THE ROLE OF IFITM3 IN MAMMARY GLAND DEVELOPMENT AND MAMMARY STEM CELLS

Le cellule staminali della ghiandola mammaria sono un modello per lo studio dei processi molecolari che regolano lo sviluppo fisiologico dell\u2019organo e lo sviluppo tumorale. Diversamente da molti organi, la ghiandola mammaria incorre in cicli di rigenerazione e involuzione associati alla gravidanza. Per questo la ghiandola mammaria \ue8 uno dei primi organi nel quale si \ue8 ipotizzata la residenza di cellule staminali. Dal concetto che le cellule staminali regolano la crescita e la differenziazione della ghiandola con l\u2019avvento della gravidanza \ue8 stato ipotizzato che cellule tumorali con propriet\ue0 staminali possano contribuire all\u2019eterogeneit\ue0 del tumore. Al fine di scoprire come il normale sviluppo dell\u2019architettura della ghiandola mammaria sia originata dalle cellule staminali e il ruolo delle cellule staminali tumorali nella progressione tumorale, in questo lavoro di tesi \ue8 stata indagata la funzione di IFITM3. IFITM3 \ue8 stato inizialmente identificato dal laboratorio della dott.ssa Zucchi nelle cellule LA7, cellule staminali della ghiandola mammaria di ratto capaci di differenziare in dome, strutture 2D simili agli alveoli che si formano nella ghiandola mammaria durante la gravidanza. \uc8 stato dimostrato dal gruppo Zucchi che la funzione di IFITM3 dipende dal cambio di localizzazione della proteina dal citoplasma alla membrana plasmatica, dove risiede nei lipid raft. Questo \ue8 stato il punto di partenza di questa tesi di ricerca con lo scopo di identificare se IFITM3 partecipi anche alla formazione di strutture tridimensionali complesse della ghiandola mammaria (strutture tubulo-alveolari) e se svolga una funzione nel mantenimento delle cellule staminali della ghiandola mammaria. Ho utilizzato sistemi transienti e stabili per la modulazione dei livelli di espressione di IFITM3 in cellule LA7 e in cellule MCF7, una linea cellulare umana di ghiandola mammaria. Mentre le cellule LA7 possiedono sia la capacit\ue0 di generare sfere sia di differenziare morfologicamente e funzionalmente in tutti i tipi cellulari e strutture 3D della ghiandola mammaria, si ritiene che cloni MCF7 utilizzati in questo studio non possiedano propriet\ue0 di cellule staminali, non formano sfere e possono differenziare unicamente in un tipo di struttura 3D, le cisti simili agli alveoli della ghiandola mammaria. Abbattendo i livelli di espressione di IFITM3 con siRNA/oligo e tecnologia lenti virale ad RNA a forcina corta (shRNA) in cellule impiegate in saggi funzionali in condizioni di coltura 3D, ho scoperto che IFITM3 \ue8 necessario per la formazione di strutture tubulo-alveolari complesse in cellule MCF7 e per il mantenimento delle propriet\ue0 di auto-rinnovamento delle cellule staminali LA7. Dato che le mammosfere e le cisti mammarie rappresentano strutture 3D associate rispettivamente a cellule staminali o differenziate, e dato che la sotto-regolazione di IFITM3 inibisce la formazione di entrambe, possiamo ipotizzare che IFITM3 abbia una funzione diversa in base al tipo cellulare. La sotto-regolazione di IFITM3 in cellule staminali LA7 in condizioni di coltura aderenti e non aderenti ha portato ad una graduale perdita delle cellule, suggerendo che IFITM3 svolga una funzione necessaria al mantenimento dell\u2019auto-rinnovamento delle cellule staminali LA7. In cellule MCF7 la sotto-regolazione di IFITM3 ha portato all\u2019incapacit\ue0 delle cellule di formare alveoli senza effetti sulla proliferazione cellulare. La sovra-regolazione di IFITM3 sia in cellule LA7 sia MCF7 ha portato alla rapida morte cellulare per un meccanismo che \ue8 ancora sotto indagine. Complessivamente ho dimostrato che la sotto-regolazione di IFITM3 porta alla perdita delle propriet\ue0 di auto-rinnovamento II e della capacit\ue0 di essere propagate come cellule staminali delle cellule LA7. La sotto-regolazione di IFITM3 in cellule che non hanno propriet\ue0 staminali di auto-rinnovamento porta all\u2019incapacit\ue0 delle cellule di formare strutture 3D. Lo studio dei profili di espressione genica con tecnologia micorarray ottenuti da cellule LA7 trattate per la sotto-regolazione di IFITM3 supporta il ruolo di IFITM3 nella regolazione del ciclo cellulare, nel trasporto vescicolare e nella modificazione dello stato cromatinico. Geni coinvolti nella proliferazione cellulare sono stati trovati sotto-regolati in seguito alla perdita di IFITM3, insieme a geni del trasporto vescicolare (che coinvolgono proteine che mediano la fusione vescicolare, come le SNARE) che possono essere collegati a IFITM3 in quanto il