Atualmente os gliomas representam cerca de 81% dos tumores cerebrais malignos, com aumento na incidência tanto em crianças, como em adultos acima dos 45 anos. Um número elevado de receptores neuroquinina tipo 1 (NK-1) estão expressos em células de glioma, estando a ligação da Substância P (SP) a esses receptores, envolvida no desenvolvimento e progressão desse tipo de tumor. A SP quelada ao DOTA (SP-DOTA), radiomarcada, vem sendo testada para utilização na terapia de gliomas, sendo o lutécio-177 (177Lu), devido a seu menor alcance tecidual, o radioisótopo mais indicado para tumores localizados em áreas críticas do cérebro. No entanto, estudos indicam a necessidade da adição de um excesso de metionina para prevenção da oxidação peptídica da SP-DOTA-177Lu, visando aumentar a estabilidade e a capacidade de ligação às células tumorais. Para superar esse desafio, surge a perspectiva da utilização de um novo análogo da SP, com estrutura modificada, para prevenir a oxidação peptídica. Neste contexto, o objetivo desse trabalho foi estudar a marcação de um novo análogo da SP com 177Lu e caracterizar suas propriedades in vitro e in vivo, visando a obtenção de um radiofármaco inédito e com potencial aplicação na terapia de tumores cerebrais e realizar estudos preliminares de marcação deste novo análogo com Ítrio-90 (90Y). O novo análogo foi obtido pela troca do aminoácido metionina (Met) pelo aminoácido norleucina (Nle) na posição 11 da cadeia peptídica da SP, sendo esses peptídeos denominados respectivamente SP(Met11)-DOTA e SP(Nle11)-DOTA. Após análise da oxidação peptídica dos dois peptídeos, os parâmetros da radiomarcação da SP(Nle11)-DOTA, com 177LuCl3, foram estudados para determinar a melhor condição de marcação. As estabilidades in vitro da SP(Nle11)-DOTA-177Lu sob refrigeração (2-8°C), no freezer (-20°C) e em soro humano (37°C) foram determinadas após radiomarcação com alta atividade, quanto ao uso de agentes estabilizantes e após diluição. A SP(Nle11)-DOTA também foi radiomarcada com 90Y, utilizando-se a condição padrão determinada, sendo a estabilidade in vitro da SP(Nle11)-DOTA-90Y sob refrigeração (2-8°C) e em freezer (-20°C), avaliada após radiomarcação com alta atividade e quanto a utilização de agente estabilizante. A capacidade de ligação in vitro às células tumorais (U-87 MG e M059J) e a biodistribuição in vivo em camundongos BALB/c sadios foram determinadas para a SP(Nle11)-DOTA-177Lu e comparadas à SP(Met11)-DOTA-177Lu. A ligação às proteínas plasmáticas e a biodistribuição em camundongos Nude com modelo tumoral também foram avaliadas. Os resultados obtidos na análise da oxidação peptídica comprovaram a importância da adição de excesso de metionina para prevenção da oxidação peptídica e indicaram uma alta estabilidade da SP(Nle11)-DOTA, durante e após o processo de radiomarcação. A adição de 148 MBq (4 mCi) da solução de 177LuCl3 em HCl 0,05N à 10 μg de SP(Nle11)-DOTA diluída em tampão acetato de sódio 0,4 M pH 4,5 seguida pela incubação a uma temperatura de 90ºC por 30 minutos, sob agitação de 350 rpm foi definida com condição padrão de marcação. O congelamento (-20°C), o uso de agentes estabilizantes e a diluição apresentaram-se como métodos efetivos para garantir uma alta estabilidade in vitro da SP(Nle11)-DOTA-177Lu, após a marcação com alta atividade. Bons resultados também foram observados para a marcação da SP(Nle11)-DOTA com 90YCl3 e para a estabilidade in vitro da SP(Nle11)-DOTA-90Y, após congelamento (-20°C) e quando utilizado ácido gentísico como estabilizante. A SP(Nle11)-DOTA-177Lu apresentou uma boa especificidade pelas células tumorais, principalmente pelas células de glioma humano M059J, sugerindo que a substituição do aminoácido metionina por norleucina na posição 11 não compromete a capacidade de ligação da SP(Nle11) às células tumorais. Uma baixa porcentagem de ligação às proteínas plasmáticas e um rápido clareamento sanguíneo foram observados para a SP(Nle11)-DOTA-177Lu, sendo esse radiofármaco eliminado preferencialmente por via renal. A SP(Nle11)-DOTA-177Lu apresentou uma boa estabilidade in vivo e se mostrou incapaz de atravessar a barreira hematoencefálica, sendo seu uso indicado por injeção intratumoral ou intracavitária. O estudo de biodistribuição em animais com modelo tumoral, mostrou que esse radiofármaco se liga às células tumorais por ligações receptor específicas. Com base nesse dados conclui-se que a SP(Nle11)-DOTA-177Lu, apresenta-se como um radiofármaco inédito que devido às suas propriedades in vitro e in vivo favoráveis, apresenta potencial aplicação na terapia de tumores