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Transcript
Laboratory Strategies for Investigating Disease
By
Charles J. Dimitroff, Ph.D.
Associate Professor of Dermatology
Associate Director for Laboratory Research
Department of Dermatology
Brigham and Women’s Hospital
Harvard Medical School
Faculty, Ph.D. Program in Immunology
The Graduate School of Arts and Sciences
Division of Medical Sciences,
Harvard University
Outline
1.) Establishing a Disease’s Threat to Human Health
• Does a disease impact a human’s lifespan or quality of life?
Outline
1.) Establishing a Disease’s Threat to Human Health
• Does a disease impact a human’s lifespan or quality of life?
2.) Identifying Gaps in Our Knowledge of Disease
• Biomarkers of disease development/progression
• Mechanism(s) of disease development/progression
Outline
1.) Establishing a Disease’s Threat to Human Health
• Does a disease impact a human’s lifespan or quality of life?
2.) Identifying Gaps in Our Knowledge of Disease
• Biomarkers of disease development/progression
• Mechanism(s) of disease development/progression
3.) Approaches to Studying Disease
• Exploring tissue, cellular, molecular and genetic features of disease
Outline
1.) Establishing a Disease’s Threat to Human Health
• Does a disease impact a human’s lifespan or quality of life?
2.) Identifying Gaps in Our Knowledge of Disease
• Biomarkers of disease development/progression
• Mechanism(s) of disease development/progression
3.) Approaches to Studying Disease
• Exploring tissue, cellular, molecular and genetic features of disease
4.) Seeking Funding to Conduct for Studies on Disease
1.) Establishing a Disease’s Threat to Human Health
Examples of Diseases:
Autoimmune Diseases (Rheumatoid Arthritis, Lupus, Psoriasis, MS)
Neuro-degenerative/-developmental Disorders (Alzheimer's, Muscular dystrophy, ALS)
Drug Addiction/Cognitive/Depression Disorders
Infectious Diseases (viral/fungal/bacterial/parasitic)/Immune Cell Function
Cardiovascular Disease
1.) Establishing a Disease’s Threat to Human Health
Examples of Diseases:
Autoimmune Diseases (Rheumatoid Arthritis, Lupus, Psoriasis, MS)
Neuro-degenerative/-developmental Disorders (Alzheimer's, Muscular dystrophy, ALS)
Drug Addiction/Cognitive/Depression Disorders
Infectious Diseases (viral/fungal/bacterial/parasitic)/Immune Cell Function
Cardiovascular Disease
Cancer
Lifetime Risk:
Nearly 1 in 2 men get cancer
Nearly 1 in 3 women get cancer
Nearly 1 in 4 will die from cancer
1.) Establishing a Disease’s Threat to Human Health
Examples of Diseases:
Autoimmune Diseases (Rheumatoid Arthritis, Lupus, Psoriasis, MS)
Neuro-degenerative/-developmental Disorders (Alzheimer's, Muscular dystrophy, ALS)
Drug Addiction/Cognitive/Depression Disorders
Infectious Diseases (viral/fungal/bacterial/parasitic)/Immune Cell Function
Cardiovascular Disease
Cancer Metastasis
2.) Identifying Gaps in Our Knowledge of Disease
Normal (or benign) vs. Cancer
Non-metastatic vs. Metastatic
Transformation (Carcinogenesis)
Intravasation/Extravasation
Invasion
Survive/Grow in Distant Tissue
Tumor Angiogenesis
Tumor Angiogenesis
Immune Evasion
Immune Evasion
2.) Identifying Gaps in Our Knowledge of Cancer
Cancer Discovery/Basic Science
Biomarkers of Cancer/Metastasis
Cancer-host genes/proteins as markers
of early disease or progression
Functional Factors in Cancer/Metastasis
Cancer-host genes/proteins as causal
factors in progression pathways
Cancer Therapeutics
Cancer Treatment
Anti-Cancer/-Metastatic Drugs and Immune Boosting Agents
3.) Approaches to Studying Disease
• Exploring tissue, cellular, molecular and genetic features of disease
3.) Approaches to Studying Disease
Experimental Contexts:
In vitro (Latin: “In glass”)
Studies on cells or molecules outside their biological context
• Proteins in solution; Cells in artificial culture medium
3.) Approaches to Studying Disease
Experimental Contexts:
In vitro (Latin: “In glass”)
Studies on cells or molecules outside their biological context
• Proteins in solution; Cells in artificial culture medium
In situ (Latin: “On site”)
Studies on cells or molecules within tissues (live or fixed)
H&E
S100
Gal-1hFc
DAPI
Merged
Laboratory Methods: Immunohistochemistry/-fluorescence; Fluorescence In situ Hybridization
3.) Approaches to Studying Disease
Experimental Contexts:
In vitro (Latin: “In glass”)
Studies on cells or molecules outside their biological context
• Proteins in solution; Cells in artificial culture medium.
