -synuclein

is the expected outcome.

mailto:[email protected]

Project leader: Giuseppina Caretti ([email protected])



Role of the SMYD3 methylase in embryonic stem cells differentiation and zebrafish development

SMYD3 is a methylase that is expressed in the embryo and its levels are reduced in adult tissues (Hamamoto et al., 2004). SMYD3 is required for heart and muscle formation in zebrafish, since zebrafish embryos in which SMYD3 is suppressed using SMYD3-specific antisense morpholino-oligonucleotides show pericardial edema and defects in the trunk structure (Fujii et al., 2010). Embryonic stem cells (ES) are pluripotent cells that have the ability to differentiate into all the embryonic and extra-embryonic tissues. Because of this feature, they are considered an in vitro model to study some of the molecular mechanisms underlying embryonic differentiation (Keller, 2005). Preliminary data from our group show that the methylase SMYD3 is expressed at high levels in ES cells and modulates the transcription of a subset of developmental genes, during mouse embryonic stem cells differentiation. Additionally, we have knocked-down SMYD3 in zebrafish embryos and reproduced the phenotipic effects reported by Fujii et al. (Fujii et al., 2010). The aim of this project is to better define the functional role of SMYD3 in the differentiation of embryonic

stem cells and during zebrafish development.

As a first aim of the project, we will use differentiation protocols that induce enrichment in lineage

commitment towards specific germ layers, in control and SMYD3-depleted ES cells. We will induce the

differentiation of ES cells towards the ectodermal fate (through the addition of retinoic acid), the

mesodermal (addition of BMP4, activin and VEGF), or the endodermal lineage (high doses of activin), using

protocols well described in the literature (Murry and Keller, 2008). These experiments will help us assess

which markers for the three germ layers are more influenced by the decreased levels of SMYD3.

Furthermore, we will perform rescue experiments with different SMYD3 mutants to evaluate which

domains of SMYD3 play a role in modulating the transcription of differentiation markers in ES cells.

Importantly, we will define if SMYD3 enzymatic activity is necessary to modulate ES cells differentiation.

To confirm our data in an in vivo model system, we will study SMYD3 expression pattern and SMYD3

function in the early stages of zebrafish development. We will verify whether the genes affected by SMYD3

depletion in embryonic stem cells display an altered expression pattern in zebrafish embryos, in which

SMYD3 levels are reduced using morpholino oligonucleotides.

Data obtained from this project will help us to better understand SMYD3-dependent molecular

mechanisms during lineage commitment and embryo formation, and the molecular dynamics leading to

cardiac defects in SMYD3-depleted zebrafish embryos.

References Hamamoto R, Furukawa Y, Morita M et al. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat Cell Biol. 2004;6:731-740. Fujii T, Tsunesumi S, Yamaguchi K et al. Smyd3 is required for the development of cardiac and skeletal muscle in zebrafish. PLoS One. 2011;6:e23491. Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev.

2005; 15;19 (10):1129-55.

Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from

embryonic development. (2008) Cell.;132(4):661-80.

http://www.ncbi.nlm.nih.gov/pubmed?term=Keller%20G%5BAuthor%5D&cauthor=true&cauthor_uid=15905405

http://www.ncbi.nlm.nih.gov/pubmed?term=gordon%20keller%20embryonic%20stem%20cell%20genes%20dev%202005%2019%201129-1155&cmd=correctspelling

http://www.ncbi.nlm.nih.gov/pubmed/18295582

http://www.ncbi.nlm.nih.gov/pubmed/18295582

Project leader: Elena Cattaneo ([email protected])

Department of Biosciences (Via Viotti 3), University of Milan


Stem cells in human neurodegenerative disorders Huntington’s disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder that usually onsets in midlife. It is characterized by motor, cognitive, and psychiatric symptoms. Once symptomatic, patients are rapidly disabled and require increasing multidisciplinary care. HD is a tremendous burden for medical, social, and family resources. The symptoms and the progression of HD can be linked to its neuropathology, which is characterized by loss of specific neuronal populations in many brain regions. Several studies have shown that medium spiny neurons (MSN) are severely affected in HD. MSN are inhibitory projection neurons and are the primary source of striatal projections. The laboratory is actively involved in international research programmes aiming at deriving specific and robust differentiation protocols for the generation of MSN. Most recently we have developed a protocol to obtain such neurons from human embryonic stem (hES) or from induced pluripotent stem cells (hiPS) using a defined in vitro neural induction system and quantitative assessment tools (Delli Carri et al., 2013). Moreover, we are using stem cells to study the normal fuction of huntingtin and its polyglutamine tract along evolution (Lo Sardo et al., 2012). In this project we aim at developing strategies to further improve the recovery and quality of fully functional human MSN from hES/hiPS cells with the goal of future transplantation studies in HD. We will combine morphogens treatment with transcription factors inducible over-expression. We plan to develop doxycycline-inducible hES lines that over-express critical combinations of transcription factors (TFs) known to be important for striatal specification and differentiation. Quality of the neurons obtained at the end of the differentiation protocol will be verified by a convergence of features such as expression of neuronal markers as well as neurochemical and bioelectrical properties. We will also use knock-out strategies to inactivate the HD gene and produce new lines expressing huntingtin protein with mutation in critical aminoacidic sites. In conclusion this project aims at (i) developing new hES cell lines over-expressing critical striatal TFs; (ii) characterizing the identity of the neural progenitors and post-mitotic neurons derived from differentiation studies; (iii) generating and characterizing HD hiPS lines; (iv) developing functional assays to test huntingtin domains important for brain function(s).

Project leader: Lucia Colombo ([email protected])



Network controlling seed size and quality

The seed represents the latest stage of ovule ontogeny. A complex genetic network coordinated by cell

specific identity determination factors controls seeds size and quality. Therefore the coordination of the

activities of these factors is required for successful reproduction. The first phase of seed development is

characterized by several morphological and molecular changes that determine i) the final size of the seed

and ii) the seed quality. The relationship between cell differentiation and the regulation of metabolic

pathways is already known and the molecular mechanisms that connect these two programs are

beginning to be studied. Interestingly, it is becoming clear that regulatory mechanisms that control cell

identity and therefore cell differentiation also influence cell size and consequently seed dimension.

Understanding of these networks is of fundamental importance for breeding program that aim to

modulate seed metabolic content and/or size.

This project aim, using molecular, cellular, genetic and biochemical approaches, to identify key regulators

of seed differentiation and growth. Many of the key regulators in development and differentiation are

highly conserved across plant species therefore we plan to use the knowledge gained from Arabidopsis to

study the network controlling legumes seed development.

Project leader: Fabio Fornara ([email protected])



Molecular control of flowering in rice

Rice is a tropical plant that flowers when exposed to short day lengths (SDs), typical of tropical regions. However, many varieties are known that can be grown in temperate areas of the world, including Mediterranean Europe. Upon perception of a favourable day length, leaves produce the Florigens. Such proteins, encoded by Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), are mobile signals that move from the leaves to the shoot apical meristem to induce profound developmental reprogramming of the stem cell population. Our laboratory is interested in understanding the molecular mechanisms responsible for reprogramming a group of undifferentiated cells to become an inflorescence. To this aim, we generated transgenic rice plants that express the florigens Hd3a and RFT1 under the control of an inducible promoter. With such tools we are able to trigger developmental reprogramming, independently of the day length conditions in which plants are grown. The candidate will use these available genetic tools as basis for his/her PhD project that will follow this rationale.

1. Monitoring flowering at the phenotypic level by measuring flowering time of induced and non-induced plants, and the robustness of the system.