cerebrais, representando uma nova possibilidade dentro do limitado arsenal terapêutico para esse tipo de tumor. / Currently gliomas represent about 81% of malignant brain tumors with increased incidence in children and in adults over 45 years. A large number of type 1 neurokinin receptor (NK-1) are expressed in glioma cells, being the binding of substance P (SP) to these receptors, involved in the development and progression of this tumor type. The SP conjugated at DOTA chelator (SP-DOTA), radiolabeled, have been tested for use in the treatment of gliomas, and the lutetium-177 (177 Lu), due to its lower tissue range, has been the most suitable radioisotope for tumors located in critical areas brain. However, studies indicate the necessity of adding an excess of methionine to prevent the peptide SP-DOTA-177Lu oxidation in order to increase the stability and capacity to bind to tumor cells. To overcome this challenge, there is the prospect of using a new analog of SP with a modified structure, to prevent peptide oxidation. In this context, the aim of this work was study the labeling of a new analog of SP with 177Lu and characterize their properties in vitro and in vivo, in order to obtain a novel radiopharmaceutical with potential application in brain tumor therapy, and perform preliminary studies labeling of this new analog with yttrium-90 (90Y). The new analog was obtained by replacement of the amino acid methionine (Met) by the amino acid norleucine (Nle) at position 11 of the peptide chain of SP, and these peptides were called SP(Met11)-DOTA and SP(Nle11)-DOTA respectively. After analysis of the oxidation for the two peptides, the radiolabeling parameters of the SP(Nle11)-DOTA with 177LuCl3 were studied to determine the best labeling condition. The SP(Nle11)-DOTA was also radiolabeled with 90Y, using standard condition, and the stability in vitro of the SP(Nle11)-DOTA-90Y assessed under refrigeration (2-8 °C) and under freezing (-20° C), after radiolabeling with high activity and use of stabilizing agent. The stabilities in vitro of the SP (Nle11)-DOTA-177Lu under refrigeration (2-8 °C), under freezing (-20 °C) and in human serum (37 °C) were determined after radiolabeling with high activity, with use of stabilizing agents and after dilution. The ability of in vitro binding to tumor cells (U-87 MG and M059J) and the biodistribution in vivo in healthy BALB/c mice were determined for the 177Lu-DOTA-SP(Nle11) and compared to 177Lu-DOTA-SP(Met11). The plasma protein binding and biodistribution in Nude mice with tumor model were also evaluated. The results obtained from analysis of oxidation for the two peptides confirmed the importance of adding excess methionine to prevent peptide oxidation and indicated a high stability of the DOTA- SP(Nle11), during and after the radiolabeling process. The addition of 148 MBq (4 mCi) of 177LuCl3 solution in 0.05N HCl at 10 μg DOTA-SP(Nle11) diluted in 0.4 M sodium acetate buffer pH 4.5 followed by incubation at a temperature of 90 °C for 30 minutes under constant agitation to 350 rpm was defined as standard labeling condition. The freezing (-20 °C), the use of stabilizing agents and the dilution were presented as effective methods to ensure high stability in vitro 177Lu-DOTA-SP(Nle11), after labeling with high activity. Good results were also observed for labeling DOTA-SP(Nle11) with 90YCl3 and for stability in vitro of the 90Y-DOTA-SP(Nle11) after freezing (-20 °C) and when gentisic acid was used as a stabilizer. The 177Lu-DOTA-SP(Nle11) showed good specificity to tumor cells, particularly human glioma cells (M059J), suggesting that substitution of the amino acid norleucine for methionine at position 11 does not compromise the capacity of SP(Nle11) binding to tumor cells. A low percentage of plasma protein binding and rapid blood clearance were observed for the 177Lu-DOTA-SP(Nle11), being this radiopharmaceutical preferably eliminated by the kidney. The 177Lu-DOTA-SP(Nle11) showed good stability in vivo and inability to cross the blood brain barrier, being its use indicated through intratumoral or intracavitary injection. The biodistribution studies in animals with tumor model showed that the radiopharmaceutical binds to the tumor cells by specific receptor binding. Based on this data was concluded that the 177Lu-DOTA-SP(Nle11), can be presented as a novel radiopharmaceutical that due to its favorable properties in vitro and in vivo, presents a potential application in the therapy of brain tumors, representing a new possibility within the limited therapeutic options for this type of tumor.