In situ (Latin: “On site”)
Studies on cells or molecules within tissues (live or fixed)
H&E
S100
Gal-1hFc
DAPI
Merged
Laboratory Methods: Immunohistochemistry/-fluorescence; Fluorescence In situ Hybridization
In vivo (Latin: “Within the living”)
Studies on biological/pathobiological pathways in living organisms
• Humans; Mice; Rats; Rabbits; Fish; Dogs; Flies; Worms
3.) Approaches to Studying Cancer
A. Correlative Analysis (Biomarker/Target Identification)
Normal
vs.
Cancer
vs.
Metastasis
Molecular Expression
Gene/Genome
Protein/Proteome
Glycome, Lipidome &
Protein Modifications
Glycans
Lipids
Sulfation
Phosphorylation
3.) Approaches to Studying Cancer
A. Correlative Analysis (Biomarker/Target Identification)
Normal
vs.
Cancer
vs.
Metastasis
Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein Modifications
• Gene expression (Relative gene levels/mutations)
Laboratory Methods: Hybridization DNA microarrays, Whole genome/exome sequencing,
qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis
3.) Approaches to Studying Cancer
A. Correlative Analysis (Biomarker/Target Identification)
Normal
vs.
Cancer
vs.
Metastasis
Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein-Modifications
• Gene expression (Relative gene levels/mutations)
Laboratory Methods: Hybridization DNA microarrays, Whole exome sequencing,
qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis
• Protein expression (Relative protein levels/variants)
Laboratory Methods: Western blot, 2D-electrophoresis, flow cytometry, Immunobeads,
Immunohistochemistry/-fluorescence, High-performance liquid chromatography, Mass
spectrometry, Immuno/lectin affinity chromatography, ELISAs, SDS-PAGE
3.) Approaches to Studying Cancer
A. Correlative Analysis (Biomarker/Target Identification)
Normal
vs.
Cancer
vs.
Metastasis
Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein-Modifications
• Gene expression (Relative gene levels/mutations)
Laboratory Methods: Hybridization DNA microarrays, Whole exome sequencing,
qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis
• Protein expression (Relative protein levels/variants)
Laboratory Methods: Western blot, 2D-electrophoresis, flow cytometry, Immunobeads,
Immunohistochemistry/-fluorescence, High-performance liquid chromatography, Mass
spectrometry, Immuno/lectin affinity chromatography, ELISAs, SDS-PAGE
• Lipid expression (Relative lipid levels/variants)
Laboratory methods: Organic extractions; Thin-layer chromatography, HPLC, MS
3.) Approaches to Studying Cancer
A. Correlative Analysis (Biomarker/Target Identification)
Normal
vs.
Cancer
vs.
Metastasis
Comparative Analysis of Host/Cancer Genes/Genome, Proteins, Lipids & Protein-Modifications
• Gene expression (Relative gene levels/mutations)
Laboratory Methods: Hybridization DNA microarrays, Whole exome sequencing,
qPCR, Real-time RT-qPCR, Nanostring Technology, RNA sequencing, SNP analysis
• Protein expression (Relative protein levels/variants)
Laboratory Methods: Western blot, 2D-electrophoresis, flow cytometry, Immunobeads,
Immunohistochemistry/-fluorescence, High-performance liquid chromatography, Mass
spectrometry, Immuno/lectin affinity chromatography, ELISAs, SDS-PAGE
• Lipid expression (Relative lipid levels/variants)
Laboratory methods: Organic extractions; Thin-layer chromatography, HPLC, MS
• Protein-Modifications (Relative modification levels)
Laboratory Methods: Western blot, flow cytometry, Immuno-beads, Immunohistochemistry/-fluorescence , HPLC, MS, Immuno/lectin affinity chromatography
3.) Approaches to Studying Cancer
B. Molecular Function Assessments (Establish a Pro/Anti-tumor Role)
X
Pro-Invasion
Malignant
Transformation
Pro-Adhesion
P
A
X
Normal Melanocyte
Pro-Angiogenic
A
Pro-Immune Evasion
Melanoma Cell
Y
Z
X
Genes “A” •
Proteins “X”
•
Lipids “Y”
•
Protein Modifications “Z”
Hypothesis-driven Research
Examples:
1.) Gene “A” triggers melanomagenesis.
2.) Protein “X” or Modification “Z” causes intravascular adhesion and metastasis.
3.) Cancer cell invasion is dependent on Lipid “Y” expression.