ruolo di IFITM3 nel prevenire il rilascio delle particelle virali dai compartimenti endosomiali \ue8 associato alla formazione di un poro di fusione e alla fusione delle membrane. I geni associati con la regolazione epigenomica e la proliferazione cellulare suggeriscono che IFITM3 possa avere differenti ruoli in cellule staminali e cellule della ghiandola mammaria differenziate, dato che questi geni sono coinvolti nel cancro, nella crescita cellulare o apoptosi e nel differenziamento cellulare.Mammary stem cells (MaSCs) are a model to understand molecular processes that regulate both normal and cancer development. In contrast to many organs, the mammary gland undergoes cycles of re-generation and involution associated with pregnancy. For this reason, the mammary gland was one of the first organs in which stem cells (SCs) were hypothesized to reside. From the concept that SCs regulate normal mammary gland growth and differentiation with onset of pregnancy, arose the hypothesis that cancer cells with SC properties may contribute to tumor heterogeneity. To understand how normal development of the mammary gland architecture arises from SCs and the role of CSCs in tumor progression, the function of the IFITM3 gene was investigated in this thesis research. IFITM3 was initially identified by Zucchi\u2019s lab in LA7 cells, rat mammary SCs that differentiate into domes, 2D structures similar to alveoli that form in mammary gland at pregnancy. It was demonstrated by the Zucchi group that the function of IFITM3 was dependent on its shuttling from the cytoplasm to the plasma membrane and being part of lipid rafts. This was the starting point of the thesis research with the aims to identify whether IFITM3 also plays a role in the formation of more complex three dimensional (3D) mammary structures (tubule-alveolar structures) and has a role in MaSCs. I used transient and stable IFITM3 expression modulating systems in the LA7 and the human MCF7 mammary cell line. In contrast to the rat LA7 SCs that have both the capacity to generate spheres and differentiate morphologically and functionally into all the mammary cell types and generate tubule-alveolar structures iin 3D culture conditions, the MCF7 clone used in this study are not considered to have SC properties, do not form spheres and can only form one type of 3D differentiated structure called cysts. Targeting IFITM3 in these cells, with siRNA/oligos and short hairpin RNA lentiviral technology in order to down-regulate the expression of the protein, and by performing functional assays in 3D culture conditions, I found that IFITM3 is necessary for the formation of complex alveolar structures in MCF7 cells and for self-renewal of LA7 SCs. As mammary spheres and mammary cysts represent different 3D structures associated with stem and differentiated cells respectively, and since IFITM3 down-regulation inhibits the formation of both, this suggests that IFITM3 may have different functions depending on the cell type. Down-regulation of IFITM3 in LA7 SCs in both adherent or in non-adherent cultures resulted in a gradual loss in the number of cells, suggesting that IFITM3 function is necessary for self-renewal of LA7 SCs. In contrast down regulation of IFITM3 in MCF7 cells resulted in the inability of the cells to form alveoli with no effect in cell proliferation. IFITM3 up-regulation in both LA7 and MCF7 cells resulted in rapid cell death by a mechanism that is still under investigation. Collectively, I demonstrate, that IFITM3 down-regulation results in loss of self-renewal and loss of the ability of LA7 to be propagated as SCs. While IFITM3 down-regulation in mammary cells that do not IV have SC self-renewal capacity results in the inability of the cells to generate 3D structures. Microarray expression experiments, obtained with down-regulation of IFITM3 in LA7 cells, support the IFITM3 function in cell cycle regulation, vesicular transport and in the modification of the chromatin state. In addition to genes involved in cell proliferation found down-regulated with loss of IFITM3, a link among vesicle-transport genes (involving proteins mediating vesicle fusion, such as SNARE), and IFITM3 was also determined, supporting a role of IFITM3 in preventing the release of viral particles from endosomal compartments, associated with membrane fusion and the formation of a fusion pore. Genes associated with epigenomic regulation and cell proliferation suggests that IFITM3 may have multiple and different roles in SCs and in differentiated mammary cells since these genes are involved in cancer, cell growth or apoptosis and in cell differentiation

