Techniques & Applications in Biomedical Science – Research Showcase

Event Context & Pedagogic Goals

• Research Showcase hosted by Manchester Metropolitan University (MMU); two sessions featuring 10 speakers (PhD students, post-docs, MSc/BSc students).

• Emphasis on how specific biomedical techniques address hypotheses, generate data, and feed into conclusions.

• Intended learning outcomes for attending students:

  • Recognise that techniques are chosen to test explicit hypotheses.

  • Practise critical evaluation of data and methodology.

  • Enrich exam revision and plan final-year projects/summer placements.

  • Engage in discussion with active researchers.

General Study Prompts for Each Talk (slide-provided framework)

• Summarise: Hypothesis → Technique(s) → How data were gathered → How data were analysed → Conclusions.

• Consider future applications to your own proposal topics.

Speaker 1 – Chloe Stephenson-Deakin (2nd yr PhD)

Topic: Differential DNA Methylation of ZFHX3 & Stroke Risk

• Background

  • Cardioembolic stroke ≈40 % of all ischaemic strokes.

  • Atrial fibrillation (AF) elevates risk \sim 5\text{-fold}.

  • ZFHX3 (zinc-finger homeobox 3) is a transcriptional regulator linked to AF, stroke, myocardial infarction (GWAS, EWAS data).

• Hypothesis

  • Over-expressing ZFHX3 in megakaryocyte-like cells will reveal downstream pathways influencing stroke susceptibility.

• Key Techniques

  • Transient transfection to over-express ZFHX3–GFP in Meg01 cells.

  • Flow cytometry to quantify transfection efficiency and identify ZFHX3+ population.

  • Light-scatter principles: forward scatter (FSC) ∝ cell size, side scatter (SSC) ∝ granularity.

  • Gating strategy: live-cell gate → singlet gate → GFP-positive gate; baseline set with unstained controls.

• Data & Analysis

  • GFP+ fraction in controls: 0.54\%.

  • GFP+ fraction post-transfection: 59.03\% (n≈10,000 events).

• Conclusion

  • Hypothesis supported; transfection robustly increases ZFHX3 expression (≈\times110 enrichment).

• Next Steps

  • Validate protein over-expression (e.g., Western blot).

  • Nanopore RNA-Seq to map transcriptomic changes.

• Advice Nuggets

  • Network, enjoy conferences, leverage freebies.

Speaker 2 – Dr Chrysa Koukorava (PDRA)

Topic: 3-D in vitro Neurovascular Unit (NVU) Model for Dementia

• Background on iPSC Technology

  • iPSC generated from adult fibroblasts → pluripotent → differentiate into neurons, astrocytes, pericytes, endothelial cells, microglia.

  • Critical for neurodegeneration research where fresh CNS biopsies are infeasible.

• Biological Question

  • How do maturation stages (day 4 → day 61) alter protein expression and cell morphology in patient- vs healthy-derived NVU cells?

• Technique Highlight: Immunocytochemistry (ICC)

  • Indirect immunofluorescence with GFP fluorophore; fixation with paraformaldehyde.

  • Confocal imaging every 10–20 days.

  • Quantification via Fiji/ImageJ.

• Representative Markers & Observations

  • Day 4: neural stem cell markers.

  • Day 52–61: neurons (Tuj1, MAP2), astrocytes (GFAP, Kir4.1, AQP4); spatial organisation visualised (30–200 µm scale bars).

• Conclusion

  • Morphological & molecular maturation confirmed; data support staged differentiation premise.

• Future/Complementary Methods

  • RT-qPCR gene-expression panels (concordant mRNA vs protein).

  • Western blot for absolute quantitation.

• Advice to Undergrads

  • Ask questions; university resources are costly—use them.

  • No need to rush career decisions; transferable skills + passion drive trajectory.

Speaker 3 – Dr Amita Arora (Post-Doc, Helsinki)

Topic: Membrane Contact-Site Protein Protrudin & Endothelial Migration

• Biological Rationale

  • Membrane contact sites (MCS) link ER to endosomes/mitochondria; Protrudin promotes neurite outgrowth.

  • Endothelial migration crucial for angiogenesis & wound healing.

• Hypothesis

  • Protrudin activates focal adhesion kinase (FAK) phosphorylation, thereby enhancing migration.

• Experimental Workflow

  1. shRNA knockdown of Protrudin (vs non-target shNT control).

  2. VEGF stimulation (or basal).

  3. Western blot with phospho-FAK (pY397)–specific antibody.

• Results (n = 3)

  • Protrudin KD confirmed (p<0.001).

  • pFAK levels ↓ significantly in KD both basal and VEGF-stimulated (***p<0.001, ##p<0.01).

