Skeletal muscle fibre-type comparison of whole tissue and subcellular membrane phospholipids and fatty acids

Membranes are dynamic structures that affect cell structure and function. Compositional changes ofmembranes have been shown with the application of a perturbation; however these are limited to whole tissue analysis. The purpose of this thesis was to compare the phospholipid (PL) fatty acid (FA) composition of rat whole muscle (Wm) to 1) purified and non-purified subsarcolemmal (SS) mitochondria in soleus, plantaris, and red gastrocnemius, and 2) sarcolemma, transverse-tubules, SS and intermyofibrillar (IMF) mitochondria fix)m whole hindlimb. The major findings were that 1) contamination significantly altered the PL FA composition of the SS mitochondrial membrane fraction, 2) Wm and SS mitochondria compositions differed between muscle types, and 3) Wm did not accurately reflect the PL FA composition of any isolated subcellular membranes, with each being unique from each other. As such, the relevancy of the trends reported in the literature of the effects of perturbations on Wm may be limited.

Openness and privacy in Ontario: an evaluation of the implementation of FIPPA : (The Freedom of Information and Protection of Privacy Act, 1987, Chapter 25 and Ontario Regulation 532/87) /

Since the early 1970's, Canadians have expressed many concerns about the growth of government and its impact on their daily lives. The public has requested increased access to government documents and improved protection of the personal information which is held in government files and data banks. At the same time, both academics and practitioners in the field of public administration have become more interested in the values that public servants bring to their decisions and recommendations. Certain administrative values, such as accountability and integrity, have taken on greater relative importance. The purpose of this thesis is to examine the implementation of Ontario's access and privacy law. It centres on the question of whether or not the Freedom of Information and Protection of Privacy Act, 1987, (FIPPA) has answered the demand for open access to government while at the same time protecting the personal privacy of individual citizens. It also assesses the extent to which this relatively new piece of legislation has made a difference to the people of Ontario. The thesis presents an overview of the issues of freedom of information and protection of privacy in Ontario. It begins with the evolution of the legislation and a description of the law itself. It focuses on the structures and processes which have been established to meet the procedural and administrative demands of the Act. These structures and processes are evaluated in two ways. First, the thesis evaluates how open the Ontario government has become and, second, it determines how Ill carefully the privacy rights of individuals are safeguarded. An analytical framework of administrative values is used to evaluate the overall performance of the government in these two areas. The conclusion is drawn that, overall, the Ontario government has effectively implemented the Freedom of Information and Protection of Privacy Act, particularly by providing access to most government-held documents. The protection of individual privacy has proved to be not only more difficult to achieve, but more difficult to evaluate. However, the administrative culture of the Ontario bureaucracy is shown to be committed to ensuring that the access and privacy rights of citizens are respected.

Metabolite changes during Riesling icewine fermentation when yeast micronutrients are used /

Icewine is an intensely s\veet dessert \vine fermented from the juice of naturally frozen grapes. Icewine fermentation poses many challenges such as failure to reach desired ethanol levels and production of high levels of volatile acidity in the fonn of acetic acid. This study investigated the impact of micronutrient addition (GO-FERM® and NATSTEP®) during the rehydration stage of the commercial \vine yeast Saccharomyces cerevisiae KI-VIII6 during Ice\vine fermentation. Sterile-filtered and unfiltered Riesling Ice\vine juice was inoculated \vith yeast rehydrated under four different conditions: in water only; with GO-FERM®; with NATSTEP®; or the combination of both micronutrient products in the rehydration water. Using sterile-filtered Icewine juice, yeast rehydration had a positive impact of reducing the rate of acetic acid produced as a function of sugar consumed, reducing the ratio of acetic acid/ethanol and reducing the ratio of acetic acid/glycerol. In the sterile-filtered fermentation, yeast rehydrated with micronutrients generated 9-times less acetic acid per gram of sugar in the first 48 hours compared to yeast rehydrated only \vith water and resulted in a 17% reduction in acetic acid in the final \vine \vhen normalized to sugar consumed. However, the sterile-filtered fermentations likely became stuck due to the overc1arification of the juice as evidenced from the low sugar consumption (117 gIL) that could not be completely overcome by the micronutrient treatments (144 gIL sugar consumed) to reach a target ethanol of IO%v/v. Contrary to \vhat \vas observed in the sterile-filtered treatements, using unfiltered Ice\vine juice, yeast micronutrient addition had no significant impact of reducing the rate of acetic acid produced as a function of sugar consumed, reducing the ratio of acetic acid/ethanol and reducing the ratio of acetic acid/glycerol. However, in the unfiltered fermentation, micronutrient addition during yeast rehydration caused a reduction in the acetic acid produced as a function of sugar consumed up to 150 giL sugar consumed.. In contrast to the sterile-filtered fermentations, the unfiltered fermentations did not become stuck as evidenced from the higher sugar consumption (l47-174g1L). The largest effects of micronutrient addition are evident in the first two days of both sterile and unfiltered fermentations.