Killing the invaders: NK cell impact in tumors and anti-tumor therapy

Natural Killer cells belong to group 1 innate lymphoid cells, which also includes ILC1s. NK/ILC1s are highly heterogeneous cell types showing distinct phenotypes across tissues and conditions. NK cells have long been described as innate lymphocytes able to directly and rapidly kill tumor cells without antigen-restriction. Different mechanisms were shown to modulate NK cell activation and tumor resistance, mainly based on cytokine stimulation and receptor-ligand interactions, and several strategies have been developed to target NK cells in tumor immunotherapy to promote NK cell function and overcome tumor evasion. The characterization of ILC1 distinct phenotype and function and the specific role in tumors still needs further investigation and will be essential to better understand the impact of innate lymphoid cells in tumors. Here, we review key aspects of NK cell biology that are relevant in tumor immune surveillance, emphasizing the most recent findings in the field. We describe the novel therapeutical strategies that have been developed in tumor immunotherapy targeting NK cells, and we summarize some recent findings related to NK cell/ILC1 transition in tumor models

Analyzing psychotherapeutic failures: A research on the variables involved in the treatment with an individual setting of 29 cases

The effectiveness of psychotherapeutic treatments has been widely demonstrated and confirmed by many studies in recent decades. The research focused on the factors of change influencing the positive outcomes of a psychotherapy, putting those that are crucial in cases of failure into the background. The dimensions of this phenomenon are relevant as well as the side effects of the psychotherapeutic interventions that reach the same percentages of the pharmacotherapeutic treatments. The study of the variables involved in failure cases therefore seems important to prevent or moderate the negative effects of treatments with a negative outcome. Impasse and deadlock situations, which may result in an early interruption of psychotherapy, are often complex and involve situational, relational, and personal factors at different levels and with different weight. A research was conducted, with a mixed approach, aimed at exploring the situational factors involved in dropout cases. In addition, the evaluation of the psychotherapist's emotional responses related to patients who terminated psychotherapy prematurely was investigated. The study was attended by a sample of 29 psychologists, experienced psychotherapists from different frameworks. Recent or salient cases of a hesitated psychotherapy with an early interruption were examined. For the first objective, a structured interview (Impasse Interview) was used, while the second one was reached by the administration of the TRQ (Therapist Response Questionnaire). The transcripts of the interviews were analyzed through a textual analysis software and five salient thematic clusters were identified. These were then assimilated to different areas of meaning: severity of the diagnosis, procedural aspects and lack of understanding of the stall in progress. Two other important themes emerged: the critical aspects concerning relational dynamics and a focus on maternal theme. Overall these five thematic areas seem to play an important and specific role compared to dropout cases. Finally, statistical analysis on emotional responses have highlighted some values above the average in these four countertransference factors: Helpless/Inadequate, Parental/Protective, Positive/Satisfied, and Overwhelmed/Disorganized. It is hypothesized that particular emotional responses of the psychotherapist may be prognostic with respect to the outcome of psychotherapy

