Objective: Anterior cruciate ligament (ACL) injuries are increasing in adolescents and increase the risk for early-onset knee osteoarthritis (OA). Biomarkers can be a non-invasive measure to assess physiological properties following knee injury or trauma. The objective of this thesis was to i) perform a systematic review to determine the most studied biomarkers of knee healing following ACL reconstruction (ACLR), and age of these patients, and ii) explore the feasibility of measuring these biomarkers in adolescents with ACL injuries. Design: Studies were included if i) participants underwent ACLR, and ii) at least one biomarker of healing was measured. Participant age, sample(s) collected, and biomarker(s) studied were recorded. Interleukin-6 (IL-6), c-terminal crosslinking telopeptide of type II collagen (CTX-II) and procollagen type II collagen propeptide (PIICP) were then measured using ELISA in adolescents prior to ACLR in urine (u) and synovial fluid (sf). Spearman’s Rho (rs) coefficients were calculated to determine the association between uCTX-II/sfCTX-II, and uIL-6/sfIL-6. A ratio of PIICP: CTX-II was calculated to represent the ratio of cartilage synthesis to degradation. Results: The review produced six studies evaluating healing following ACLR. IL-6 and CTX-II were the most studied (3/6 studies), and only one study included adolescents (age 19.6±4.5). Due to multiple undetectable biomarker levels, we could only report rs for uCTX-II/sfCTX-II (rs = -.200, p-value = .800, n=4). We also reported a ratio for sfPIICP: sfCTX-II (23.06 ±19.23). Conclusion: Exploring biomarkers in adolescents was motivated by their unique physiology due to puberty, and this was the first study to do so. The findings from this pilot study indicate that further analysis is required to determine optimal sample preparation. This will allow for reliable results while studying the feasibility of these biomarkers during ACLR recovery. This insight can ensure more informed decision making by clinicians clearing patients for return-to-activity.

It has long been recognized that course and efficiency of a chemical reaction is largely mediated by the short-lived transient species (excited state or radicals) which are formed as reactive intermediates during a chemical reaction. Subtle changes not only in the bonding and electronic distributions but also in the conformations and geometries of these intermediates have a dramatic influence on the reactivity. A detailed understanding of the structural and dynamical aspects of electronic excited states is therefore essential towards unraveling photoinduced natural processes and for designing novel photonic materials. Time-resolved techniques have been widely used to study the transient species (or intermediates) formed during photochemical and photophysical reactions for better understanding of the reaction mechanism and dynamics. Time-resolved absorption spectroscopy is a promising tool to study the temporal dynamics and the kinetics of photophysical processes. But the absorption spectra of species in solution usually consist of broad spectral band revealing little or no information about the structure of the transient species under investigation. Time-resolved resonance Raman (TR3) spectroscopy, on the other hand, is a potential sensitive modality, which not only allows one to study the dynamics but also provides the vibrational structure of the transient species of interest in microsecond to picosecond time scale. Moreover, by choosing the wavelength of excitation one can selectively probe the particular transient species from a complex molecular system especially a biological molecule. Thioxanthone (TX) is well known for its dramatic solvatochromic behavior and has drawn enormous attention in the recent years. The objective of present thesis has been to understand the solvent-induced structural changes on the lowest excited triplet state of TX and its derivatives. We have primarily employed nanosecond TR3 spectroscopy, a pump-probe technique, to investigate structure of the lowest excited triplet state. Transient absorption experiments have also been carried out to study the excited electronic states. In order to substantiate our experimental findings and also to get more insight into the triplet-state structure, we have performed density functional theory (DFT) calculations. The polarizable continuum solvation model has been employed to account for the solvent effect into the computation. Time dependent (TD) -DFT calculations have also been performed to get the energy and the structure of the excited states. The present thesis has been divided into eight chapters. Chapter 1 gives brief literature review on photochemistry and photophysics of TX and the introduction to the TR3 technique. In this chapter we have briefly introduced key concepts which form the basis of the thesis. Chapter 2 covers the experimental and theoretical methodologies used in the present thesis work. The major components of the TR3 spectrometer as well as the important technical