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Cancer Research (Fach) / Cancer Research (Lektion)

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• Which method is suitable to visualize gene expression in tissues and cells? fluorescence in situ hybridization (FISH) FISH --> detection/quantification of nucleic acid sequences in fixed cells / tissues / tumors
• You would like to test, if the small GTPase Ras interacts with its downstream effector protin Raf. Which biochemical method can be utilized to study this protein-protein interaction? Provide also a brief explanation of the technique in bullet points. Co-IP and Western blot [Co-Immunoprecipitation --> technique for identifying/isolating protein-protein interactions Western blot --> protein transfer on membrane Immunoprecipitation --> biochemical method for collecting an antigen via pull-down assay]
• Definition of Photoswitching direct, light-induced isomerization of the fluorophor
• Definition of HTC High-Throughput Chemistry conduction of many chemical / genetic / pharma tests and identifying active compunds / genes / antibodies
• Definition of RNAi screen RNA interference screen --> Identifying gene products whose knockdown is associated with phenotypic changes / regulators
• Definition of NGS Next-Generation Sequencing --> quantitative analyses of miRNomes and transcriptomes
• Defintion of FACS Fluorescence-Activated Cell Sorting -- isolation of populations of intact cells
• Definition of CGH Comparative Genomic Hybridization --> analysis of CNV´s (copy number variations) relative to ploidy level in the DNA + compare two genomic samples
• Definition of aCGH Array Comparative Genomic Hybridization --> detection of aberrations + mapping
• Defintion of HR-CGH High-resolution Comparative Genomic Hybridization --> detection of structural variations, microdeletions and duplications
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• Definition of microarray Genetic method for analysis of gene expression
• Function of an Affymetrix chip Parallel synthesis of oligonucleotide probes on a slide with photolithographics
• Definition of FRET Fluorescence Resonance Energy Transfer --> process of energy transfer without radiation
• Function of qrPCR detection and monitoring of mRNA expression
• Function of Bioanalyzer Agilent qualitative and quantitative analysis of RNA
• Function of protein microarrays Tracking of protein interactons, their activities and function.
• Definition of MS/MS tandem mass spectrometry --> instrumental analysis of coupling two mass spectrometers together to analyze chemical samples better.
• Name three types of epitopes that can be recognized by antibodies. -primary sites -secondary sites -posttranslational modification
• Explain the physical basis of fluorescence. A form of luminiscence / emission of light by a substance that has absorbed light or other electromagnetic radiation. A molceule gets excited into a state of higher energy. In the process of relaxation, the molecule falls back into a state of lower energy through emission of a photon.
• Which strategy / treatment is often used to change excitation and / or emission characteristics of fluorescent proteins? Irradiation / photoswitching (reversible)
• In PET tomography, two photons are generated upon collision of positrons (gamma-rays) emitted by the radioactive isotope 18F. How are those photons called? What is the radioactive tracer substance in this method? The photons are called Annihilation photons. The radioactive tracer substance is called Radio-pharmacon Fluodeoxyglucose.
• What are the two original sources for the multiple genetically encoded fluorescent proteins? GFP --> jellyfish RFP --> corals
• Name two general types of protein mutations that can affect a proteins activity. -truncation (cut the protein) -point-mutation
• How would you find out if the kinase activity of a protein is important for a specific biological function? Point mutation of the kinase to destroy its activity
• Which is the light-sensing domain that is derived from the phototrophin receptor in plants (abbreviation and written out)? LOV domain = Light-oxygen-voltage-sensing domain
• On which 3 levels can we manipulate a protein´s function? -genomic level -mRNA level -protein level
• The CRISPR technology is used for precise genome editing. What is the major enzyme class that cleaves the crRNA? Cas endonucleases like Cas9
• What is the cellular consequence if a dominant active mutant version of the small GTPase Ras is overexpressed (such as in a cancer cell)? Enhanced proliferation or proliferation independence of the the signal pathway.
• Name the 3 components of a qRT-PCR analyzer. 1) PC 2) PCR-machine 3) optic module
• What is an aptamer and how are aptamers generated? Aptamers are oligonucleotides or peptide molecules that bind to a specific molecule --> a sequence is selected from a large sequence pool.
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• Name at least 2 methods to fix protein on an array surface. -electric spotting / robotic spotting --> contact printing -Pilezzo-electro spotting -Photolithography
• In mass spectrometry analysis, which 2 steps are breaking up the proteins into peptides and later on into single amino acids? 1) protein digestion by proteases 2) CID-module (Collision-Induced Dissociation module)
• In a tandem mass spectrometry analysis, how are the ions called in the first and the second MS (both names)? -parent-ions = precursor ions -daughter-ions = product ions
• Describe the generation of monoclonal antibodies. Monoclonal antibodies (mAbs) are produced by introducing an antigen to a mouse and then fusing polyclonal B cells from the mouse’s spleen to myeloma cells. The resulting hybridoma cells are cultured and continue to produce antibodies to the antigen. Hybridomas producing the desired mAb are then grown in large numbers on a selective medium that is periodically harvested to obtain the desired mAbs.
• Repetetive elements are difficult to sequence. Why? From a computational perspective, repeats create ambivalence in alignment and assembly, which, in turn, can produce biases and errors when interpreting results. They are difficult to sequence, because a definite starting point of the sequence can´t be ensured, due to an average read length of sequencing. You can´t be sure where the repetetive sequence starts and ends.
