Projects 2020/21

Projects 2020/21

 

LIST OF PROJECTS 2020/21  – assigned to PhD students:

 

– Bone marrow modelling for platelet production

-Molecular determinants of diaphragm muscle impairment in an intermediate mouse model of spinal muscular atrophy

– Mapping the spatiotemporal coordination of cellular signals and nuclear function

– Functional and molecular adaptations of skeletal muscle oxidative metabolism to repeated periods of high-intensity interval training interspersed with a detraining period

– Targeting cell differentiation mechanisms to reduce cancer cell pathogenesis.

– Ocular genetics (and “oculomics”) in developmental eye disorders and uveal melanoma

-Structural and functional characterization of the Voltage-gated proton channel (Hv1) channel

Evolution of HCMV non-primary infection and host immune response in HCMV-seropositive mothers of children attending day-care centers. An observational prospective study


 

Research project proposed by: Christian Di Buduo and Alessandra Balduini
Carolina Paula Miguel, PhD student.

Title of the research project: Bone marrow modelling for platelet production

Description of the research project:
Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood. Under- or over-production of platelets has major clinical implications for many diseases, including thrombocytopenia and myeloproliferative neoplasms, where life-threatening side effects with incurable outcomes are common. The scientific and clinical communities are actively searching for new modes to generate functional platelets ex vivo to address clinical needs as well as for insight into fundamental studies of mechanisms. We hypothesize that engineering a 3D bone marrow mimic will propel mechanistic understanding of platelet shedding and determine future protocols for therapeutic inquiry.

On the basis on our previous publications, the candidate will utilize non-thrombogenic silk protein biomaterial to perfect an ex vivo three dimensional (3D) tissue model of the bone marrow to study platelet release from megakaryocytes derived from human induced pluripotent stem cells. Megakaryocytes receive cues from the bone marrow environment including cell-cell contact, contact with extracellular matrix components, and physical characteristics of the tissue (topography and rigidity of the extracellular space) as well as shear stress generated by the blood flow in the vessels. By refining the environment in the 3D silk-based bone marrow system the aim will be to provide all the physical and biochemical characteristics necessary to improve ex vivo platelet release by megakaryocytes. The outcome of these studies is expected to be the design of new tools to mimic the bone marrow niches ex vivo gaining insight into the mechanisms that control platelet release in a physiological relevant manner.

References

1. Di Buduo CA, Wray LS, Tozzi L, Malara A, Chen Y, Ghezzi CE, Smoot D, Sfara C, Antonelli A, Spedden E, Bruni G, Staii C, De Marco L, Magnani M, Kaplan DL, Balduini A. Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies. Blood (2015) 125(14): 2254-64.
2. Abbonante V, Di Buduo CA, Gruppi C, De Maria C, Spedden E, De Acutis A, Staii C, Raspanti M, Vozzi G, Kaplan DL, Moccia F, Ravid K, Balduini A. A new path to platelet production through matrix sensing. Haematologica (2017) 102(7): 1150-1160.
3. Giannini S, Lee-Sundlov MM, Rivadeneyra L, Di Buduo CA, Burns R, Lau JT, Falet H, Balduini A, Hoffmeister KM. ?4GALT1 controls ?1 integrin function to govern thrombopoiesis and hematopoietic stem cell homeostasis. Nat Commun. 2020 Jan 17;11(1):356.


 

Research project proposed by: Monica Canepari

Francesca Cadile, PhD student.

Title of the research project: Molecular determinants of diaphragm muscle impairment in an intermediate mouse model of spinal muscular atrophy

Description of the research project:

