7/22/14ScheduleM/W/F: 10:30AM-2:30PM

Contact InformationJacqueline: (650)-922-5104Raquel: (415)-813-9516

Basic Brain Anatomy Caudate-Putamen

- The putamen is a round structure located at the base of the forebrain. It comes together with the caudate nucleus to form the dorsal striatum. The last part of the system is the globus pallidus. The three form the basal ganglia. Functions for this system include voluntary movements, procedural learning, routine behaviors, eye movements, cognition, and emotion.

Pons- A part of the brainstem, the pons is responsible for hearing, equilibrium, taste, facial

touch/pain/expressions, eye motor movements, chewing, swallowing, and secretion of saliva, and tears.

Brain Cancer Primary vs. Secondary

- Primary brain cancer or tumors start within the brain, while secondary (brain metastasis tumors) tumors occur due to spreading from another part of the body.

Glioma (GBM)- This is a type of tumor that starts in the brain or spine that arises from glial cells.

Convection Enhanced Delivery (CED) Human Models Mouse Models

- Probe/Cannula construction with silica tubing, a rigid metal needle, and flexible Teflon tubing. After CED, administer inhalation anesthesia before placing the mouse in a sterotaxic frame.

Analgesics Buprenorphine (Buprinex -- IV/Intramuscular/Subcutaneous)

- Narcotic that treats severe chronic pain. Bupivacaine (Topical)

- Causes numbness or loss of feeling in a local area of the body. Ketamine/Xylazine (Peritoneal/Intravenous)

- Least likely to cause hypotension and suppresses breathing much less than most. Used to start and maintain general anesthesia. Xylazine is often used with ketamine in veterinary anesthesia. It is also an Alpha-2 receptor agonist that most importantly (in respect to anesthesia) induces a negative feedback loop for the presynaptic inhibition of noradrenalin (NA) release in CNS. Slows down the heart and the body.

Isoflurane/100% Oxygen (Inhalation)- A halogenated ether and a respiratory depressant. However, both its induction and recovery are

very rapid.

Brain Stem Viral Injection (60 Day Lethality) Cell Line DIPG-SF-8628 (Diffuse Intrinsic Pontine Glioma) 15 Mice imaging (8/6/14) 15 Mice injection (8/11/14)

1. 1x106 cells per microliter2. 1x105 cells per microliter3. Cell Pallet per microliter

Vocabulary1. Cell Culture: The maintenance or growth of dispersed cells in a medium after removal from the

body.2. Cannula: A metal tube for insertion into the body to draw off fluid or to introduce medication.3. Aspiration: The process of drawing breath (medicine).4. Analgesic: Any family of drug used to achieve analgesia, relief from pain.5. Peritoneal: Via the stomach.6. Systemic: Pertaining to the body as a whole or system.7. Stereotaxic: Pertaining to the three-dimensional study of the brain.8. Glioma: A tumor of the brain composed of neuroglia (glial cells).9. DIPG: Glioma that primarily affects children, spreading through the brain stem and growing between

normal nerve cells. This causes difficulty with attempted removals. Surgery typically cause neural damage.

10. Agonist: a chemical that binds to a receptor and activates the receptor to produce a biological response.

7/26/14Breakdown Types of Human Glioma

- Benign (I, II)- Anaplastic Glioma (III)- Glioblastoma (IV, GBM)

Efficacy Studies- Bioluminescent Imaging (BLI)- Animal Survival

Variables- Cell Lines (U87MG, GBM6, etc.)- Anti-Cancer Agent (VEGF, Small Molecular Inhibitor, DNA*, etc.)- Routes of Delivery (Oral, Intra Peritoneal, Intravenous, Subcutaneous, Intra Tumoral, etc.)

Vocabulary1. Telomere: A region of repetitive nucleotide sequences that cap ends of chromatids. However, the

telomeres in cancer cells are not shortened after duplication as in normal cells. This feature renders them "immortal".

2. Phosphor Buffered Saline (PBS): A water-based salt solution containing sodium phosphate, sodium chloride, and sometimes potassium chloride and potassium phosphate. It is isotonic with human blood, having the same osmolarity and ion concentrations, and thus used for biological research. it is generally used for substance dilution and rinsing/cleaning.