Nossa hipótese é de que os efeitos pró-inflamatórios iniciais da ativação do receptor ativado por protease 2 (PAR2) na articulação temporomandibular (ATM) sejam mediados por mecanismos neurogênicos. A análise por imunofluorescência revelou um alto grau de imunorreatividade ao PAR2 em aferentes primários trigeminais da ATM. Além do mais, a imunorreatividade ao PAR2 também foi observada na camada íntima da sinóvia, além de co-localizar com o marcador neuronal PGP9.5 e o neuropeptídeo substância P. A injeção intra-articular de agonistas PAR2 na ATM induziu um aumento dependente da dose no extravasamento plasmático, influxo de neutrófilos e indução de alodinia mecânica. O bloqueio farmacológico de receptors NK1 inibiu o aumento no extravasamento plasmático, influxo de neutrófilos e alodinia induzido pela ativação do PAR2. Em conclusão, a ativação do PAR2 é pró-inflamatório na ATM, via mecanismos neurogênicos envolvendo receptores NK1, sugerindo que o PAR2 é um importante componente da resposta imunológica inata na ATM. / We hypothesised that the early pro-inflammatory effects of proteinase-activated receptor 2 (PAR2) activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR2 in retrogradely labelled trigeminal ganglion neurons. Furthermore, PAR2 immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance P-containing peripheral sensory nerve fibres. The intra-articular injection of PAR2 agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx and induction of mechanical allodynia. The pharmacological blockade of NK1 receptors abolished PAR2-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR2 activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK1 receptors. This suggests that PAR2 is an important component of innate neuro-immune response in the TMJ.

Pneumonia is the second leading cause of disability-adjusted life-years lost worldwide and the eighth leading cause of death in the United States. Traumatic brain injury (TBI) patients have classically been considered immunosuppressed, but recent research reported that mild head trauma patients have reduced incidence of pneumonia compared to blunt trauma patients. Using our mild TBI model followed by bacterial pneumonia, we investigated the effect of neuronal signaling on innate immune function. To test whether any mild injury primes host immune responses to pneumonia, we generated a mild tail trauma (TT) model. mTBI mice showed protection from bacterial pneumonia while TT mice did not. Using an FDA-approved neurokinin-1 receptor (NK1R) antagonist, aprepitant, we confirmed our previous findings that substance P (SP) is a key mediator of enhanced resistance to pneumonia. Blocking NK1R showed that mTBI-induced release of SP augments pulmonary neutrophil recruitment and microbicidal activity to pulmonary bacterial pathogens. In TT mice, NK1R agonism enhanced the same neutrophil functions, further supporting the hypothesis. No differences were found between mTBI and TT neutrophils’ ability to phagocytose, generate oxidative burst, or acidify phagosomes. However, neutrophils from mTBI mice produced more neutrophil extracellular traps in response to bacterial challenge. These studies show that neurokinin signaling in our model contributes to enhanced bacterial clearance. Cholinergic anti-inflammatory pathway signaling though the α7 nicotinic acetylcholine receptor (α7 nAChR) is also a critical component of improved survival. Blockade of α7 nAChR abrogated the mTBI survival benefit. Mimicking cholinergic signaling using α7 nAChR agonist recapitulated the mTBI reduced pro-inflammatory cytokine production and improved survival. No physiologic differences emerged within 24h following pneumonia, but mTBI and α7 agonist treated mice had significantly lower TNFα in bronchoalveolar fluid, suggesting reduced injurious pulmonary inflammation. However, replacing early TNFα during pneumonia did not increase mortality. Western blot analysis showed downregulation of HMGB1 release in mTBI mice, suggesting that vagal cholinergic signaling reduces late mediators of organ damage. Our experiments show that mTBI enhances resistance to pneumonia by activating the vagus nerve signaling through neurokinin and cholinergic pathways. Translation of these findings could be innovative solutions to fighting or preventing infections.

