BN5101 Biomedical Engineering Systems Lecture Notes PDF

Restricted Go to https://classpoint.app Enter 5-digit class code DOI: 10.21276/ijircst.2018.6.4.9 BN5101: Biomedical Engineering Systems Week 2: Introduction to Nucleic Acids &...

Restricted Go to https://classpoint.app Enter 5-digit class code DOI: 10.21276/ijircst.2018.6.4.9 BN5101: Biomedical Engineering Systems Week 2: Introduction to Nucleic Acids & Proteins Class Sessions @ Utown Auditorium 1 Monday 6pm – 9pm Restricted Copyrights  Restricted Recall: Molecular Composition of the Cell Lipids Nucleic acids Proteins Carbohydrates Restricted 2  Restricted Recall: Macromolecules are composed of smaller subunits Hydrolysis Dehydration/condensation Subunits Macromolecules FATTY ACID, GLYCEROL LIPIDS (DNA and RNA) Restricted 3  Restricted Lecture Outline Introduction to Nucleic Acids Overview of DNA Structure Introduction to Amino acids Overview of 4 levels of protein structure & function Restricted 4  Restricted Learning Objectives and Readings At the end of the video, you will be able to: Identify the three primary components of DNA and RNA nucleotides, namely the triphosphate, base and sugar. Describe the key structural features of the DNA double helix Appreciate the differences between DNA and RNA structure Describe the basic structure of an amino acid and categorize the 20 naturally-occurring amino acids by their chemical characteristics (non-polar, polar, acidic, or basic) Describe the 4 levels of protein structure and the chemical interactions that stabilize each level to give rise to a fully folded functional protein Restricted 5  Restricted Relevant Readings Alberts et al. Essential Cell Biology, 4th Edition, Garland Science, 2014. Chapter 2, Pg 56 – 58; Chapter 4, Pg 121-150; Chapter 5, Pg 171-181 Campbell Biology, 10th Edition, Chapter 5, Pg 125- 137; Chapter 16, Pg 372-374 Restricted 6  Restricted Nucleic Acids and Nucleotides Nucleic acids play important roles in the storage, transmission and use of genetic information (more details later) DNA and RNA are two types of nucleic acids in living things – DNA = deoxyribo-nucleic acid – RNA = ribo-nucleic acid Nucleic acids are polymers called polynucleotides Polynucleotides are made from monomers called nucleotides Restricted 7  Restricted Components of Nucleotides Each nucleotide consists of three components: 1. Sugar 2 2. Nitrogenous base 3. Phosphate group(s) 3 1 Restricted 8  Restricted 1. Sugar (5C pentose ring) Ribose (in RNA) has a hydroxyl (OH) group on the 2’ carbon Deoxyribose (in DNA) has a hydrogen (H) group on the 2’ carbon 5’ 5’ HOCH2 HOCH2 O OH O OH 4’ C C 1’ 4’ C C 1’ H H H H H H H H C C 2’ 3’ C C 2’ 3’ OH OH OH H ribose deoxyribose RNA sugar DNA sugar Restricted 9  Restricted 2. Nitrogenous Base Attached to 1’-C of each Pyrimidines (one ring) nucleotide 5 different types (right) DNA bases: A, C, G, T RNA bases: A, C, G, U Cytosine Thymine Uracil 5’ (C) (T, in DNA) (U, in RNA) HOCH2 O BASE Purines (two rings) 4’ C C 1’ H H H H 3’ C C 2’ Adenine (A) Guanine (G) OH H Restricted 10 10  Restricted 3. Phosphate Group(s) (H3PO4) One or more phosphate groups attached at 5’-C of the sugar. A free nucleotide usually has three phosphate groups – two phosphates removed in the formation of a polynucleotide (more later) O O O γ β α 5’ HO P O P O P O CH2 O OH O- O- O- triphosphate group 4’ C C 1’ H H H H 3’ C C 2’ OH H Gives DNA and RNA an overall negative charge (‘acidic’) Restricted 11  Restricted Nucleotide Nomenclature Nomenclature: 1 (Sugar)  2 (base)  3 (phosphate) 2 (which base?) 3 (how many?) 1 (deoxy or not?) Restricted 12  Restricted Nucleotide Nomenclature Nucleotide = Sugar + Base + Phosphate = (deoxy)nucleoside triphosphate, (d)NTP Full names are often abbreviated: *Nucleoside = Sugar + Base Restricted 13  Restricted Practice Example Nomenclature: 1 (Sugar)  2 (base)  3 (phosphate) 2 (Purine or Pyrimidines?; which base?) 3 (how many?) 1 (deoxy or not?) Restricted 14  Restricted Test Yourself What is the name of the following molecule? 