Biochemical Analysis of the Regulation of Vesicle Fusion, Peroxisomal Protein Import and Proteolytic Processing in Eukaryotes.

My dissertation research provides major contributions to the current understanding of intracellular protein trafficking in two important areas: vesicular fusion and peroxisome biogenesis. I began with a study of SNAREs, highly conserved proteins that form the core fusion machinery within secretory and endosomal trafficking systems. I examined the regulation of an integral member of this protein machinery, syntaxin 5, by its mammalian endoplasmic reticulum/Golgi Sec1/Munc18 protein binding partner rSly1. To address their functional relationship, I produced a conformation-specific monoclonal antibody to use in immunostaining experiments and in vitro ER-to-Golgi transport assays. Results from the manipulation of rSly1/syntaxin 5 interactions indicate that rSly1 function is intimately associated with syntaxin binding, not promoting availability of the SNARE motif, but perhaps supporting a later step in SNARE complex formation. My research continued with an examination of peroxisome matrix protein import, a mode of protein transport that utilizes two distinct peroxisomal targeting signals (PTS1 and 2) to bind exclusively to two different cytosolic receptors that define separate import pathways. A few matrix enzymes contain a PTS2 that is cleaved off after import, in plants and mammals. The identity of the PTS2 processing protease is unknown. Bioinformatic analysis of the Arabidopsis genome revealed a 76kDa Deg-protease, AtDEG15, predicted to be peroxisomal. Analysis of protein extracts from Arabidopsis plants with a knockout mutation in atdeg15 reveals that the precursor form of the PTS2 protein thiolase (THL) was not processed. Using reverse transcription, I isolated and cloned AtDEG15. In vitro peroxisome import, protease assays, and mutagenic analysis demonstrated that AtDEG15 is a novel PTS2-specific processing protease. Two proteins found in Arabidopsis thaliana have been identified that possess both a putative PTS1 and a PTS2, long-chain acyl-CoA synthetase 7 (LACS7) and alpha-crystallin domain protein 31.2 (ACD31.2). While both proteins have been localized to the peroxisome, the PTS responsible for their localization remains unclear. Mutagenic analysis and standard in vitro import assays showed that either the PTS1 or PTS2 is sufficient to direct ACD31.2 and LACS7 import. Thus, using cellular and molecular biological techniques, my research has expanded the current understanding of intracellular protein trafficking.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60813/1/roonie_1.pd

A Canine Model to Assess the Biochemical Stress Response to Laparoscopic and Open Surgery

Purpose: To develop an animal model to assess the stress response to open and laparoscopic surgery. Such a model would allow objective physiologic assessment of the putative benefits of laparoscopy and provide a framework in which to compare modifications in operative and anesthetic technique that might decrease the stress of surgery. Materials and Methods: Mongrel dogs underwent laparoscopic (N = 12) or open surgical (N = 12) left nephrectomy. In 11 control animals, after induction of anesthesia and line placement, the animal underwent either no intervention (open surgery sham; N = 6) or pneumoperitoneum only (laparoscopic sham; N = 5). Serum glucose and cortisol were measured preoperatively, at skin closure, and at 4, 8, and 24 hours postoperatively. Values at each time point were compared in the laparoscopic and open surgical nephrectomy groups and in each of the two nephrectomy groups and their respective shams. Results: Compared with baseline, there was a sharp rise in serum cortisol at the time of skin closure, with a gradual decline to baseline values by 24 hours, in all experimental animals. Significantly lower serum cortisol concentrations were seen at 4 and 8 hours postoperatively in the laparoscopic group than in the open surgery group. Cortisol was significantly higher in the open group than in the sham-open group at all time points, whereas cortisol was greater in the laparoscopic group than in the pneumoperitoneum-only group only at the 4-hour time point. No differences were seen in serum glucose between groups. Conclusions: The serum cortisol concentration appears to be a good measure of surgical stress in the canine model. The rapid decline in serum cortisol after laparoscopy compared with open surgery may indicate a lesser degree, or quicker resolution, of surgical stress in the former. Furthermore, the similarity in cortisol curves between laparoscopy and pneumoperitoneum only suggests that surgical stress in laparoscopic surgery may be attributable mainly to the effects of pneumoperitoneum.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63212/1/089277901317203100.pd