• Conclusion

  • Hypothesis upheld: Protrudin required for FAK activation and hence migration.

• Auxiliary Techniques

  • Immunofluorescence imaging of pFAK.

Speaker 4 – Mr Joel Malungu (Summer Student)

Topic: PDLIM Gene Expression in Hepatic Stellate Cells (HSCs)

• Clinical Relevance

  • Liver fibrosis = excess ECM after chronic injury; HSC activation via mechanosignalling (YAP-1).

  • PDLIM proteins scaffold mechanotransduction pathways.

• Hypothesis

  • PDLIM family members are expressed during HSC activation.

• Technique: Quantitative PCR (qPCR)

  • Workflow: TGF-β treatment → RNA isolation → cDNA synthesis → qPCR for COL1A1, ACTA2, 7 PDLIM genes, YAP1.

• Findings

  • PDLIM1, PDLIM5, PDLIM7, and YAP1 expressed in LX-2 cell line.

• Interpretation

  • Supports role for PDLIM5, merits functional assays (siRNA, over-expression) to dissect contribution.

Speaker 5 – Dr Alysha Burrows (PDRA)

Topic: RNA Sequencing in Alzheimer’s Disease (AD) Neural Specification

• Clinical Split

  • Familial (early-onset) vs Sporadic (late-onset) AD; hallmark = premature depletion of neural stem cells in dentate gyrus (DG).

• Hypothesis

  • AD-derived neural stem cells (NSCs) mature prematurely; thus show altered gene-expression trajectories.

• Methods

  • Bulk RNA-Seq on NSCs from controls vs AD patients at ‘younger’ and ‘older’ in-vitro ages.

  • Principal Component Analysis (PCA) using DESeq2; volcano plots for differential expression.

• Key Results

  • PCA: older AD samples diverge more from controls than younger.

  • More differentially expressed genes & maturation-linked pathways dysregulated in AD.

• Significance

  • Genes identified represent targets to delay premature specification—potential for drug screens.

• Integrative Techniques

  • Single-cell RNA-Seq to resolve heterogeneity.

  • ATAC-Seq on isolated nuclei for chromatin accessibility.

  • Flow cytometry to enrich sub-populations pre-sequencing.

• Advice

  • Explore diverse techniques; maintain work-life balance to prevent burnout.

Speaker 6 – Ms Freja Steinke (BSc FYP)

Topic: Effect of Azacytidine (DNMT Inhibitor) on Platelet Integrity

• Background

  • Azacytidine (Aza) irreversibly binds DNA methyltransferase; used in myelodysplastic syndromes (MDS).

  • Reports of thrombocytopenia/bleeding in patients raise concern that Aza impairs platelet function.

• Hypothesis

  • Aza treatment will modulate platelet adhesion & spreading on fibrinogen.

• Techniques

  • Immunostaining with FITC-phalloidin (actin) on platelets incubated with graded Aza concentrations; fluorescent microscopy to quantify adhesion (well-bottom coverage) and spreading (cytoskeletal rearrangement).

  • Photobleaching precautions: store plates dark before imaging.

• Results & Analysis

  • Imaging at 20 min & 60 min showed no significant differences across doses; graphically confirmed.

• Conclusion

  • Hypothesis rejected: Aza did not impair adhesion/spreading of healthy donor platelets.

  • Bleeding in patients may stem from megakaryocyte dysfunction, not platelet defects.

• Future Directions

  • Treat megakaryocyte precursors with Aza, generate platelets in vitro, then assess function.

Speaker 7 – Ms Saarah Mazhar (MSc, now MRC PhD)

Topic: Immune-Cell Contribution to Collagen I During Wound Healing (WH)

• Biological Premise

  • Proper ECM balance critical: excess → fibrosis; deficit → chronic wounds.

  • Zebrafish/mouse data indicate macrophages may directly deposit collagen.

• Hypothesis

  • Neutrophils & macrophages drive collagen I deposition by stimulating fibroblasts or via autonomous secretion.

• Technique Focus: RT-qPCR (SYBR Green)

  • CT values → 2^{-\Delta\Delta C_T} fold-change calculations; melt-curve validation for primer specificity.

• Experimental Overview

  • Cell lines: HDF, THP-1 (mCherry), HL-60.

  • Differentiation/polarisation confirmed via qPCR markers:

  • Macrophage M1 (TNF-α), M2 (TGF-β, VEGF), pan-macrophage CD68.

  • Neutrophil CD11b, CD16, CXCL8.

  • Scratch assay (Incucyte) + immunofluorescence for collagen I.

• Selected Findings

  • Successful differentiation of THP-1 and HL-60 derivatives.

  • M2 macrophages accelerated wound closure; co-culture ± ascorbic acid altered collagen deposition (n = 1 pilot).