1.Deoxyadenosine triphosphate 2.Deoxyadenosine diphosphate 3.Adenosine triphosphate 4.Deoxyribose triphosphate Restricted 15  Restricted Polymerization of nucleotides Two nucleotides are linked via a condensation reaction to form a covalent phosphodiester bond – Linkage is between the 3’C hydroxyl group of one nucleotide and the 5’C phosphate group of the adjacent nucleotide Phosphodiester bond (links 2 adjacent nucleotides) Restricted 16 Two nucleotides are linked via a condensation reaction Restricted to form a covalent phosphodiester bond Nucleotides have 3 components: Phosphodiester bond Two nucleotides are linked via a condensation reaction between the hydroxyl group on the sugar of one nucleotide and the phosphate group of the next nucleotide to form a phosphodiester bond Restricted 17  Restricted Building up to a Polynucleotide A polynucleotide chain is typically made up of hundreds to millions 5’ of nucleotide subunits end Polynucleotide chain has: – Alternating phosphate and sugars linked by phosphodiester bonds (i.e., sugar-phosphate backbone) – Nitrogenous bases projecting out from the sugar backbone – Two chemically distinct ends 5’C phosphate group (5’ P) 3’C hydroxyl group (3’ OH) 3’ end Restricted 21  Restricted Directionality of the Polynucleotide Chain 5’ end Two distinct ends: 5’ phosphate group A 3’ hydroxyl group There is a C directionality to the polynucleotide sequence: C 5’-ACCT-3’ Next nucleotide will be added to the 3’ T OH end of the growing chain (5’  3’ elongation) 3’ end Restricted 22  Restricted James Watson & Francis Crick (FYI) April 25, 1953 Supplementary Restricted 23  Restricted Unravelling DNA: The Double Helix Two polynucleotide chains coil round each other to form a double helix Outer sugar-phosphate backbone with bases facing inwards (like rungs of a ladder) One complete turn of the double helix  3.4 nm in length and comprises 10 base pairs. Restricted 24  Restricted Helix Bridge, Marina Bay, Singapore (FYI) Restricted 25  Restricted Key Features of DNA: Two Antiparallel Strands Two antiparallel strands (i.e., chains run in opposite directions) Restricted 26  Restricted Key Features of DNA: Complementary Base-Pairing Complementary Base-Pairing (i.e., H-bonding between bases on adjacent strand) Restricted 27  Restricted Complementary Base Pairing Two bases from adjacent strands pair via hydrogen bonding: A – T pair: 2 H-bonds C – G pair: 3 H-bonds H-bonding is always between 1 purine + 1 pyrimidine so constant backbone width of 2 nm Restricted 28  Restricted Base-pairing and Chargaff’s Rule.. Even though different organisms have differing amounts of DNA, Amount of G = C Amount of A = T Restricted 29  Restricted Test Yourself If one strand of DNA reads 5’-GGAGTCT-3’, what is the sequence of the complementary strand? 1.5’-CCTCAGA-3’ 2.5’-TCTGAGG-3’ 3.5’-AGACTCC-3’ 4.5’-GGAGTCT-3’ Recall the key features of DNA: - Antiparallel - Complementary base pairing Restricted 30  Restricted Unravelling DNA: The Double Helix Restricted 31  Restricted RNA vs. DNA Structure RNA = ribonucleic acid DNA = deoxyribonucleic acid – 4 bases: A, C, G, U – 4 bases: A, C, G, T – Ribose sugar (2’ OH) – Deoxyribose sugar (2’ H) – Single-stranded – Double-stranded Khan Academy Restricted 32  Restricted Video: The Structure of DNA (MITx Bio) Copyrights https://www.youtube.com/watch?v=o_-6JXLYS-k 33 Restricted  Restricted Lecture Outline Introduction to Nucleic Acids Overview of DNA Structure Introduction to Amino acids Overview of 4 levels of protein structure & function Restricted 34  Restricted Proteins are built up from Amino Acids Proteins are constructed from the same set of 20 amino acids A polypeptide is a linear polymer chain built from these amino acids A protein is a biologically functional molecule that consists of one or more polypeptides or protein (polypeptide) (1 or more polypeptide chains) Each functional protein has a unique 3D structure (i.e., native conformation) Copyrights Restricted 35  Restricted Amino Acid Structure Amino acids have four groups attached to a central carbon atom Each amino acid is distinguished by a unique R group (side chain) E.g., Alanine α R group non-ionized form ionized form (at pH 7.0) Different representations of chemical structure: 'line structure' convention H O H O H2N Cα C H2N OH OH R R Copyrights 36 Restricted  Restricted Nonpolar side chains; hydrophobic Figure 5.16 Side chain (R group) The 20 amino acids (@pH 7.0) Glycine