Monitoring compliance of CITES lion bone exports from South Africa

From 2008 to 2018, South Africa permitted the export of captive-bred African lion (Panthera leo) skeletons to Southeast Asia under CITES Appendix II. Legal exports rose from approximately 50 individuals in 2008 to a maximum of 1,771 skeletons in 2016, and has led to ongoing concerns over possible laundering of non-lion, multiple-source and wild-sourced bones. South Africa is required under its obligations to CITES to employ mechanisms for monitoring and reporting trade, and to limit the potential for illegal trade and laundering of lion and other large felid bones. Monitoring tools for legal trade are critical to compliance with CITES. Here we evaluate the CITES-compliance procedure implemented by South Africa for export of lion bones and identify six essential general points for consideration in the implementation of animal export quota compliance protocols. We provide specific insight into the South African lion bone export monitoring system through: i) outlining the protocols followed; ii) assessing the utility of cranial morphology to identify species; iii) evaluating skeleton consignment weight as a monitoring tool; and iv) presenting molecular (DNA) species assignment and pairwise-comparative sample matching of individuals. We describe irregularities and illicit behaviour detected in the 2017 and 2018 lion bone quotas. Notably, we report that the compliance procedure successfully identified and prevented the attempted laundering of a tiger (P. tigris) skeleton in 2018. We emphasise the utility of mixed-method protocols for the monitoring of compliance in CITES Appendix II export quota systems

Neandertal Mandibular Molars from Hortus Cave, France: A Comparison of Crown Shapes Using Elliptical Fourier Analysis

Neandertal permanent mandibular molars are well represented at Hortus Cave, France, Sub-Phase IVb and Vb, providing an opportunity to assess intrapopulation and intersite shape variation. We expected the mandibular molars from Hortus cave, comprising Hortus II, Hortus IV, Hortus V and Hortus VI to cluster together, compared to Neandertals close in proximity, such as Malarnaud and La Quina 5 as well as those more distantly located such as Arcysur- Cure 1, Engis 2 and Scladina 4A-1. A single Neolithic cave burial from Maurenne Caverne de la Cave, Belgium (n = 11) is used to contextualize the degree of variation in the Hortus assemblage. From photographic images of the permanent molars, crown shapes were digitized and binarized. The resulting images were processed in R using elliptical Fourier analysis and the resulting amplitudes of the harmonics were subjected to principal components analysis and hierarchical clustering. Hortus II and Hortus V, both from Sub-Phase Vb, are relatively similar to one another in M1 crown shape, and are secondarily grouped with Hortus IV from Sub-Phase IVb. Maurenne Caverne de la Cave has a larger degree of variation in crown shape than the Hortus assemblage, and the Neandertals imperfectly cluster together and apart from the Neolithic sample for some multivariate comparisons. Variance in Neandertal molar crown shape can be primarily explained as the result of chronology. Hortus consistently groups with other MIS 3 Neandertals such as La Quina 5 and Engis 2, while those from MIS 5, represented by Scladina 4A-1 and Malarnaud are distinct as is Arcy-sur-Cure 1 from late MIS 3. Across the molars, the Hortus assemblage is most similar to La Quina 5 from Charente, Southwest France

The diet of young Neandertals from France, Pech de l’Azé I and Hortus II, reconstructed using dental microwear texture analysis

