During the period 2003-2006 I became involved in two projects, which were already underway in the Laboratory in Padua, and I initiated a third one.

The first project was a study of mutations of the plasma-membrane calcium-pump isoform 2 causing digenic deafness, which was funded by the European Community Project EuroHear and developed in collaboration with the Laboratories of Dr. Fabio Mammano at The Venetian Institute of Molecular Medicine, and of Prof. Paolo Gasparini at the University of Trieste, Italy. The study resulted in the publication in the Proceedings of the National Academy of Science of the USA (Ficarella et al., 2007).

The second project dealt with the control of neuronal Ca²⁺ homeostasis by the transcriptional repressor DREAM, which was funded by the NeuroNE European Network and developed in collaboration with the Laboratory of Prof. Jose R. Naranjo in Madrid, Spain. We are presently preparing the manuscript for publication.

The third project, which then became the main theme of my research, dealt with calcium homeostasis and mitochondrial dysfunction in striatal neurons in Huntingtons disease. We have demonstrated that mutant huntingtin induces systemic transcriptional downregulation of Ca²⁺-related signaling genes and consequent decrease in cell Ca²⁺. We propose that this downregulation represents possible compensatory mechanism developed by the neurons to prevent the calcium overload that would lead to their demise. We have also demonstrated that in the absence of stressing insults mutant huntingtin produces a silent damage to mitochondria, which becomes fully evident when striatal neurons are stressed with Ca²⁺ overload. These results are now have been published in the Journal of Biological Chemistry (Lim et al., 2007).

One important aspect of my work during these years became the use of the calcium sensitive recombinant photoprotein aequorin to measure Ca²⁺ signaling directly within cell organelles, and of the lentiviral gene transfer technology, particularly to study Ca²⁺ signaling in cells poorly amenable to conventional transfection techniques, e.g., neurons.

Lentiviral vectors:

Cloning of gene of interest into lentiviral vector. Use of different promoters and bicistronic dual gene expression. Fusion with fluorescent and biochemical tags.

Production of viral particles:

I improved protocol for production of lentiviral particles and made it easily adjusteble to experimental requirements. To quantify virus titer I use Real-Time PCR, which is quick and reliable. We routinely obtain viral titer 10E8-10E9/ml.

Generation of stably infected cell lines:

I have generated several cell lines stebly expressing different transgenes.

• Isolation of total RNA from cell cultures and animal tissues;
• Preparation of samples for microarray, retrotranscription for PCR and Q-PCR.
• Transcription of mRNA in vitro and its intracellular microinjection.

• Cloning of DNA of interest into various vectors using standard cloning techniques;
• Polymerase Chain Reaction (PCR) which can be designed and tuned for a particular purpose;
• RNA interference: desing of hairpin oligos, subcloning into pSUPER, cloning into lentiviral vector, virus production, infection and analysis protein nock-down.

• Desing of hairpin oligos,
• Subcloning into pSUPER and cloning into a lentiviral vector,
• Generation of cell lines stabely expressing shRNA,
• Analysis of the protein knock-down.

• Bioinformatic survey on expression pattern and genomic organization of gene of interest, analysis of its isoforms and splice variants;
• Design of primers;
• Preparation of samples: RNA extration, retrotranscription;
• Real-time PCR
• Analysis of raw data

• Isolation and mantainment of starfish oocytes, sea urchin and ascidian eggs.
• In vitro fertilization of gametes of marine animals.

• HEK293T, HeLa, CHO, SH-SY5Y, ST-Hdh^Q111.

• Rat and mouse cerebellar granule neurons and striatal neurons.

Member of the Secretariat of two international meetings:

• EMBO workshop on Calcium Signaling and Disease, 2004, Capri, Italy;
• International Meeting on Calcium in Health and Disease, 2004, Rovaniemi, Finland.

Supervision of the work of Carmelina Chiarello leading to the Master degree in Sanitary Biology, which was completed in October 2007.

Supervision of the work of Paola Rebellato towards the Master degree in Pharmaceutical Chemistry and Technology, which was completed in March 2007.

Supervision of the experimental work leading to the Bachelors degree of Roberto Sommavilla in Experimental Biotechnology. The work was successfully completed in September 2004.

BIORAD Seminar on PCR Real time and Microarrays in genomic research, Padova, Italy.

NeuroNE Workshop on Protein Aggregation and Neurodegeneration. Soelden, Austria.

14th International Symposium on Calcium-Binding Proteins and Calcium Function in Health and Disease. La Palma, Canary Islands, Spain.

Oral presentation "Calcium homeostasis and mitochondrial dysfunction in striatal neurons of Huntington's disease". D. Lim, L. Fedrizzi, M. Tartari, E. Cattaneo, M. Brini, and E. Carafoli.

NeuroNE Workshop on Neuronal Calcium in Health and Disease. La Palma, Canary Islands, Spain.

Oral presentation "Mitochondrial Defects and Calcium Signaling Dysfunction in a striatal cell model of Huntington's disease". D. Lim, L. Fedrizzi, M. Tartari, C. Zuccato, E. Cattaneo, M. Brini, and E. Carafoli.

20th IUBMB International Congress of Biochemistry and Molecular Biology. Kyoto, Japan.

Poster "Altered ATP and Bradykinin (BK)-mediated calcium signaling in striatal cells expressing mutated huntingtin". D. Lim, M. Tartari, E. Cattaneo, L. Fedrizzi, M. Brini, and E. Carafoli.

International Symposium on Calcium in Health and Disease, Rovaniemi, Finland.

Poster "The role of the Ca2+-dependent transcriptional repressor DREAM on Ca2+ homeostasis" M. Brini, L. Fedrizzi, D. Lim, J.R. Naranjo, and E. Carafoli.

12th International Symposium on Calcium-Binding Proteins and Calcium Function in Health and Disease. Cavalese, Italy.

Poster "NAADP+ initiates the Ca2+ response during fertilization of starfish oocytes". D. Lim, K. Kyozuka, G. Gragnaniello, E. Carafoli, and L. Santella.