While tremendous advances have been made in prosthesis technology, a greater understanding of amputee gait is needed, especially among amputees in developing countries. Field studies as well as prosthesis technology in developing countries are limited due to barriers associated with equipment and resources availability. Furthermore, individuals with lower limb amputation experience increased difficulty walking and a higher fall rate compared to non-amputees, which may be exacerbated by environment, terrain, or prosthesis componentry. Due to the importance of walking on various terrain for increased quality of life as well as the differences between prosthesis technology available in developing and developed countries, a better understanding of amputee gait on underdeveloped outdoor terrain is needed. We began to address these needs with three studies that explored factors that influenced and predicted amputee gait on realistic end-user outdoor terrain. First, we investigated the effects of environment (i.e. indoor laboratory or outdoor natural walking path), terrain, and amputation level on energy expenditure and dynamic stability while walking among lower limb amputees and non-amputees. We found that terrain and amputation level affected amputee energy expenditure and stability while environment and specific uneven terrain type had minimal effects. These results may guide future work investigating the effects of terrain in laboratory-based studies. Second, we investigated the ability to predict quantitative measures of amputee gait on outdoor underdeveloped terrain from laboratory-based measurements. We found individual participant characteristics and easily accessible measures of indoor gait were as or more effective at predicting energy expenditure and dynamic stability than gait measures requiring greater experimental and analytical resources. These results may offer a tool for researchers to assess performance among amputees in various settings without the need for expensive and technical equipment. Third, we examined the effect of a low-cost prosthetic knee joint on amputee gait. Specifically, we investigated the effects of on energy expenditure, gait stability, and perceptions of the low-cost prosthetic knee joint while walking on indoor and outdoor terrains. We found evidence that the low-cost knee increased energy expenditure and increased some characteristics of dynamic stability while decreased others. Furthermore, we also identified key insights among amputees about the performance of the low-cost prosthetic knee joint that could aid in future design modifications of the knee. Together, these studies help to clarify differences in walking performance between laboratory and outdoor terrains among lower limb amputees, help circumvent the challenges of obtaining quantitative gait measures during field studies in developing countries and may help guide the future design and use of low-cost prosthetic knee technology. / Doctor of Philosophy / While tremendous advances have been made in prosthesis technology, a greater understanding of how lower limb amputees walk (i.e. amputee gait) is needed, especially among amputees in developing countries. Studies in the field as well as the devices that amputees where to walk (prosthesis technology) in developing countries are limited due to barriers associated with equipment and resources availability. Furthermore, individuals with lower limb amputation experience increased difficulty walking and a higher fall rates compared to non-amputees, which may be exacerbated by environment, terrain, or components of the prosthesis. Due to the importance of walking on various terrain for increased quality of life as well as the differences between prosthesis technology available in developing and developed countries, a better understanding of how amputees walk on uneven outdoor terrain is needed. We began to address these needs with three studies that explored factors that influenced and predicted how amputees walk on realistic end-user outdoor terrain. First, we investigated the effects of environment (i.e. indoor laboratory or outdoor natural walking path), terrain, and amputation level on energy expenditure and walking stability among lower limb amputees and non-amputees. We found that terrain and amputation level affected amputee energy expenditure and stability while environment and specific uneven terrain type had minimal effects. These results may guide future work investigating the effects of terrain in laboratory-based studies. Second, we investigated whether we could predict amputee walking performance on outdoor underdeveloped terrain from laboratory-based measurements. We found individual participant characteristics and easily accessible performance measures were as or more effective at predicting energy expenditure and stability than performance measures requiring greater experimental and analytical resources. These results may offer a tool for researchers to assess performance among amputees in various settings without the need for expensive and technical equipment. Third, we examined the effect of a low-cost prosthetic knee joint on amputee gait. Specifically, we investigated the effects of on energy expenditure, gait stability, and perceptions of the low-cost prosthetic knee joint while walking on indoor and outdoor terrains. We found evidence that the low-cost knee increased energy expenditure and increased some characteristics of stability while decreased others. Furthermore, we also identified key insights among amputees about the performance of the low-cost prosthetic knee joint that could aid in future design modifications of the knee. Together, these studies help to clarify differences in walking performance between laboratory and outdoor terrains among lower limb amputees, help circumvent the challenges of obtaining quantitative gait measures during field studies in developing countries and may help guide the future design and use of low-cost prosthetic knee technology.