2. Assaying expression of candidate genes, known to be targets regulated by Hd3a and RFT1 proteins. 3. Sampling the stem cell population of rice meristems from induced and non-induced plants to

perform global transcript profiling of genes differentially expressed during reprogramming. Profiling will be performed through next generation sequencing technologies.

4. Validate candidate genes by functional analysis, also developing transgenic rice plants.

The candidate will be supervised during his/her PhD project by the PI and by an experienced post-doc of the lab that will provide technical and intellectual guidance.

Project leader: Carmela Gissi ([email protected])



Evolutionary dynamics of nuclear genes involved in replication and repair of the mitochondrial genome in fast-evolving chordates

The mitochondrial genome (mtDNA) of Metazoa is the molecule of choice in animal phylogenetic reconstructions but is also regarded as a model system for studying the processes governing the evolution of an entire genome (Gissi et al. 2008). As peculiarity, this genome is characterized by co-evolution with the nuclear genome, in fact the biogenesis and maintenance of mitochondria depends on tightly regulated interactions between the nuclear and mt genetic systems (Garesse and Vallejo 2001; Cannino et al. 2007). For example, the mtDNA of metazoans encodes only for some subunits of the respiratory complexes and for few components of the mt protein synthesis machinery, while the overwhelming majority of mt proteins are encoded by the nucleus, including those involved in replication, transcription and repair of the mtDNA as well as in the formation of the mt nucleoid. In general, we can expect that these nuclear-encoded mt proteins will evolve in different way depending on the details of the mtDNA organization and functionality in the different taxa, and then on the overall mtDNA evolutionary trends. At present, the mtDNA has been completely sequenced in more than 2000 metazoan species belonging to the most diverse phyla, from sponges to mammals. Interestingly, among Chordata, the mtDNA of vertebrates shows low evolutionary rate and almost frozen structural and compositional features, while the mtDNA of Tunicata, the sister taxon of vertebrates, is characterized by fast nucleotide substitution rate, hypervariability of the gene order (with genes nevertheless all located on the same strand), apparent absence of a major regulatory region for transcription and replication, and strong variability of base composition and asymmetry (Gissi et al. 2010; Rubinstein et al. 2013).

The aim of this project is to study the evolutionary dynamics of the above-mentioned nuclear-encoded gene categories in representative of vertebrates and tunicates, and in amphioxus (the only representative of Cephalochordata), in order to predict which proteins and protein-regions are mainly responsible of the differences observed between Tunicata, Vertebrata and Cephalochordata in the mt genome organization and functionality. This study will also allow the candidate to participate to new genome and transcriptome projects of tunicate species.

References Cannino G, Di Liegro CM, Rinaldi AM (2007) Nuclear-mitochondrial interaction. Mitochondrion. 7: 359-366.

Epub 2007 Aug 2002. Garesse R, Vallejo CG (2001) Animal mitochondrial biogenesis and function: a regulatory cross-talk between

two genomes. Gene. 263: 1-16. Gissi C, Iannelli F, Pesole G (2008) Evolution of the mitochondrial genome of Metazoa as exemplified by

comparison of congeneric species. Heredity 101: 301-320 Gissi C, Pesole G, Mastrototaro F, Iannelli F, Guida V, Griggio F (2010) Hypervariability of ascidian

mitochondrial gene order: exposing the myth of deuterostome organelle genome stability. Mol Biol Evol. 27: 211-215.

Rubinstein ND, Feldstein T, Shenkar N, Botero-Castro F, Griggio F, Mastrototaro F, Delsuc F, Douzery EJ, Gissi C, Huchon D (2013) Deep sequencing of mixed total DNA without barcodes allows efficient assembly of highly plastic ascidian mitochondrial genomes. Genome Biol Evol. 5: 1185-1199

Project leader: Nerina Gnesutta ([email protected])



NF-Y partners in the regulation of cell cycle promoters

Transcriptional regulation is at the heart of all biological processes, and it is governed by transcription factors -TFs-, which bind to discrete genomic regions. Many proto-oncogenes and tumor suppressors are TFs and their misregulation leads to changes in gene expression patterns that result in uncontrolled cell growth and cancer. The CCAAT box is a DNA element that is found enriched in promoters of growth controlling genes, and is specifically bound by the trimeric transcription factor NF-Y. Recently, the locations of several TF binding sites have been mapped in vivo at the genomic level by the ENCODE consortium project. Bioinformatic analyses of these data, performed by our group and others, have shown that NF-Y locations significantly overlap, within short distances, with a defined set of TFs, among which the proto-oncogenes Myc and Fos. Such analyses suggest that specific configurations of the relative binding sites in promoters can underlie the rules of biochemical and functional interactions of NF-Y with its partners to control gene expression. Such information, together with the knowledge of the crystal structure of NF-Y bound to DNA, recently solved by our group in collaboration with proff Nardini and Bolognesi, is the foundation of the proposed project.

The research project aims at understanding, at the biochemical, structural and functional levels, the interactions of NF-Y with “fellow” TFs, e.g. Fos (Fos/Jun), based on “prototypical” cell cycle promoters bound by NF-Y and its genomic partners, in which two or more CCAAT boxes are spaced by 32/33 nucleotides. Such informations will allow us to analyse larger sets of promoters, and could provide useful information to understand and predict TFs interactions in the regulation of CCAAT promoter genes.

Specific aims of the project will include: in vitro biochemical analyses of purified proteins to evaluate TFs cooperativity in DNA binding by EMSA; isolation of TFs ternary complexes with DNA for structural analyses by x-ray crystallography or SAXS analyses, to visualise surfaces involved in protein-protein interactions; in vivo studies of promoter occupancy by ChIP, following TFs inactivation, to understand possible hierarchy in DNA binding; in vivo studies by transient expression of wt and mutant proteins to analyse their relevance in the regulation of cell cycle, and gene promoter-reporter assays to evaluate functional interactions in gene expression regulation.

References Dolfini D, Gatta R, Mantovani R. NF-Y and the transcriptional activation of CCAAT promoters. Crit. Rev. Biochem. Mol. Biol. 2012; 47: 29-49. Fleming JD, Pavesi G, Benatti P, Imbriano C, Mantovani R. Struhl R. NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors. Genome Res 2013; 23:1195-209 Nardini M, Gnesutta N, Donati G, Gatta R, Forni C, Fossati A, Vonrhein C, Moras D, Romier C, Bolognesi M, Mantovani R. Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination. Cell 2013; 152: 132-143.

Project leader: Luisa Guerrini ([email protected])


Analysis of the molecular mechanisms at the basis of thalidomide-induced limb defects

in zebra fish embryos

Severe developmental malformations were detected in human feti in the 1950s, when women used the

antinausea and sedative drug thalidomide in the first trimester of pregnancy (1,2).

The striking similarities between the phenotypic abnormalities of babies born from mothers exposed to

thalidomide during pregnancy and patients affected by syndromes associated to mutations in the p63 gene

(3), prompted us to verify whether p63 could be a molecular target for the thalidomide drug. Our results

indicate that the Np63α and Np63ß proteins, but not Np63 and p53, are degraded through the

proteasome upon thalidomide exposure in several human cell lines expressing either the endogenous or

the transfected p63 proteins. By serial deletions analysis, we identified serine 383 in p63 as necessary for

thalidomide mediated degradation of p63, since mutation of S383 to alanine abolished thalidomide action

on p63. GSK3 kinase is responsible for S383 phosphorylation since the use of a specific GSK3 inhibitor also

abolished thalidomide action on p63 (4 and Lopardo et al. in preparation).