Les adultes néandertaliens présentent des différences de régime alimentaire en fonction de leur paléohabitat. Nous examinons si les jeunes enfants néandertaliens de France datant du stade isotopique marins 3 montrent également ces distinctions alimentaires à partir de l’étude de la texture des micro-usures dentaires des deuxièmes molaires déciduales des individus Pech de l’Azé I, datant d’une phase froide-tempérée, et Hortus II, datant d’une phase froid-aride extrême. L’échantillon comparatif (n=76) comprend des molaires déciduales d’agriculteurs-cueilleurs provenant de trois grottes néolithiques de Belgique, dont Caverne de la Cave à Maurenne (n=5), Sclaigneaux (n=7) et Bois Madame (n=6), ainsi que des agriculteurs de l’époque romaine d’Herculanum (n=15) et médiévaux de Canterbury, Angleterre (n=43). Lorsque la complexité est comparée à l’anisotropie, Pech de l’Azé I présente une valeur élevée provenant de la mastication de plantes à parties dures ou de particules adhérentes, ou de la consommation d’aliments mal transformés ou chargés de silicates abrasifs. En revanche, Hortus II a une valeur faible, peut-être en raison de l’accès limité aux parties dures des plantes comme les graines et les noix. Toutefois, Pech de l’Azé I et Hortus II se ressemblent en présentant une faible valeur d’anisotropie qui est révélatrice de mouvements complexes des mâchoires lors de la mastication et sont différents des enfants humains néolithiques, romains et médiévaux qui ont tendance à avoir des valeurs plus élevées. Les régimes alimentaires des enfants néandertaliens diffèrent en fonction du paléohabitat et sont généralement plus diversifiés que ceux des producteurs d’aliments, qu’ils habitent des environnements boisés ou ouverts.Neandertal adults show differences in diet with respect to paleohabitat. To examine whether Neandertal children of France during Marine Isotope Stage 3 also show these dietary distinctions, the deciduous second molars of Pech de l’Azé I, from a cold-temperate period, and Hortus II from Sub-Phase Vb, an extreme cold-arid interval, were examined using dental microwear texture analysis. The comparative sample (n=76) includes deciduous molars from Neolithic forager-farmers of Belgium, including Caverne de la Cave at Maurenne (n=5), Sclaigneaux (n=7) and Bois Madame (n=6), Roman-era farmers from Herculaneum (n=15) and Medieval agriculturalists from Canterbury, England (n=43). When complexity is compared to anisotropy, Pech de l’Azé I exhibits an elevated value from the mastication of plants with hard parts or adherent particles, or the consumption of foods that were poorly processed or grit-laden, whereas Hortus II presents a low value, perhaps from limited access to hard plant parts such as seeds and nuts. However, Pech de l’Azé I and Hortus II resemble each other in having a low value for anisotropy, which is indicative of complicated movements of the jaws during mastication and are dissimilar to Neolithic, Roman and Medieval human children who tend to have higher values. The diets of Neandertal children differ with respect to paleohabitat and typically were more diverse than those of food producers regardless of whether they inhabited wooded or open environments

Long-term consequences of an intensive care unit stay in older critically ill patients: design of a longitudinal study

<p>Abstract</p> <p>Background</p> <p>Modern methods in intensive care medicine often enable the survival of older critically ill patients. The short-term outcomes for patients treated in intensive care units (ICUs), such as survival to hospital discharge, are well documented. However, relatively little is known about subsequent long-term outcomes. Pain, anxiety and agitation are important stress factors for many critically ill patients. There are very few studies concerned with pain, anxiety and agitation and the consequences in older critically ill patients. The overall aim of this study is to identify how an ICU stay influences an older person's experiences later in life. More specific, this study has the following objectives: (1) to explore the relationship between pain, anxiety and agitation during ICU stays and experiences of the same symptoms in later life; and (2) to explore the associations between pain, anxiety and agitation experienced during ICU stays and their effect on subsequent health-related quality of life, use of the health care system (readmissions, doctor visits, rehabilitation, medication use), living situation, and survival after discharge and at 6 and 12 months of follow-up.</p> <p>Methods/Design</p> <p>A prospective, longitudinal study will be used for this study. A total of 150 older critically ill patients in the ICU will participate (ICU group). Pain, anxiety, agitation, morbidity, mortality, use of the health care system, and health-related quality of life will be measured at 3 intervals after a baseline assessment. Baseline measurements will be taken 48 hours after ICU admission and one week thereafter. Follow-up measurements will take place 6 months and 12 months after discharge from the ICU. To be able to interpret trends in scores on outcome variables in the ICU group, a comparison group of 150 participants, matched by age and gender, recruited from the Swiss population, will be interviewed at the same intervals as the ICU group.</p> <p>Discussion</p> <p>Little research has focused on long term consequences after ICU admission in older critically ill patients. The present study is specifically focussing on long term consequences of stress factors experienced during ICU admission.</p> <p>Trial Registration</p> <p><a href="http://www.controlled-trials.com/ISRCTN52754370">ISRCTN52754370</a></p

Height and timing of growth spurt during puberty in young people living with vertically acquired HIV in Europe and Thailand.