Motherhood in the Time of Coronavirus: The Impact of the Pandemic Emergency on Expectant and Postpartum Women\u2019s Psychological Well-Being

The birth of a child is a critical and potentially stressful experience for women, entailing several changes both at the individual and interpersonal level. This event can lead to different forms of distress, ranging in intensity and duration. Many studies highlighted medical, psychological, and social variables as risk factors potentially influencing the onset or aggravation of perinatal maternal conditions. The current pandemic emergency and the restrictive measures adopted by local governments to prevent the spread of the coronavirus infection may negatively affect mothers-to-be and new mothers potentially increasing the likelihood of anxiety, depressive or post-traumatic symptoms to develop. Moreover, the forced quarantine combined with the limited access to professional or family support may increase feelings of fatigue and isolation. The present study aims to investigate women\u2019s psychological well-being during pregnancy and in the first months after childbirth, integrating the evaluation of some traditionally studied variables with the specificities of the current situation. 575 Italian women have been administered an online self-report questionnaire assessing the presence of anxiety disorders, depressive and post-traumatic symptoms as well as the expectations toward childbirth (for mothers-to-be) or the subjective experience of childbirth (for postpartum women). Findings revealed a higher percentage of women than that reported in the literature scored above the clinical cut-off both during pregnancy and postpartum on a series of measures of psychological well-being, thus demonstrating that this period was perceived as particularly challenging and stressful and had significant impact on the women\u2019s well-being. Moreover, some socio-demographic, medical, and pandemic-related variables, especially the lack of presence and support from one\u2019s partner during labor and delivery as well as in the first days postpartum was found to predict women\u2019s mental health. These findings suggest the need for developing specific interventions targeted at women who cannot benefit from the support of their partners or family

Dropout and narcissism: An exploratory research about situational factors and personality variables of the psychotherapist

The premature interruption of psychotherapeutic treatments is a significant phenomenon, with rates that can be above 50%, although differences emerge among studies. In general, the variables related to the psychotherapists have a significant effect on the treatment and a greater extent than the situational variables. In this study a multi-method research has been conducted to test the situations of impasse ending in dropout and the relationship between these situations and the covert narcissistic dimension of the therapist. Each participant was administrated the Impasse Interview, asking the therapist to focus on a salient or recent dropout case, and the Hypersensitive Narcissism Scale. The linguistic analysis of the interviews carried out with T-Lab produced 4 thematic clusters. Furthermore, as for the covert narcissistic dimension, 11 therapists reported a score below the average (Group A) and 9 scoring in the average (Group B). Finally, the association between the four clusters and the two groups of therapists was investigated. The therapeutic alliance and the emotional reaction of the therapist, influenced by his narcissistic dimension, represent key variables for the outcome of the therapy

Inositol phosphatase INPP4B sustains ILC1s and intratumoral NK cells through an AKT-driven pathway

Innate lymphoid cells (ILCs) are a heterogeneous population of lymphocytes that coordinate early immune responses and maintain tissue homeostasis. Type 1 innate immune responses are mediated by natural killer (NK) cells and group 1 ILCs (ILC1s). Despite their shared features, NK cells and ILC1s display profound differences among various tissue microenvironments. Here, we identify the inositol polyphosphatase INPP4B as a hallmark feature of tissue-resident ILC1s and intratumoral NK cells using an scRNA-seq atlas of tissue-associated and circulating NK/ILC1s. Conditional deletion of Inpp4b in ILC1s and NK cells reveals that it is necessary for the homeostasis of tissue-resident ILC1s but not circulating NK cells at steady-state. Inpp4b-deficient cells display increased rates of apoptosis and reduced activation of the prosurvival molecule AKT. Furthermore, expression of Inpp4b by NK/ILC1s is necessary for their presence in the intratumoral environment, and lack of Inpp4b impairs antitumor immunity. These findings highlight INPP4B as a novel regulator of tissue residency and antitumor function in ILC1s and NK cells

