Vincenzo Cinella

REFERENCES:
Professor Ellis Reinherz; email: Ellis_Reinherz@dfci.harvard.edu
Doctor Aoi Akitsu; email: Aoi_Akitsu@dfci.harvard.edu
The current agreement is for 2 years of full-time research from August 2022 to August 2024.
I am listed as a co-author in a manuscript entitled: "Parsing digital or analog TCR performance through piconewton forces."
LAB SKILLS:
• Presentation and critical discussion during journal club meetings of peer-reviewed publications of general or pertinent relevance to our research projects. The main topics discussed include basic T cell biology with a focus on how T-cell receptor repertoires evolve during the primary infection to generate memory and exhausted T cells, downstream interactome studies of the TCR signaling, mechanosensing properties of T cells, and new design of TCR-based immunotherapy. The topics discussed serve as a reference to design experiments and data analysis relevant to our research goals
• Critical reading and written evaluation reports of research topics that might be useful for the interpretation of our data and planning of the new experiment
• Basic mice husbandry and handling ( colony monitoring, weaning, bleeding, monitoring of cross-breeding mice strains and their phenotype monitoring )
• Habilitation to work in ABSL2 to infect mice
• Mice irradiation and intravenous tail injections
• Mice anesthesia by intraperitoneal injection of ketamine and infection by nasal injection of a PBS solution containing Influenza-A PR8 virus
• Mice lungs and lymphoid organs processing to extract T cells to perform Single-cell RNA-seq or FACS immunophenotyping
Multiparameter flow cytometry and FACS analysis to generate reports for the post-doc
• Cell culture
• TCR Knock-out by electroporation of Single-guide RNA-directed CAS9 Ribonucleoprotein complex
• Retroviral vector production using the Plat-E cells packaging cell line
• Purification, handling, and transduction optimization of Lineage-negative cells with vectors encoding several T cell receptors
• In vitro stimulation of TCR expressing cell lines with peptide-pulsed DC cell line and ELISA to detect cytokines production
• Molecular cloning of TCR to generate ecotropic-pseudotyped retroviral transfer vectors
• Bone Marrow Dendritic Cell purification from mice bone marrow
•Immunophenotyping of circulating T cells generated using the Retrogenic mice system. Briefly, T cell-deficient RAG knock-out mice are irradiated. The next day, to carry out the bone marrow transplantation, lineage-negative cells transduced with a retroviral vectors encoding for a TCR are administered by intravenous tail injection. After six weeks, blood samples are taken from mice to verify the presence of circulating naive T cells expressing the single clonotypic TCR of interest. Naive T cells are harvested and sorted by processing the spleen and lymph nodes. This procedure allows for the creation of Naive T cells expressing different TCRs and their use for adoptive transfer studies to confirm the relationships between specific TCRs and their functional properties in vivo.

I am collaborating in research projects studying the in Vivo and in Vitro functional properties of T cells expressing different T-cell receptors binding to the same peptide-MHC. The lab exploits a multi-disciplinary approach allowing for the characterization of each TCR's biophysical and conformational properties. Thanks to our work, we aim to establish the basis to understand how different TCR's biophysical and conformational properties can influence the transcriptome and phenotype of T cells (generation of memory, resident memory, or exhausted cells) and their potential to clear viral infection at different pMHC densities on infected cells. Our research have implications for rational vaccine design and guidelines for choosing specific TCR-based Immunotherapy.