Thalidomide has been recently been shown to have teratogenic effects also in the zebra fish (5); we have

evidences that thalidomide modulated p63 protein levels also in vivo in developing Zebra fish embryos,

with concomitant teratogenic limb defects.

The aim of the project will be to dissect the molecular pathways altered by thalidomide treatment at the

basis of the observed limb defects, using zebrafish as animal model.

References

1) N. Vargesson (2009) Thalidomide-induced limb defects; resolving a 50-year old puzzle. Bioessays

31: 13271336.

2) Stephens TD, Fillmore BJ. (2000). Hypothesis: thalidomide embryopathy-proposed mechanism of action. Teratology. 61:189-195.

3) Schmidt M, FM Salzano. (1988). New case of an EEC-like syndrome in twins. Acta Genet Med Gemellol (Roma). 37:347-350

4) Galli F, Rossi M, D'Alessandra Y, De Simone M, Lopardo T, Haupt Y, Alsheich-Bartok O, Anzi S, Shaulian E, Calabrò V, La Mantia G, Guerrini L.MDM2 and Fbw7 cooperate to induce p63 protein degradation following DNA damage and cell differentiation. J Cell Sci. 2010 Jul 15;123(Pt 14):2423-33.

5) Ito T, Ando H, Suzuki T, Ogura T, Hotta K, Imamura Y, Yamaguchi Y, Handa H. (2010) Identification

of a primary target of thalidomide teratogenicity. Science.;327(5971):1345-50.

Project leader: Martin Kater ([email protected])]



Yield Increase in Rice by Altering Inflorescence Architecture

Rice is one of the most important crops for feeding the world. Rice yield increase over the next 25

years is of enormous importance to meet the demand of a rapidly growing world population. This rise in yield will have to be sustainable and without increase of cultivated land. This project focuses on improving rice yield through altering the inflorescence structure. The inflorescence or panicle has a primary axis on which primary branches develop. From the primary branches the secondary branches arise from which the spikelets are born. The number of secondary branches varies between rice varieties and by that the number of seeds that develop on a panicle. Panicle branching is therefore an important character for rice crop improvement.

Very little is known about the genetic control of panicle branching. Recently, Yoshida et al. (2013) identified a regulator of rice panicle branching called TAWAWA1, however the molecular mechanism is still far from understood. We have in the frame of the French-Italian EVOREPRICE project isolated by laser micro-dissection microscopy isolated the apical, primary and secondary meristems of rice inflorescences. This material has been used for RNA extraction and next generation sequencing analysis. The transcriptomes are now under study and are the starting point for this project to identify regulators of branching.

Recently we obtained using the same strategy the transcriptomes of the Arabidopsis inflorescence meristem and floral meristems (Mantegazza et al., 2014). The PhD student will also use this information to compare these data with the ones obtained in rice and will perform an evolutionary study on the development of reproductive meristems in monocot and dicot plants.

Yoshida et al. (2013). TAWAWA1, a regulator of rice inflorescence architecture, functions through the

suppression of meristem phase transition. Proc Natl Acad Sci U S A 110, 767-772.

Mantegazza, et al (2014). Gene Coexpression Patterns During Early Development of the Native Arabidopsis

Reproductive Meristem: Novel candidate developmental regulators and patterns of functional redundancy.

Plant J. doi: 10.1111/tpj.12585. [Epub ahead of print].

Project leader: Paolo Landini ([email protected])



Identification of RNA thermosensors in the opportunistic pathogens Pseudomonas aeruginosa and

Escherichia coli.

Regulatory mechanisms affecting mRNA translatability via trans-acting factors such as small regulatory RNAs (sRNAs) and/or riboswitches are widespread in Eubacteria, controlling a variety of cellular processes including virulence factors and pathogenesis. However, the identification of genes subjected to post-transcriptional regulation is not straightforward: indeed, as modulation of translation efficiency is not always reflected by changes in the mRNA amount, transcriptomic-based methodologies often fail to detect genes regulated through these mechanisms. In the frame of this general topic, we propose a research project for a PhD student on RNA thermosensors, i.e., a mechanism of post-transcriptional regulation involving temperature-dependent secondary structures in the mRNA.

We have recently identified four new putative thermosensor elements in the opportunistic pathogen P. aeruginosa. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, 3 out of 4 thermosensors appear to control virulence-related genes, such as ptxS, a regulatory gene implicated in P. aeruginosa pathogenesis. We plan to identify the RNA sequence/structure elements involved in temperature-dependent control and characterize the molecular mechanism and the physiological role underlying this regulation. In addition to further characterization of the thermosensors already identified in P. aeruginosa, we intend to carry out a genomic survey for new RNA thermosensors in enteropathogenic Escherichia coli. Indeed, since temperature sensing is known to be important in virulence regulation in enterobacteria, we expect to identify an even higher number RNA thermosensors in virulence related genes in pathogenic E. coli. This project will be carried out as collaboration between the groups led by Prof. P. Landini e Dr. F. Briani of the Department of Biosciences.


Project leader: Federico Lazzaro ([email protected])



Role of Translesion Synthesis (TLS) polymerases in rNMPs incorporation during DNA replication.

Ribonuclease H (RNase H) are evolutionary conserved enzymes capable of removing the RNA moiety in RNA:DNA hybrid molecules. Mutations in RNase H2 are found in a subset of patients suffering of a rare genetic disease, called Aicardi-Goutières Syndrome (AGS). AGS is a genetic encephalopathy whose clinical features mimic congenital viral infection. Initiation of autoimmunity is caused by interferon (IFN)-stimulatory nucleic acids derived from exogenous (e.g. viral infection) or endogenous sources (i.e. DNA replication, repair or retrotranscription) (Cerritelli & Crouch, 2009). It has been found that replicative DNA polymerases can incorporate rNTPs in place of dNTPs during DNA replication with an unexpected high frequency (~ 1/1000 nt) (McElhinny, Kumar, et al., 2010a; McElhinny, Watts, et al., 2010b). rNMPs embedded in chromosomal DNA can represent an imprint, positioned in S-phase, that regulates DNA transactions (Dalgaard, 2012). RNase H enzymes are crucial for the removal of these rNMPs from genomic DNA and for the maintenance of chromosome integrity. Recently we have found that impairment of RNase H1 and RNase H2 in yeast causes rNMPs accumulation in the genome and chronic activation of the post-replication repair (PRR) system which is becoming essential for cell survival (Lazzaro et al., 2012). The high rate of rNTPs mis-incorporation observed under normal conditions (1/1000 dNTPs) suggests possible physiological functions for the presence of rNMPs in newly replicated DNA. In a collaborative study we recently demonstrated that the presence of rNMPs during leading strand DNA synthesis acts as a strand discrimination signal for the Mismatch DNA repair machinery(Ghodgaonkar et al., 2013). Moreover our preliminary results show that TLS polymerases have an important role not only in bypassing rNMPs incorporated but can also incorporate rNMPs during DNA replication. In particular the evolutionary conserved TLS polymerase Pol-eta shows elevated predisposition to misincorporate rNMPs when the dNTPs pools became extremely limited. Here we will investigate in yeast and human cells how these particular events are regulated, extending the results to any processes where the DNA synthesis is essential even though dNTPs are deficient. Evidence in yeast indicates that the simultaneous deletion of all genes coding RNase H enzymes is tolerated by the cell, suggesting that rNMPs normally incorporated by DNA polymerases are maintained at sub-lethal levels by other repair pathways and these pathways may be new candidates involved in AGS. In order to identify these possible new pathways we applied the “synthetic genetic array” SGA approach to search for new genes functionally interacting with RNases H (Costanzo et al., 2010). An initial analysis identified promising candidate genes involved in recombination/repair 38%; replication/transcription 24%; chromatin 7%; meiosis/mitosis 7%; mitochondria 10%; RNA metabolism 3%; uncharacterized 10%. In this project we will validate and characterize these positives with particular attention to candidates conserved in human cells. References Cerritelli, S. M., & Crouch, R. J. (2009). Ribonuclease H: the enzymes in eukaryotes. FEBS J, 276(6), 1494–