3. Euthanize (Animal): Putting an animal to death for the purpose of dissection and research.4. Vascular Endothelial Growth Factor (VEGF): A signal protein that stimulates vasculogenesis and

angiogenesis (production of blood vessels). Over-expressed VEGF provides the blood supply for cancer cells to proliferate.

5. Subcutaneous: "Beneath the skin". It is the lowest part of the integumentary system, right below the dermis (hypoderm).

People- Cut it out/Surgery- Targeted/Localized Apoptosis- Changing the Cancer Cells

Me- Molecular Processes (Cell Cycle/Protein Cascade)- Cell Replacement vs. Cell Repair- Prevention (develop overgrowth resistant cell lines)- Induce Apoptosis- Changing Environment/Homeostasis of Cancer Cells- The cells need something to live. They are different from normal cells. Use their peculiarity

against them.- Telomeres don't shorten because of DNA damage? Prevent DNA damage? Target the telomeres

of cancer stem cells (assuming cancer stem cell theory)- Forkhead Box O (FoxO) transcription factors in PI3-Kinase/AKT Signaling pathway is inhibited by

AKT (ubiquitinated) and destroyed by proteasome. FoxO is a tumor suppressant factor, so any step in this process could be focused on to treat tumors and further growth/metastasis.

7/30/14Cause Oncogene

Characteristics Invasiveness/Infiltration

- Seen through division between regular brain cell and cancer cells.

Cell Culture Normal Cell

- Isolation ex vivo -- blood, enzymes, explant culture (growth media)- Maintain- Cell line cross contamination

Cancer Cell Manipulation

- Media Change (adherent/aspiration vs. non-adherent/centrifuge and resuspension)- Passaging Cells involve transferring a small number of cells into a new vessel, preventing

senescence.

Vocabulary1. Senescence: Loss of a cell's power of division and growth.2. Oncogene: A gene that has potential to cause cancer, in which it is usually mutated or

overexpressed. An activated oncogene can help a cell to survive apoptosis and continue to divide.3. Transfection: A non-viral method of introducing DNA into cells 4. Transduction: A method of introducing DNA into cells that involves a virus-mediated transfer.

8/4/14Meeting Formalin is used to soak brains in order to produce blocks. These are in turn used for staining

samples. Melanoma Cell Adhesion Molecule (MCAM): An anti-body has been produced to deal with invasive

cancer. The CD146 protein is encoded by the MCAM gene. This gene is highly expressed in parts of blood vessel walls.- MCAM gene is a marker for Mesenchymal Stem Cells (MSCs).

BRAF 645 cell line experiment (Jackie) with viruses. Older disease samples are less representative because of additional mutations and general

evolution/progression of newer samples.

Changing Media Check if media needs to be changed.

- Detached (dead) cells- Yellow media (too acidic/lack of nutrients)

Pipette (glass) out old media into beaker (bleach), and dispose in glass container. Pipette 15mL of media into the cell culture container. Check the cells under the microscope. Put the container back into the incubator. Check on the cells in three days.

Checking On Cell Cultures Check temperature for incubator (37 degrees Celsius). Check/refill water.

- Lack of water results in media use, leading to cell death.

Vocabulary1. Mesenchymal Stem Cells (MSCs): Tissue cells that lack polarity and surrounded by an extracellular

matrix. This includes lymphatic, circulatory, and connective tissue.2. Hemacytometer: A device used to estimate the number of cells being dealt with. It consists of two

empty (semi-rectangle) counting chambers. Averaging with two chambers allows for greater accuracy, especially with the possible error due the estimation itself.

- Total cells/mL: Total Cells Counted x (dilution factor/# of squares) x 104cells/mL- By using 4 squares and a dilution factor of 4 (e.g. 400mL/100mL sample), the equation can be

significantly simplified.3. Dimerization: Joining of two molecular subunits into a single dimer.4. Dimer: Oligimer or a molecular complex with a few subunits as opposed to a polymer with many to

unlimited subunits.5. Autophosphorylation: A process in which a phosphate group binds with a protein kinase (type of

enzyme).6. Blood Brain Barrier (BBB): A highly selective permeability barrier that separates the circulating blood

from the brain extracellular fluid (BEF) in the CNS. It is formed by capillary endothelial cells, connected by tight junctions. - Astrocytes are necessary to create BBB. This is direct relation to GBM.