Alanine Valine Leucine Isoleucine (Gly or G) (Ala or A) (Val or V) (Leu or L) (Ile or I) Polypeptides are made up of 20 amino acids R group determines the Methionine (Met or M) Phenylalanine (Phe or F) Tryptophan (Trp or W) Proline (Pro or P) chemical characteristics Polar side chains; hydrophilic of each amino acid: Non-polar Polar Acidic (-) Basic (+) Serine Threonine Cysteine Tyrosine Asparagine Glutamine (Ser or S) (Thr or T) (Cys or C) (Tyr or Y) (Asn or N) (Gln or Q) 1-letter or 3-letter form Electrically charged side chains; hydrophilic Basic (positively charged) E.g., Alanine, Ala, A Acidic (negatively charged) Aspartic acid Glutamic acid Lysine Arginine Histidine (Asp or D) (Glu or E) (Lys or K) (Arg or R) (His or H) 37 Copyrights 37 Restricted  Restricted Don’t Panic! You will be given the structures (do not memorize) You should understand how to classify amino acids – Polar (hydrophilic) or non-polar (hydrophobic) side chains – Neutral or charged side chains You should be able to identify the types of interactions the amino acid is capable of forming based on its side chain You should be able to predict where in a protein you would expect the amino acid to be found (surface vs. interior) Copyrights Restricted 38  Restricted Non-polar (Hydrophobic) Side Chains Side chain (R group) Glycine Alanine Valine Leucine Isoleucine (Gly or G) (Ala or A) (Val or V) (Leu or L) (Ile or I) Methionine Phenylalanine Tryptophan Proline (Met or M) (Phe or F) (Trp or W) (Pro or P) Tend to be located in the interior of soluble proteins Glycine: Flexible (smallest side chain) Proline: Rigid (ringed side chain) Restricted  Restricted Polar (Hydrophilic) Side Chains Can form hydrogen bonds Tend to be located on the exterior of soluble proteins Serine Threonine Cysteine (towards water) (Ser or S) (Thr or T) (Cys or C) Ser and Thr hydroxyl groups can be phosphorylated Cysteine can form disulfide bonds via thiol (S-H) groups Tyrosine Asparagine Glutamine (Tyr or Y) (Asn or N) (Gln or Q) Restricted  Restricted Two cysteines can form a covalent disulfide bond COO- COO- H3N+ CH H3N+ CH CH2 CH2 SH S Cysteine Disulfide bond (Cys, C) + between R groups SH S CH2 CH2 H3N+ CH H3N+ CH COO- COO- Disulfide bonds can further constrain protein conformation: Copyrights Restricted 41  Restricted Example: Hair protein contains many disulfide bonds  Disulfide bonds in your hair proteins contribute to its mechanical strength  The science behind your perm: 1. Add a chemical to break disulfide bonds. 2. Roll hair around curlers, which places different thiol groups in proximity. 3. Add another chemical (usually hydrogen peroxide, H2O2) to reform bonds. Copyrights Restricted 42  Restricted Test Yourself Leucine and valine have side chains consisting entirely of C and H; therefore, these amino acids: A. are hydrophilic. B. are non-polar. C. have sulfur atoms in their side chains. D. are electrically charged. Copyrights Restricted 43  Restricted Two amino acids are linked by a peptide bond Two amino acids undergo a dehydration reaction to yield a dipeptide linked by a covalent peptide bond Dehydration reaction Direction of growth In cells, the next amino acid is added to the carboxyl terminus. Hence, polypeptide growth occurs in N  C direction Copyrights Restricted 44  Restricted Figure 5.17 Making a polypeptide chain. Peptide bond What is the sequence of the resulting New peptide bond forming tripeptide? Side (Use the 3-letter code) chains Back- bone Amino end Peptide Carboxyl end (N-terminus) bond (C-terminus) 45 Copyrights Restricted Copyrights 45  Restricted Figure 5.17 Making a polypeptide chain. Peptide bond Resulting tripeptide: New peptide bond forming N – Met – Tyr – Cys - C Side chains Must always indicate directionality of the polypeptide chain Back- bone Amino end Peptide Carboxyl end (N-terminus) bond (C-terminus) 46 Copyrights 46 Restricted  Restricted BREAK Copyrights Restricted 47  Restricted Concept Check a) Which amino acids make up the following tripeptide? b) Identify the peptide bonds. How many are there? c) Which atoms are lost in the formation of one peptide bond? What is a possible byproduct of this reaction? d) What groups are located at both ends of the structure? e) Indicate the