1)Explain the most and least effective uses of a viable plate count.

2)Determine if complement is part of innate immunity or acquired immunity or both and defend your decision.

3)Explain how the results of a diagnostic test could be completely reversed if the test is given to the same patient several months/years apart.

The sirtuins are a class of NAD-dependent deacetylases that are thought to play a role in the cellular response to nutritional status. Some sirtuins are involved in remodeling chromatin structure. A) How would you expect deacetylation of histones to affect the transcriptional activity of genes? B) Sirtuin 3 (Sirt3) deacetylates a specific lysine in the active site of acetyl-CoA synthetase, activating the enzyme. Why would this particular deacetylation reflect nutritional status? Remember, acetyl-CoA synthetase catalyzes the reaction: ATP + acetate + CoA <=> AMP + PPi + acetyl-CoA

Describe the sexual reproduction cycle in fungi (a general process, not for a specific type of fungi). Is the fungal sexual life cycle a type of alternation of generations seen in plants and some algae? Explain.

1. You are studying a particular type of lung cancer, small-cell lung carcinoma. You are interested on driver mutations of this cancer, that is mutations that provide strong selection advantage and thus have strong potential in initiating tumorigenesis. You have access to primary tumor biopsies from patients, and healthy surrounding tissue: how you can best use these tissue samples to identify mutations underlying this particular type of lung cancer?

2. The mutation you isolated has not been characterized before; how can the crystal structure of the protein bound on DNA help you understand how the mutation affects the function of the protein? What are some common mutations of the p53 protein that drive cancers?

3. You believe DNA binding is affected by the identified mutation, and your next goal is to characterize the functional perturbations of the mutant p53. a. Describe an in vitro approach to study the function of the mutant protein. b. Describe an experimental approach using cultured cells that can illuminate the altered function of the mutant protein

4. You find in #3 that the expression levels of certain miRNAs are changed as a result of the p53 mutation. a. What experimental approach can reveal how altered miRNA levels change Ago/miRNA targeting of mRNAs at the transcriptome wide level? b. What experimental approach can quantify altered translation levels for the mRNAs identified in a)?

Knockout mice are created by incubating embryonic stem (ES) cells with a DNA construct that contains a portion of the gene with a disrupted exon. The exon is disrupted by inserting a gene for neomycin resistance (neo^r) into the exon. When the DNA construct carrying the disrupted exon is taken up by an ES cell, a knockout allele results when the added DNA construct

(a)        replicates as an independent DNA molecule in the ES cell and is maintained in all cells derived from it.

(b)       undergoes homologous recombination with the wild type allele of the gene in the ES cell.

(c)        inserts randomly in the genome of the ES cell.

(d)       is processed in the ES cell such that the neo^r gene is excised and pasted at random sites in the genome of the ES cell.

Depending on the vector used for gene therapy, the therapeutic gene can integrate into the chromosome or it can stay as an extrachromosomal piece of DNA (episome). Some long-lived cells (for example, neurons, myocytes, hepatocytes) can have stable long-term expression of the therapeutic gene without integration of the introduced gene into the host genome. Which vectors will be most suitable for introduction of a gene into long-lived cells, where the introduced gene does not integrate into the host genome?

(a) Vectors derived from retroviruses

(b)       Vectors derived from Adeno-associated viruses (AAVs)

(c)        Vectors derived from plant viruses

(d)       Vectors derived from bacterial viruses

For genome editing of a crop plant by CRISPR-Cas9, you have designed a sgRNA (single guide RNA) that contains a sequence that recognizes the target sequence in the genome of the plant, and a region that binds to the Cas9 protein. Given the following target sequence in the genome, the sequence of the sgRNA that recognizes the target sequence is ______________________________. Binding of the sgRNA-Cas9 complex to the target site will lead to a ______________ (single or double)-stranded cut in the DNA. Fill in the blanks using the following choices.

3’-------------GATCGGATCGATGGAACAGA-------------5’

     5’-------------CTAGCCTAGCTACCTTGTCT-------------3’

(a)        3’ – CUAGCCUAGCUACCUUGUCU – 5’; single

(b)       3’ – CUAGCCUAGCUACCUUGUCU – 5’; double

(c)        5’ – CUAGCCUAGCUACCUUGUCU – 3’; single

(d)       5’ – CUAGCCUAGCUACCUUGUCU – 3’; double

What is the number of ATP and ATP equivalents that can be produced via oxidative phosphorylation per cycle of the Citric Acid Cycle (CAC), starting from pyruvate? B) What is the number of ATP and ATP equivalents that can potentially be produced if Complex I of the Electron Transport System (ETS) is inhibited? C) If Complex II of the ETS is inhibited? D) If Complex III of the ETS is inhibited? E) If Complex IV of the ETS is inhibited?

Which single category of gene function has the most representation in both bacterial and eukaryotic genomes?

Which list of elements below is present in all biomolecules in plants?