Accelerometers are objective monitors of physical activity (PA) that can be used to estimate energy expenditure (EE). Most accelerometer EE estimation equations are based on steady-state data and do not consider excess post-exercise oxygen consumption (EPOC) after exercise. PURPOSE: To quantify the error in accelerometer EE estimates due to EPOC after varying durations of high-intensity treadmill running. METHODS: Nine young, healthy, recreationally active males participated in three study visits. Visit 1 included a treadmill VO2 peak test to determine the treadmill speed correlating to 80% VO2 peak for visits 2 and 3. Visit 2 included a seated 20-min baseline and three short (30s, 60s, 120s) vigorous treadmill running bouts each followed by 20 minutes of seated rest. Visit 3 included a supine 60-min baseline and a 30-min treadmill running bout followed by 3 hours of supine rest. Twelve EE estimation equations each using either a non-dominant wrist or right hip ActiGraph GT3X+ accelerometer were compared to the true EE measured by the Parvomedics TrueOne 2400 indirect calorimeter. RESULTS: The Freedson 1998 EE estimation equation overestimated EE during the 20min post-exercise period after each exercise bout (mean kCals [95% CIs]; 30s: 19.3 [11.4, 27.2], 60s: 16.6 [8.5, 24.7], 120s: 13.4 [5.74, 21.1], 30min: 15.1 [6.69, 23.5]). The Crouter 2009 branching algorithm underestimated EE during the 20min post-exercise period after each exercise bout (mean kCals [95% CIs]; 30s: -8.59 [-10.6, -6.62], 60s: -11.6 [-13.7, -9.38], 120s: -15.0 [-18.1, -11.8], 30min: -11.0 [-14.3, -7.77]), but was partially corrected by adding in the measured EPOC. CONCLUSION: Estimated EE during lying or seated rest from linear accelerometer equations was heavily dependent on the y-intercept of the equation, which represents the estimated resting EE of the wearer, with the Crouter calibration study being the only one to directly measure resting EE. More sophisticated approaches, like the Crouter 2009 and newer machine learning algorithms, have better potential to more accurately estimate EE across various activity types. New accelerometer EE estimations should include resting in their calibration protocols in order to more accurately estimate EE during rest.

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Obesity is a chronic disease caused by an imbalance of energy intake and expenditure resulting in excessive accumulation of adipose tissue (AT) either in major adipose depots like subcutaneous (SAT) or visceral (VAT), or ectopic lipid deposition in other organs and tissues such as liver or muscle. In 2019 obesity was ranked globally under the top five death causes and increases the risk for suffering from non-communicable diseases such as stroke, diabetes and various types of cancer. Current therapeutic strategies implement dietary interventions and increased physical activity, application of incretin-based drugs such as dual or triple agonists, or bariatric surgery. The recruitment and activation of brown adipose tissue (BAT) represents an intriguing therapeutic approach to combat obesity by increasing energy expenditure via thermogenesis. BAT is a highly metabolically active organ and its activity is induced by cold. To maintain body temperature, BAT is specialized in the dissipation of energy to produce heat by a high demand of oxygen and substrates such as lipids and glucose. Myoglobin (MB) expression was detected in BAT of cold-exposed rodents and is increased during brown adipocyte differentiation suggesting an unrecognized physiological role in BAT contributing to thermogenesis. Since BAT and muscle are both highly metabolic active organs and are derived from the same myogenic factor 5 positive progenitor, it is likely, that BAT MB might exert similar functions as in muscle tissue. In addition to facilitating oxygen supply, further contributions of MB have been assigned to scavenging ROS and regulating cellular NO levels. Furthermore, a role of MB as a lipid shuttle was proposed, as MB seems to enable energy production via beta-oxidation and prevent myocardial lipid accumulation. This project addressed the hypothesis that MB expression is upregulated in active (brown) AT to support thermogenesis by serving as lipid shuttle from the cytosol into the mitochondria, by contributing and sustaining oxygen supply and/ or by acting as ROS scavenger during thermogenesis. To investigate consequences of MB expression in BAT on mitochondrial function and thermogenesis in vitro and in vivo, MB overexpressing, knockdown or knockout adipocytes and global myoglobin-knockout (Mb-KO) mice were used. Initially, temperature- and differentiation-dependent changes in MB gene and protein expression were investigated in vivo and in vitro. MB expression was upregulated in BAT of cold-exposed C57BL/6N mice and during adipogenesis of brown adipocytes as confirmed in previous findings. Furthermore, BAT Mb gene expression correlated positively with Ucp1 suggesting MB in vivo being regulated by -adrenergic signaling. Surprisingly, in vitro Mb expression was inversely correlated to Ucp1 expression in immortalized and primary brown adipocytes after -adrenergic stimulation with norepinephrine or CL316,243. Since MB expression is increased during adipogenesis, the regulation by PPARG was investigated in immortalized brown adipocytes. Neither the stimulation by PPARG agonists such rosiglitazone or fatty acids nor cell-autonomous effects induced by hypothermia changed Mb gene expression concluding that