DESIGN, SYNTHESIS AND APPLICATIONS OF FLUORESCENT AND ELECTROCHEMICAL PROBES

“Seeing is believing” the proverb well suits for fluorescent imaging probes. Since we can selectively and sensitively visualize small biomolecules, organelles such as lysosomes, neutral molecules, metal ions, anions through cellular imaging, fluorescent probes can help shed light on the physiological and pathophysiological path ways. Since these biomolecules are produced in low concentrations in the biochemical pathways, general analytical techniques either fail to detect or are not sensitive enough to differentiate the relative concentrations. During my Ph.D. study, I exploited synthetic organic techniques to design and synthesize fluorescent probes with desirable properties such as high water solubility, high sensitivity and with varying fluorescent quantum yields. I synthesized a highly water soluble BOIDPY-based turn-on fluorescent probe for endogenous nitric oxide. I also synthesized a series of cell membrane permeable near infrared (NIR) pH activatable fluorescent probes for lysosomal pH sensing. Fluorescent dyes are molecular tools for designing fluorescent bio imaging probes. This prompted me to design and synthesize a hybrid fluorescent dye with a functionalizable chlorine atom and tested the chlorine re-activity for fluorescent probe design. Carbohydrate and protein interactions are key for many biological processes, such as viral and bacterial infections, cell recognition and adhesion, and immune response. Among several analytical techniques aimed to study these interactions, electrochemical bio sensing is more efficient due to its low cost, ease of operation, and possibility for miniaturization. During my Ph.D., I synthesized mannose bearing aniline molecule which is successfully tested as electrochemical bio sensor. A Ferrocene-mannose conjugate with an anchoring group is synthesized, which can be used as a potential electrochemical biosensor.

Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can’t be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications. Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.

Bionano Electronics: Magneto-Electric Nanoparticles for Drug Delivery, Brain Stimulation and Imaging Applications

Nanoparticles are often considered as efficient drug delivery vehicles for precisely dispensing the therapeutic payloads specifically to the diseased sites in the patient’s body, thereby minimizing the toxic side effects of the payloads on the healthy tissue. However, the fundamental physics that underlies the nanoparticles’ intrinsic interaction with the surrounding cells is inadequately elucidated. The ability of the nanoparticles to precisely control the release of its payloads externally (on-demand) without depending on the physiological conditions of the target sites has the potential to enable patient- and disease-specific nanomedicine, also known as Personalized NanoMedicine (PNM). In this dissertation, magneto-electric nanoparticles (MENs) were utilized for the first time to enable important functions, such as (i) field-controlled high-efficacy dissipation-free targeted drug delivery system and on-demand release at the sub-cellular level, (ii) non-invasive energy-efficient stimulation of deep brain tissue at body temperature, and (iii) a high-sensitivity contrasting agent to map the neuronal activity in the brain non-invasively. First, this dissertation specifically focuses on using MENs as energy-efficient and dissipation-free field-controlled nano-vehicle for targeted delivery and on-demand release of a anti-cancer Paclitaxel (Taxol) drug and a anti-HIV AZT 5’-triphosphate (AZTTP) drug from 30-nm MENs (CoFe2O4-BaTiO3) by applying low-energy DC and low-frequency (below 1000 Hz) AC fields to separate the functions of delivery and release, respectively. Second, this dissertation focuses on the use of MENs to non-invasively stimulate the deep brain neuronal activity via application of a low energy and low frequency external magnetic field to activate intrinsic electric dipoles at the cellular level through numerical simulations. Third, this dissertation describes the use of MENs to track the neuronal activities in the brain (non-invasively) using a magnetic resonance and a magnetic nanoparticle imaging by monitoring the changes in the magnetization of the MENs surrounding the neuronal tissue under different states. The potential therapeutic and diagnostic impact of this innovative and novel study is highly significant not only in HIV-AIDS, Cancer, Parkinson’s and Alzheimer’s disease but also in many CNS and other diseases, where the ability to remotely control targeted drug delivery/release, and diagnostics is the key.

Detection of Carbapenemase Production in a Collection of Enterobacteriaceae with Characterized Resistance Mechanisms from Clinical and Environmental Origins by Use of Both Carba NP and Blue-Carba Tests

Rapid-screening methods to confirm the presence of resistance mechanisms in multidrug-resistant bacteria are currently recommended. Carba NP and Blue-Carba tests were evaluated in carbapenemase-producing Enterobacteriaceae from hospital (n = 102) and environmental (n = 57) origins for detecting the different molecular classes among them. Both methods showed to be fast and cost-effective, with high sensitivity (98% to 100%) and specificity (100%), and may be easily introduced in the routine laboratory.

