BFMV - Teses de Doutoramento
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- Safebed : improving safety and sustainability of recycled manure solids as bedding material for dairy cowsPublication . Pires, Ana José de Oliveira Nunes; Oliveira, Maria Manuela Castilho Monteiro de; Bexiga, José Ricardo Dias; Fangueiro, David PauloABSTRACT - SAFEBED: IMPROVING SAFETY AND SUSTAINABILITY OF RECYCLED MANURE SOLIDS AS BEDDING MATERIAL FOR DAIRY COWS - Antimicrobial resistance (AMR) and the environmental impact of livestock production pose significant challenges to sustainable dairy farming. The use of recycled manure solids (RMS) as bedding material offers economic and environmental benefits but raises concerns regarding microbial safety, including the persistence of antimicrobial-resistant bacteria and potential pathogens. This study evaluated the effects of biochar supplementation in RMS to mitigate AMR risks, enhance microbial safety, and reduce environmental emissions. Fresh RMS samples were collected from a commercial dairy farm and divided into five treatments: non-supplemented RMS (C-), acidified RMS (C+), and RMS supplemented with 2.5%, 5%, or 10% biochar. Samples were incubated for 30 days at ambient temperature, with assessments on Days 0, 5, 15, and 30. Two assays were conducted: one in a humid period (April–May) and another in a dry period (June–July). Microbial analyses included bacterial quantification, Escherichia coli and Enterococcus spp. isolation, AMR and virulence profiling, and 16S rDNA sequencing for microbial community shifts. In parallel, the environmental study examined RMS characteristics, nutrient retention, and greenhouse gas (GHG) emissions. Results from the bacterial quantification revealed no statistically significant reduction in E. coli and Enterococcus spp. counts with biochar supplementation. However, trends suggested a potential decrease in AMR and virulence factors, with reductions observed in oxytetracycline resistance and biofilm formation. In contrast, the microbiome analysis demonstrated a significant reduction in bacterial loads, with 5% biochar leading to a 59.50% decrease under humid conditions. However, Brucella spp. and Pseudomonas spp. increased in biochar amended RMS. Environmentally, the supplementation with 10% biochar significantly reduced CO₂ (32%) and N₂O (47%) emissions, stabilized nitrogen, and minimized volatilization losses. However, methane emissions were largely unaffected. Addicionally, Biochar supplementation increased micronutrient and heavy metal concentrations (except for molybdenum), as well as potassium and magnesium levels, while decreasing sodium, phosphorus, and total organic carbon content. This study highlights biochar’s potential for improving microbial safety and environmental sustainability in dairy farming. Further research should optimize biochar concentrations, assess long-term effects, and evaluate field-scale applications to fully harness its role in sustainable manure management
- Unconventional Cohesin-Dockerin binding mechanisms reveal the complexity of cellulosome assemblyPublication . Santos, Marlene Rafaela Cardoso Duarte; Gomes, Pedro Miguel Bule Dias; Prates, José António MestreABSTRACT - The plant cell wall is a vast reservoir of carbon and energy, yet its recalcitrant nature presents a significant challenge to efficient biomass conversion. In order to overcome this hurdle, certain anaerobic bacteria have evolved sophisticated multi-enzyme complexes for the efficient degradation of plant cell wall polysaccharides, termed cellulosomes. Cellulosomes are assembled via calcium-dependent, high-affinity interactions between the cohesin (Coh) modules present in non-catalytic scaffoldins and the enzyme-borne dockerin (Doc) modules. The majority of these interactions display a dual-binding mode (DBM), which enhances the structural flexibility of the cellulosome by allowing Docs to bind Cohs in two opposing orientations, thereby minimizing steric hindrance. This study focuses on the Coh-Doc systems of Bacteroides cellulosolvens