A técnica de Mobilização Neural (MOB) é um método não invasivo que demonstrou tanto na pesquisa básica, como na pesquisa clinica ser eficaz na redução da sensibilidade à dor. O presente, estudo visa examinar os efeitos da MOB na disfunção locomotora, na força muscular, nas alterações morfológicas no nervo isquiático e nas alterações moleculares induzida pela constrição crônica (CCI) do nervo isquiático de ratos Wistar. Para analisar a disfunção locomotora utilizamos o índice funcional do nervo Isquiático (IFC). Para analisar a força muscular, o sistema Biopac System. A ultraestrutura do nervo foi analisada pela técnica de microscopia eletrônica de transmissão e as alterações moleculares por meio de ensaios de Western blot. Ao finalizarmos os tratamentos com MOB os animais foram eutanasiados e os tecidos como, nervo isquiático, gânglios das raizes posteriores (DRG L4-L6) e Susbtância Cinzenta Periaquedutal (PAG) foram retirados. Os DRG´s foram processados pela técnica de Western Blot para a detecção da substância P (SP), receptor de potencial transitório vanilóide tipo I (TRPV1) e receptores opióides dos tipos µ (MOR), δ (DOR) e k (KOR). Com relação a PAG, analisamos somente os receptores opióides por Western Blot. Nossos resultados demonstraram uma reverção da disfunção locomotora induzida pela CCI após a MOB e aumentou 172% a força do músculo tibial anterior nos animais tratados quando comparado com os animais do grupo CCI. Nossos estudos sobre a ultraestrutura do nervo isquiático demonstraram intenso processo de degeneração Waleriana após a CCI e regeneração após a MOB. Podemos sugerir um papel importante da MOB na modulação da expressão da SP e do TRPV 1. Sobre os receptores DOR e KOR no DRG, não encontramos alterações estatísticas entre os grupos, mas observamos um aumento da expressão de MOR após a MOB. Na PAG, nós observamos uma diminuição de DOR e KOR no grupo CCI e aumento após a MOB. Por outro lado, não encontramos alterações estatíticas para o receptor MOR. Baseado nestes achados, podemos sugerir que a MOB reverte a disfunção locomotora, aumenta a força muscular, induz a regeneração do nervo isquiático, modula a SP e TRPV 1 e aumentou a expressão de MOR no DRG´s. Sugerimos ainda que, a analsegia induzida pela técnica de MOB possa ter um envolvimento também com o sistema inibitório descendente de dor resultando na inibição da transmissão do estímulo nociceptivo aferente e assim, diminuindo a dor neuropática devido influência da MOB sobre os opióides na PAG. / Neural mobilization technique (MOB) is a noninvasive method that demonstrated to be effective in reducing pain sensitivity in both clinical and research study. The present study aims to examine the effects of MOB in locomotors dysfunction, muscle strength, morphological changes in sciatic nerve and molecular changes induced by chronic constriction (CCI) of the sciatic nerve in Wistar rats. To analyze locomotors dysfunction we used the Sciatic nerve functional index (SFI). To analyze muscle strength, was used Biopac System. The nerve morphology was analyzed using electron microscopy and molecular changes through western blot assays. After MOB treatments, animals were euthanized and tissues such as, sciatic nerve, the posterior root ganglions (DRG L4-L6) and substance periaqueductal gray (PAG) were removed. The DRG were processed by western blot for detection of substance P (SP), transient receptor potential vanilloid type I (TRPV1) and opioids receptors (MOR, DOR, KOR). Regarding PAG, we analyze only opioids receptors. Our results demonstrated a full reversal of locomotors dysfunction-induced by CCI after MOB treatment and an increase of 172% on maximal tetanic muscle strength in animals treated with MOB when compared to the CCI group. Our studies on photomicrography of sciatic nerve showed an intense Wallerian degeneration process in CCI animals and an intense regeneration of myelinated fibers. In western blot assays, we identified, in DRG, an increase of SP and TRPV1 expression after CCI and a decrease of optical density after MOB treatment. Regarding opioid receptor, we did not identify statistical changes on