Water quality and fish populations of the Welland River, Ontario /

The water quality and fish populations of the Welland River were observed to decline with distance downstream. This coincided with increased agricultural , domestic and industrial waste loadings. The river upstream of the City of Welland received considerable loadings from agricultural sources. Centrarchids, sciaenids, ictalurids, cyprinids and esocids characterized this upper section of the river. Most of these species were tolerant of low dissolved oxygen concentrations and the high turbidity which prevailed there . The river near Port Robinson receives many industrial and domestic wastes as evidenced by the water quality data. The fish in this section were less abundant and the observed population was comprised almost solely of cyprinids. Further downstream, near Montrose, the Welland River received shock loads of chemical wastes that exceeded a specific conductance of ISiOOO ;umhos/cm. Few fish were captured at this site and those that were captured were considered to be transients. A review of the literature revealed that none of the common indices of water quality in use today could adequately predict the observed distributions. In addition to the above, the long-term trend (l3 yrs) of water quality of the lower Welland River revealed a gradual improvement. The major factor thought to be responsible for this improvement was the operation of the Welland Sewage Treatment Plant. The construction of the New Welland Ship Canal coincided with large fluctuations of the total solids and other parameters downstream. These conditions prevailed for a maximum of three years (1972- 1975)' Furthermore, spawning times and temperatures, geographic distributions, length-weight regressions and many other descriptive aspects of the ecology of some 26 species/ taxa of fish were obtained. Several of these species are rare or new to southern Ontario.

Secretory immunity in overweight and obese versus normal-weight early-and late-pubertal females