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease with an incidence in humans of approximately 1 in 10.000 births per year. SMA is caused by the deletion/mutation of the survival of motor neuron gene (SMN). The genetic defects occurring in SMA determine the degeneration of spinal motoneurons (MNs), leading to progressive muscular atrophy weakness and respiratory failure. Indeed increasing evidence suggest that SMA pathogenesis is more complex than expected: many authors have recently speculated that, even though MNs are the most affected cells in SMA, their loss might not only depend from the lack of SMN: retrograde signals coming from muscles and NMJs can be crucial players of the MN alteration.(1). A more extensive analysis is therefore needed to better clarify the molecular, functional and temporal defects affecting skeletal muscles and NMJs in SMA. In this context, diaphragm muscle is a muscle relatively poorly studied, notwithstanding is one of the earliest affected muscles. The central goal of this project is to assess intrinsic diaphragm muscle defects, the molecular causes underlying them and their pathogenic role in an intermediate SMA mouse model (SMNΔ7mice or Jackson Laboratory stock #005025) with median survival of 13 days. This goal is of primary importance for understanding the pathogenesis of SMA and for the development of appropriate pharmacological strategy. In this way, the project will have the potential to identify the causes and the factors influencing the course of the disease leading to respiratory failure and early death. To achieve its goal, the project will combine morphological, biochemical, functional and molecular analyses (2,3,4,5) on isolated diaphragm muscle. The project is supported by the BLUE SKY RESEARCH Grant.

References

1) Bottai D, Adami R (2013) Spinal muscular atrophy: new findings for an old pathology. Brain Pathol.23(6):613-22.
2) Carnio S, LoVerso F, Baraibar MA, Longa E, Khan MM, Maffei M, Reischl M, Canepari M, Loefler S, Kern H, Blaauw B, Friguet B, Bottinelli R, Rudolf R, Sandri M. (2014) “Autophagy Impairment in Muscle Induces Neuromuscular Junction Degeneration and Precocious Aging”. Cell Rep. 8(5):1509-21
3) Tirone M., Lan Tran N., Ceriotti C., Andrea Gorzanelli A., Canepari M, Bottinelli R., Raucci A., Di Maggio S., Santiago C., Mellado M, Saclier M., François S., Careccia G., He M., Marchis F., Conti V.; Ben Larbi S., Cuvellier S., Casalgrandi M., Preti A, Chazaud B., Al-Abed Y., Messina G., Sitia G., Brunelli S., Marco Emilio Bianchi M.E. and Vénéreau E. (2018) “High Mobility Group Box 1 orchestrates tissue regeneration via CXCR4” J Exp Med 215(1):303-318.


 

Research project proposed by: Konstantinos Lefkimmiatis

Muhammad Ali Muslimani, PhD student.

Title of the research project: Mapping the spatiotemporal coordination of cellular signals and nuclear function

Description of the research project:
A fundamental aspect of intracellular communication is the ability of the cell to translate into function environmental cues. This is achieved mainly thanks to signalling molecules called second messengers. First discovered among these, cyclic AMP (cAMP) regulates a variety of cellular functions ranging from movement to more fine processes such as transcription. It is not surprising therefore that unbalanced cAMP signalling has been connected to many human pathologies including cancer, neurodegeneration and cardiovascular disease. The ability of cAMP to affect many cellular tasks simultaneously, depends on the spatial and temporal organization of its signalling cascade. According to this principle each signal activates the cAMP cascade only within specific regions of the cell and consequently is coupled to a specific function. However, our understanding on how the functional center of the cell, the nucleus, interprets cAMP signals is unknown.
The overarching goal of this PhD project is to dissect the nuclear cAMP signalling axis and to understand how its defects are connected to human disease. The successful candidate will develop and validate an array of (Fluorescence Resonance Energy Transfer) FRET-based sensors that will complement the state-of-the-art toolbox available to our laboratory. The multidisciplinary nature of the project provides a unique training opportunity both experimentally (live cell imaging, molecular biology and biochemistry) and conceptually, since the candidates will be challenged with long standing questions in the field of cellular signalling. The scientific outcomes of this project will be integrated in a broader framework aiming to unveil the spatiotemporal map that connects cAMP to nuclear function and human disease.