In the Eye of the Covid-19 Storm: A Web-Based Survey of Psychological Distress Among People Living in Lombardy

In March 2020, the World Health Organization announced the Covid-19 outbreak a pandemic and restrictive measures were enacted by the Governments to fight the spread of the virus. In Italy, these measures included a nationwide lockdown, with limited exceptions including grocery shopping, certain work activities, and healthcare. Consistently with findings from previous studies investigating the psychological impact of similar pandemics [e.g., Severe Acute Respiratory Syndrome (SARS)], there is evidence that Covid-19 is associated with negative mental health outcomes. Given this background, we conducted a cross-sectional study aimed at investigating the impact of the Covid-19 pandemic and the subsequent restrictive measures imposed by the Government on the psychological health of Italian men and women aged = 18 years and living in Lombardy, one of the worst-hit regions. The study also aimed at identifying what factors are associated with specific psychological outcomes. Thus, we developed an online survey that included a researcher-made questionnaire to collect sociodemographic, household, general health, and pandemic-related information. The Generalized Anxiety Disorder-7, the Patient Health Questionnaire-9 and the Perceived Stress Scale were used to assess anxiety, depression, and perceived stress, respectively. We found that younger age, greater concerns about the pandemic, female gender, being unmarried, not having children, and being a student were associated with worse psychological health. These findings may provide further insight into the risk factors associated with negative psychological outcomes during the current pandemic, with identification of vulnerable groups. This body of evidence may help professionals implement targeted psychosocial treatment and prevention programs

Perinatal Parenting Stress, Anxiety, and Depression Outcomes in First-Time Mothers and Fathers: A 3- to 6-Months Postpartum Follow-Up Study

Objective: Although there is an established link between parenting stress, postnatal depression, and anxiety, no study has yet investigated this link in first-time parental couples. The specific aims of this study were 1) to investigate whether there were any differences between first-time fathers’ and mothers’ postnatal parenting stress, anxiety, and depression symptoms and to see their evolution between three and 6 months after their child’s birth; and 2) to explore how each parent’s parenting stress and anxiety levels and the anxiety levels and depressive symptoms of their partners contributed to parental postnatal depression. Method: The sample included 362 parents (181 couples; mothers’ MAge = 35.03, SD = 4.7; fathers’ MAge = 37.9, SD = 5.6) of healthy babies. At three (T1) and 6 months (T2) postpartum, both parents filled out, in a counterbalanced order, the Parenting Stress Index-Short Form, the Edinburgh Postnatal Depression Scale, and the State- Trait Anxiety Inventory. Results: The analyses showed that compared to fathers, mothers reported higher scores on postpartum anxiety, depression, and parenting stress. The scores for all measures for both mothers and fathers decreased from T1 to T2. However, a path analysis suggested that the persistence of both maternal and paternal postnatal depression was directly influenced by the parent’s own levels of anxiety and parenting stress and by the presence of depression in his/her partner. Discussion: This study highlights the relevant impact and effects of both maternal and paternal stress, anxiety, and depression symptoms during the transition to parenthood. Therefore, to provide efficacious, targeted, early interventions, perinatal screening should be directed at both parents

Atención socioemocional a niños con rezago escolar y en situación de vulnerabilidad

ITESO, A.C

The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell-mediated resistance to metastasis

Fondazione Cariplo (grant no. 2015–0564 to A.M.)Cluster Alisei (grant no. MEDINTECH CTN01_00177_962865 to A.M.)European Research Council (grant no. 669415-PHII to A.M.)Italian Association for Cancer Research (AIRC IG-2016 grant no. 19014 to A.M.; AIRC 5 × 1000 grant no. 21147 to A.M.; AIRC IG-2016 grant no. 19213 to M.L.)Medical Research Council (Pathobiology of Early Arthritis Cohort grant no. 36661 to C.P.)Arthritis Research UK Experimental Treatment Centre (grant no. 20022 to C.P.