• Proposed Next Steps

  • CRISPR-Cas9 to tag HDF collagen (HiBiT) for real-time tracking.

  • Flow cytometry to grade neutrophil maturity (N1 vs N2 polarisation).

  • 3-D fibrin-based culture to recapitulate in vivo ECM.

• Personal Advice

  • Balance hard work with rest; research is demanding but rewarding.

Speaker 8 – Mr Elliot Bowes (Summer Intern)

Topic: Sub-Cellular Localisation of CLN7 in Neuronal Cells

• Disease Context

  • Batten disease (Neuronal Ceroid Lipofuscinosis) caused by mutations in \ge 13 CLN genes; CLN7 encodes a lysosomal transmembrane protein—substrate unknown.

• Hypothesis

  • CLN7 localises to Golgi, ER, or lysosomal membranes.

• Methodological Pipeline

  1. Lentiviral transfection of SH-SY5Y cells with mScarlet-CLN7.

  2. Organelle-specific live dyes: LysoTracker, ER-Tracker, Golgi-Tracker.

  3. Fluorescent microscopy to assess co-localisation (Pearson’s coefficient planned).

• Foundational Skill: Cell Culture

  • Thawing, passaging, media refresh, confluency management outlined step-by-step.

  • Will transition to iPSC-derived neural progenitor cells for final-year project.

• Rationale

  • Cell culture underpins virtually all downstream assays (qPCR, Western, RNA-Seq, ICC, live-cell imaging).

Speaker 9 – Dr Xenia Sawkulycz (PDRA)

Topic: Linking Gene X to Inherited Thrombocytopenia via Knockout Mouse

• Clinical Background

  • Inherited thrombocytopenia: genetically driven low platelet count, autosomal dominant/recessive.

• Hypothesis

  • Gene X expression will be absent or markedly reduced in knockout (fl/fl PF4-Cre) mice vs wild type.

• Technique: qPCR with Genotype Groups

  • WT/WT (+/+), WT/fl (heterozygote), fl/fl (homozygote PF4-Cre).

  • Primer design checked via in silico melt-curve prediction.

• Result

  • Relative expression: fl/fl group ≈0 vs WT baseline 1; statistically verifies successful knockout.

• Conclusion & Future Methods

  • Data support hypothesis; next: Western blot & ICC to confirm protein loss.

Cross-Cutting Techniques & Concepts

• Flow Cytometry: quantitative single-cell analysis; gating and fluorescence thresholds critical.

• qPCR: C_T values converted to fold-change; primer specificity via melt curve; reference genes (GAPDH, S18).

• Western Blotting: antibody-based detection of total vs phosphorylated proteins; densitometry for quantitation.

• Immunostaining/ICC: spatial protein localisation; choice of direct vs indirect, fluorophore selection (FITC, Cy5, GFP).

• RNA Sequencing: bulk vs single-cell; library prep, QC, alignment, differential expression; PCA for variance.

• Cell Culture Fundamentals: thaw, passage, maintain sterility, manage confluency.

Ethical, Practical & Philosophical Notes

• Translational relevance: linking bench assays (e.g., FAK activation, platelet biomechanics) to patient outcomes (bleeding, stroke, dementia).

• Ethical dimension: balancing treatment efficacy (Azacytidine) against adverse effects; ensuring patient quality of life.

• Scientific mindset: value of negative data (Aza study), persistence (multiple techniques to triangulate evidence), inquiry (ask questions early).

Connections to Previous & Foundational Principles

• Central dogma (DNA → RNA → Protein) underlies qPCR, RNA-Seq, Western blot.

• Signal transduction (VEGF → VEGFR2 → FAK pY397) exemplifies kinase cascades.

• Epigenetics (DNA methylation inhibition by Aza, differential methylation in ZFHX3).

• Stem-cell biology (pluripotency vs lineage commitment in iPSC models).

Statistical & Numerical References

• Cardioembolic stroke proportion: 40\%.

• AF risk increase: 5\times baseline.

• Flow-cytometry transfection efficiency: 59.03\% vs 0.54\%.

• qPCR cycling: 25\text{–}35 cycles; significance markers p<0.05\rightarrow p<0.0001.

• Sample replication: typically n=3 biological replicates in Western/qPCR.

Study Recommendations for Exam & Project Prep

• Map each biomedical question to the most informative technique(s).

• Practise interpreting flow-cytometry plots, Western lanes, qPCR melt curves, PCA plots.

• Remember controls: unstained cells, shNT, WT mice, vehicle treatments.

• Always articulate hypothesis → prediction → experimental design → expected vs observed outcome.

• When data refute hypothesis (Aza study), propose mechanistic alternatives and next experiments.