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.

Anti-inflammatory effect of exercise training in subjects with type 2 diabetes and the metabolic syndrome is dependent on exercise modalities and independent of weight loss

We investigated the effect of different exercise modalities on high sensitivity-C reactive protein (hs-CRP) and other inflammatory markers in patients with type 2 diabetes and the metabolic syndrome. Eighty-two patients were randomized into 4 groups: sedentary control (A); receiving counseling to perform low-intensity physical activity (B); performing prescribed and supervised high-intensity aerobic (C) or aerobic + resistance (D) exercise (with the same caloric expenditure) for 12 months. Evaluation of leisure-time physical activity and assessment of physical fitness, cardiovascular risk factors and inflammatory biomarkers was performed at baseline and every 3 months. Volume of physical activity increased and HbA1c decreased in Groups B–D. VO2max, HOMA-IR index, HDL-cholesterol, waist circumference and albuminuria improved in Groups C and D, whereas strength and flexibility improved only in Group D. Levels of hs-CRP decreased in all three exercising groups, but the reduction was significant only in Groups C and D, and particularly in Group D. Changes in VO2max and the exercise modalities were strong predictors of hs-CRP reduction, independent of body weight. Leptin, resistin and interleukin-6 decreased, whereas adiponectin increased in Groups C and D. Interleukin-1β, tumor necrosis factor-α and interferon-γ decreased, whereas anti-inflammatory interleukin-4 and 10 increased only in Group D. In conclusion, physical exercise in type 2 diabetic patients with the metabolic syndrome is associated with a significant reduction of hs-CRP and other inflammatory and insulin resistance biomarkers, independent of weight loss. Long-term high-intensity (preferably mixed) training, in addition to daytime physical activity, is required to obtain a significant anti-inflammatory effect.

Urinary osteopontin predicts incident chronic kidney disease, while plasma osteopontin predicts cardiovascular death in elderly men

Background and objectives The matricellular protein osteopontin is involved in the pathogenesis of both kidney and cardiovascular disease. However, whether circulating and urinary osteopontin levels are associated with the risk of these diseases is less studied. Design, setting, participants and measurements A community-based cohort of elderly (Uppsala Longitudinal Study of Adult Men [ULSAM; n=741; mean age: 77 years]) was used to study the associations between plasma and urinary osteopontin, incident chronic kidney disease, and the risk of cardiovascular death during a median of 8 years of follow-up. Results There was no significant cross-sectional correlation between plasma and urinary osteopontin (Spearman rho=0.07, p=0.13). Higher urinary, but not plasma osteopontin, was associated with incident chronic kidney disease in multivariable models adjusted for age, cardiovascular risk factors, baseline glomerular filtration rate (GFR), urinary albumin/creatinine ratio, and inflammatory markers interleukin 6 and high sensitivity C-reactive protein (Odds ratio for 1-standard deviation (SD) of urinary osteopontin, 1.42, 95% CI (1.00-2.02), p=0.048). Conversely, plasma osteopontin, but not urinary osteopontin, was independently associated with cardiovascular death (multivariable hazard ratio per SD increase, 1.35, 95% CI (1.14-1.58), p<0.001, and 1.00, 95% CI (0.79-1.26), p=0.99, respectively). The addition of plasma osteopontin to a model with established cardiovascular risk factors significantly increased the C-statistics for the prediction of cardiovascular death (p<0.002). Conclusions Higher urinary osteopontin specifically predicts incident chronic kidney disease while plasma osteopontin specifically predicts cardiovascular death. Our data put forward osteopontin as an important factor in the detrimental interplay between the kidney and the cardiovascular system. The clinical implications, and why plasma and urinary osteopontin mirror different pathologies, remains to be established.

The catheter to vein ratio and rates of symptomatic venous thromboembolism in patients with a peripherally inserted central catheter (PICC): a prospective cohort study.