1505. doi:10.1111/j.1742-4658.2009.06908.x

Costanzo M, Baryshnikova A, Bellay J, Kim Y, Spear ED, Sevier CS, et al. The Genetic Landscape

of a Cell. Science. 2010;327:425–31

Dalgaard, J. Z. (2012). ScienceDirect.com - Trends in Genetics - Causes and consequences of ribonucleotide

incorporation into nuclear DNA. Trends Genet, 1–6. doi:10.1016/j.tig.2012.07.008

Ghodgaonkar, M. M., Lazzaro, F., Olivera-Pimentel, M., Artola-Borán, M., Cejka, P., Reijns, M. A., et al.

(2013). Ribonucleotides Misincorporated into DNA Act as Strand-Discrimination Signals in Eukaryotic

Mismatch Repair. Molecular Cell, 50(3), 323–332. doi:10.1016/j.molcel.2013.03.019

Lazzaro, F., Novarina, D., Amara, F., Watt, D. L., Stone, J. E., Costanzo, V., et al. (2012). RNase H and

postreplication repair protect cells from ribonucleotides incorporated in DNA. Mol Cell, 45(1), 99–110.

doi:10.1016/j.molcel.2011.12.019

McElhinny, S. A. N., Kumar, D., Clark, A. B., Watt, D. L., Watts, B. E., Lundstr o m, E.-B., et al. (2010a).

Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol, 6(10), 774–781.

doi:10.1038/nchembio.424

McElhinny, S. A. N., Watts, B. E., Kumar, D., Watt, D. L., Lundstr o m, E.-B., Burgers, P. M. J., et al. (2010b).

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases. Proc Natl Acad Sci U

S A, 107(11), 4949–4954. doi:10.1073/pnas.0914857107

Project leader: Federica Marini ([email protected])



DAXX protein and the human DNA damage response: chromatin remodeling, gene expression, genome stability and tumorigenesis

The DNA damage response (DDR) functions in cells to detect, signal and repair lesions to the nuclear DNA

structure. The apical transducer of the DDR in human cells is the kinase ATM, which in response to double

strand breaks (DSBs) phosphorylates a broad range of targets impacting on DNA repair, cell cycle arrest,

senescence or apoptosis. ATM gene is mutated in ataxia telangiectasia, a rare disease characterized by

neurological and immunological features and by cancer predisposition (1). Since DNA repair takes place

within the complex organization of the chromatin, the DDR must be able to detect lesions within

nucleosome-DNA template, remodel the local chromatin architecture for processing and repair lesions and

restore the initial organization.

DAXX is a chromatin-associated factor involved in transcriptional regulation working together with the DNA

helicase ATRX, as a dedicated chaperone for the replication-independent deposition of the histone H3.3

variant onto gene regulatory regions as well as pericentric and telomeric heterochromatin (2). Mutations in

DAXX, ATRX and H3.3 underlie paediatric/adult glioblastoma (3) and can associate in cancer with

alternative lengthening of telomeres (ALT). Furthermore, DAXX interacts with several transcription factors,

directly regulating gene expression.

DAXX was initially linked to DNA damage for its role in p53 regulation and apoptosis (4), but the recent

description of H3.3 deposition at UV damaged DNA sites by HIRA (5), an histones chaperone, suggests that

DAXX histones chaperon activity could be involved in the DDR.

Coherently, we have found that ATM and its mediator, Chk2, both phosphorylate DAXX on multiple

residues, mutations of which impinge on DAXX activities, including DAXX/H3.3 interaction, suggesting that

the ATM/Chk2/DAXX/H3.3 axis plays a role in chromatin dynamics in DDR, with potential implications for

cancer pathogenesis.

This project aims to provide a deep understanding of the molecular bases and functionality of DAXX in the

cellular response to DNA DSBs. We will explore the hypothesis that DAXX could be targeted by DDR kinases

redirecting its activity to modify chromatin and transcription at DNA damage sites and/or to modulate

transcription of genes involved in DDR. This could occur through H3.3 deposition or directly through the

functional interaction with specific transcription factors.

Initially, we will determine how DAXX loss and expression of DAXX phospho-mutants affect genome

stability, proliferation and survival after genotoxic stress. A specific role at telomeres or centromeres will

also be assessed. Successively, we will evaluate if DAXX phosphorylations affect protein stability or

localization and post-transcriptional modifications at other sites relevant for the activity of this protein.

Finally, we will evaluate H3.3 deposition and chromatin modifications at DSBs or other genomic regions (i.e.

telomeres). In the meantime human cell lines knock in for DAXX mutants will be produced to approach

gene expression studies in presence of DNA damage.

References

1. Shiloh Y, Ziv Y. Nat Rev Mol Cell Biol. 2013 Apr;14(4):197-210.

2. Drané P, Ouararhni K, Depaux A, Shuaib M, Hamiche A. Genes Dev. 2010 Jun;24(12):1253-65.

3. Schwartzentruber J, et al., Nature. 2012 Jan 29;482(7384):226-31

4. Tang J, et al. Nat Cell Biol. 2006 Aug;8(8):855-62

5. Adam S, Polo SE, Almouzni G. Cell. 2013 Sep 26;155(1):94-106.

Project leader: Roberto Mantovani ([email protected])



NF-YA isoforms in ES cells and cancer

Regenerative medicine has taken the center stage in medical sciences since the discovery of embryonic

stem cells (ES). ES cells express “stemness” genes, many of which code for transcription factors. NF-Y is a

trimeric CCAAT-binding factor, composed of NF-YA, NF-YB and NF-YC (1). We showed that one of the

splicing isoform of NF-YA plays a crucial role in maintaining the mouse ES stemness potential (2). The

CCAAT box is often present in promoters of genes overexpressed in different types of cancer, and it is

believed that NF-Y plays an important role in mediating high levels of expression (3). The mechanisms are

related to the capacity to connect with the circuitry of stem cells transcription factors and their regulated

genes. In general, two splicing isoforms -long and short- are produced from the NF-YA locus, and their

expression is apparently quite regulated. They differ in 28 AA in the Q-rich transcriptional activation

domain. Somewhat surprisingly, it has recently emerged that the two isoforms have different, often

opposing roles in important cellular processes.

The aim of the project will be to investigate the mechanistic role of NF-YA isoforms by overexpression and

functional inactivation. The expansion of the stem cells compartment(s) has been associated to NF-YAs, and

it is possible that NF-YAl is involved in differentiation. We will evaluate the interplay of NF-YA with

important ES regulators, by analysis of profilings and ChIP-Seq data in mouse ES cells in which the two

isoforms are overexpressed. In parallel, we will inactivate NF-Y isoforms in mouse as well as in human iPS,

which are equivalent to human ES cells. The expected results are a better understanding of the molecular

mechanisms that lead to differentiation or the expansion of the stem cells pools, as well as the interplay

between NF-Y and other TFs on common targets.