References

1. Burdyga A, Surdo N, Monterisi S, Di Benedetto G, Grisan F, Penna L, Pellegrini L, Bortolozzi M, Swietach P, Pozzan T, Lefkimmiatis K. Phosphatases control PKA dependent functional microdomains at the outer mitochondrial membrane. Proc Natl Acad Sci U S A 115(28) : E6497-E6506, 2018
2. Grisan F, Burdyga A, Iannucci LF, Surdo NC, Pozzan T, Di Benedetto G, Lefkimmiatis K. Studying β1 and β2 adrenergic receptor signals in cardiac cells using FRET-based sensors. Prog Biophys Mol Biol. 2019 Jun 29. pii: S0079-6107(19)30060-4. doi: 10.1016/j.pbiomolbio.2019.06.001.
3. Lefkimmiatis K, Leronni D, Hofer AM. The inner and outer compartments of mitochondria are sites of dist


 

Research project proposed by: Maria Antonietta Pellegrino

Emanuela Crea, PhD student.

Title of the research project: Functional and molecular adaptations of skeletal muscle oxidative metabolism to repeated periods of high-intensity interval training interspersed with a detraining period

Description of the research project:

Skeletal muscle is a remarkably plastic tissue, with mitochondria responding rapidly to either exercise training interventions or a reduction/cessation of physical activity. High-intensity interval training have been reported to improve mitochondrial respiration and increase molecular signalling related to mitochondrial biogenesis. However, these mitochondrial adaptations are quickly reversed following detraining periods. Further studies are needed to better understand the magnitude and time-course of skeletal muscle oxidative metabolism adaptations in response to repeated training stimuli. This project aims to investigate functional and molecular biomarkers of skeletal muscle oxidative metabolism respond to repeated training interventions interspersed with a detraining period. In-vivo functional biomarkers related to adaptations of skeletal muscle oxidative metabolism to training and detraining will be obtained from the evaluation of human respiratory, cardiovascular and metabolic responses during exercise. Molecular analyses for mitochondrial biogenesis and autophagy/mitophagy will be performed on muscle samples obtained by percutaneous biopsies. Ex-vivo functional indexes of mitochondrial respiration will be determined by high-resolution respirometry. The results of this project will have important implications on exercise training prescription for both healthy subjects and patients.

References

1. R. Wibom, E. Hultman, M. Johansson, K. Matherei, D. Constantin-Teodosiu, P.G. Schantz, Adaptation of mitochondrial ATP production in human skeletal muscle to endurance training and detraining, J. Appl. Physiol. 73 (1992) 2004–2010.
2. Bishop DJ, Granata C, Eynon N. Can we optimise the exercise training prescription to maximise improvements in mitochondria function and content? Biochim Biophys Acta. 2014 Apr;1840(4):1266-75.
3. Lindholm ME, Marabita F, Gomez-Cabrero D, Rundqvist H, Ekstrom TJ, Tegner J, et al. An integrative analysis reveals coordinated reprogramming of the epigenome and the transcriptome in human skeletal muscle after training. Epigenetics. 2014;9(12):1557–69.


 

Research project proposed by: Virginie Sottile

Paola Fulghieri, PhD student.

Title of the research project: Targeting cell differentiation mechanisms to reduce cancer cell pathogenesis.

Description of the research project:

BACKGROUND: There is growing evidence that molecular pathways regulating the differentiation of stem cells during normal tissue development are often impaired or mis-regulated in the case of cancer development. The capacity of cancer cells to remain undifferentiated and to undergo uncontrolled proliferation, mesenchymal migration and yield metastatic growth could therefore represent be therapeutically targeted by forcing them to differentiate into post-mitotic lineages. By developing approaches to control and force differentiation of undifferentiated cancer cells, arises the possibility to deplete tumours from their most dangerous component.
AIM: This project will use normal can cancer stem cell models to study the process of differentiation in cancer, compared to normal non-cancerous cells. The overall aim is to develop molecular and cellular interventions to force differentiation in cancer cells, and test the hypothesis that this would result in reduced tumorigenic features. Molecular targets include pathways involved in epithelial-mesenchymal transition, and stem-cell specific regulators.
EXPERIMENTAL OUTLINE: The project uses advanced stem cell and cancer cell culture, including 2D and 3D organoid models to assess migration, differentiation and gene expression changes in target cells. Cell differentiation will be induced and their resulting tumorigenic capacity monitored using advanced microscopy, flow cytometry and real-time imaging. Mouse and human cultures, combined with molecular reporters, will be used to identify key regulatory pathways controlling cell differentiation, and drive undifferentiated cancer cells towards a less tumorigenic phenotype.