BACKGROUND: Peripherally inserted central catheters (PICCs) are a common vascular access device used in clinical practice. Their use may be complicated by adverse events such as venous thromboembolism (VTE). The size of the vein used for PICC insertion and thus the catheter to vein ratio is thought to be a controllable factor in the reduction of VTE rates in patients who have a PICC. However, an optimal catheter to vein ratio for PICC insertion has not previously been investigated to inform clinical practice. OBJECTIVES: To determine the effect of the catheter to vein ratio (proportion of the vein measured at the insertion point taken up by the catheter) on rates of symptomatic VTE in patients with a PICC and identify the optimal ratio cut-off point to reduce rates of this adverse event. METHOD: Adult patients waiting for PICC insertion at a large metropolitan teaching hospital were recruited between May and December 2013. Vein diameter at the PICC insertion site was measured using ultrasound with in-built callipers. Participants were followed up at eight weeks to determine if they developed symptomatic VTE. RESULTS: Data were available for 136 patients (50% cancer; 44% infection; 6% other indication for PICC). Mean age was 57 years with 54% males. There were four cases of confirmed symptomatic VTE (two involving the deep veins, one peripheral vein and one pulmonary embolism). Receiver operator characteristic (ROC) analysis determined that a 45% catheter to vein ratio was the ideal cut off point to maximise sensitivity and specificity (AUC 0.761; 95% CI 0.681-0.830). When a ratio of 46% or above was compared to one that was less than or equal to 45% using a log binomial generalised linear model it was found that participants with a catheter to vein ratio >45% were 13 times more likely to suffer VTE (relative risk 13, p=0.022; CI 1.445-122.788). CONCLUSION: It was found that a 45% catheter to vein ratio was the optimal cut off with high sensitivity and specificity to reduce the risk of VTE. However, further research is needed to confirm these results as although adequately powered; the number of cases of VTE was comparatively small, resulting in wide confidence intervals.

A practical MEMS gravimeter

The ability to measure tiny variations in the local gravitational acceleration allows – amongst other applications – the detection of hidden hydrocarbon reserves, magma build-up before volcanic eruptions, and subterranean tunnels. Several technologies are available that achieve the sensitivities required (tens of μGal/√Hz), and stabilities required (periods of days to weeks) for such applications: free-fall gravimeters, spring-based gravimeters, superconducting gravimeters, and atom interferometers. All of these devices can observe the Earth tides; the elastic deformation of the Earth’s crust as a result of tidal forces. This is a universally predictable gravitational signal that requires both high sensitivity and high stability over timescales of several days to measure. All present gravimeters, however, have limitations of excessive cost (£70 k) and high mass (<8 kg). In this thesis, the building of a microelectromechanical system (MEMS) gravimeter with a sensitivity of 40 μGal/√Hz in a package size of only a few cubic centimetres is discussed. MEMS accelerometers – found in most smart phones – can be mass-produced remarkably cheaply, but most are not sensitive enough, and none have been stable enough to be called a ‘gravimeter’. The remarkable stability and sensitivity of the device is demonstrated with a measurement of the Earth tides. Such a measurement has never been undertaken with a MEMS device, and proves the long term stability of the instrument compared to any other MEMS device, making it the first MEMS accelerometer that can be classed as a gravimeter. This heralds a transformative step in MEMS accelerometer technology. Due to their small size and low cost, MEMS gravimeters could create a new paradigm in gravity mapping: exploration surveys could be carried out with drones instead of low-flying aircraft; they could be used for distributed land surveys in exploration settings, for the monitoring of volcanoes; or built into multi-pixel density contrast imaging arrays.

A micro-extraction technique using a new digitally controlled syringe combined with UHPLC for assessment of urinary biomarkers of oxidatively damaged DNA

The formation of reactive oxygen species (ROS) within cells causes damage to biomolecules, including membrane lipids, DNA, proteins and sugars. An important type of oxidative damage is DNA base hydroxylation which leads to the formation of 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG) and 5-hydroxymethyluracil (5-HMUra). Measurement of these biomarkers in urine is challenging, due to the low levels of the analytes and the matrix complexity. In order to simultaneously quantify 8-oxodG and 5-HMUra in human urine, a new, reliable and powerful strategy was optimised and validated. It is based on a semi-automatic microextraction by packed sorbent (MEPS) technique, using a new digitally controlled syringe (eVolH), to enhance the extraction efficiency of the target metabolites, followed by a fast and sensitive ultrahigh pressure liquid chromatography (UHPLC). The optimal methodological conditions involve loading of 250 mL urine sample (1:10 dilution) through a C8 sorbent in a MEPS syringe placed in the semi-automatic eVolH syringe followed by elution using 90 mL of 20% methanol in 0.01% formic acid solution. The obtained extract is directly analysed in the UHPLC system using a binary mobile phase composed of aqueous 0.1% formic acid and methanol in the isocratic elution mode (3.5 min total analysis time). The method was validated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), extraction yield, accuracy, precision and matrix effect. Satisfactory results were obtained in terms of linearity (r2 . 0.991) within the established concentration range. The LOD varied from 0.00005 to 0.04 mg mL21 and the LOQ from 0.00023 to 0.13 mg mL21. The extraction yields were between 80.1 and 82.2 %, while inter-day precision (n=3 days) varied between 4.9 and 7.7 % and intra-day precision between 1.0 and 8.3 %. This approach presents as main advantages the ability to easily collect and store urine samples for further processing and the high sensitivity, reproducibility, and robustness of eVolHMEPS combined with UHPLC analysis, thus retrieving a fast and reliable assessment of oxidatively damaged DNA.