References Nardini M., Gnesutta N., Donati G., Gatta R, Forni C., Fossati A., Vonrhein C., Moras D., Romier C., Bolognesi M., Mantovani R. Cell, 152, 132-143 (2013) Dolfini D, Minuzzo M, Pavesi G, Mantovani R. Stem Cells. 30, 2450-9 (2012) Dolfini D. and Mantovani R. Targeting the Y/CCAAT box in cancer: YB-1 or NF-Y? Cell Death and Differentiation, 20, 676-685 (2013)

Project leader: Graziella Messina ([email protected])



Role of the anthocyanins in slowing down the progression of Muscular Dystrophies

Muscular dystrophies (MDs) are clinically and molecularly heterogeneous diseases, characterized by

primary wasting of skeletal muscle that compromises patient mobility and, in the most severe cases, respiratory and cardiac functions, leading to wheelchair dependency, respiratory failure and premature death 1 . In many cases, the mutation affects proteins that form a link between the cytoskeleton and the basal lamina. Absence of one protein often causes the disassembly of the whole multiprotein complex associated with dystrophin, leading to increased fragility of the sarcolemma, especially during intense contractile activity. Damaged or dead fibers can be repaired or replaced by satellite cells (SCs), the adult stem cells of skeletal muscle. However, dystrophic SCs share the same molecular defect and produce fibers that are also prone to degeneration. With time, the population of SCs is exhausted and the muscle tissue is progressively replaced by connective and adipose tissue. Muscular dystrophies are among the most difficult diseases to treat, although the underlying pathogenesis is well understood. Skeletal muscle is the most abundant tissue of the body and is composed of large multinucleated fibers, whose nuclei cannot divide. Consequently, any cell or gene replacement strategy must restore proper gene expression in hundreds of millions of post-mitotic nuclei, which are embedded in a highly structured cytoplasm and surrounded by a thick basal lamina. It is therefore clearly evident that, although caused by absence, partial deletion or mutations in a single gene, these pathologies could be considered as multifactorial: misregulation of associated sarcoplasmic proteins, severe inflammation and consequent macrophage infiltration, resulting in fibrosis, are the final outcome. Among the different approaches, many efforts are directed to slow down the progression of the disease to counteract progressive degeneration 2.

Dietary flavonoids have received considerable attention since epidemiological studies have suggested that regular consumption of flavonoid-rich foods ameliorates muscular pathology, due primarily to their antioxidant properties and by modulating cell signalling and metabolic pathways3. Among the different classes of flavonoids, anthocyanins are the most recognized, visible members. In these last years, there are increasing evidences that seem to confirm that many biological effects of anthocyanins are related not only to their antioxidant properties but also to their ability to modulate mammalian cell signalling pathways. Aim of this research proposal is to study the protective effects that an anthocyanin-enriched diet has on a mouse model of muscular dystrophy and, more in general, in slowing down the progression of the disease.

References Emery, A.E. The muscular dystrophies. Lancet 359, 687-695 (2002). Cossu, G. & Sampaolesi, M. New therapies for Duchenne muscular dystrophy: challenges, prospects

and clinical trials. Trends in molecular medicine 13, 520-526 (2007). Hori, Y.S. et al. Resveratrol ameliorates muscular pathology in the dystrophic mdx mouse, a model

for Duchenne muscular dystrophy. The Journal of pharmacology and experimental therapeutics 338, 784-794 (2011).

Project leader: Anna Moroni ([email protected]).



HCN channel dysfunction in Alzheimer's disease

In this research project, we aim to investigate pathogenic factors which may play a role early in the development of Alzheimer’s disease (AD), and may represent new targets for early pharmacological intervention. There is growing recognition that the pathophysiological process of AD begins many years prior to clinically obvious symptoms. Several studies both in animal models and human patients have indeed argued that abnormal hyperactivity at key nodes in the olfactory-limbic pathway is prodromal to the development of the disease (e.g. Bakker et al., 2012). Thus, increased hippocampal excitability and altered neuronal activity within the entorhino-hippocampal loop have received growing attention as a potential target for early therapeutic intervention, which may effectively curb disease progression. Based on these premises, we will focus our investigation on a distinct class of voltage-gated ion channels, the hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels, and in particular the HCN1 isoform, which is expressed at very high levels in key limbic structures affected early in AD pathology (entorhinal cortex layer II stellate cells, hippocampal CA1 pyramidal neurons). Recent work has shown that HCN1 channel levels in the temporal lobe are severely reduced in the course of aging, as well as in Alzheimer patients. Loss of HCN1 in stellate cells and CA1 pyramidal neurons is known to result in increased dendritic excitability, altered grid and place field maps, and increased susceptibility to limbic seizures. Thus, altered HCN channel function may be expected to have a significant impact both on the development and expression of AD pathology, as it may contribute to the cognitive deficits observed in this condition, but also drive altered network excitability in early or prodromal stages of the disease, and thus represent a risk factor for AD. The project will address the molecular mechanisms that underlie the age- and AD-related loss in HCN1 channel expression; assess its functional consequences on the development of AD pathology; and lay the basis for the development of drug screening programs aimed at targeting HCN channel function in vivo for the treatment of Alzheimer’s disease.

References

Bakker A, Krauss GL, Albert MS, Speck CL, Jones LR, Stark CE, Yassa MA, Bassett SS, Shelton AL, Gallagher M.

(2012) Reduction of hippocampal hyperactivity improves cognition in amnestic mild cognitive impairment.

Neuron. 74(3):467-74. doi: 10.1016/j.neuron.2012.03.023.


Project leader: Marco Muzi Falconi ([email protected])



Function and regulation of nuclease activities in the maintenance of genome stability

Carcinogenesis is associated to alterations in the sequence and organization of the genome.

Maintenance of genome integrity is essential for all living organisms and genomic instability is a

hallmark of cancer cells. DNA metabolism is integrated with cell cycle progression through checkpoint

mechanisms. Understanding the mechanistic details of the cellular response to DNA damage is thus

critical for the comprehension of tumor development and for the design of better therapeutic

approaches. We have concentrated our research largely on the lesions generated to UV light, which are

repaired by NER, as a paradigm for all bulky DNA lesions. We have shown that UV lesions do not

directly trigger a checkpoint response. They are processed by NER and, when repair is not efficiently

completed, Exo1 nuclease can process the NER intermediates triggering the checkpoint. We have

suggested that closely opposing lesions are likely responsible for preventing the completion of NER

and thus trigger the checkpoint response. These lesions form as a consequence of UV irradiation and

the mayor cause of cytoxic and mutagenic effects of UV.

In general, this project aims at understanding how cells maintain genome integrity and what happens

when these controls are lost. The project is based on integrated approaches to identify genes and

pathways, define the molecular mechanisms of these pathways, determine what happens when they

are misfunctional and, in the long term, connect such defects to human pathologies. We will analyze

the regulation nucleases involved in checkpoint activation, and determine the mechanism and factors

repairing closely opposing lesions. We will characterize two new factors that influence genome

stability. We will employ genetics, cellular and molecular biology to study how protein interactions

and post-translational modifications modulate Exo1. Using mutants and cells expressing fluorescently

tagged proteins, we will study how cells deal with closely opposing lesions. Through genetic,

biochemical and NGS approaches, we will address the physiological role of two new factors involved in

repair.