7. Bioluminescence: Production and emission of light by a living organism.8. Fluorescence: Emission of light by a substance that has absorbed light or other electromagnetic

radiation.

8/7/14Genetics Epigenetics: Anything other than DNA sequence that influences development of an organism

(Contemporary definition by Robin Holliday). The study of changes in gene expression caused by certain base pairs in DNA, or RNA, being "turned off" or "turned on" again through chemical reaction.- Genetics to the alphabet as epigenetics to punctuation.- Most common form is DNA Methylation.

Cell Differentiation- Gene Expression is the process by which information from a gene is used in the synthesis of a

functional gene product (often proteins).- Transcription: The process of making an RNA copy of a gene sequence. This copy, called a

messenger RNA (mRNA) molecule, leaves the cell nucleus and enters the cytoplasm, where it directs the synthesis of the protein, which is encodes. This is carried through by RNA polymerase.

- Translation: The process of translating the sequence of a mRNA molecule to a sequence of amino acids during protein synthesis. The genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence that I encodes. In the cell cytoplasm, the ribosome reads the sequence of the mRNA in groups of three bases to assemble the protein.

- Regulatory Elements- cis-/trans-: non-coding parts of DNA that regulate transcription of nearby/distant genes.

Stem Cell Theory - Tumor-propagating potential of GBM stem-like cells (TPCs).

- TPCs produce differentiated cancer cells.- Results from experiments show that 50 TPCs could propagate but 100,000 DGCs could not.- TFs (OLIG2, SALL2, POU3F2, SOX2) reprogram differentiated glioblastoma cells (DGCs) into

TPCs.- This theory focuses around the idea that only certain cancer cells have the ability to reproduce

and sustain tumors.- This theory also focuses on a distinct epigenetic state that enables GBM cells to propagate

tumors in vivo.

8/18/14Puncture Nikolai Begg Trocar focuses on single location targets with purely puncture safety in mind. Drills used for mice skulls focus on precision and accuracy, generally utilizing spread out locations. Spreading out the pressure allows for more freedom and control, giving the user more room for

adjustment. This also prevents heating damage from single location punctures.

Cell Growth Curve (Seeding) Follow passaging procedure with flask of cells. After neutralizing 3mL sitting trypsin with 7mL media, transfer to a 50mL falcon tube. Place a 100µL sample and 300µL trypan blue into a test tube. Pipette 10µL sample/trypan blue into each side of the hemacytometer. Repeat with other flask. Count, average, and record the data. Calculate the amount of sample needed for acquired cell number (1 x 105 cells per 5mL in T25

flasks). After seeding the appropriate amount of cells to desired number of flasks (10), add media to achieve

desired total volume (5mL). Incubate the seeded samples. Tips: Try not to splash or at least splash on the side of cell growth. Pipette into the corners if possible

or down a slope to avoid splashing.

Meeting Brain Stem Pons Injection (DIPG)

- Signals going down and or no signals.- Brain stem injections generally have complications.

CED Experiment- No significant difference between controlled and treated groups.- Possibility that the drug didn't work (FasL and VEGF), especially with so many factors affecting

angiogenesis.- Possible improvement is injecting luciferin into tumor (luciferase) and image to make sure the

drug is injected into the correct area.

Vocabulary1. Nolvasan (2% Chlorohexidine): A topical disinfectant and for wound healing in terms of animal use.2. Antigen: An antibody generator; any substance that provides an adaptive immune response. Often

foreign or toxic to the body. Attracts and binds to specific antibodies.

Neuroscience and Circuitry

During the course of my internship/volunteering, I've had a good amount of free time on the computer. The time I get to myself also allows me to think and think. Then think some more. Ideas fly by, and the ones that catch my interest develop into something more.

Two of the fields I've found most interesting these past couple of weeks are neuroscience and circuitry. It all started from basic review of the CNS and PNS, supplemented by slides from Jackie. My questions and her answers brought me to further curiosity. We had a discussion about multi-tasking that also involved some new material in psychology Jackie taught me.

I have a few games/apps on my iPhone that seem to promote multi-tasking. One is a simple game that involves a series of arrows. If the arrow is black, you swipe in the direction of the arrow to proceed to the next arrow. If the arrow is striped, you swipe in the opposite direction. Exercises like this stimulate the brain to adapt and respond more quickly to the complex task (or some deem multi-task). This further led to my interest in neuron stimulation. Details, details, details. Everything starts somewhere, and the building blocks interest me the most.