1 year of full-time research experience
REFERENCE:
Professor Pietro Genovese; email: Pietro_Genovese@DFCI.HARVARD.EDU
I am currently listed as a co-author in a publication in Nature Communication entitled: " IL-12 Reprograms CAR-Expressing Natural Killer T cells to Long-Lived Th1-Polarized Cells with Potent Antitumor Activity"
Lab skills:
• Multi-parameter flow cytometry
• Design of molecular cloning constructs using the New England Biolab Hi-fidelity system to create constructs for Lentiviral and Adeno-Associated viral vector production. The Lentiviral vectors were used to transduce primary human and murine B cells, while the Adeno-associated viral vectors were used to deliver the DNA template to induce the Homology-directed repair after the Double-strand break induced by the single-guide RNA-directed Cas9 delivered in the cells by electroporation
• Single-guide RNA design to perform Knock-out and Homology-Directed Repair experiments using the CRISPR/Cas9 system. I used the Tide analysis to estimate the efficiency of the Non-Homologous End Joining
• Basic mice husbandry, retro-orbital bleeding, mammary fat-pad injection of tumor cells, and organ processing for FACS analysis
• Purification from PBMC and handling of primary human B cells
• Primary B cells phenotype analysis by FACS
• Transduction optimization of human and murine primary B cells using lentiviral and retroviral vectors with different pseudotyping
• PCR
• Digital Droplet PCR to estimate the copy number per cell of integrated lentiviral and retroviral vectors
• Presentation of data during Lab meeting
I collaborated on projects aimed at creating novel cellular immunotherapies for Acute Myeloid Leukemia patients and HER2-positive tumors.
I was also involved in a gene therapy project to create safer lentiviral vectors to treat patients affected by Adrenoleukodystrophy.

6 months and 2 weeks of full-time research experience
The work performed in my thesis consisted of the basis to develop data for a publication in press in the journal International Journal of Cancer entitled " GNAS mutation induces phosphodiesterase 4D expression and inhibits growth in colorectal cancer cell line". Currently, I am listed as the fifth co-author.
THESIS TITLE: " Molecular alterations in SW48 and HCT-116 Colorectal cancer cell line with a focus on GNAS R201C activating mutation".
REFERENCES in the Helsinki University:
Professor Ari Ristimaki; email: ari.ristimaki@helsinki.fi
Doctor Pirjo Nummela; email: pirjo.nummela@helsinki.fi
LAB SKILLS:
• Data analysis, p-value calculation, and data presentation in lab meeting
• Cell culture
• In-vitro functional assays: proliferation, adhesion, cAMP measurement, phosphodiesterase inhibition, and transwell invasion assay on membranes coated with matrigel. The transwell invasion assay was optimized based on published protocols
•PCR
• qPCR
• Western Blotting
• Basic RNA-seq differentially expressed gene and enriched pathways analysis
• Study of pathway alteration in the context of colorectal cancer and in general carcinogenesis
THESIS ABSTRACT:
The Guanine Nucleotide binding protein gene (GNAS) encodes for the alpha-subunit of the stimulatory G protein (Gs-alpha), a key component for the signal transduction mediated by the G protein-coupled receptor (GPCR). After stimulation, the Gs-alpha activates the adenylyl cyclase which generates cAMP, a secondary messenger that activates the Protein Kinase A (PKA). When active, the PKA can translocate into the nucleus and phosphorylate its target transcription factors, such as the cAMP-response element binding protein (CREB), thus altering the gene expression in the cell.
GNAS-activating mutations are recurrent in approximately 2% of Colorectal cancer patients.
cAMP levels are reported to be low in several CRC cell lines, while other studies suggest that higher levels of cAMP result in lower malignant features. PDE4D is a cAMP selective phosphodiesterase that hydrolyzes cAMP, thus inactivating its effect. Several papers report that PDE4D inhibition correlates with cAMP level enhancement, thus leading to reduced oncogenic features.
To study the potential roles played by GNAS activating mutation and the related downstream interactome, cell lines edited by Horizon Discovery (Waterbeach, England) were purchased with SW48 and HCT-116 bearing GNAS R201H mutation, respectively. Each of the mutated cell lines was purchased with its respective parental cell line. This mutated GNAS subunit is constitutively activated, thus stimulating adenylate cyclase to produce more cAMP.
RNA-sequencing was performed, thus highlighting differential expression of genes and gene ontology pathway enrichment for genes up-regulated between each mutated cell line and the respective parental cell line.
No significant difference in adhesion was detected between any of the mutated cell lines compared to their parental ones.
No significant difference in the proliferation rate was detected between the two SW48 mutated cell lines compared to their respective parental cell line.
The HCT-116 GNASm cell line was significantly less proliferative and invasive than the parental one.
No difference in PKA activation, measured by western blotting with phospho PKA-substrate antibody, was detected. Moreover, the HCT-116 basal cAMP level between mutated and parental cell line was not significantly different, although forskolin induction showed a much higher cAMP level enhancement in the HCT GNAS mutated cell line.
No significant difference in HCT-116 GNAS mutated cell line cAMP basal level can be explained by a strong induction of PDE4D, which was detected by the RNA-seq data and further validated with qPCR.
Similar PDE4D induction was validated in the SW48 GNAS mutated cell line.
HCT-116 proliferation assay with Ro (unspecific PDE4inhibitor) and GEBR-7b (selective PDE4D inhibitor) treatment was performed, showing that the proliferation rate was lower only for the treated GNAS mutated cell line compared to the control treatment with DMSO, while the HCT-116 parental cell line was insensitive to PDE4D inhibition. These results are in line with the fact that there is a strong expression of PDE4D only in the GNAS mutated cell line and that PDE4D inhibition should lead to an increase of the cAMP level due to the activity of the constitutively activated GNAS subunit.
More experiments are needed to prove that PDE4D inhibition leads to a higher cAMP level enhancement and, that it is directly related to the decrease in the proliferation rate. Further functional assays with PDE4D inhibition, such as invasion and colony formation assay, could be performed to verify the reduction of other malignant features.
Then, the pathway components' down-regulation related to the cAMP/PDE4D signaling could be studied to better understand the mechanisms behind the phenotypic effect related to cAMP level enhancement.
Further studies about the PDE4D-specific inhibitor GEBR-7b effect in GNAS mutated Colorectal cancers are needed to better understand a potential tumor-suppressive effect in combination with other chemotherapy or forskolin.