3.) Approaches to Studying Cancer
B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role)
1.) Turn on genes (Gain of Function)
- Elevate gene and corresponding protein, lipid or protein-modification levels
2.) Turn off genes (Loss of Function)
- Silence gene expression, ablate the gene or replace with drug-resistance gene
3.) Approaches to Studying Cancer
B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role)
1.) Turn on genes (Gain of Function)
- Elevate gene and corresponding protein, lipid or protein-modification levels
2.) Turn off genes (Loss of Function)
- Silence gene expression, ablate the gene or replace with drug-resistance gene
Laboratory Methods:
• Overexpress gene expression in host/cancer cells
- Transfection (plasmid vector)/ transduction (virus) of cDNA
• Silence gene expression in host/cancer cells
- Transfection (plasmid)/ transduction (virus) of cDNA
- mRNA interference (destruction of mRNA species)
- short-hairpin interfering RNAs
- small-interfering RNAs or microRNAs
- Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas9
- Targeted gene resection
3.) Approaches to Studying Cancer
B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role)
1.) Turn on genes (Gain of Function)
- Elevate gene and corresponding protein, lipid or protein-modification levels
2.) Turn off genes (Loss of Function)
- Silence gene expression, ablate the gene or replace with drug-resistance gene
Laboratory Methods:
• Overexpress gene expression in host/cancer cells
- Transfection (plasmid vector)/ transduction (virus) of cDNA
• Silence gene expression in host/cancer cells
- Transfection (plasmid)/ transduction (virus) of cDNA
- mRNA interference (destruction of mRNA species)
- short-hairpin interfering RNAs
- small-interfering RNAs or microRNAs
- Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas9
- Targeted gene resection
• Construction of mutant mice (germ line transmission)
- Targeted gene disruption (Knock-out) - whole animal or conditional (tissue-specific)
- Targeted gene insertion (Knock-in) - inducible or tissue/cell-specific
3.) Approaches to Studying Cancer
B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role)
1.) Turn on genes (Gain of Function)
- Elevate gene and corresponding protein, lipid or protein-modification levels
2.) Turn off genes (Loss of Function)
- Silence gene expression, ablate the gene or replace with drug-resistance gene
Laboratory Methods:
• Overexpress gene expression in host/cancer cells
- Transfection (plasmid vector)/ transduction (virus) of cDNA
• Silence gene expression in host/cancer cells
- Transfection (plasmid)/ transduction (virus) of cDNA
- mRNA interference (destruction of mRNA species)
- short-hairpin interfering RNAs
- small-interfering RNAs or microRNAs
- Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas9
- transcriptional repression (blocks DNA → RNA)
• Construction of mutant mice (germ line transmission)
- Targeted gene disruption (Knock-out) - whole animal or conditional (tissue-specific)
- Targeted gene insertion(Knock-in) – inducible or tissue/cell-specific
• Block protein, lipid or protein-modification function in cell assays
- Antibodies/Lectins; Dominant negative mutants; Structural variants;
Metabolic/enzymatic inhibitors; Chemicals; Structural mimetics/antagonists
3.) Approaches to Studying Cancer
B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role)
Host/Cancer Cell Virulence
In vitro Studies
• Cell proliferation/death (apoptosis)
• Induce immunoregulatory molecules
• Induce invasion/signaling molecules
• Alter adhesion/molecules
3.) Approaches to Studying Cancer
B. Molecular Function Assessments (Establish a Pro-/anti-tumor Role)
Host/Cancer Cell Virulence
Role in Metastasis Pathways
In vitro Studies
• Cell proliferation/death (apoptosis)
• Induce immunoregulatory molecules
• Induce invasion/signaling molecules
• Alter adhesion/molecules
• Promote growth/angiogenesis
In vivo
• Induce invasion/adhesion
tumorigenicity
• Encourage immune evasion
• Promote metastasis (dissemination/seeding/growth)
4.) Seeking Funding to Conduct for Studies on Disease
4.) Seeking Funding to Conduct for Studies on Disease
• Hypothesis-driven research
4.) Seeking Funding to Conduct for Studies on Disease
• Hypothesis-driven research
• Address a public health problem
4.) Seeking Funding to Conduct for Studies on Disease
• Hypothesis-driven research
• Address a public health problem
• Address a gap in our knowledge/Advance the field
4.) Seeking Funding to Conduct for Studies on Disease
• Hypothesis-driven research
• Address a public health problem
• Address a gap in our knowledge/Advance the field
• A translational aspect – human/animal studies
4.) Seeking Funding to Conduct for Studies on Disease
• Hypothesis-driven research
• Address a public health problem
• Address a gap in our knowledge/Advance the field
• A translational aspect – human/animal studies
• Collaborative/Innovative
4.) Seeking Funding to Conduct for Studies on Disease
• Hypothesis-driven research
• Address a public health problem
• Address a gap in our knowledge/Advance the field
• A translational aspect – human/animal studies
• Collaborative/Innovative
• Training/mentorship is lauded
Laboratory Research on Cancer
in
BWH Dermatology
Host Immune
System
Host Microenvironment
Melanocyte
Melanoma Cell
Laboratory Research on Cancer
in
BWH Dermatology
Host Immune
System
Host/Tumor Factors
Host Microenvironment
Melanocyte
Melanoma Cell
Laboratory Research on Cancer
in
BWH Dermatology
Host Immune
System
Host/Tumor Factors
Host Microenvironment
Melanocyte
Melanoma Cell
Tumor
Membrane
Receptor
Laboratory Research on Cancer
in
BWH Dermatology
Host Immune
System
Host/Tumor Factors
Host Microenvironment
Melanocyte
Tumor Cell Signals
Genes
Melanoma Cell
Tumor
Membrane
Receptor
Laboratory Research on Cancer
in
BWH Dermatology
Host Immune
System
Host/Tumor Factors
Host Microenvironment
Melanocyte
Tumor Cell Signals
Genes
Melanoma Cell
Tumor
Membrane
Receptor
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