direction of polypeptide growth (in the cell). H O CH3 H H O + NH3 C C N C C N C C - O H2C H H O HC CH3 H2C H2C - COO CH3 Copyrights Restricted 13  Restricted Clicker Question If tyrosine (Tyr) is the next amino acid linked to the tripeptide from before, what will be the sequence of the resulting tetrapeptide? Alanine, Ala A. N-Ile-Ala-Glu-Tyr-C H O CH3 H H O + B. N-Tyr-Ile-Ala-Glu-C NH3 C C N C C N C C - O C. N-Glu-Ala-Ile-Tyr-C H2C H H O HC CH3 H2C H2C D. N-Tyr-Ile-Ala-Glu-C - COO CH3 Glutamic acid, Glu Isoleucine, Ile Copyrights Restricted 49  Restricted Recall: Proteins are built up from amino acids or protein (polypeptide) (1 or more polypeptide chains) How do we get to the folded 3D conformation? Copyrights Restricted 50  Restricted Four Levels of Protein Structure Primary Secondary Tertiary Quaternary Regions of secondary structure stably Higher order complex of Linear sequence Local structure interact to form multiple individually of amino acids adopted by stretch folded chain folded polypeptide of AAs chains Copyrights Restricted 51  Restricted Primary Structure (1º) Figure 5.20a Primary structure (1°) is the Campbell Biology linear sequence of amino acids, much like the order of letters in a long word Recall: Amino acids are linked by peptide bonds N  C directionality of polypeptide chain Example: Primary Structure of Transthyretin (127 aa) Copyrights Restricted 52  Restricted Secondary Structure (2º) Secondary structure (2°) are local structures adopted by a stretch of amino acids due to hydrogen bonding between polypeptide backbone groups Two most common motifs are the alpha-helix and beta-sheet: Note: R groups are NOT involved in the formation of secondary structure! Copyrights Restricted 54  Restricted Animation: Secondary Structure (2o) Restricted  Restricted Tertiary Structure (3º) Tertiary structure (3°) is formed when regions of secondary structure stably interact to form a folded polypeptide structure Note: R group interactions drive the formation of tertiary structure! Copyrights Restricted 56  Restricted Tertiary structure is determined by interactions between R groups Figure 5.20f Campbell Biology R-group interactions ? ? ? ? Polypeptide backbone Tertiary structure is held together by R-group Copyrights interactions within the polypeptide chain 57 Restricted  Restricted Tertiary structure is determined by interactions between R groups Figure 5.20f Campbell Biology R-group interactions Hydrogen bond London dispersion forces (ID-ID) Disulfide bond Ion-ion interactions Polypeptide backbone Tertiary structure is held together by R-group Copyrights interactions within the polypeptide chain 58 Restricted  Restricted Animation: Tertiary Structure (3o) Restricted  Restricted Quaternary Structure (4º) Quaternary structure (4°) results Hemoglobin subunit when two or more polypeptide (tertiary structure) chains form one fully functional protein Subunits can be alike or different Quaternary structure is held together by R-group interactions between polypeptide chains - hydrogen bonds - ion-ion interactions - London dispersion forces - disulfide bonds Example: Hemoglobin is Hemoglobin protein (α2β2) made up of 4 polypeptide (quaternary structure) subunits, 2 alpha-chains and 2 beta-chains Figure 5.20a Copyrights Restricted Campbell Biology 60 Disulfide bonds can stabilize tertiary (intra-chain) and Restricted quaternary (inter-chain) structures Cysteine Example: Insulin has three disulfide bonds which stabilizes (Cys, C) the 3D protein structure http://nursingpharmacology.info Restricted  Restricted Animation: Quaternary Structure (4o) Restricted  Restricted Summary: Four Levels of Protein Structure Restricted 63  Restricted Summary: Protein Structure and Function Four levels of protein structure – Primary structure: linear amino acid sequence – Secondary structure: local structures: α-helices and β-sheets – Tertiary structure: overall 3-D folded conformation of one chain – Quaternary structure: association of multiple chains/subunits Each level is stabilized by different bonds and interactions. – Primary structure: peptide bond – Secondary structure: regular H-bonding between backbone groups – Tertiary structure: multiple interactions (intrachain) – Quaternary