DOR and KOR in DRG, but we observed an increased expression of MOR in CCI after MOB treatment group. In PAG analyses, we observed a decrease in DOR and KOR expression after MOB treatment when compare with CCI animals. On the other hand, we did not identify any changes on MOR receptor. Based on our findings, we suggest that treatment with neural mobilization technique it is able to reverses the locomotors dysfunction and increases maximum tetanic force of the tibialis anterior muscle after CCI. Furthermore, the same treatment was also able to induce a severe regeneration in the sciatic nerve after treatment. Still, we can suggest an important role of MOB in modulating SP and TRPV 1 expression. We suggest that antinociceptive effect induced by MOB technique can also be involved with descending pain inhibitory system resulting in inhibition of the transmission of afferent nociceptive stimulus and thereby reducing neuropathic pain because of the influence of MOB opioids in the PAG.

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease. Neuropeptides, especially vasoactive intestinal peptide (VIP) and substance P (SP), have long been considered to play key roles in UC. Among other effects, these neuropeptides have trophic and growth-modulating as well as wound-healing effects. Furthermore, whilst VIP has anti-inflammatory properties, SP has pro-inflammatory effects. It is generally assumed that the main source of SP and VIP in the intestine is the tissue innervation. It is not known whether or not they are produced in the epithelial layer. The details concerning the expressions of their receptors in UC are also, to a great extent, unclear. Apart from the occurrence of peptidergic systems in the intestine, there are also neuronal as well as non-neuronal cholinergic systems. The pattern concerning the latter is unknown with respect to UC. The studies in this thesis aimed to investigate the expression of SP and VIP and their major receptors (NK-1R and VPAC1) in UC colon, compared to non-UC colon. The main emphasis was devoted to the epithelium. A second aim was to examine for levels of these neuropeptides in blood plasma in UC. Another aim was to examine for the non-neuronal cholinergic system in UC, thus, to investigate whether there is acetylcholine production outside nerves in the UC colon. Methods used in the thesis were immunohistochemistry, in situ hybridization, enzyme immunosorbent assay, and in vitro receptor autoradiography. For the first time, mRNA for VIP and SP has here been found in the colonic epithelium. That was especially noted in UC mucosa showing a rather normal morphology, and in non-UC mucosa. Marked derangement of the mucosa was found to lead to a distinct decrease in VIP binding, and also a decrease in the expression level of VIP receptor VPAC1 in the epithelium. In general, there was an upregulation of the SP receptor NK-1R in the epithelium when the mucosa was deranged. The plasma levels of SP and VIP were higher for UC patients compared to healthy controls. There were marked correlations between the levels of the peptides in plasma, their levels in the mucosa and the degree of mucosal derangement/inflammation. A pronounced nonneuronal cholinergic system was found in both UC and non-UC colon. Certain changes occurred in this system in response to inflammation/derangement in UC. The present study shows unexpectedly that expressions for VIP and SP are not only related to the nerve structures and the inflammatory cells. The downregulation of VPAC1 expression, and the tendencies of upregulation of NK-1R expression levels when there is marked tissue derangement, may be a drawback for the intestinal function. The study also shows that there is a marked release of neuropeptides to the bloodstream in parallel with a marked derangement of the mucosa in UC. The cholinergic effects in the UC colon appear not only to be associated with nerverelated effects, but also effects of acetylcholine produced in local non-neuronal cells. The thesis shows that local productions for not only acetylcholine, but also SP and VIP, occur to a larger extent than previously considered.

Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.

Background: Achilles tendinopathy is a common clinical syndrome of chronic Achilles tendon pain combined with thickening of the tendon and impaired tendon function. Tendinopathy is often, but not always, induced by mechanical overload, and is frequently accompanied by abnormalities at the tissue level, such as hypercellularity and angiogenesis, in which case the condition is called tendinosis. In tendinosis, there are no signs of intratendinous inflammation, but occasionally increased apoptosis is observed. Tendinosis is often hard to treat and its pathogenesis is still not clear. Recently, a new hypothesis has gained support, suggesting a biochemical model based on the presence of a non-neuronal production of classically neuronal signal substances by the primary tendon cells (tenocytes) in tendinosis. The possible functional importance of these signal substances in tendons is unknown and needs to be studied. In particular, the neuropeptide substance P (SP) and catecholamines are of interest in this regard, since these substances have been found to be up-regulated in tendinosis. As both SP and catecholamines are known to exert effects in other tissues resulting in changes similar to those characteristic of tendinosis, it is possible that they have a role in tendinosis development. It is furthermore unknown what elicits the increased intratendinous neuropeptide production in tendinosis, but given that tendon overload is a prominent riskfactor, it is possible that mechanical stimuli are involved. The hypothesis of this thesis work was that intratendinous production of SP is up-regulated in response to load of Achilles tendons/tenocytes, and thatstimulation of the preferred SP receptor, the neurokinin-1 receptor (NK-1 R), aswell as stimulation of the catecholamine α2 adrenoreceptors, contribute to the hypercellularity seen in tendinosis, via increased proliferation and/or decreased apoptosis, and that SP stimulates tendon angiogenesis. The purpose of the studies was to test this hypothesis. To achieve this, two model systems were used: One in vivo (rabbit Achilles tendon overload model of tendinosis) and one in vitro (human primary Achilles tendon cell culture model). Results: In the rabbit Achilles tendon tissue, SP and NK-1 R expression was extensive in the blood vessel walls, but also to some extent seen in the tenocytes. Quantification of endogenously produced SP in vivo confirmed intratendinous production of the peptide. The production of SP by human tendon cells in vitro was furthermore demonstrated. The catecholamine synthesizing enzyme tyrosine hydroxylase (TH), as well as the α2A adrenoreceptor (α2A AR), were detected in the tenocytes, both in vivo in the rabbit tissue and in vitro in the human tendon cells. As a response to mechanical loading in the in vivo model, the intratendinous levels of SP increased, and this elevation was found to precede distinct tendinosis changes. The in vitro model demonstrated the same response to load, i.e. an increased SP expression, but in this case also a decrease in the NK-1 R expression. In the in vivo model, exogenously administered SP, as well as clonidine (an α2 AR agonist), accelerated tenocyte hypercellularity, an effect that was not seen when administrating a specific α2A AR antagonist. Exogenous administration of SP also resulted in intratendinous angiogenesis and paratendinous inflammation. In the in vitro model, both SP and clonidine had proliferative effects on the human tenocytes, specifically mediated via NK-1R and α2A AR, respectively; both of which in turn involved activation/phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Exogenously administered SP, in Anti-Fas induced apoptosis of the tenocytes in vitro, confirmed SP to have an anti-apoptotic effect on these cells. This effect was specifically mediated via NK-1 R and the known anti-apoptotic Akt pathway. Conclusions: In summary, this thesis concludes that stimulation of NK-1 R and α2A AR on tenocytes, both in vitro and in vivo, mediates significant cell signalling effects leading to processes known to occur in tendinosis, including hypercellularity. The pathological role of the hypercellularity in tendinosis is still unclear, but it is likely to affect collagen metabolism/turnover and arrangement, and thereby indirectly tendon biomechanical function. Additional evidence is here provided showing that SP not only causes tenocyte proliferation, but also contributes to anti-apoptotic events. Furthermore, it was concluded that SP may be involved in the development of tendinosis, since its production is increased in response to load, preceding tendinosis, and since SP accelerates tendinosis changes, through some mechanistic pathways here delineated. These findings suggest that inhibition of SP, and possibly also catecholamines, could be beneficial in the reconstitution/normalization of tendon structure in tendinosis.