and Ruminococcus flavefaciens, two bacteria with highly intricate cellulosomal architectures. While B. cellulosolvens makes extensive use of the DBM, facilitating the assembly of up to 110 enzymes into a single complex, R. flavefaciens was traditionally thought to rely solely on single-binding mode interactions, forming smaller, albeit complex, cellulosomes. Additionally, Ruminococcus albus, a ruminal bacterium that possesses several Doc-encoding genes with high homology to R. flavefaciens, lacks the necessary cohesins to form canonical cellulosomes, raising questions about its cellulose degrading mechanism. In this work, the molecular mechanisms underlying the Coh-Doc interactions in these three microorganisms were explored, revealing several key insights. First, it was discovered that type II Coh-Doc interactions in B. cellulosolvens, previously thought to function only in cell anchoring, also exhibit DBM, underscoring the organism's reliance on this versatile mechanism to assemble its highly complex cellulosome. Contrary to earlier reports, R. flavefaciens was also found to incorporate DBM through the action of the adaptor scaffoldin ScaH, introducing structural flexibility to avoid steric clashes between cellulosomal units. Furthermore, a novel binding mechanism was uncovered in which a single Coh domain could bind two Doc modules simultaneously. This novel 2:1 Coh-Doc interaction mode involves smaller, naturally truncated Doc modules. The study also demonstrated interspecies hybridization potential, with cross species Coh-Doc interactions occurring between R. flavefaciens and R. albus, suggesting a cooperative role in the rumen ecosystem. Additionally, through structure guided protein engineering, enhanced Coh-Doc binding affinities were achieved by modulating the electrostatic profiles of the binding surfaces. This research advances our understanding of cellulosome assembly, with implications for the efficient conversion of lignocellulosic biomass into biofuels, as well as potential biotechnological applications in protein engineering and molecular biology RESUMO - A biomassa vegetal representa a maior fonte de carbono orgânico do planeta. Contudo, a parede celular vegetal é altamente recalcitrante, o que dificulta o acesso e ação das enzimas envolvidas na sua degradação. Estas enzimas, genericamente designadas de Cazymes (carbohydrate active enzymes), são produzidas por diversos microrganismos e desempenham um papel crucial na conversão da biomassa vegetal. Elas encontram se organizadas em dois sistemas distintos. Os microrganismos aeróbios secretam enzimas livres no espaço extracelular, onde estas encontram e degradam o seu respetivo substrato. Em contraste, os microrganismos anaeróbios, que habitam ambientes altamente competitivos, desenvolveram uma estratégia distinta ao secretarem complexos multi-enzimáticos, denominados celulossomas, que mantêm as enzimas funcionalmente integradas, trabalhando em sinergia e acopladas à parede celular. Este mecanismo promove uma degradação mais eficiente e coordenada da parede celular vegetal, destacando-se como uma estratégia evolutiva para maximizar a conversão da biomassa. A montagem do celulossoma, que se baseia em interações proteína-proteína, tem vindo a ser alvo de estudos de natureza estrutural e funcional que conduziram à elucidação da forma como os celulossomas se organizam. A montagem destes complexos é mediada por interações de alta afinidade dependentes de cálcio entre duas proteínas modulares: as coesinas (Cohs) e as doquerinas (Docs). As coesinas são componentes modulares repetidas localizadas em proteínas estruturais não catalíticas, denominadas escafoldinas, enquanto as doquerinas estão predominantemente presentes na extremidade C-terminal das CAZymes. A interação Coh-Doc serve para fixar firmemente as CAZymes às escafoldinas, que por sua vez também podem conter um módulo doquerina que facilita a ancoragem à parede celular ou a articulação com outras escafoldinas portadoras de coesinas, formando estruturas ainda mais complexas. [...].