structure: multiple interactions (interchain) Shape and function is ultimately specified by a protein’s amino acid sequence (primary structure) – 20 different a.a. gives rise to a multitude of protein sequences and shapes – Loss/change of structure (e.g., denaturation) results in a loss/change of function Restricted 64  Restricted Video – Protein Structure and Function Copyrights https://www.youtube.com/watch?v=2Rmy7wfPvmQ (up to 04:15) 65 Restricted  Restricted Proteins have important and diverse biological functions Keratin Hemoglobin (structural protein) (transport protein) Rhodopsin (receptor protein) Collagen + Elastin (structural protein) TAS1R2 receptor (receptor protein) Salivary amylase (enzymatic protein) Myosin + Actin Insulin (contractile/motor protein) (signaling protein) Copyrights Restricted 66  Restricted Each protein has a unique 3D conformation A functional protein has a stable 3D shape (i.e., native conformation) There is great diversity in protein structures – Mostly classified into fibrous or globular proteins – Some contain all helices, some all sheets, some mixed (secondary) – Can comprise a single polypeptide (tertiary) or multiple subunits (quaternary) A protein’s 3D structure is essential to its function Collagen (structural protein) Lysozyme (enzyme) GFP (fluorescent protein) Fibrous Globular Other (e.g., β-barrel) 67 Copyrights Restricted  Restricted Representations of 3D Protein Structure Wire diagram Ribbon diagram Ball and stick Surface Space-filling representation Copyrights Restricted 68  Restricted Proteins fold into a minimal-energy configuration Each protein spontaneously folds into a single stable conformation of lowest energy (i.e., ‘native’ conformation) Multiple interactions within the protein molecule help to stabilize its folded shape Hydrophobic interactions (left) and H-bonds (right) help to stabilize the folded protein shape Copyrights Restricted Fig. 4.5 and 4.6 Essential Cell Bio 69  Restricted Proteins – the Workhorses of the Cell Figure 4.11 Essential Cell Bio Proteins account for more than 50% of the dry mass of most cells Proteins come in all shapes, sizes, and types; each has a specific job Proteins do a whole lot of everything! – Structural proteins (e.g., keratin) – Transport proteins (e.g., hemoglobin) – Enzymatic proteins (e.g., amylase) – Signaling proteins (e.g., insulin) – Motor proteins (e.g., actin and myosin) – Receptor proteins (e.g., rhodopsin) – Storage proteins (e.g., casein) Proteins make the chemistry in the Estimated to be 42 million protein cell happen in order for life to exist molecules in a single cell, with 1000s – 10,000s of each protein type Copyrights Restricted 70  Restricted Proteins have diverse structures and functions Enzymatic proteins Defensive proteins Function: Selective acceleration of chemical reactions Function: Protection against disease Example: Digestive enzymes catalyze the hydrolysis Example: Antibodies inactivate and help destroy of bonds in food molecules. viruses and bacteria. Antibodies Enzyme Virus Bacterium Storage proteins Transport proteins Function: Storage of amino acids Function: Transport of substances Examples: Casein, the protein of milk, is the major Examples: Hemoglobin, the iron-containing protein of source of amino acids for baby mammals. Plants have vertebrate blood, transports oxygen from the lungs to storage proteins in their seeds. Ovalbumin is the other parts of the body. Other proteins transport protein of egg white, used as an amino acid source molecules across cell membranes. for the developing embryo. Transport protein Ovalbumin Amino acids for embryo Cell membrane Figure 5.15-a Campbell Biology Copyrights Restricted 71  Restricted Proteins have diverse structures and functions Hormonal proteins Receptor proteins Function: Coordination of an organism’s activities Function: Response of cell to chemical stimuli Example: Insulin, a hormone secreted by the Example: Receptors built into the membrane of a pancreas, causes other tissues to take up glucose, nerve cell detect signaling molecules released by thus regulating blood sugar concentration other nerve cells. Receptor Signaling protein Insulin High secreted Normal molecules blood sugar blood sugar