Two months of full-time research traineeship entitled "Molecular mechanism of transformation of acute myeloid leukemia with focus on FLT3".
REFERENCES in Lund University:
Professor Lars Ronnstrand; email: lars.ronnstrand@med.lu.se
Doctor Kazi Uddin; email: kazi.uddin@med.lu.se
Lab skills: cell culture, transfection, immunoprecipitation, and western blotting.
FLT3 mutations are recurrent in around 20% to 30% of patients with acute myeloid leukemia (AML). Since many FLT3 direct inhibitors showed emergent resistance in patients, research needs to focus on FLT3 downstream targets that play a role in AML.

The traineeship aimed to verify if there is a direct binding between activated FLT3 and PLC-gamma.

Cos1 cells were transfected with control, WT FLT3, WT FLT3 ( later stimulated with its ligand for 15 minutes), internal tandem duplication mutated FLT3 (constitutively activated form), and KIT (positive control) plasmids for 24 hours. Immunoprecipitations were performed using PLC-gamma antibody, followed by western blotting. No FLT3 was detected in the immunoprecipitate, while KIT (positive control) was detected with PLC-gamma. Therefore, no binding between activated or mutated FLT3 and PLC-gamma was detected.

Research in AML still needs to study FLT3 downstream targets to constantly discover new potential therapeutic targets to overcome chemotherapy resistance.

4 months of full-time research experience

REFERENCE at the University of Modena and Reggio Emilia:

Doctor Forti Luca; email: luca.forti@unimore.it

Enzyme immobilization is often studied to improve the yield of industrial processes carried out in bioreactors.

Lipases hydrolyze the substrate p-nitrophenyl acetate, thus leading to an increase in absorbance. Lipase immobilization on spheric supports made of clay and glass showed a significantly increased enzyme activity, which was tested by OD measurement.

This study proved that lipase immobilization on glass and clay spheric supports could enhance the biodiesel production yield in bioreactors from the conversion of organic wastes.