References

1. Ishay-Ronen D, Diepenbruck M et al. Gain fat-lose metastasis: converting invasive breast cancer cells into adipocytes inhibits cancer metastasis. Cancer Cell 2019;35(1):17-32.e6.
2. Lourenco AR, Ban Y et al. Differential contributions of pre- and post-EMT tumor cells in breast cancer metastasis. Cancer Res 2020;80(2):163-69.
3. Chen T, You Y, et al. Epithelial-mesenchymal transition (EMT): A biological process in the development, stem


 

Research project proposed by: Edoardo Errichiello

Mauro Lecca, PhD student.

Title of the research project: Ocular genetics (and “oculomics”) in developmental eye disorders and uveal melanoma

Description of the research project:

Congenital ocular anomalies, including congenital cataract (CC), microphthalmia/anophthalmia/coloboma (MAC), microcornea, disorders of the anterior segment and primary congenital glaucoma (PCG) are among the most common causes of severe visual impairment and blindness in newborns. On the other side, uveal melanoma (UM) represents the most common intraocular malignancy in adults with currently no effective therapeutic options. Unfortunately, most patients remain undiagnosed because of the high genetic heterogeneity of all these conditions. The main directions of this research project are: I) identification of novel causative genes and molecular pathways related to eye anomalies as well as the investigation of novel pathogenic mechanisms (e.g. chromothripsis in UM) through a range of Next Generation Sequencing (NGS) technologies; II) expanding the phenotypic spectrum associated with known ocular developmental genes; III) functional assessment of variants of uncertain significance; IV) implementation of a multifaceted approach to study rare eye disorders (“oculomics”). A secondary aim of the project is the building up of a regional network for ocular genetics. A subset of patients who will be investigated in this project have already been characterized clinically over the past years and first-tier genetic tests have been performed in almost all cases with negative results. Notably, 52 pedigrees with congenital eye anomalies (mainly CC, MAC, and PCG) and 5 significant UM pedigrees among 12 families with multiple affected generations have been already recruited. Additional familial and sporadic cases will be collected during the first year of the project. The PhD candidate will deal with the most recent NGS approaches (such as whole-exome/genome sequencing), as well as bioinformatics and functional assays. The project will be supported by the Association “Sguardi per la vita Onlus” (a first grant was already allocated in July 2019) and will be carried out in collaboration with the Niguarda Hospital and the AUSL-IRCCS Reggio Emilia.

References:

Errichiello E, Gorgone C, Giuliano L, Iadarola B, Cosentino E, Rossato M, Kurtas NE, Delledonne M, Mattina T, Zuffardi O. SOX2: Not always eye malformations. Severe genital but no major ocular anomalies in a female patient with the recurrent c.70del20 variant. Eur J Med Genet. 2018;61(6):335-340.
Errichiello E, Mustafa N, Vetro A, Notarangelo LD, de Jonge H, Rinaldi B, Vergani D, Giglio SR, Morbini P, Zuffardi O. SMARCA4 inactivating mutations cause concomitant Coffin-Siris syndrome, microphthalmia and small-cell carcinoma of the ovary hypercalcaemic type. J Pathol. 2017;243(1):9-15.
Kurtas N, Arrigoni F, Errichiello E, Zucca C, Maghini C, D’Angelo MG, Beri S, Giorda R, Bertuzzo S, Delledonne M, Xumerle L, Rossato M, Zuffardi O, Bonaglia MC. Chromothripsis and ring chromosome 22: a paradigm of genomic complexity in the Phelan-McDermid syndrome (22q13 deletion syndrome). J Med Genet. 2018;55(4):269-277.


Research project proposed by: Marco Lolicato

Alessandro Bontà, PhD student.