So what happens if you stimulate neurons? What happens if you increase the speed at which signals are sent and received? Theoretically (at least with the limited amount of knowledge I possess at the moment), speeding up neuron signaling could increase a variety of functions and be applied to many medical concerns. This could range from increasing regular brain activity to fixing the lethargic neurons in MS.

Next, I decided to look for analogies. I stumbled upon two crucial ones mentioned in a few articles. They linked the axon with electrical wiring and ion channels with capacitors. Redrawing the axon, I end up with a rough electric circuit. Well, circuitry is certainly something under human control, especially with respect to speed and voltage. I then found the concept of voltage multipliers (more specifically, the Cockcroft-Walton Generator). If I treat the axon as simply part of a circuit, attaching a voltage multiplier could potentially increase the signaling.

Now I had to consider logistics, no matter how shallow. Size. Nano. Perfect because I'm a nanoengineer, and nanocircuits are already viable. However, nanocircuits are only viable in the sense that all the required parts have been built. Errors that occur at that level are still being tested.

I'm very excited to see where all these ideas go. Whether or not this is actually viable, this is a great research opportunity and experience.

8/20/14Vocabulary1. BRAF: Proto-oncogene BRAF makes a protein called protein serine/threonine-protein kinase B-RAF.2. Xenograft: Otherwise known as xenotransplantation, this is the transplantation of living cells, tissue,

or organs from one species to another.

50-60% Snu449 (top) and 100% Snu449 Confluence (bottom).

8/25/14

Media/Trypsin Recipe ATCC provides media concoctions for specific cell lines. Different media is used based on cell sensitivity, which is in turned gauged through experience and

work with each specific cell line. Trypsin is activated by heat, generally through a water bath or incubator.

- Cell line U251 should be used 0.25 trypsin, while 0.05 works with most other cell lines.

Blocking Procedure After brain extraction, let the brain sit in formalin for at least 24 hours. Cut the brain directly in front of the tumor, and cut off the cerebellum (frontal/coronal planes). Sit the brain flat in the blocking container (either side works), and leave it in alcohol. Tissue Core will prepare paraffin finish for the block. Returned blocks can be reused for staining and slides.

Vocabulary1. Reestablish (Cell Line): To make sure cell line is able to grow again in vivo (mice). This process opens

up options such as cell injection of cryopreservation.2. Xenograft Panel: A selection of xenografts. Creating a xenograft panel requires implanting more

human cell lines in mice.3. Subcutaneous Tumor Measurement: (Length x Width)2/24. Lentivirus: A subset of retrovirus that integrates into host chromosome. It infects dividing AND non-

dividing cells. It has the ability to transduce with high efficiency but requires a long incubation time.5. Adenovirus: A non-enveloped virus with a double-stranded DNA genome. It demonstrates stability

and uses host replication machinery for increased reproduction.6. Ecotropic: Having a narrow host range.7. Amphotropic: Having a wide host range.8. Vectors (molecular biology): A DNA molecule used as a vehicle to artificially carry foreign genetic

material into another cell, where it can be replicated and/or expressed. In this specific case, we are working with recombinant DNA (rDNA) or a vector containing foreign DNA.

9. Wild type: Refers to the phenotype of the typical form of a species as it occurs in nature.10. Pyrophoric: Describes substances that ignite spontaneously in air at or below 55 degrees Celsius 11. Liquid Nitrogen (LN2): A colorless, clear and diatomic liquid with no smell. In lab, it is generally used

for cryogenics and cryopreservation.12. DNA Fingerprinting: A method of isolating and making images of sequences of DNA. DNA is

extracted (skin, blood, hair, etc.) and purified. The DNA is cut along restriction enzymes, and the segments are run through electrophoresis. The fragments are subject to a blotting technique before undergoing autoradiography. A piece of X-ray film is exposed to the fragments, producing dark marks at points of attachment. The resultant pattern of these marks can then be analyzed.

13. Assay: An investigative or analytic procedure.14. Polymerase Chain Reaction (PCR): A biochemical technology in molecular biology used to amplify a

single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. This process relies on thermal cycling.

15. Autoclave: A pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 degrees Celsius for around 15-20 minutes depending on the size of the load and the contents.