Obesity is a condition associated with a wide variety of health problems including hypertension, dyslipidemia, diabetes mellitus, certain forms of cancer, cardiovascular disease, and gallstones (157). TTiere is growing evidence that obesity may also be related to compromised immune function due to altered metabolic, psychological, and physical attributes (93). The aim of this study was to compare: a) immunity-related variables such as frequency of upper respiratory tract infections (URTI) and salivary secretory immunoglobulin A (sIgA) levels between overweight/obese (OB) and normal weight (NW) early-pubertal and late-pubertal girls, and b) stress-related variables such as Cortisol, melatonin, the melatonin/cortisol ratio, testosterone and the testosterone/cortisol ratio. Physical activity levels, stress indicators, and fatigue were used to explain potential differences in the dependent variables. It was hypothesized that the OB females would have lower melatonin (M) and higher Cortisol (C) and testosterone (T) levels compared with NW girls, regardless of maturity status. The altered levels of melatonin, Cortisol, and testosterone, would result in decreased M/C and T/C ratios, despite the increase in testosterone in OB females. It was hypothesized that this altered hormonal status results in a compromised immunity marked by higher frequency of upper respiratory tract infections (URTI) and decreased levels of secretory immunoglobulin A (sIgA). It was also hypothesized that OB girls would participate in less hours of physical activity than their NW counterparts and that this would relate to their stress and immunity levels. Forty (16 early- and 24 late-pubertal) overweight and obese females were compared to fifty-three (27 eariy- and 26 late-pubertal) age-matched normal-weight control subjects. Participants were categorized as early-pubertal (EP) or late-pubertal (LP) using Tanner self-staging of secondary sex characteristics. Subjects were classified into the two adiposity groups according to relative body fat (%BF), where normal weight (NW) subjects had a %BF less than 25%, and overweight and obese (OB) subjects had a %BF greater than 27.5%. Participants completed a number of questionnaires and information was collected on menstrual history, smoking history, alcohol and caffeine consumption, and medical history. Following the determination of maturity status, a complete anthropometric assessment was made including height, body mass, and body composition. All questionnaires and measurements were completed during a one-hour visit between 1 500 and 1900 hours Relative body fat was assessed using bioelectrical impedance analysis. Resting saliva samples were obtained and assayed (ELISA) for testosterone, Cortisol, melatonin and secretory immunoglobulin A. Physical activity was self-reported using the Godin- Shephard Leisure time questionnaire, and quantified using Actigraph GTIM accelerometers, which participants wore for seven consecutive days from the time they woke up in the morning, until the time they went to bed. Late-pubertal girls also completed questionnaires on their perceived stress and fatigue. Finally, all participants also filled out a one-month health log to record frequency of symptoms of upper respiratory tract infections (URTI). Significant age effects were found for testosterone, Cortisol, incidence of sickness, and sIgA when controlling for physical activity, however there were no significant effects of adiposity on any of the variables. There was a trend which neared-significance for an effect of adiposity on sIgA (p=0.01). There were no significant differences between the groups on the total selfreported leisure-time physical activity in METs per week, however EP girls recorded significantly greater levels of moderate, hard, and very hard physical activity from accelerometers. Results of the perceived stress and fatigue questionnaires in late-pubertal girls demonstrated that contrary to what was hypothesized, NW girls reported more stress and more fatigue than OB girls. Results of the present study suggest that excess adiposity in early- and latepubescent girls may not have a negative impact on immunity as hypothesized.

Coaches' use of and impressions of computer-mediated communication (CMC) media /

The study examined coaches' usage of text-based computer-mediated communication (CMC) media (e.g., text-messaging, email) in the coach-player relationship. Data were collected by surveying Ontario-based male baseball coaches (n = 86) who coached players between 15 and 18 years old. Predictions were made regarding how demographic factors such as age and coaching experience affected coaches' CMC use and opinions. Results indicated that over 76% of respondents never used any CMC media other than email and team websites in their interactions with players. Results also revealed that coaches' usage rates contrasted with their opinion of the usefulness of the media, and their perception of players' use of the media. Coaches characterized most CMC media as limited, unnecessary, and sometimes inappropriate. Additional research should explore players' CMC usage rates and possible guidelines for use of the new media in authority relationships. Academia needs to keep pace with the developments in this area.

The validation of heart rate variability in individuals with spinal cord injury

The current classification system for spinal cord injury (SCI) considers only somatic information and neglects autonomic damage after injiuy. Heart rate variability (HRV) has the potential to be a valuable measure of cardiac autonomic control after (SCI). Five individuals with tetraplegia and four able-bodied controls underwent 1 min continuous ECG recordings during rest, after Metoprolol administration (max dose=3x5mg) and after Atropine administration (0.02mg/kg) in both supine and 40° head-up tilt. After Metoprolol administration there was a 61.8% decrease in the LF:HF ratio in the SCI participants suggesting that the LF:HF ratio is a reflection of cardiac sympathetic outflow. After Atropine administration there was a 99.1% decrease in the HF power in the SCI participants suggesting that HF power is highly representative of cardiac parasympathetic outflow. There were no significant differences between the SCI and able-bodied participants. Thus, HRV measures are a valid index of cardiac autonomic control after SCI.

