Thesis Defense

What is the purpose of using a commercial olive leaf extract rather than purified oleuropein?

• It is standardized to 20% oleuropein for consistent dosing (contains 300mg of OLE per tsp)

• More translationally relevant

• Affordable and accessible

Why is glycerol used as a vehicle control in this study?

• Glycerol is the excipient in the olive leaf extract, prepared at 0.3% v/v to match the concentration in the highest OLE dilution

• Isolating treatment from solvent effects

What limitations exist when using an immortalized cell line instead of primary patient-derived cells?

• Patient heterogeneity

• Altered signaling

• Stress responses

• Findings are preclinical requiring validation in primary cells

Why does scratch assay closure not exclusively reflect migration?

Scratch closure reflects both migration and proliferation; without mitomycin C, the contributions cannot be separated, leading to results described as impaired wound closure.

What does mitomycin C provide in a scratch assay?

• DNA crosslinking agent

• Blocks cell division without impairing motility,

• Helps attribute changes in wound closure to migration rather than proliferation

Why did you choose these timepoints for the migration assay (24h and 48h)?

• 24h and 48h are standard for capturing early and sustained effects on wound closure

• observing both initial migration and longer-term dynamics

• AKA its standard protocol in literature

What is the rationale for the selected concentration ranges in this study?

• Concentrations are designed to span a physiologically relevant range while avoiding overt cytotoxicity

• Although possible active ranges may not be fully captured.

What do the black dots represent in the scratch assay image?

• Sharpie marks to ensure imaging of the same wound region at each time point

• Maintaining consistency across scratch assay imaging

Why might the OLE+CAL be weaker than OLE alone at 48 hours?

• Calcitriol may attenuate oleuropein's anti-migratory effects over time

• Alter cellular metabolism, allowing partial recovery of migration

Could calcitriol interfere with oleuropein's mechanism?

• Possibility we can't rule out

• Both compounds converge but in different ways

• CAL acts through VDR-mediated transcriptional regulation, while oleuropein acts through antioxidant and direct pathway inhibition.

• It's possible that calcitriol-induced changes in gene expression or metabolism alter the cellular context

• Modifying how cells respond to oleuropein.

• Might explain the 48-hour attenuation in the combination group.

What is the most important takeaway from this study?

• Endometriotic cell behavior is more significantly altered in migration than cytotoxicity

• Targeting inflammation to impair migration may be more relevant in future studies

What is the difference between stromal and epithelial cells?

• Epithelial cells form structured, tightly connected layers and are primarily involved in barrier function and glandular activity

• Stromal cells are part of the connective tissue and provide structural support, secrete extracellular matrix, and regulate the local microenvironment

• In endo, epithelial cells drive lesion formation and surface behaviors such as migration

• while stromal cells contribute to inflammation, fibrosis, and hormone responsiveness.

How is the 12Z cell line immortalized?

• SV40 large T antigen, which inactivates key tumor suppressor proteins, allowing the cells to bypass normal cell cycle regulation and divide indefinitely

• Because of this modification, their behavior may differ from primary endometriotic cells, a limitation in vivo conditions.

What is the chemistry behind OLE and its mechanism of action?

• OLE is a polyphenol, meaning it has aromatic rings with hydroxyl (–OH) groups

• Donates hydrogen to reactive oxygen species

• Stabilizing free radicals and converting them to less reactive forms

• The aromatic ring then stabilizes the leftover radical via resonance, which is why it doesn’t become damaging itself

What distinguishes Vitamin D3 from calcitriol, and why is it relevant?

• Vitamin D3: inactive prohormone, made in skin via UVB

• Magnesium: essential cofactor for all vitamin D enzymes

• Vitamin D3 is a secosteroid — synthesized from 7-dehydrocholesterol in the skin when UVB breaks open the cholesterol ring structure

• The liver adds a hydroxyl group, and then the kidneys add a second hydroxyl group to make calcitriol, which is the form that can actually bind the vitamin D receptor and do something.

• Calcitriol binds VDR, affects inflammation and proliferation

• Used calcitriol directly, no liver or kidneys in cell culture, bypasses conversion

How did you verify the concentration of oleuropein in your extract?

• Relied on the manufacturer's standardization of 20% oleuropein

• Acknowledge the lack of independent verification as a limitation

Why was DMSO used as a solvent for calcitriol?

• DMSO is budget-friendly and effective for solubilizing fat-soluble calcitriol while maintaining non-cytotoxic levels.

Why were 12Z cells specifically chosen for this study?

• Well-characterized immortalized human endometriotic epithelial cell line derived from peritoneal tissue

• Donated

• Maintaining a stable phenotype

• Easy to culture

Why are PBMCs not suitable for comparing epithelial selectivity?

• PBMCs differ in biology and signaling

• they cannot effectively assess the selectivity of compounds on epithelial cells compared to a proper cell line.

Why can't changes in viability be construed as changes in cell number?

• Viability assays reflect metabolic activity, not cell number

• metabolically active cells can produce varying signals even if their numbers are unchanged.

Why was a dose-dependence not observed?

• The tested concentrations may be below thresholds for graded effects, and the active range might not be fully captured.

What does the treatment by time interaction signify statistically?

• It indicates the effect of treatment on wound area varied over time

• Showing non-uniform closure dynamics between treatment groups.

Why did OLE+CAL show viability above 100%?

• Values above 100% mean those wells produced more metabolic signal than the control, not that more cells exist

• Calcitriol is known to modulate mitochondrial function and cellular redox balance through VDR signaling

• This can increase resazurin reduction per cell without any increase in cell count

• Interpreted as calcitriol-induced alteration in cellular metabolism rather than increased proliferation

• Would need a complementary direct cell count assay to confirm

Why was NF-kB not directly measured in this study?

• Measuring NF-kB activation requires additional molecular biology techniques such as western blotting, ELISA, or immunofluorescence which were outside the scope of this study

• The two aims were specifically defined around cellular behavior not intracellular signaling

• Mechanistic interpretation grounded in existing literature

• Direct pathway validation including NF-kB, AKT, ROS, cytokines, and VDR expression is identified as the most important future direction

How can a mechanistic link be suggested without measuring pathways?

• plausible interpretations based on published evidence are presented

• with functional outcomes established.

What is the difference between migration and invasion?

• Migration is movement across a surface, while invasion involves moving through an extracellular matrix

• This study focuses only on migration

Why was a calcitriol-only control not included?

• Dr. Hartmann's previous graduate student Kim Tran had already conducted substantial research on calcitriol's independent effects in the context of endometriosis

• This study was therefore designed to evaluate oleuropein as the primary novel compound, building on that existing calcitriol foundation

• The combination arm was included to test whether the two compounds together produced greater effects than oleuropein alone

• Identified as a priority for future work

How does this study advance the field beyond previous work?

It is the first study in human endometriotic epithelial cells, examining oleuropein with calcitriol, and employing quantitative statistical modeling.

How does the in vitro dilution series relate to in vivo exposure?

In vitro testing does not directly reflect physiological conditions, as oleuropein is metabolized rapidly, affecting actual tissue concentrations.

How does rapid in vivo metabolism of oleuropein affect relevance?

Rapid metabolism limits direct extrapolation of findings, as metabolites may retain activity but not replicate the parent compound's effects.

Would results differ with primary cells?

Yes, primary cells would reflect biological heterogeneity and variability in behavior that the stable phenotype of 12Z cells may not capture.