• What are „Liquid Biopsies“ and how could they help to detect cancer? Diagnostic detection of tumor cells or tumor DNA in the blood. By detaching tumor cells from their tissue, tumor DNA gets into the peripheral blood. This DNA has characteristic changes in its sequence that make it possible to differentiate it from the DNA of normal body cells. Tumor DNA comes in two forms: -in intact tumor cells as ctcDNA ("circulating tumor cell DNA") and-detached from tumor cells as ctDNA ("circulating tumor DNA"), a fraction of the cfDNA ("cell free DNA") Liquid Biopsy enables the detection of tumors whose location is unknown or which are difficult or difficult to reach for patients with classic biopsy techniques (e.g. fine needle puncture).
• Which two factors are most critical when evaluating next generation sequencing libraries? -quantity and physical characteristics of the RNA or DNA source material  -desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) 
• Next generation sequencing can be performed using different technologies. Name at least three of them (technologies, not brand names). 1) Sequencing by synthesis in flowcells 2) Pyrosequencing: sequencing by synthesis 3) Sequencing by ligation 4) Real-time single molecule sequencing 5) Sequencing by a non-optical semiconductor device 6) Sequencing by ion flow perturbations
• By which sensitive method can microarray-derived gene expression data be verified (abbreviation AND written out)? How is this method different from the “original” or conventional method? New method: qRT-PCR = Quantitative real time PCR -can observe very low levels of expressions -verification of microarray data -requires very small amounts of mRNA Conventional method: PCR = Polymerase chain reaction -most sensitive -discriminates closely related mRNA´s -technically simple -BUT difficult to get truly quantitative results OR another conventional method: Northern Blot - analysis of gene expression via detection of RNA -detection of fragments by specific probes
• In qRT-PCR, which two main principles allow detection of the amplification product? How do they work? Name one example each. -specific sequence detection --> beads etc. / FRET or TaqMan samples -non-specific sequence detection --> detects any double stranded DNA 1) Specific sequence detection (Fluorescence resonance transfer=FRET probes) --> distinguishes between a specific sequence of interest and non-specific products. Can be used to detect different alleles --> Fluorescently labeled nucleic acid probes 2) Non-specific detection (SYBR-Green)--> detects any dsDNA produced during the reaction --> uses a dsDNA intercalating dye that fluoresces once bound to DNA The use of fluorescently labeled oligonucleotide probes or primers or fluorescent DNA-binding dyes to detect and quantitate a PCR product allows quantitative PCR to be performed in real time. Specially designed instruments perform both thermal cycling to amplify the target and fluorescence detection to monitor PCR product accumulation. DNA-binding dyes are easy to use but do not differentiate between specific and nonspecific PCR products and are not conducive to multiplex reactions. Fluorescently labeled nucleic acid probes have the advantage that they react with only specific PCR products, but they can be expensive and difficult to design. Some qPCR technologies employ fluorescently labeled PCR primers instead of probes.
• Name at least 2 different types of posttranslational protein modifications with one biological function each. 1) Phosphorylation --> critical role in the regulation of many cellular processes, including cell cycle, growth, apoptosis and signal transduction pathways.  2) Glycosylation --> effects on protein folding, conformation, distribution, stability and activity. 3) Ubiquitination --> degradation 4) SUMOylation --> mark a protein for targeting 5) Disulfide bond --> stabilize the tertiary and/or quaternary structures of proteins 6) Acetylation --> regulating gene transcription 7) Lipidation --> method to target proteins to membranes in organelles, vesicles and the plasma membrane. 8) Methylation --> increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when bound to carboxylic acids 9) Hydroxylation
• p-Werte werden oft als absolute Wahrscheinlichkeit oder als Tatsache interpretiert. Nennen Sie zwei alternative Methoden oder Interpretationen um zwei verschiedene Konditionen zu vergleichen -Bayesisian interpretation -absolute effect strength -correlation between two measurements or variables (Pearson´s r = predictive model)
• Sie haben X und Y. Welche nicht verknüpft sind aber korrelieren. Zeichnen Sie eine Skizze zur Erklärung. x <-- z --> y
• How can you distinguish casual relationship from correlation? Through acute perturbation of X combined with measurements of Y
• Welche Behandlung / Methode verwendet man um schrittweise Epitheliale Zellen anzureichern? Erklären Sie die Methode. Surgery --> mince tissue --> separation of cells based on cell-cell or cell-substrate adhesion, as fibroblasts dissociate faster than ephitelial cells [incubation with Collagenase for 20min --> removing the supernatant with fibroblast and repeating the procedure --> cultivation of the epithelial cells from the pellet]
• Wofür wendet man diese FISH an? FISH (=Fluorescence in situ hybridization) enables the detection/quantification of nucleic adid sequences (mRNA or DNA sequences) in fixed cells, tissues and tumors.
• Die Fixierung mit Formaldehyd findet durch eine Vernetzungsreaktion an den reactive sites der Aminosäuren statt. Nennen Sie zwei Typen von reactive sites die in dieser Reaktion mit Formaldehyd reagieren. 1) Primary amines (e.g. in Lysine, Glutamine) 2) Thiols (e.g. in Cysteine)
• Welche Metallart wird als Quelle für Röntgenstrahlen in einem Röntgengerät verwendet? Wolfram Tungsten Wire 
• Nenne die Aminosäuresequenz von GTP und den Mechanismen der Strukturveränderung der zum flureszierenden GFP führt. The primary sequence SYG is auto-catalytically converted into the chromophor. MANY double bonds coupled
• Name the steps of FISH. 1) fixation + permeabilization 2) hybridization of probe with fluorescently labeled oligonucleotides 3) washing 4) imaging under fluorescence microscope 5) localization and detection of fluorescent signal

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