Case Study 2: Bill (please answer question 2 and 5 only)

Bill is 52 and has been previously diagnosed with Type 2 Diabetes Mellitus. He admits that he often does not take his medications which are metformin and glyburide.

1. What types of medications are metformin and glyburide? Briefly describe their general mechanisms as well as their potential side effects/drug-nutrient interactions

Bill has come into hospital because he has been diagnosed with HHS. Hyperosmolar hyperglycemic state/ syndrome (HHS) is when a diabetes patient has an abnormally high plasma glucose level (greater than 30 mmol/L) due to inadequate amounts of insulin to maintain normoglycemia. HHS is very common amongst T2DM older adults, and symptoms tend to include dehydration due to reduced thirst recognition—this causes older adults to be slightly confused. This happens when T2DM is uncontrolled for a long time. Symptoms may include poluyuria and polydipsia. Patients must be hospitalized.

The treatment of HHS consists of correction of the dehydration with intravenous fluids, reduction of the blood sugar levels with insulin, and management of any other underlying conditions that might have precipitated the condition

In hospital insulin therapy is started for Bill.

2. Outline the basic principles for Bill’s nutrition therapy to assist in control of his DM.

Bill’s measurements and typical daily diet is recorded as follows:

Weight: 97.3 kg; Height: 175

Daily Diet:

BF: 2 crumpets, 1 tbspn each of honey and cream cheese, diet cola

MT: 1 coffee scroll, 1 coffee with milk, no sugar

Lunch: Fast-food roll from subway (full size), 9 Chips

Dinner: 1 breast grilled chicken/beef, 1 cup salad, 1 potato/rice

Supper: 4 biscuits and a hot chocolate made with full cream milk

3. Assess Bill’s weight and BMI. What would be a healthy weight range for Bill?

4. Determine Bill’s energy and protein requirements for weight maintenance. What energy and protein intakes would you recommend to assist with weight loss?

5. Prioritize two nutrition problems and discuss how you would implement solutions to these. What foods would you change? Write out some daily food choices

You are given a sample containing two proteins in phosphate-buffered saline: Protein A (pI value 8.4) and Protein B (pI value 7.0).

You wish to separate this protein mixture by ion-exchange and have available a column of CM­Sephadex. To carry out the procedure, you have prepared an appropriate MES buffer (50 mM, pH 6.0, with 20 mM NaC1). We will call this Buffer M.

(i) Suggest a preparatory procedure and materials needed to change the phosphate buffer of the protein sample with the MES buffer (Buffer M) required for ion-exchange.

(ii)Once the proteins are introduced into Buffer M, comment on the overall charge states for each of Protein A and Protein B.

(iii)The column of CM-Sephadex is equilibrated with Buffer M. What is the overall charge state of the matrix under these buffer conditions?

(iv)The protein mixture is now introduced to the column. Describe the process of adsorption that will occur. Which of the two proteins, Protein A or Protein B, will experience strongest adsorption under these conditions? Justify your answer.

(v) What should be done next to effect the best separation of Protein A and Protein B from the matrix? Give a full molecular description of events.

(vi) Consider a different separation procedure in which a different buffer choice was made to carry out the separation on CM-Sephadex. In this second scenario, Buffer T, containing 50 mM Tris buffer, pH 7.5 with 50 mM NaC1 was used for starting conditions. Outline how a separation of Protein A and Protein B can still be effected in Buffer T utilizing CM-Sephadex matrix. Explain the order of elution that would occur for each of the Protein A and Protein B species with your new procedure.

(vii) Comment on whether it would be possible to separate Protein A from Protein B again using Buffer T, but with a different column chemistry, one containing Mono-Q matrix.

6. (4pts) Indicate if you “AGREE” or DISAGREE” with the following statements. Explain your reasoning.

A) (1pt) ATP hydrolysis is necessary for actin treadmilling. (4 sentence max)

B) (1pt) Late endosomes are converted to mature lysosomes by an increase in their internal pH. (4 sentence max)

C) (1pt) Lysosomal membranes contain a proton pump that utilizes the energy of ATP hydrolysis to pump protons out of the lysosome, thereby maintaining the lumen at a low pH. (4 sentence max)

D) (1pt) If cells were treated with a weak base which neutralizes the pH of organelles, it is expected that M6P receptors would accumulate in the Golgi due to the M6P receptors inability to bind lysosomal enzymes. (4 sentence max)

Which of the following is the source of new, novel alleles in a population?

A) mutation

B) crossing over in meiosis

C) gene flow

D) mitosis

What does it mean for a population to be in Hardy-Weinberg equilibrium?

Multiple Choice

• There are no changes in gene frequencies from one generation to the next.

• Natural selection is occuring and selecting a particular phenotype in the population.

• Evolution is occurring in the population.

• Allele frequencies in the population are occurring over time.

In a sample population of people, 36% have the recessive trait of "free" earlobes (q2). What is the frequency of the dominant allele for attached earlobes, p?

Multiple Choice

• 0.40

• 0.64

• 0.10

• 0.24