## 4 Discussion  
In this study, the differentiation-inducing effect of the pure polyphenol tyrosol at the concentration found in the 'Bidni' Maltese EVOO variety is described. Evidence of significant differentiation and a change in cell maturation was shown through biochemical assays, the assessment of cell surface marker expression, the observation of morphological features, and transcription analysis. Critically, we also observed that the anti-proliferative action of tyrosol on HL-60 cells is specific, as no cytotoxicity was observed when the same polyphenol was tested on non-malignant human lymphocytes. The differentiating activity and inhibition of cell proliferation of HL-60 cells has been reported elsewhere using EVOO crude polyphenol extracts. [PERSON] et al., 2006 suggested that this effect is partially a result of dialdehyde forms of elenoic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) and tyrosol (pHPEA-EDA) present in the phenolic extract while [PERSON] et al., 2009 reported that the composition of their crude extract contained 3,4-DHPEA, pHPEA, 3,4-DHPEA-EDA, pHPEA-EDA, and 3,4-HPEA-EDA (oleuropein aglycone) [21,32]. In this study, the pure compound pHPEA was used allowing the confirmation that differentiation was due to a single bioactive agent. In addition, an extended testing time-frame of 3 and 5 days, respectively, was used. Since marked oxidative activity and expression of cell surface markers was seen on day 3, this suggests that tyrosol is driving the HL-60 cells to the monocyte stage as opposed to that of the granulocyte. The capacity of polyphenols to inhibit cell cycle progression is an important feature shared with many drugs currently used as cancer treatments and was therefore assessed here. Previous work by [PERSON] et al., 2008 demonstrated that hydroxytyrosol treatment of HL-60 cells over a period of 25 h resulted in an increase in cells in the G\({}_{0}\)/G\({}_{1}\) phase and a decline in cells in the S phase, causing an increase in apoptotic cells [24]. This result is compatible with our findings for tyrosol, where a marked increase in the cells recorded in the sub G\({}_{\text{o}}\)/G\({}_{1}\) stage was observed, although a direct comparison is not possible as the experimental time-plan differed. We also report an increase in cells undergoing late apoptosis, as well as necrosis on day 5. These results are in contrast with those recorded by [PERSON] et al., 2000 who reported an absence of anti-proliferative activity and apoptosis induction by tyrosol at a concentration of 100 \(\upmu\)M for 2 days, however, the timeframe may have been a contributing factor [37]. In a further step, we also analysed the HL-60 transcriptome following polyphenol application in an effort to describe the genetic changes seen during tyrosol-induced monocytic differentiation. The transcriptome analysis, which is reported here for the first time, further confirms the upregulation of myeloid differentiation genes such as OSCAR, RELB, VEGFA, GAB2, JUNB, DNASE2, ICAM, and CCL3. OSCAR is a cell surface receptor expressed on monocytes, overexpression of which is linked to monocyte adhesion and migration [38,39]. The importance of VEGFA in myeloid cell differentiation has also been reported by [PERSON] et al., 2017 while [PERSON] et al., 2011 discussed its expression in two monocyte subsets [40,41], showing its role in monocyte chemotaxis. JUNB has been found to regulate the responses of monocytes to different immunostimulatory ligands, and its levels have also been reported to increase during monocyte maturation [42]. Moreover, ICAM that was upregulated across all time points, is a cell surface marker that is expressed by macrophages. The adhesion molecule binds to CD11b, which was observed to increase using fluorescent antibody analysis (Figure 5a). ALDH codes for aldehyde dehydrogenase, an enzyme important for the regulation of HSC differentiation. [PERSON] et al. 2006 reported that ALDH inhibition in NOD/SCID mice resulted in HSC expansion and a delay in HSC differentiation [43].