Unilateral exercise can produce certain contralateral strength effects. Deleterious events can be cross-transferred as well, as illustrated by a strict symmetry in some chronic inflammatory diseases. To date, knowledge on the effects of marked overuse of skeletal muscles is limited, and there is largely no information if unilateral overuse affects the contralateral muscles. In view of this, the present study was undertaken to test the hypothesis that unilateral muscle overuse causes alterations in tissue structure and the tachykinin system, with a focus on substance P (SP), not only in the exercised muscles, but also in the contralateral muscles. SP is a well-known neuromodulator that is known to be proinflammatory. An experimental rabbit model with unilateral muscle overuse of the soleus and gastrocnemius muscles caused by exercise via electrical muscle stimulation (E/EMS) was used. In total, 40 rabbits were randomly divided into seven groups of which two groups served as controls. The rabbits were anaesthetized and then set on a “kicking machine” to perform exercise via EMS for 2h every second day. Experimental periods for groups 1-3 were 1, 3 and 6w, respectively, whereas groups 4-6 were exercised for 1w but also subjected to injections in the peritendinous tissue with SP, NaCL, Captopril (C), an ACE inhibitor, and DL-Thiorphan (Th) which inhibits the activity of neural endopeptidase. One group was not subjected to the experiment at all. The day after the last session of E/EMS, the soleus muscle and the gastrocnemius muscle from both legs were collected for analysis. Alterations in muscle structure and the tachykinin system were analyzed with enzyme and immunohistochemical techniques, in situ hybridization and EIA methods. After 1w of E/EMS, focal areas of the exercised muscles contained a mild infiltration of inflammatory cells (myositis) and small morphological changes. After 3 and 6w of E/EMS, distinct myositis and muscle changes were bilaterally present in focal areas of both muscles. The structural changes, which mainly were observed in myositis areas, consisted of increased fiber size variability, split fibers, internal myonuclei, necrotic fibers, fibrosis, fat infiltration, and small fibers containing developmental MyHCs. Bilateral morphological changes, such as loss of axons, were also observed in nerves. In addition, expressions of tachykinin and the SP-preferred receptor, the neurokinin-1 (NK-1R), were bilaterally upregulated in nerve structures and blood vessel walls.  Infiltrating white blood cells exhibited tachykinin–like and NK-1R immunoreactivity. NK-1R immunoreactions were also found in necrotic and regenerating muscle fibers. The concentration of tachykinin (SP) was significantly increased in both soleus and gastrocnemius muscles after E/EMS. There was a significant correlation between the two sides in concentration of tachykinin and in the intensity of tachykinin-like immunoreaction in blood vessel walls. The muscle fiber size and capillary supply of fibers were bilaterally decreased after 3w of EMS. The myositis areas contained an increased number of vessels with a larger size than capillaries, while areas with increased amount of connective tissue contained a very low number of capillaries. A bilateral fiber type shift against a lower proportion of slow MyHCI fibers and higher proportion of fast MyHCII fibers was observed in both muscles. The local injections of C+Th and SP+C+Th led to marked structural changes in the muscle tissue and marked increased NK-1R and tachykinin-like immunoreactivity in the myositis areas and increased tachykinin concentration in the tissue. In conclusion, the repetitive unilateral muscle overuse caused by E/EMS led overtime to muscle injury and myositis. The affected areas contained both degenerative and regenerative alterations in the muscle tissue and nerves, and an upregulation of the tachykinin system. Most interestingly, the changes not only occurred in the exercised side, but also in the homologous contralateral muscles. The tachykinin system appears to be an important factor in the processes of crossover effects.