Project leader: Marco Nardini ([email protected])

Location: Department of Biosciences, University of Milan


Structural analysis of transcription factor/DNA complexes

One of the key issues in biology is how the genetic information is transferred to biological functions. Binding of transcription factors (TFs) to discrete sequences in gene promoters and enhancers, is crucial to the process, which needs to interface with chromatin, whose fundamental unit is the nucleosome, formed by core histones wrapped by 146 bp of DNA. Binding of TFs entails the recruitment of histone modifying and chromatin remodeling machines, thus helping to define the chromatin status (“euchromatin” vs “heterochromatin”). TFs fall in essentially two categories: (i) “pioneer” TFs, with intrinsic chromatin association capacity; (ii) "activating" TFs, binding to a favorable chromatin landscape pre-set by pioneers. In this context, the present PhD project focus on NF-Y, a histone-like TF that binds and activates the CCAAT box [1], and on MYC, a proto-oncogene that binds the E-box (5’-CACGTG-3’), whose altered expression transforms cells [2]. NF-Y and MYC are deemed to be paradigms of pioneer and activating factors, respectively, and indeed they were shown to interact directly. Furthermore, the availability of the 3D structures for both NF-Y and MYC [3, 4] makes both TFs potential targets for development of anti-cancer drugs. Because of their direct binding, the strong correlation between the NF-Y and MYC loci in vivo, and the fact that regulation of MYC and CCAAT genes is crucial to oncogenic transformation, understanding of their interplay at the structural level will further increase druggable surfaces. The present PhD project will be carried out in the Nardini’s (structural biology) lab as a

continuation of an ongoing research project that led recently to the successful determination of the X-ray

structure of the NF-Y in complex with its target DNA [3]. The project will pursue analyses of NF-Y in

complex with DNA containing multiple CCAAT boxes, and the NF-Y/MYC/MAX-DNA complex, both by X-ray

crystallography and solution scattering methods (Small/Wide Angle X-ray Scattering, SAXS/WAXS). The

SAXS/WAXS experiments, being performed on solution samples, are always practicable, provided that the

sample is sufficiently pure and monodisperse. For the X-ray crystallography approach, the Nardini lab

experience in growing protein-DNA complex crystals will be crucial for the achievement of this step:

several E-box and CCAAT-containing fragments will be designed and tested in an approach that proved

successful for the NF-Y/CCAAT complex [3].

The 3D structure of the NF-Y/DNA complex, will also allow to search rationally for potential

inhibitors. As a part of the PhD project, inhibitor/NF-Y docking simulations will be carried out to screen

virtual chemical libraries of low molecular weight compounds, searching for inhibitors of the NF-Y

quaternary assembly and of its DNA-binding capacity. X-ray crystallography will be then applied to

characterize the structure of the complexes between NF-Y and the best inhibitors. A similar approach will

be eventually applied to interfere with the interactions between NF-Y and MYC/MAX. Potential inhibitors

selected through the in silico approaches will be cross-validated in vitro through Thermal shift and

electrophoretic mobility shift assay (EMSA) experiments, and in cells by ChIPs.

References:

Dolfini D, Gatta R, Mantovani R. Crit Rev Biochem Mol Biol. (2012) 47: 29-49. Prendergast GC, Lawe D, and Ziff EB. Cell (1991) 65: 395-407. Nardini M, Gnesutta N, Donati G, Gatta R et al. Cell (2013) 152: 132-143. Nair SK, Burley SK. Cell (2003) 112: 193-205.

Project leader: Paolo Pesaresi ([email protected])



FRUIT of the Plastid: a new strategy to uncover genes involved

in fruit development and ripening Given the fundamental nature of both the dietary and biological significance of fruit, molecular

dissection of fruit maturation has considerable interest. The yield and quality factors associated with fruits are of key importance to agricultural production and future improvements of fruit characteristics will depend on deep knowledge of the mechanisms that control fruit maturation.

The research program has the objective of deepening our understanding of the molecular processes responsible of the chloroplast dedifferentiation into chromoplast/gerontoplast at the onset of fruit maturation. The structural and molecular changes that plastids undergo during fruit maturation are in common between dry (Arabidopsis) and fleshy (tomato) fruits and require an extensive exchange of information between plastids and the nucleus. The chloroplast-nucleus crosstalk has been deeply characterised in leaves, however it is poorly understood in fruits.

The proposed project aims to fill this gap of knowledge:

by shedding light into the molecular networks controlling plastid-nucleus communication, with a particular focus on the retrograde communication, during the onset of fruit maturation in the model species Arabidopsis thaliana;

by transferring the gained knowledge to the model species Lycopersicon esculentum (tomato);

by obtaining novel genetic information to be used for improving fruit shelf life and conservation That will be pursued by a dual functional genomic strategy: i) a candidate gene approach, that is

aimed to verify the possible involvement in fruit maturation of factors already known to have a role in leaf anterograde and retrograde signalling; ii) a genetic screen in Arabidopsis thaliana that has the objective to isolate rare mutants characterised by altered plastid-to-nucleus retrograde communication.

The identified genes will be firstly characterised in Arabidopsis and their role and biotechnological relevance in fruit maturation will be verified in tomato.

Project leader: Achille Pellicioli ([email protected])



Targeting Slx4-Esc4 complex to prevent adaptation to DNA damage, genetic instability and cancer

The drive to proliferate and the need to maintain genome integrity are two of the most powerful forces

acting on biological systems. When these forces enter in conflict, such as in the case of cells experiencing

DNA damage, feedback mechanisms are activated to ensure that cellular proliferation is stopped and no

further damage is introduced while cells repair their chromosomal lesions. In this circumstance, the DNA

damage response dominates over the biological drive to proliferate, and may even result in programmed

cell death if the damage cannot be repaired efficiently. Interestingly, the drive to proliferate can under

specific conditions overcome the DNA damage response and lead to a reactivation of the proliferative

program in checkpoint-arrested cells. This phenomenon is known as adaptation to DNA damage and is

observed in all eukaryotic species where the process has been studied, including normal and cancer cells in

humans. Indeed, it has been suggested that checkpoint adaptation can increase the risk of genomic

instability and cancer development (Syljuasen RG, Oncogene 2007, 26, 5833-39). Saccharomyces cerevisiae

is an ideal model organism to study checkpoint adaptation following a sustained cell cycle arrest in the

presence of unrepairable DNA breaks, and by using this amenable system several genes have been involved

to switch off the DNA damage checkpoint signaling.

We recently found that Slx4-Esc4 complex is a new player in checkpoint adaptation, following one

persistent double strand DNA break (DSB) in yeast (manuscript in preparation), according to its role in DNA

replication checkpoint (Ohouo PY et al. (2013) Nature. 3;493(7430):120-4.).

Interestingly, Slx4-Esc4 complex is functionally highly conserved from yeast to humans and participates in

many different DNA repair pathways such as resolving replication fork blocks, homologous recombination

and inter-strand crosslink repair. Importantly, SLX4 was recently shown to be a component of the Fanconi

anemia pathway (FA), a rare recessive disorder characterized by chromosomal instability, increased cancer

susceptibility, developmental of abnormalities, bone marrow failure, and childhood cancers (Kim Y, et al.

(2013) Blood 3;121(1):54-63.). The main characterized function of SLX4 is to act as a scaffold for several

nucleases involved in different steps of DSB repair, however this repair function seems to be separated

from its role in checkpoint inactivation (Ohouo PY et al. (2013) Nature. 3;493(7430):120-4; and our

unpublished observation).

We aim to further characterize the molecular mechanism by which Slx4-Esc4 counteracts the DNA damage

checkpoint signaling, also by screening for specific mutations that separate genetically its DNA repair and

checkpoint signaling functions. Preliminary experiments will be done in yeast; then the PhD student will

transfer the obtained information to investigate the role of Slx4-Esc4 in the maintenance of genomic

stability in different human cell lines. Particularly useful to characterize SLX4 roles in checkpoint signaling

and DSB repair will be cell lines, already available in our laboratory, that have been derived from FANCP

patients.