Contractile and motor proteins Structural proteins Function: Movement Function: Support Examples: Motor proteins are responsible for the Examples: Keratin is the protein of hair, horns, undulations of cilia and flagella. Actin and myosin feathers, and other skin appendages. Insects and proteins are responsible for the contraction of spiders use silk fibers to make their cocoons and webs, muscles. respectively. Collagen and elastin proteins provide a fibrous framework in animal connective tissues. Actin Myosin Collagen Muscle tissue Connective 100 µm tissue 60 µm Figure 5.15-b Campbell Biology Copyrights Restricted 72  Changes at the amino acid level can Restricted greatly impact protein structure and function Sickle-cell anemia results from a single amino acid substitution (Position 6) Campbell Fig. 5.19 Restricted  Restricted Proteins interact with other molecules to carry out specific functions All proteins interact (or bind) to other molecules in a specific manner Any substance that interacts with a protein, forming a stable association (“complex”), is called a ligand for that protein Protein: p53 Protein: lactase Protein: hemoglobin Lactase Lactose Ligand: DNA Ligand: lactose Ligand: oxygen Initiates DNA repair Hydrolysis of lactose to Transports O2 in the glucose and galactose bloodstream Restricted 74  Proteins interact with their ligands via specific Restricted three dimensional non-covalent interactions Non-covalent interactions Ligands can be ions, small molecules, macromolecules, etc. Molecular binding: an attractive interaction between two molecules that results in a stable association at close proximity Effective binding when surface contours of ligand match those of protein (e.g., hand in glove) Binding is stabilized via multiple intermolecular interactions in 3D. Binding specificity and affinity vary: – Affinity: strength and stability of protein-ligand binding – Specificity: number of ligands that a protein can recognize and bind Proteins typically show high specificity Fig. 4.31 Essential Cell Bio Restricted When a protein folds, side chains are brought together Restricted in close proximity to form a binding site Binding site: cavity in the protein surface that interacts with the ligand via a specific 3D arrangement of amino acids Fig. 4.32 Essential Cell Bio Ion-ion interaction Side chains are often the functional components of proteins Restricted 76  Restricted Example: Antibodies (Immune Defense Proteins) Antibodies (Ab) are large Y-shaped immunoglobulins produced by the immune system in response to foreign molecules Each antibody recognizes and binds to its target molecule (i.e., antigen) with extremely high affinity and specificity This binding event acts to either inactivate the target directly or mark it for immune destruction Antibody structure: Y-shaped protein composed of four polypeptide chains (2 heavy chains, 2 light chains) held together by disulfide bonds, with two identical antigen-binding sites (red) An antibody binds to a specific region on an antigen called an epitope. A single antigen Antigen-binding sites can have multiple epitopes for different, specific antibodies. Credit: CUSABIO Copyrights Restricted Credit: Lumen Learning 77  Restricted E.g., Sweet Taste Receptor and Engineering of Sugar Substitutes Sucrose (table sugar) Brain Stabilized by interprets non-covalent as interactions “Sweet” Taste receptor Restricted  Restricted Applications in clinical instrumentations Blood Glucose Monitor Copyrights Restricted 79  Restricted That’s all folks!! Have a Great Week Ahead! Restricted 80  Restricted Announcement In-class briefing for examsoft 26 Aug 2024 (Monday, Week 3), 6-6:30pm, UTown Auditorium 2 Bring your laptop/device! Restricted 81  Restricted Recall this question last week What is your goal after completing the degree (eg: MSc) in BME? A. Get a job in an industry company B. Get a job in the research lab C. Further studies for PhD D. Start-up a company E. Not sure yet and see where life takes me Restricted 82  Restricted Pre-session questionnaire Submit your response on any key topics/ questions that they would like the facilitators to cover during the session: https://share.hsforms.com/1t2yDV5q8Tm6yHOXC5NQiJQ1szrj Restricted 83