Metabolic syndrome and associated factors in a population-based sample of schoolchildren in Colombia: The FUPRECOL Study

In contrast to the definition of metabolic syndrome (MetS) in adults, there is no standard definition of MetS in pediatric populations. We aimed to assess the differences in the prevalence of MetS in children and adolescents aged 9–17 years in the city of Bogota (Colombia) using four different operational definitions for these age groups and to examine the associated variables. A total of 673 children and 1,247 adolescents attending public schools in Bogota (54.4% girls; age range 9–17.9 years) were included. The prevalence of MetS was determined by the definitions provided by the International Diabetes Federation (IDF) and three published studies by Cook et al., de Ferranti et al., and Ford et al. The prevalence of MetS was 0.3%, 6.3%, 7.8%, and 11.0% according to the IDF, Cook et al., Ford et al., and de Ferranti et al. definitions, respectively. The most prevalent components were low high-density lipoprotein cholesterol and high triglyceride levels, whereas the least prevalent components were abdominal obesity and hyperglycemia. Overall, the prevalence of MetS was higher in obese than in non-obese schoolchildren. In conclusion, MetS diagnoses in schoolchildren strongly depend on the definition chosen. These findings may be relevant to health promotion efforts for Colombian youth to develop prospective studies and to define which cut-offs are the best indicators of future morbidity.

QUANTIFICATION OF CALCIUM IN SPARKLING WINES BY INFRARED SPECTROSCOPY

Markets are increasingly competitive and the companies feel the urge to improve their manufacturing processes. Blending that with a larger control of quality and safety it was created a need to develop new methods of analysis each time more accurate, faster and with lower costs. Alentejo is a region with a wide variety of soils, most of them are rich in calcium and potassium. In the production of sparkling wine many wineries use encapsulated yeast in alginate beads, instead of the traditional method, champenoise. The first method is faster, allowing a more versatile production, reducing the risk of contamination and features organoleptic characteristics similar to the traditional method (yeast free). However, encapsulated yeast spheres should be only used if the base wine matches a number of features, among them calcium content. In this study the calcium content in the wine was determined by atomic absorption spectroscopy (AAS) and by near-infrared spectroscopy. The AAS is a high sensitivity method clearly produces a reliable result, however it is very time consuming and produces great quantities of environmental waste, therefore the possibility of using near-infrared spectroscopy as a method was studied to be a fast, simple and clean alternative to the AAS. It was obtained a calibration model with a variation coefficient higher than 0.80 which indicates that the near-infrared spectroscopy as an adequately alternative the ASS.

Oral food perception and polyphenol-rich foods acceptance - the importance of knowing individuals saliva characteristics for promoting consumption

Polyphenols are widely present in fruits, vegetables, cereals and beverages. Their study gained scientific interest because of their beneficial effects on health. Although there is currently no official dietary recommendation for polyphenol intake, health professionals recommend the consumption of 5-8 daily portions of fruits and vegetables. This is not always achieved and, despite possible causes associated to practical schedule difficulties, the aversive bitter and astringent sensations associated to polyphenols may also lead to avoidance. As such, a better understanding on mechanisms responsible for differences among people, in polyphenol oral perception, is needed for promoting healthier choices. Saliva has been linked to polyphenol consumption. We have previously observed, in animal models, changes in salivary proteome induced by tannin-enriched diets. Moreover, differences in astringency perception were attributed to differences in salivary protein composition. In a recent experiment, we observed differences among individuals with dissimilar tannic-acid perception: people with high sensitivity for the oral sensations elicited by tannins have higher amounts of salivary cystatins and lower capacity to maintain their levels after tannic-acid ingestion. Additionally, and similarly to previous studies, salivary amylase was observed to be involved in tannin perception. In this presentation, oral cavity characteristics influencing the perception of polyphenol-containing foods will be discussed.