other pathways regulate MB expression. Evaluating various MB expression levels (high, low, none) on mitochondrial respiration and responsiveness to adrenergic stimulation, Mb knockdown, knockout and overexpression experiments in immortalized and primary brown adipocytes were performed. Herein, a MB expression level dependent increase in maximal mitochondrial respiratory capacity and acute response to adrenergic stimulation, signaling and lipolysis was observed. Also in white adipocytes, metabolic activity was improved by MB overexpression. Since -adrenergic stimulation is accompanied with enhanced ROS production and MB acts as ROS scavenger in cardio myocytes, effects of MB expression on ROS and superoxide levels were determined. However, no impacts were detected, although cold-induced genes were found related to ROS in BAT of Mb-KO mice in vivo, thus a function as ROS scavenger in BAT cannot be excluded. MB binds fatty acids and acylcarnitines, therefore proposed lipid binding residues of MB were mutated. At first, the ability of MB’s lipid binding property was evaluated by dot blot lipid overlay assays. As a result, palmitic and oleic acid were bound by oxygen-carrying MB. The mutant instead showed a reduced binding capacity. In functional assays, the non-lipid binding property abolished the beneficial effects in substrate flux, mitochondrial respiration and thermogenesis of MB in immortalized brown adipocytes. At the end, this data clearly demonstrated that MB’s lipid binding is essential to augment substrate flux and permit increased mitochondrial respiration and thermogenesis. To investigate consequences of MB-deficiency on thermogenesis in vivo, whole-body Mb-KO mice models were exposed to thermoneutrality (30 °C), room temperature (23 °C, mild cold stress) and cold (8 °C) for seven days. Lack of MB resulted in impaired thermoregulation at temperatures below thermoneutrality and diminished the response to pharmacological BAT activation after intraperitoneal CL316,243 application. To address the translational potential, MB is differentially expressed in subcutaneous (SC) and visceral (VIS) depots of human adipose tissue (AT). In lean patients MB was significantly lower expressed in SC AT compared to VIS AT, whereas in obese patients the opposite was overserved. Further analyses revealed that MB expression was more pronounced in AT samples with higher thermogenic potential. As a conclusion, the present study demonstrates for the first time a functional relevance of MB’s lipid binding property and suggests MB as a previously unrecognized player in BAT biology that increases mitochondrial respiratory capacity, a crucial aspect for rapid adaptation to metabolic changes. The exact mechanisms how MB contributes to lipid transport, remain to be elucidated.

Nescient Helix Loop Helix 2 (Nhlh2) is a member of the basic helix-loop-helix transcription factor family. Mice with a targeted deletion of Nhlh2, called N2KO mice, show adult onset obesity in both males and females. Nhlh2 regulates other genes by binding to the E-box in the promoter region of these genes. This transcription factor regulates many other transcription factors including MC4R and PC1/3 which are associated with human obesity. The Nhlh2 promoter has been analyzed for putative transcription factors binding sites. These putative binding sites have been tested to be the regulators of Nhlh2 by transactivation assays with mutant promoters, Electrophoretic Shift Assay (EMSA), and Chromatin Immunoprecipitation Assay (ChIP) as methods to investigate the DNA-protein binding. The results of these experiments showed that the Nhlh2 promoter has five Signal Transducer and Activator of Transcription 3 (Stat3) binding site motifs at -47, -65, -80, -281, -294 and two Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB) binding site motifs at -67 and -135. While NFκB acts as a negative regulator of Nhlh2, this research showed that Stat3 acts as a regulator for the Nhlh2 basal expression and leptin stimulation. The ChIP assay using chromatin from mouse hypothalamus and antibodies against Stat3 and the NFκB subunits P50, P65, and c-Rel demonstrated that all of these antibodies were able to pull down the part of the Nhlh2 promoter containing the binding sites of Stat3 and NFκB. The EMSA results not only demonstrated that NFκB and Stat3 binding site motifs are real binding sites, but also exists the possibility of a relationship between these transcription factors to regulate Nhlh2 expression with leptin stimulation. An effort in analyzing the human NHLH2 3'UTR showed that one of the SNPs located at position 1568 in the NHLH2 mRNA (NHLH2A^1568G) which converts adenosine to guanine might have the potential to decrease the mRNA stability. For more investigation about this SNP, the mouse Nhlh2 tail was cloned into 2 different vectors and these vectors were subjected to site directed mutagenesis to create the 3'UTR SNP that convert A to G. One of these vectors used luciferase as a reporter gene for expression while the other one was used to measure Nhlh2 mRNA stability. These vectors were transfected into hypothalamic cell line N29/2 to test the effect of this SNP on Nhlh2 expression. This study demonstrated that this SNP down regulated luciferase expression and also decreased Nhlh2 mRNA stability. Taken together, this study demonstrated that Nhlh2 could be regulated transcriptionally by both NFκB and Stat3 transcription factors and post-transcripitionally by the 3'UTR SNP that converts adenosine to guanine. / Ph. D.

