PhD Oral Exam - Brigitte Desharnais, Chemistry & Biochemistry

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

In postmortem cases, forensic toxicologists perform analyses for legal and illegal drugs, volatile substances, poisons and biochemical parameters in order to determine the causes and circumstances of death [1, 2].

Evaluation of the metabolic capacity of an individual might help to achieve this goal [3–5]. Knowledge that the deceased had a poor metabolic capacity might help differentiate between medical error and accidental overdose, for example.

Traditionally, DNA analysis of genes encoding for metabolizing enzymes of has been used for this purpose [5, 6]. However, the genotype can be quite a poor predictor of phenotype [5]; intervening factors such as sex, age, presence of inducers or inhibitors [3, 7–11] muddy the water.

A proof-of-concept methodology estimating the postmortem metabolic capacity through characterization and quantification of cytochrome P450 (CYP) enzymes in liver tissue is presented here [12]. Combining quantitative proteomics with detection of the peptides bearing mutation sites allowed for a more accurate estimation of the metabolic capacity than genotyping alone.

The current regulatory environment, and best practices, requires forensics bioanalytical methods to be validated. Anticipating the validation of this method, several methodological issues were foreseen.

In order to properly validate the quantitative part of the CYP analysis method, a simple, analyst-independent, and systematic procedure to choose and validate a calibration model (order, weighting) based on statistical analysis was developed [13, 14]. Additionally, the omnipresence of the target analyte(s) in authentic matrix (human liver) calls for a methodology allowing to deal with endogenous concentration(s) of analytes in matrices used to prepare calibration standards and quality control samples. An automated tool was developed to correct for the endogenous analytes’ concentration [15].

Finally, characterization of the CYP enzymes, via the monitoring of peptides bearing a mutation site, requires validation via a qualitative decision point method. Current guidelines about this type of analysis [16–20] are ill adapted to deal with the binary nature of the results. A more suitable set of guidelines was developed [21] and tested [22].

These mathematical method validation tools, in combination with the CYP analysis method, provide the necessary framework for metabolic capacity estimation in postmortem cases.