The obtained results may explain at the molecular level the mechanism of checkpoint adaptation both in

yeast and human cells, also suggesting Slx4-Esc4 as a novel target to selectively inhibit proliferation of

cancer cells.

Project leader: Katia Petroni ([email protected])



Role of anthocyanin-enriched diet on cardioprotection

Dietary flavonoids have received considerable attention since epidemiological studies have suggested that regular consumption of flavonoid-rich foods or beverages is associated with a decreased risk of cardiovascular mortality [1–3], attributed primarily to their antioxidant properties and by modulating cell signaling and metabolic pathways. Among the different classes of flavonoids, anthocyanins are the most recognized, visible members, that contribute to the cardioprotection. In these last years, recent studies have suggested that dietary flavonoids, and more specifically regular anthocyanin consumption, induce a state of myocardial resistance evidenced by a reduced infarct size following regional ischemia and reperfusion [4] that is related, at least in part, to an improvement in the antioxidant defenses of the heart (i.e. cardiac glutathione). Moreover, there are increasing evidences that seem to confirm that many biological effects of anthocyanins are related not only to their antioxidant properties but also to their ability to modulate mammalian cell signaling pathways. For instance, recent studies in rats have shown that an anthocyanin-rich diet modulate the metabolism of (n-3) PUFA and to induce a marked increase in plasma EPA and DHA, fatty acids known to be protective against heart disease complication [5,6].

Aim of this research proposal is to study the cardioprotective effects that an anthocyanin-enriched diet has on the myocardial muscle and also its role in the prevention of drug cardiotoxicity such as in the case of many antitumor drugs. With this aim, we will investigate as a dietary strategy whether using functional foods, as anthocyanin-rich corn, can have muscle protective properties and can reduce the incidence and prognosis of myopathies [6-7].

The project will be divided in three different tasks, including i) the role of dietary anthocyanins from corn in the prevention of cardiotoxicity induced by chemotherapic agents, ii), to investigate the effects of dietary anthocyanins on specific microRNAs involved in cardiac regeneration and aging, iii) to establish the molecular mechanism underpinning the cardioprotective action of anthocyanins in murine cardiomyocytes.

With these activities, we expect to contribute to the understanding of how and why anthocyanins contribute to promote cardioprotection.

References: [1] Lancet 342:1007-11, 1993.

[2] BMJ 312: 478-81, 1996

[3] Am J Clin Nutr 85:895-909, 2007.

[4] FEBS J 273:2077-2099, 2006.

[5] J Nutr 138:747-752, 2008.

[6] J Nutr 141:37-41, 2011.

[7] Lancet 374:1849-56, 2009.

Project leader: Stefano Ricagno ([email protected])



Amyloid Light-chain amyloidosis: uncovering the molecular bases of cardiotoxicity

Light chain amyloidosis (AL), caused by tissue deposition of misfolding-prone monoclonal immunoglobulin

light chains as amyloid fibrils, is the most common systemic amyloidosis, with an incidence of 10 patients

per million/year. As in most protein misfolding diseases, the incidence of AL increases with age. Recent

solid clinical and experimental evidence indicates that the cell dysfunction observed in this pathology is due

to soluble, oligomeric light chains (LC) [1]. Most AL patients present multiorgan involvement. Cardiac

involvement is present in up to 75% of patients and is the main prognostic determinant, leading to death

for chronic heart failure or fatal arrhythmias. Patients with severe heart dysfunction are often too fragile to

receive aggressive treatments and do not survive long enough to benefit from any responses to therapy [1].

Understanding the bases of protein toxicity towards cardiac cells and finding a way to efficiently block it

would be of paramount importance to ameliorate life expectancy for patients and offer more efficacious

therapeutic options.

Unpublished data by collaborators at University of Pavia cell suggest that damage may be mediated by dys-

functional interactions of cardiotoxic light chains (CT-LC) with cellular components. By a functional

proteomic approach and using a set of LCs with well-defined pathogenic profiles in patients, it was

observed that CT-LCs specifically interact with a set of proteins involved in key cellular functions, whereas

the non cardiotoxic light chains (NT-LC) do not show such interactions. Cell alterations consistent with

perturbation of these proteins function are observed (Lavatelli et al under submission and [2]). The project

here presented aims to characterise the protein/protein complexes composed of the CT-LCs and two

mitochondrial interactors, as the molecular and biochemical bases for future drug development. Such

interactors are enzymes involved in the fatty acid metabolism.

The two interactors will be crystallised both alone and in complex with the CT-LCs in case they are forming

stable complexes. The crystal structures of the two interactors alone will provide an atomistic description

of these two proteins, which is still lacking. However, for the scope of this project, such structures will be

particularly useful to gain insight on which regions of the protein are mostly exposed, hydrophobic or

flexible, and could therefore be the regions involved in the interactions with the CT-LCs. Once these regions

are identified, interactor variants mutated in such regions will be produced. The ability of the mutated

variants to form stable interactions with CT-LCs will be tested by GF. Moreover the interactor structures will

be used as models for small angle X-ray scattering (SAXS) complex reconstruction. Any stable complex

between CT-LCs and the interactors will be structurally characterised using both X- ray crystallography and

SAXS. After the characterisation in vitro, extensive crystal screening will be performed for each stable

complex, to find suitable crystallisation conditions. Crystal hits will be optimised to produce diffraction

quality crystals. Once diffraction data are collected, the structures will be determined by molecular

replacement using the structure of the corresponding interactor and of CT- LC.

References

Palladini, G., et al. Blood, 2006. 107(10): p. 3854-8.

Diomede, L., et al. Blood, 2014. 123(23): p. 3543-52.

Project leader: Paola Riva ([email protected])

Department of Medical Biotechnologies and Translational Medicine, via Viotti 3/5, 20133 Milano


Molecular mechanisms involved in phenotype expression variability of Neurofibromatosis type 1

Neurofibromatosis type 1 (NF1 [OMIM + 162200]) is a common autosomal dominant disorder affecting

1/3500 individuals and caused by the point mutations or deletion of NF1, a tumor-suppressor gene with a

Ras-GTPase activity, encoding neurofibromin. NF1 is characterized by a highly variable expressivity with

multisystemic symptoms that may manifest at birth and evolve during lifetime. The clinical signs, including

café au lait spots, axillary and inguinal freckling, dermal or plexiform neurofibromas, iris Lisch nodules, but

also an increased risk of other benign and malignant tumors, highlight altered developmental pathways and

provide insights into the close relationship between development and cancer. Central to these

interconnected aspects is neurofibromin defective deregulation and the resulting hyperactivation of the

Ras signal transduction machinery. The heterogeneous clinical expression is hardly conceivable on the basis

of a great majority of NF1 deletions or truncating mutations. In fact, a genotype-phenotype correlation has

never been proven until now, suggesting that additional genes or unexplored gene expression regulatory

mechanisms are probably involved in the high variability of NF1 phenotype complexity.

The spinal NF1, together with the NF1 forms associated with gliomas and MPNSTs entail greater morbidity

than the other ones and they often can be diagnosed only at late stages of the disease. The availability from

Policlinico of Milan and Besta Neurological Institute of a cohort of patients affected by familial NF1 some

including some peculiar trios (father/mother with classical NF1 and son/daughter with NF1 with glioma or

spinal neurofibromas), prompted us to verify whether additional genes, besides NF1, predispose certain

NF1 patients to develop spinal NF1 or gliomas.