aspect of the TR3 technique have been discussed in detail. In the section of the theoretical method, basic concepts of the computational method, density functional theory and key concepts related to the solvation are briefly discussed. Chapter 3 focuses on a systematic vibrational study of the ground and lowest triplet states of TX. TR3 experiments have been carried out and the observed vibrational frequencies have been assigned. It has been observed that electronic excitation distorts the molecule, enabling the increased electron delocalization in the central ring keeping the ground state symmetry intact. The largest structural reorganization is observed in the central ring of TX, consisting of an oxygen atom. Normal mode analyses show that the normal mode composition is significantly influenced by the electronic excitation. The C=C stretching and C=O stretching modes are coupled to a greater extent in the triplet state as compared to the ground state. In the ground state, the two high-frequency modes can be assigned almost exclusively to the C=O stretching and C=C stretching, whereas in the triplet state, both of these coordinates have comparable contributions to the two totally symmetric modes. Chapter 4 deals with a very unique observation of simultaneous detection of two triplets. This is the first time when two triplet states have been simultaneously deleted using TR3 experiments. We have performed TR3 experiments in wide variety of solvents differing in their polarities and hydrogen atom donor abilities. The transient Raman signal has been observed from both n - π∗ and π - π∗ triplet states simultaneously. The population ratio of the two triplet states has been found to be dependent on the solvent polarity. Additionally, the excitation wavelength study has revealed that the relative ratios of the transient Raman peaks (assigned to two different triplet states) change with the excitation wavelength. Our claim of simultaneous detection of two triplets has been reconfirmed by triplet quenching experiments carried out at different temperature. It has also been observed that the CO bond length is very sensitive to the solvent polarity and specific interactions play an important role in determining the structure of lowest triplet-state. In Chapter 5, we focus on the understanding of the effect of chlorine substitution on the lowest excited triplet state of TX. TR3 spectroscopy has been used as an experimental tool to study the vibrational structure of 2-chlorothioxanthone (CTX). TR3 results indicate the coexistence of two lowest triplet states in the thermal equilibrium akin to the parent compound. The above observation has been further substantiated by probe wavelength dependent study. The configuration of the T1 state has been assigned to π – π∗, whereas the T2 state has been ascribed as n - π∗. The population ratio of 3n - π∗ to 3 π - π ∗ triplet states has been found to be more for CTX as compared to TX which has been substantiated by the flash photolysis experiments. Chapter 6 highlights the influence of solvent effect on lowest triplet state structure of CTX. Transient absorption spectroscopy has been employed to understand the triplet state electronic structure; whereas solvent induced changes in the structure of the lowest triplet state have been studied using TR3 spectroscopy. Time-resolved absorption measurements show that solvent polarity has dramatic dependence on the wavelength of T1 - Tn absorption maximum. A good correlation between the wavelength of T1 - Tn absorption maximum and ET(30) value of the solvent is observed. TR3 experiments carried out in solvents of varying polarities indicate that the contribution of n - π∗ character to the lowest excited triplet state increases with the increase in the solvent polarity. Both transient absorption and TR3 studies reveal that specific solvent effect is more pronounced in comparison to the nonspecific solvent effect. Chapter 7 of the thesis deals with the study on the triplet state structure and solvent effect on 2-trifluoromethyl Thioxanthone. Flash photolysis in tandem with TR3 spectroscopy has been employed to understand both the electronic and the vibrational structures of this pharmaceutically important thioxanthone derivative. Experiments have also been carried out in solvents of varying polarities to study solvent-induced changes in the triplet-state electronic spectra. We have observed the coexistence of two lowest triplet states alike the parent compound. The T1 state has been assigned to π - π∗ state, whereas n - π∗ configuration has been attributed to the T2 state. The wavelength of triplet-triplet absorption maximum of the lowest triplet state has been found to be sensitive to the solvent polarity and good correlation has been observed with the ET(30) value. The transient Raman results indicate that the CF3 substitution leads to increase in the population ratio of n - π∗ and π - π ∗ triplet states. Finally, Chapter 8 contains overall summary of the thesis and future directions of the present investigation.