OBJECTIVE: The aim of this study was to describe growth during puberty in young people with vertically acquired HIV. DESIGN: Pooled data from 12 paediatric HIV cohorts in Europe and Thailand. METHODS: One thousand and ninety-four children initiating a nonnucleoside reverse transcriptase inhibitor or boosted protease inhibitor based regimen aged 1-10 years were included. Super Imposition by Translation And Rotation (SITAR) models described growth from age 8 years using three parameters (average height, timing and shape of the growth spurt), dependent on age and height-for-age z-score (HAZ) (WHO references) at antiretroviral therapy (ART) initiation. Multivariate regression explored characteristics associated with these three parameters. RESULTS: At ART initiation, median age and HAZ was 6.4 [interquartile range (IQR): 2.8, 9.0] years and -1.2 (IQR: -2.3 to -0.2), respectively. Median follow-up was 9.1 (IQR: 6.9, 11.4) years. In girls, older age and lower HAZ at ART initiation were independently associated with a growth spurt which occurred 0.41 (95% confidence interval 0.20-0.62) years later in children starting ART age 6 to 10 years compared with 1 to 2 years and 1.50 (1.21-1.78) years later in those starting with HAZ less than -3 compared with HAZ at least -1. Later growth spurts in girls resulted in continued height growth into later adolescence. In boys starting ART with HAZ less than -1, growth spurts were later in children starting ART in the oldest age group, but for HAZ at least -1, there was no association with age. Girls and boys who initiated ART with HAZ at least -1 maintained a similar height to the WHO reference mean. CONCLUSION: Stunting at ART initiation was associated with later growth spurts in girls. Children with HAZ at least -1 at ART initiation grew in height at the level expected in HIV negative children of a comparable age

UBC-Nepal Expedition: Haemoconcentration underlies the reductions in cerebralblood flow observed during acclimatization to high-altitude

At high‐altitude, increases in haematocrit (Hct) are achieved through altitude‐induced diuresis and erythropoiesis, both of which result in increased arterial oxygen content (CaO2). Given the impact alterations in Hct have on CaO2, haemoconcentration has been hypothesized to partly mediate the attenuation of the initial elevation in cerebral blood flow (CBF) at high‐altitude. To test this hypothesis, healthy males (n = 13) ascended to 5050 m over nine days without the aid of prophylactic acclimatization medications. Following one‐week of acclimatization at 5050 m, participants were haemodiluted by rapid saline infusion (2.10 ± 0.28 L) to return Hct towards pre‐acclimatized levels. Arterial blood gases, Hct, global CBF (duplex ultrasound), and haemodynamic variables were measured following initial arrival to 5050 m, and after one‐week of acclimatization at high‐altitude, prior to and following the haemodilution protocol. Following one‐week at 5050 m, Hct increased from 42.5 ± 2.5 to 49.6 ± 2.5% (P < 0.001), and was subsequently reduced to 45.6 ± 2.3% (P < 0.001) following haemodilution. Global CBF decreased from 844 ± 160 to 619 ± 136 ml mi−1 n (P = 0.033) following one‐week of acclimatization and increased to 714 ± 204 ml mi−1n (P = 0.045) following haemodilution. Despite the significant changes in Hct, and thus CaO2, cerebral oxygen delivery was unchanged at all time points. Furthermore, these observations occurred in the absence of any changes in mean arterial blood pressure, cardiac output, arterial blood pH, or oxygen saturation pre‐ and post‐haemodilution. These data highlight the influence of Hct in the regulation of CBF and are the first to demonstrate experimentally that haemoconcentration contributes to the reduction in CBF during acclimatization to altitude

A Centennial Retrospective on Charles Williams

Edited transcript of a panel discussion (including audience contributions) at the 17th Mythopoeic Society Conference