To address the above hypothesis we will search for i) candidate genes possible involved in development of

gliomas or spinal NF1 by whole exome sequencing (WES) in trios affected by spinal NF1, or displaying

gliomas, all carrying a mutation in the NF1 gene. A total of eight trios will be included in this screening

study. ii) The occurrence of functional mutations, detected in the WES selected genes, will be verified in a

cohort of more than 80 NF1 patients developing gliomas and spinal NF1 by mutation analysis. iii) The

identified mutations, probably involved in the pathogenesis of the above diseases, will be validated by in

vitro functional studies. Furthermore, considering that new emerging levels of gene regulation, based on

competitive endogenous RNA (ceRNA) activity, plays important roles in the development of different

diseases including cancer, we hypothesize that this mechanism may be involved in the expression

regulation of the functional copy of NF1 gene and have a consequence on the variability of NF1 clinical

signs. If little is known about the NF1 gene post-transcriptional regulation, at now only a few targeting

miRNAs have been validated, the possible involvement of NF1 mRNA in ceRNA cross talk has never been

investigated. At this purpose vi) we will also study the expression profile of miRNA targeting NF1 mRNA of

the NF1 transcript and specific NF1 pseudogenes, in both constitutional and tumor mRNAs from patients

with gliomas to investigate the possible involvement of NF1 mRNA in ceRNA cross talk. This project will

provide new insights on the molecular basis of NF1 expression variability opening new perspectives on

identification of new prognostic markers and pharmacological targets.

Project leader: Giulia Soldà ([email protected])

Department of Medical Biotechnologies and Translational Medicine, via Viotti 3/5, 20133 Milano


Identification of genetics and molecular bases of inherited sensorineural hearing loss by

whole-exome sequencing

Inherited nonsyndromic sensorineural hearing loss (NSHL) shows an extremely high genetic

heterogeneity, with more than 70 genes already associated with NSHL, and many others still to be

discovered (http://hereditaryhearingloss.org).

We are currently applying whole-exome sequencing (WES) as a cost- and time-effective

strategy to search for pathogenic variants underlying deafness. Indeed, this technique has already

proven to be helpful for the discovery of novel genes/mutations responsible for recessive NSHL

(DFNB82 and DFNX4 loci) [1-3]. Nine NSHL families, with a clear recessive (autosomal or X-

linked) inheritance pattern and at least two affected individuals, have been already selected for

WES; additional families are being recruited. The WES of the first 23 patients has already been

performed through external NGS services at BGI (Beijing Genomics Institute, China), and Yale

Genome Center (New Haven, USA).

The proposed PhD project will involve a combination of both in-silico and wet-lab

approaches in order to:

1) Develop data analysis pipelines to efficiently detect and prioritize candidate variants;

2) Functionally characterize by in-vitro and in-vivo studies novel genes/mutations.

The data analysis will include, among others, the implementation of the detection of splicing

mutations, indels, and structural variants. Putative pathogenic mutations identified by WES will be

tested to evaluate their segregation with the disease within the probands’ families and their

recurrence in sporadic and familial NSHL cases. In this frame, the availability of a large non-

syndromic deafness series (about 1300 individuals) of patients/families will be a key resource in the

validation step, to screen for the identified mutations and to search for additional genetic defects in

the candidate genes pointed out by WES. A in-house database of all variants identified by WES in a

wide (>3000) cohort of Italian subjects is also available to the study, to identify population-specific

polymorphisms.

We are currently analyzing the putative pathogenic role of candidate mutations in two novel

NSHL-causing genes both at the mRNA and at the protein level, by expression experiments in

eukaryotic cell lines. In addition, the function of this newly identified genes in the auditory system

will be tested in zebrafish, by adopting the CRISPR-Cas system cutting-edge technology to

selectively and stably inactivate the gene of interest [4], thus allowing a life-long analysis of its

roles in ear development and homeostasis. Similar approaches will be adopted for the

characterization of additional genes/mutations derived by WES data analysis.

References 1. Walsh T et al. 2010. Whole exome sequencing and homozygosity mapping identify mutation in the cell

polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 87:90-4.

2. Schraders M, et al. 2011. Next-generation sequencing identifies mutations of SMPX, which encodes the small

muscle protein, X-linked, as a cause of progressive hearing impairment. Am J Hum Genet 88:628-34.

3. Huebner AK, et al. 2011. Nonsense mutations in SMPX, encoding a protein responsive to physical force, result

in X-chromosomal hearing loss. Am J Hum Genet 88:621-7.

4. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. 2013.

Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227-9.

Project leaders: Chiara Tonelli/Lucio Conti

([email protected] - [email protected])



Site of ABA action in flowering

Drought escape (DE) defines the ability of plants to complete their life cycle before severe stress

leads to death and inability to produce fruits and seeds. A DE response emerges upon the

integration of photoperiodic stimuli with the production of the phytohormone ABA. A key

question arises as to the site where this integration occurs. In particular, the distribution of ABA in

vivo is still poorly characterized. The goal of this PhD project proposal will be to set up

experiments to analyze the distribution of ABA in vivo by using a blend of immunofluorescence

approaches, confocal imaging of ABA detector probes and molecular genetic techniques. The final

goal is to understand the mechanisms and adaptive significance of flowering as a major DE

strategy, thus providing insights into the mechanisms that enable plants to adapt to changing

environments.

mailto:[email protected]%20-

Project leader: Chiara Zuccato ([email protected])

Department of Biosciences (Via Viotti 3), University of Milan


A molecular study of the impact of synaptic dysfunction in Huntington’s Disease

Huntington's disease (HD) is a genetically dominant, neurodegenerative disorder caused by an elongated

polyglutamine (polyQ) segment in the huntingtin (Htt) protein. Mounting evidence indicates that mutant

Htt disrupts normal synaptic function (contributing to HD behavioral, cognitive, and motor symptoms), and

that alteration of the cortico-striatal excitatory circuit occurs early in HD progression (Zuccato and

Cattaneo, Progress in Neurobiology 2007; Milnerwood and Raymond, TINS 2010; Zuccato et al.,

Physiological Review 2010). We believe that the search for effective strategies to help restore the activity of

the cortico-striatal synapse (regarding structure, function, and plasticity) is an important area in HD

research that requires further study.

This project focuses on the investigation of molecules that can be implicated in dysfunctions of synaptic

circuitries and will focus on metalloprotease disintegrin ADAM10. ADAM10 has only recently emerged in

the HD field on the basis of our demonstration that normal Htt inhibits its activity during brain

development (Lo Sardo and Zuccato et al., Nature Neuroscience 2012). Recent works show that ADAM10

exerts a critical role in the adult brain by controlling the structure and function of excitatory synaptic

circuitries (Malinverno et al., The Journal of Neuroscience 2010; Marcello et al., The Journal of Clinical

Investigation 2013; Saftig and Reiss, Eur J Cell Biol. 2011 review). Therefore, ADAM10 represents a good

and novel target of investigation in HD due to its inherent structural and functional role in excitatory

synaptic circuitries. Preliminary evidence in HD mice suggests that ADAM10 activity is impaired in the

disease.

The goal of this project is to evaluate whether an unbalance in ADAM10 activity and/or its targets are a

relevant component in the dysfunction of the cortico-striatal glutamatergic circuitry in HD. We will perform

in vivo studies in HD mice and employ pharmacological and genetic strategies to modulate ADAM10 activity

in the brain. Biochemical, neuropathological, electrophysiological and behavioural assays will be used to

evaluate whether manipulating ADAM10 activity normalizes synaptic circuitries and is beneficial to HD

mice.

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