Synthesis of chiral homoallylic alcohols and phthalans through the asymmetric allylation of carbonyl compounds /

The implementation of chiral centres within biologically active compounds has been a perplexing yet motivational force in chemistry. This work presents the attempted formation of a concurrent or sequential tandem catalyzed methodology of enantioselective nucleophilic addition and electrophilic cyclization. The 2'- arylalkynyl- aldehyde, ketone, and imine substrates used within were adeptly chosen with a dually activated structure; 1) for nucleophilic addition to the electrophilic substituents; and 2) for carbophilic activation of the alkyne substituent to undergo cyclization. To accomplish the nucleophilic addition, two distinct allylation methodologies were pursued: (/?)-BINOL catalyzed-allylboration and (5)- BINAP-AgF catalyzed-allylsilylation. BINAP catalyzed enantioselective allylation of 2'-arylalkynyl-aldehydes, to form chiral homoallylic alcohols, was successful. Homoallylic alcohols were isolated with high enantio-purity (>80%), which then underwent sequential cyclization to form chiral allylic phthalans, in moderate yields. An application of this methodology towards the construction of biologically active compounds was included with the partial synthesis of the natural product and H. pylori inhibitor, (+)-Spirolaxine methyl ether.

Seasonal changes in behavioural and thermoregulatory responses to hypoxia in the Eastern Chipmunk (Tamias striatus) /

Mammalian heterotherms, such as hibemators, are known to be more tolerant of low oxygen tensions than their homeothermic counterparts. It has been suggested that this relative hypoxia tolerance is related to their ability to deal with dramatic changes in body temperature during entry to and arousal from torpor. However, hibemators demonstrate dramatic seasonality in both daily heterothermy and overall torpor expression. It was of interest to test if seasonal comparisons of normothermic individuals within a single species with the capacity to hibernate produce changes in the response to hypoxia that would reflect a seasonal change in tolerance to low oxygen. In particular, the species studied, the Eastern chipmunk {Tamias striatus), is known to enter into torpor exclusively in the winter. To test for seasonal differences in the metabolic and thermoregulatory responses to hypoxia, flow-through respirometry was used to compare metabolic rate, minimum thermal conductance, body temperature, and a thermal gradient used to assess selected ambient temperature in response to hypoxia in both summer and winter acclimated animals. Although the animals periodically expressed torpor throughout the winter, no differences between season in resting metabolic rate, body temperature or minimum thermal conductance were observed in normoxia. The metabolic trials indicated that chipmunks are less responsive to hypoxia in the winter than they are in the summer. Although body temperature dropped in response to hypoxia in both seasons, the decrease was less in the winter, and there was no corresponding decrease in metabolic rate. Providing the animals with a choice of ambient temperatures in hypoxia resulted in a blunting of the drop in body temperature in both seasons, suggesting that the reported fall in body temperature set point in hypoxia is not fully manifested in the behavioural pathways responsible for thermoregulation in chipmunks. Instead, body temperature in hypoxia appears to be highly dependent on ambient temperature and oxygen concentration. The results of this study suggest that the season in which the responses to hypoxia are measured is important, especially in a heterotherm where seasonality can affect the degree to 1 which the animal is tolerant of hypoxia. Winter-acclimated chipmunks appear more capable of defending metabolic heat production in hypoxia, a response consistent with the increased thermogenic capacity observed in animals that must periodically enter and arouse from torpor during hibernation.

Glucose dynamics in rat skeletal muscle : recovery from osmotic stress

The purpose of this study was to examine cell glucose kinetics in rat skeletal muscle during iso-osmotic recovery from hyper- and hypo-osmotic stress. Rat EDL muscles were incubated for sixty minutes in either HYPO (190 mmol/kg), ISO (290 mmol/kg), or HYPER (400 mmol/kg) media (Sigma medium-199, 8 mM glucose) according to an established in vitro whole muscle model. In addition to sixty minute baseline measures in aniso-osmotic conditions, (HYPO-0 n=8; ISO- 0, n=S; HYPER-0, n=8), muscles were subjected to either one minute (HYPO-1 n=8; ISO-1, n=8; HYPER-1, n=8) or five minutes (HYPO-5 n=8; ISO-5, n=8; HYPER-5, n=8) of iso-osmotic recovery media and analyzed for metabolite content and glycogen synthase percent activation. To determine glucose uptake during iso-osmotic recovery, muscles (n=6 per group) were incubated for sixty minutes in either hypo-, iso-, or hyper-osmotic media immediately followed by five minutes of iso-osmotic media containing 3H-glucose and 14 C-mannitol. Increased relative water content/decreased [glucose] (observed in HYPO-0) and decreased water content/increased [glucose] (observed in HYPER-0) returned to ISO levels within 5 minutes of recovery. Glycogen synthase percent activation increased significantly in HYPO-5 over iso-osmotic controls. Glucose uptake measurements revealed no significant differences between groups. It was determined that [glucose] and muscle water content rapidly recovered from osmotic stress demonstrating skeletal muscle's resilience to osmotic stress.