PRISM (Polarized Radiation Imaging and Spectroscopy Mission): an extended white paper

PRISM (Polarized Radiation Imaging and Spectroscopy Mission) was proposed to ESA in May 2013 as a large-class mission for investigating within the framework of the ESA Cosmic Vision program a set of important scientific questions that require high res- olution, high sensitivity, full-sky observations of the sky emission at wavelengths ranging from millimeter-wave to the far-infrared. PRISM’s main objective is to explore the distant universe, probing cosmic history from very early times until now as well as the structures, distribution of matter, and velocity flows throughout our Hubble volume. PRISM will survey the full sky in a large number of frequency bands in both intensity and polarization and will measure the absolute spectrum of sky emission more than three orders of magnitude bet- ter than COBE FIRAS. The data obtained will allow us to precisely measure the absolute sky brightness and polarization of all the components of the sky emission in the observed frequency range, separating the primordial and extragalactic components cleanly from the galactic and zodiacal light emissions. The aim of this Extended White Paper is to provide a more detailed overview of the highlights of the new science that will be made possible by PRISM, which include: (1) the ultimate galaxy cluster survey using the Sunyaev-Zeldovich (SZ) e↵ect, detecting approximately 106 clusters extending to large redshift, including a char- acterization of the gas temperature of the brightest ones (through the relativistic corrections to the classic SZ template) as well as a peculiar velocity survey using the kinetic SZ e↵ect that comprises our entire Hubble volume; (2) a detailed characterization of the properties and evolution of dusty galaxies, where the most of the star formation in the universe took place, the faintest population of which constitute the di↵use CIB (Cosmic Infrared Background); (3) a characterization of the B modes from primordial gravity waves generated during inflation and from gravitational lensing, as well as the ultimate search for primordial non-Gaussianity using CMB polarization, which is less contaminated by foregrounds on small scales than thetemperature anisotropies; (4) a search for distortions from a perfect blackbody spectrum, which include some nearly certain signals and others that are more speculative but more informative; and (5) a study of the role of the magnetic field in star formation and its inter- action with other components of the interstellar medium of our Galaxy. These are but a few of the highlights presented here along with a description of the proposed instrument.

Canopy-scale biophysical controls of transpiration and evaporation in the Amazon Basin.

Canopy and aerodynamic conductances (gC and gA) are two of the key land surface biophysical variables that control the land surface response of land surface schemes in climate models. Their representation is crucial for predicting transpiration (λET) and evaporation (λEE) flux components of the terrestrial latent heat flux (λE), which has important implications for global climate change and water resource management. By physical integration of radiometric surface temperature (TR) into an integrated framework of the Penman?Monteith and Shuttleworth?Wallace models, we present a novel approach to directly quantify the canopy-scale biophysical controls on λET and λEE over multiple plant functional types (PFTs) in the Amazon Basin. Combining data from six LBA (Large-scale Biosphere-Atmosphere Experiment in Amazonia) eddy covariance tower sites and a TR-driven physically based modeling approach, we identified the canopy-scale feedback-response mechanism between gC, λET, and atmospheric vapor pressure deficit (DA), without using any leaf-scale empirical parameterizations for the modeling. The TR-based model shows minor biophysical control on λET during the wet (rainy) seasons where λET becomes predominantly radiation driven and net radiation (RN) determines 75 to 80 % of the variances of λET. However, biophysical control on λET is dramatically increased during the dry seasons, and particularly the 2005 drought year, explaining 50 to 65 % of the variances of λET, and indicates λET to be substantially soil moisture driven during the rainfall deficit phase. Despite substantial differences in gA between forests and pastures, very similar canopy?atmosphere "coupling" was found in these two biomes due to soil moisture-induced decrease in gC in the pasture. This revealed the pragmatic aspect of the TR-driven model behavior that exhibits a high sensitivity of gC to per unit change in wetness as opposed to gA that is marginally sensitive to surface wetness variability. Our results reveal the occurrence of a significant hysteresis between λET and gC during the dry season for the pasture sites, which is attributed to relatively low soil water availability as compared to the rainforests, likely due to differences in rooting depth between the two systems. Evaporation was significantly influenced by gA for all the PFTs and across all wetness conditions. Our analytical framework logically captures the responses of gC and gA to changes in atmospheric radiation, DA, and surface radiometric temperature, and thus appears to be promising for the improvement of existing land?surface?atmosphere exchange parameterizations across a range of spatial scales.