Introdução: A síndrome do intestino curto (SIC) representa um estado clínico de má absorção grave, e a gestão dietética de pacientes com SIC é extremamente desafiadora. Uma vez que o grau de desnutrição é frequentemente considerável, a intervenção dietética bem sucedida depende da estimativa mais exata possível das necessidades energéticas para prever as metas da terapia nutricional. Objetivo: Quantificar o gasto energético total (GET) em pacientes com SIC pelo método da água duplamente marcada (ADM). Materiais e Métodos: Neste estudo observacional, o GET foi mensurado pelo método da água duplamente marcada em 22 voluntários, 11 com SIC e 11 controles pareados por sexo, idade e IMC (grupo Controle). O GET foi estimado pela equação de Escott-Stump e a partir de acelerômetro, e foi comparado com o GET determinado pela ADM. O gasto energético em repouso (GER) foi mensurado por calorimetria indireta (CI) e comparado com o GER estimado pela equação de Harris e Benedict. O acelerômetro também foi utilizado para estabelecer o nível de atividade física. Resultados: Os participantes tinham idade (média ± DP) de 53 ± 8 anos. O GET medido por ADM foi significativamente menor no grupo SIC comparado ao grupo Controle (p < 0,01); no entanto, o GET estimado não diferiu significativamente entre os grupos. O GET medido foi significativamente maior do que o GET estimado por fórmula no grupo SIC, (respectivamente 1875 ± 276 e 1517 ± 175 kcal/dia, p < 0,01), assim como para o grupo Controle (2393 ± 445 e 1532 ± 178 kcal/dia, p < 0,01). No entanto, o GET medido foi significativamente menor do que o GET predito a partir do acelerômetro no grupo SIC (2075 ± 298 kcal/dia, p = 0,02), e não diferiu significativamente no grupo Controle (2207 ± 355 kcal/dia, p = 0,21). Não foram verificadas diferenças significantes entre o GER medido e predito para ambos, e entre os grupos. Conclusão: O GET medido em pacientes com SIC foi significativamente maior do que o GET estimado por fórmula, e foi menor quando comparado com os valores dos sujeitos controles. No entanto, o GET estimado a partir do acelerômetro, superestima o GET medido por ADM. As fórmulas atualmente utilizadas na prática clínica parecem subestimar as necessidades de energia de pacientes com SIC. Desta forma, adaptações da estimativa atual, aumentando as prescrições de ingestão energética nestes pacientes parecem ser adequadas para apoiar as necessidades diárias de energia e evitar a subnutrição. / Background: Short bowel syndrome (SBS) is a serious malabsorption disorder, and dietetic management of SBS patients is extremely challenging. Once the degree of undernutrition has been assessed, successful dietary intervention depends on the most accurate estimation and provision of energy needs to provide nutritional therapy goals. Objective: To quantify total energy expenditure (TEE) in SBS patients using the doubly labeled water (DLW) method. Design: In this observational study, TEE was measured by the DLW method in 22 participants, 11 with SBS and 11 gender-age-and BMI-matched controls (Control group). Predicted energy requirements were determined using the Escott-Stump equation and by using and accelerometer, and they were compared with TEE determined with DLW. Resting energy expenditure (REE) was measured using indirect calorimetry and compared with predict REE using the Harris and Benedict equation. The accelerometer was also used to determine physical activity level. Results: Participants were aged (mean ± SD) 53 ± 8 years. Measured TEE was significantly lower in the SBS group compared to the Control group (p < 0.01); however, predicted TEE did not differ significantly between the groups. Measured TEE was significantly higher than predicted TEE for the SBS group, (1875 ± 276 and 1517 ± 175 kcal/d, p < 0.01) and also for the Control group (2393 ± 445 and 1532 ± 178 kcal/d, p < 0.01) when determined by formula. However, measured TEE was significantly lower than predicted TEE (2075 ± 298 kcal/d, p = 0.02) for the SBS group, and did not differ for the Control group (2207 ± 355 kcal/d, p = 0.21) when determined by accelerometer. No significant differences were seen between measured and predicted REE both within and between groups. Conclusion: Measured TEE in SBS patients was significantly higher than predicted using standard equations, but also lower than values for age, BMI and gender-matched non-SBS controls. However, predicted TEE using accelerometer overestimated the measured TEE. Currently-used formulas in clinical practice appear to underestimate energy requirements of SBS patients. Therefore, adjustments to the current estimation, increasing the energy intake requirements in these patients appear to be adequate to support the daily energy requirements and avoid undernutrition.