Mechanisms of endothelin-1 induced reactive oxygen species production in vascular adventitial fibroblasts

With the relationship between endothelin-1 (ET-1) stimulation and reactive oxygen species (ROS) production unknown in adventitial fibroblasts, I examined the ROS response to ET-1 and angiotensin (Ang II). ET-1 -induced ROS peaked following 4 hrs of ET-1 stimulation and was inhibited by an ETA receptor antagonist (BQ 123, 1 uM) an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (PD98059, 10 uM), and by both a specific, apocynin (10 uM), and non-specific, diphenyleneiodonium (10 uM), NAD(P)H oxidase inhibitor. NOX2 knockout fibroblasts did not produce an ET-1 induced change in ROS levels. Ang II treatment increased ROS levels in a biphasic manner, with the second peak occurring 6 hrs following stimulation. The secondary phase of Ang II induced ROS was inhibited by an ATi receptor antagonist, Losartan (100 uM) and BQ 123. In conclusion, ET-1 induces ROS production primarily through an ETA-ERKl/2 NOX2 pathway, additionally, Ang II-induced ROS production also involves an ETa pathway.

Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

Electronic and magnetic properties of LaV1-xO3 /

A series of LaVi^xOs compounds (x=0.00, 0.02, 0.04, 0.06, 0.08) were prepeired using the standard solid reaction. The samples were chareicterized by X-ray diffraction (XRD), fourprobe resistivity, smd magnetic susceptibility studies. Powder X-ray diffraction analysis indicated the formation of a single-phase sample with a orthorhombic structure which was first found in GdFeOs (space group Pnma) . The Unit Cell program was used for calculating lattice peirameters from XFID data. The XRD spectnim could be indexed on a cubic lattice with Og = 2ap ~ (7.8578 to 7.9414 A). The lattice parameter was observed to increase as the Vanadium vacancy increased. Four-probe resistivity measurements exhibited semiconductor behavior for all sajnples from room temperature down to 19K. The resistivity of samples increased with increasing Vanadium vacancy. The resistivity of samples demonstrated activated conduction with an activation energy of approximately 0.2 eV. The activation energy increased with increasing lattice parameter. Field cool magnetic susceptibility measurements were performed with field of 500 G from 300 K to 5 K. These measurements indicated the presence of an antiferromagnetic transition at about 140 K. The data was fitted above Neel temperature to Ciurie-Weiss law yielding a negative parameignetic Curie temperature. This implies that antiferromagnetic ordering is present.