Title of the research project: Structural and functional characterization of the Voltage-gated proton channel (Hv1) channel

Description of the research project:

Ion channels are molecular machines important in regulating membrane potential and ion homeostasis. They, also, play a key role in cell proliferation and apoptosis. Uncontrolled cell growth, resistance to apoptosis and a dysregulated chemical environment are all cancer hallmarks. Therefore, ion channels are considered excellent drug targets. In metastatic breast cancer cell, the voltage gated proton channel Hv1 is involved in cell migration and in the maintenance of intracellular pH. To date, only one class of specific Hv1 inhibitor is known; mostly because only one structure of Hv1 channel is available. However, no mechanism of action has been, yet, described.
The goal of the project is to understand the pharmacology of Hv1 channel from a structural perspective. The project involves the generation of multiple structures in complex with small molecule modulators; validation of the structural findings with electrophysiological experiments and the screening of compound libraries to identify new potential drugs.The PhD candidate will be actively involved in all the project steps, from molecular cloning to data collection, structure determination and functional analysis. He/She will learn the state-of-the-art methodologies in molecular biology, biochemistry, cell biology and electrophysiology. I will guide the PhD candidate to his/her own independence by learning how to design experiments, interpret the results and present the data to an audience.

References

1. Fernández, A., Pupo, A., Mena-Ulecia, K., & Gonzalez, C. (2016). Pharmacological Modulation of Proton Channel Hv1 in Cancer Therapy: Future Perspectives. Molecular Pharmacology, 90(3), 385–402.
2. Takeshita, K., Sakata, S., Yamashita, E., Fujiwara, Y., Kawanabe, A., Kurokawa, T., et al. (2014). X-ray crystal structure of voltage-gated proton channel. Nature Structural & Molecular Biology, 21(4), 352–357.
3. DeCoursey, T. E. (2018). Voltage and pH sensing by the voltage-gated proton channel, HV1. Journal of the Royal Society, Interface, 15(141).


Research project proposed by: Daniele Lilleri
Laboratorio Genetica – Trapiantologia e Malattie cardiovascolari
Fondazione IRCCS Policlinico San Matteo
Prof Mario Mondelli – tutor interno

Piera D’angelo, PhD student.

Evolution of HCMV non-primary infection and host immune response in HCMV-seropositive mothers of children attending day-care centers. An observational prospective study.

Description of the research project:

Human cytomegalovirus (HCMV) is the leading infectious agent causing congenital disabilities. HCMV is a complex DNA virus which following primary infection undergoes latency. Latent strain may periodically restart replication causing reactivation episodes. Additionally, a new HCMV strain may also superinfect an already immune individual causing a reinfection. Reactivations and reinfections (collectively called non-primary infections) occur both in immunocompetent and immunocompromised individuals.
Evolution of and immune response to non-primary HCMV infections in immunocompetent individuals are largely unknown. We designed this study in order to: i) investigate the natural history of non-primary HCMV infection in HCMV-seropositive Italian women and the relevant humoral and cell-mediated immune response; ii) reliably distinguish between reactivation and reinfection. The study will be conducted in a population at high risk for HCMV reinfection i.e. HCMV-immune non-pregnant mothers of children attending day care centers. This study will expand our knowledge about non-primary HCMV infection in women of childbearing age and, in addition, several aspects of the antibody and T-cell response to HCMV will be analysed to provide an advancement in the comprehension of the immune protection from HCMV infection. This issue is now of great interest and future impact, since many efforts are made for the development of HCMV vaccine and several candidate vaccines are being proposed, eliciting different aspects of the B and T cell response.
Britt W. Controversies in the natural history of congenital human cytomegalovirus infection: the paradox of infection and disease in offspring of women with immunity prior to pregnancy. Med Microbiol Immunol 2015; 204:263-71.

References

Ross SA, Arora N, Novak Z, Fowler KB, Britt WJ, Boppana SB. Cytomegalovirus reinfections in healthy seroimmune women. J Infect Dis. 2010;201(3):386-389. doi:10.1086/649903
Wang D, Fu TM. Progress on human cytomegalovirus vaccines for prevention of congenital infection and disease. Curr Opin Virol. 2014;6:13 -23.
Permar SR, Schleiss MR, Plotkin SA. Advancing Our Understanding of Protective Maternal Immunity as a Guide for Development of Vaccines to Reduce Congenital Cytomegalovirus Infections. J Virol. 2018;92. pii: e00030-18.


 

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