Bioactive peptides constitute a major class of signaling molecules in animals and have been shown to play a role in diverse physiological processes, including hypertension, appetite and sleep. As a result, knowing the identity of these molecules and understanding the mechanisms by which they are regulated has basic and medical significance. In this dissertation, I describe the development and application of novel methods for discovering bioactive peptides and the molecular pathways that control their activity. Recent analyses of mammalian RNAs have revealed the translation of numerous short open reading frames (sORFs). However, it is unknown whether these translation events produce stable polypeptide products that persist in the cell at functionally relevant concentrations. In Chapter 1, I describe a study in which we used a novel mass spectrometry-based strategy to directly detect sORF-encoded polypeptides (SEPs) in human cells. This analysis identified 115 novel SEPs, which is the largest number of mammalian SEPs discovered in a single study by more than a factor of 25. We observed widespread translation of SEPs from non-canonical RNA contexts, including polycistronic mRNAs and sORFs defined by non-AUG start codons. We also found that SEPs possess properties characteristic of functional proteins, such as stable expression, high cellular copy numbers, post-translational modifications, sub-cellular localization, the ability to participate in specific protein-protein interactions and the ability to influence gene expression. Taken together, these findings provide the strongest evidence to date that coding sORFs constitute a significant human gene class. In chapter 3, I describe a study in which we combine quantitative in vivo peptidomics, classical biochemical experiments and pharmacological studies in animal models to elucidate the metabolism of the neuropeptide substance P in the spinal cord. We identified two physiological substance P metabolites: the N- terminal fragments SP(1-9) and SP(1-7). Focusing our efforts on the SP(1-9)- producing pathway, we determined that an activity sensitive to the inhibitor GM6001 is the dominant SP(1-9)-generating activity in the spinal cord. We also show that GM6001 treatment causes a nearly three-fold increase in endogenous substance P levels in the spinal cords of mice, highlighting the functional relevance of the pathway blocked by this inhibitor.

Chronic abdominal pain is a major cause of patient morbidity in inflammatory bowel diseases (IBD). A balance of pro- and anti-nociceptive factors regulating colonic dorsal root ganglion (DRG) neurons, which synapse onto second order dorsal horn neurons, are known to regulate chronic pain but the mechanisms are poorly understood. This thesis examined whether neuroanatomical remodeling of DRG central nerve terminals underlies pro-nociceptive signaling and whether subsets of immune cells source the anti-nociceptive factor, β-endorphin. To examine pro-nociceptive mechanisms, acute and chronic dextran sulfate sodium (DSS) mouse models of colitis were established and substance P (SP; marker of nociceptor terminals) immunohistochemistry used to investigate changes in immunoreactivity of DRG terminals in the thoracic dorsal horn (segments T9-T13). SP immunoreactivity was increased in the dorsal horn (4 fold; P < 0.001) and central canal (P < 0.001) following chronic colitis. In contrast, SP immunoreactivity was unchanged in acute colitis. However, five weeks later SP immunoreactivity was increased both in the dorsal horn (4 fold; P < 0.01) and central canal (P < 0.001). In the cervical spinal cord, SP immunoreactivity was not increased following colitis, suggesting that changes seen in the thoracic level were specific to signaling from colonic DRG neurons. Immunoreactivity for the SP NK1 receptor on second order neurons was also examined and a significant increase in immunoreactivity was observed on post-synaptic second order cell bodies following chronic DSS. This could provide an additional mechanism for enhanced SP neurotransmission centrally. ii The source of the anti-nociceptive mediator, β-endorphin, during chronic DSS colitis was investigated using magnetic cell sorting and flow cytometry. The number of β- endorphin expressing CD4+ (2.4 fold; P < 0.05) and CD11b+ (2.6 fold; P < 0.05) cells in mice increased following chronic colitis. These findings suggest that during colitis there is a time-dependent increase of SP immunoreactivity in thoracic DRG central terminals, which could play a role in pro- nociceptive signaling in chronic inflammation. These actions may be balanced by anti- nociceptive factors such as β-endorphin which are found in subsets of immune cells. / Thesis (Master, Physiology) -- Queen's University, 2012-08-29 16:28:41.166