Nestmate recognition in the large carpenter bee, Xylocopa virginica /

Abstract Many species of social insects have the ability to recognize their nestmates. In bees, sociality is maintained by bees that recognize which individuals should be helped and which should be hanned in order to maximize fitness (either inclusive or individual) (Hamilton 1964; Lin and Michener 1972). Since female bees generally lay eggs in a single nest, it is highly likely that bees found cohabitating in the same nest are siblings. According to the kin selection hypothesis, individuals should cooperate and avoid aggression with same sex nestmates (Hamilton 1964). However, in opposite sex pairs that are likely kin, aggression should increase among nestmates as an expression of inbreeding avoidance (Lihoreau et al. 2007). Female bees often guard nest entrances, recognizing and excluding foreign conspecific females that threaten to steal nest resources (Breed and Page 1991). Conversely, males that aggressively guard territories should avoid aggression towards other males that are likely kin (Shellman-Reeve and Gamboa 1984). In order to test whether Xy/ocopa virginica can distinguish nestmates from non-nestmates, circle tube testing arenas were used. Measures of aggression, cooperation and tolerance were evaluated to detennine the presence of nestmate recognition in this species. The results of this study indicate that male and female X virginica have the ability to distinguish nestmates from non-nestmates. Individuals in same sex pairs demonstrated increased pushing, biting, and C-posturing when faced with non-nestmates. Males in same sex pairs also attempted to pass (unsuccessfully) nOIl-nestmates more often than ncstmates, suggesting that this behaviour may be an cxpression of dominancc in males. Increased cooperation exemplified by successful passes was not observed among nestmates. However, incrcased tolerance in the [onn of head-to-head touching was observed for nestmates in female same sex and opposite sex pairs. These results supported the kin selection hypothesis. Moreover, increased tolerance among opposite sex non-nestmates suggested that X virginica do not demonstrate inbreeding avoidance among nestmates. 3 The second part of this study was conducted to establish the presence and extent of drifting, or travelling to different nests, in a Xylocopa virgillica population. Drifting in flying Hymenoptera is reported to be the result of navigation error and guard bees erroneously admitting novel individuals into the nest (Michener 1966). Since bees in this study were individually marked and captured at nest entrances, the locations where individuals were caught allowed me to determine where and how often bees travelled from nest to nest. Ifbees were captured near their home nests, changing nests may have been deliberate or explained by navigational error. However, ifbees were found in nests further away from their homes, this provides stronger evidence that flying towards a novel nest may have been deliberate. Female bees are often faithful to their own nests (Kasuya 1981) and no drifting was expected in female X virginica because they raise brood and contribute to nest maintenance activities. Contrary to females, males were not expected to remain faithful to a single nest. Results showed that many more females drifted than expected and that they were most often recaptured in a single nest, either their home nest or a novel nest. There were some females that were never caught in the same nest twice. In addition, females drifted to further nests when population density was low (in 2007), suggesting they seek out and claim nesting spaces when they are available. Males, as expected, showed the opposite pattern and most males drifted from nest to nest, never recaptured in the same location. This pattern indicates that males may be nesting wherever space is available, or nesting in benches nearest to their territories. This study reveals that both female and male X virginica are capable of nestmate recognition and use this ability in a dynamic environment, where nest membership is not as stable as once thought.

Conformational analysis of antibody binding site using EDMA methods /

Euclidean distance matrix analysis (EDMA) methods are used to distinguish whether or not significant difference exists between conformational samples of antibody complementarity determining region (CDR) loops, isolated LI loop and LI in three-loop assembly (LI, L3 and H3) obtained from Monte Carlo simulation. After the significant difference is detected, the specific inter-Ca distance which contributes to the difference is identified using EDMA.The estimated and improved mean forms of the conformational samples of isolated LI loop and LI loop in three-loop assembly, CDR loops of antibody binding site, are described using EDMA and distance geometry (DGEOM). To the best of our knowledge, it is the first time the EDMA methods are used to analyze conformational samples of molecules obtained from Monte Carlo simulations. Therefore, validations of the EDMA methods using both positive control and negative control tests for the conformational samples of isolated LI loop and LI in three-loop assembly must be done. The EDMA-I bootstrap null hypothesis tests showed false positive results for the comparison of six samples of the isolated LI loop and true positive results for comparison of conformational samples of isolated LI loop and LI in three-loop assembly. The bootstrap confidence interval tests revealed true negative results for comparisons of six samples of the isolated LI loop, and false negative results for the conformational comparisons between isolated LI loop and LI in three-loop assembly. Different conformational sample sizes are further explored by combining the samples of isolated LI loop to increase the sample size, or by clustering the sample using self-organizing map (SOM) to narrow the conformational distribution of the samples being comparedmolecular conformations. However, there is no improvement made for both bootstrap null hypothesis and confidence interval tests. These results show that more work is required before EDMA methods can be used reliably as a method for comparison of samples obtained by Monte Carlo simulations.