Chaque année, des chevaux de course décèdent de fractures complètes lors d’entrainement ou de course. A l’exercice, les charges supra-physiologiques cycliques entrainent des microfissures indiscernables par les techniques d’imagerie in vivo actuelles. Les ostéoclastes recrutés aux sites lésés induisent localement une lyse osseuse et une ostéopénie par résorption excessive, créant une concentration de contrainte à l’origine de fracture complète. Cependant, le rôle biologique des ostéoclastes dans les fractures de stress n’est à ce jour pas clairement identifié. Les objectifs de cette étude ont été d’établir des cultures in vitro d’ostéoclastes équins, corréler le nombre d’ostéoclastes avec l’isoforme 5b de l’enzyme phosphatase acide tartrate-résistante (TRACP 5b), corréler la résorption osseuse avec la TRACP-5b et le télopeptide C-terminal du collagène de type I (CTX-I) et étudier les effets de facteurs d’inflammation sur l’activité ostéoclastique. Après aspiration de moelle osseuse sternale, les cellules souches hématopoïétiques ont été isolées, conservées dans une banque cellulaire, décongelées, différenciées en ostéoclastes, avec ou sans os équin, et stimulées par des facteurs inflammatoires (IL-1β ou LPS). CTX-I et TRACP-5b ont été dosés par ELISA. Le nombre d’ostéoclastes colorés à la phosphatase acide tartrate-résistante et les aires de résorption osseuse colorées au bleu de toluidine ont été évalués. Dans les cultures d’ostéoclastes, la TRACP-5b augmentait (p < 0,0001) avec le temps et était corrélée (r = 0,63, p < 0.001) avec le nombre d’ostéoclastes. Dans les cultures d’ostéoclastes sur os, le CTX-I (p = 0,0018) et la TRACP-5b (p = 0,02) augmentaient avec le temps, étaient corrélés ensemble (r = 0,64, p < 0,002) et étaient tous deux corrélés avec la résorption osseuse (respectivement, r = 0,85, p < 0,001 et r = 0,82, p < 0,001). La stimulation inflammatoire n’a eu aucun effet significatif. Ostéoclastes équins et résorption osseuse ont été obtenus avec succès sur os équin à partir d’aspiration de moelle osseuse sternale. Pour la première fois, CTX-I et TRACP-5b ont été mesurés dans des cultures d’ostéoclastes équins. In vitro, CTX-I est un biomarqueur de résorption osseuse équine et TRACP-5b un biomarqueur du nombre d’ostéoclastes et de résorption osseuse équins. Des recherches ultérieures sont nécessaires afin d’évaluer si la TRACP 5b sérique permet la détection de toute résorption osseuse excessive chez les chevaux de course. / Every year racehorses die from complete catastrophic fractures in training and racing. Supra-physiological cyclic loads during exercise cause bone microcracks not discernible with current imaging in vivo. Osteoclasts recruited to the sites of the damage induce focal bone lysis and osteopenia by excessive resorption, creating a stress riser area susceptible to complete fracture. However, the biological impact of osteoclasts in stress fractures is not entirely understood. Our objectives were to establish in vitro cultures of equine osteoclasts, to correlate osteoclast numbers with the tartrate-resistant acid phosphatase 5b isoform (TRACP-5b), to correlate bone resorption with the TRACP-5b and the C-terminal telopeptide of type I collagen (CTX-I), as well as to investigate the effects of inflammatory factors on osteoclast activity. Following equine sternal bone marrow aspirations, hematopoietic stem cells were isolated, stored in a cell bank, thawed, differentiated into osteoclasts, with or without equine bone slices, and they finally underwent inflammatory stimulation (IL-1β or LPS). CTX-I and TRACP 5b were assayed by ELISAs. Osteoclasts stained with tartrate resistant acid phosphatase were counted and bone resorption areas stained with toluidine blue were measured. In the osteoclast cultures, the TRACP 5b increased (p < 0.0001) with time and correlated (r = 0.63, p < 0.001) with osteoclast number. In the osteoclast-bone cultures, both CTX-I (p = 0.0018) and TRACP-5b (p = 0.02) increased with time, correlated with each other (r = 0.64, p < 0.002) and both correlated with bone resorption (r = 0.85, p < 0.001 and r = 0.82, p < 0.001, respectively). The inflammatory stimuli had no significant effects. Equine osteoclasts and bone resorption were successfully induced on equine bone from sternal bone marrow aspiration. For the first time CTX-I and TRACP-5b were measured in equine osteoclast cultures. In vitro, CTX-I is a biomarker of equine bone resorption and TRACP-5b a biomarker of equine osteoclast number and bone resorption. Further investigations are required to measure the serum TRACP-5b capacity to detect excessive skeletal resorption in racehorses.