- Laparoscopic techniques for reproductive surgery in dogsPublication . Tavares, Inês Maria Tenreiro; Tenreiro Tavares, Inês; Rivera Barreno, Ramón; Luís, José Paulo Pacheco de SalesABSTRACT - Laparoscopic surgeries are already routine in human medicine. In companion animals, they are gradually being applied more and tutors are already requesting them. Sterilization is the most common surgery performed on companion animals. In this way, the interest in evaluating the sterilization of male and female dogs and the associated pain arose. In the first study, we compared the viability and the effects on pain and inflammation caused by a new technique for laparoscopic castration of male dogs with the classic technique. Surgical times, pain scale, blood and salivary cortisol and C-reactive protein were compared between the two groups. The laparoscopic group had significantly lower pain, cortisol, and C-reactive protein values than the classical orchiectomy group. The results suggest that this laparoscopic castration is a safe and beneficial alternative to traditional orchiectomy in dogs. Studies in human medicine have concluded that acetazolamide reduces pain associated with carbon dioxide insufflation during laparoscopic surgery. So far, no study has evaluated the action of acetazolamide for this purpose in companion animals. In the second study we compared the values of serum cortisol, salivary cortisol and the University of Melbourne Pain Scale in three groups of bitches subjected to: open ovariectomy, laparoscopic ovariectomy and laparoscopic ovariectomy with preoperative administration of acetazolamide. We found evidence that preoperative administration of acetazolamide may be beneficial in controlling postoperative pain in bitches after laparoscopic surgery. The third paper reports the successful resolution of an intra-surgical finding of teratoma in the left ovary during laparoscopic ovariectomy. This is the first reported case of a teratoma removed by laparoscopic ovariectomy
- Composição de leite de vaca e bebidas vegetais tidas como alternativa ao leitePublication . Antunes, Irene Cristina da Silva; Quaresma, Mário Alexandre Gonçalves; Bexiga, José Ricardo Dias; Pinto, Carlos AugustoNos últimos anos, a substituição do leite de vaca (LV) por bebidas vegetais (BV) tem vindo a tornar-se cada vez mais popular. Esta tese tem como objetivo comparar os perfis nutricionais do LV e das BV, focando o seu conteúdo em macro e micronutrientes. Os resultados indicam que algumas BV, como a de soja e de côco, apresentam teores energéticos e de hidratos de carbono (HC) semelhantes aos do LV. Além disso, enquanto o perfil de HC do LV se limitou à lactose ou galactose-glicose, as BV apresentaram um perfil de HC mais complexo, contendo diferentes tipos e teores de HC como a rafinose, glucose, maltose, frutose, sacarose e sorbitol. Em relação à fração lipídica, o colesterol e os ácidos gordos (AG) trans foram quantificados apenas no LV, estando ausentes nas BV. O LV e a BV de côco apresentaram predominância de AG saturados (SFA), enquanto as BV de avelã e amêndoa foram ricas em AG monoinsaturados (MUFA) e as BV de soja, arroz e aveia destacaram-se pelos seus AG polinsaturados (PUFA). No entanto, é crucial analisar os SFA no LV individualmente, pois alguns não representam risco para a saúde. Relativamente aos micronutrientes, a BV de soja apresentou teores mais elevados de Ca, Mg e Cu em relação ao LV, juntamente com teores semelhantes de K e P. Por outro lado, o Se foi quantificado apenas no LV. Todas as BV apresentaram o mesmo teor de vitamina B12 que o LV, e a BV de soja foi o tipo de BV com maior teor na maioria das outras vitaminas. Embora muitas BV tenham sido fortificadas com micronutrientes, a sua biodisponibilidade é desconhecida. Dada a variabilidade nutricional das BV, é difícil identificar uma BV que seja nutricionalmente equivalente ao LV
- Sharing more than friendship : dynamics of direct transmission of antimicrobial resistance between human families and their companion animalsPublication . Menezes, Juliana Cruz de Oliveira de; Pomba, Maria Constança Matias Ferreira; Fonseca, Andreia de Jesus Amaral Gomes BarbosaEnterobacterales that produce extended-spectrum β-lactamases (ESBLs), AmpC cephalosporinases and carbapenemases (CPE), as well as colistin-resistant strains, are important pathogens raising public health concerns due to their increasing prevalence. The prospective longitudinal study focusing on households in Portugal (PT) and the United Kingdom (UK), showed that ESBL/AmpC-producing Enterobacterales strains carriage in healthy companion animals occur at a significantly lower frequency (p-value <0.0001) compared to animals with active infections under antibiotic therapy. Whole genome sequencing (WGS) analysis identified the sharing of ESBL/AmpC-producing Enterobacterales strains between healthy companion animals and humans in two households from Portugal (n=41), involving Escherichia coli human pandemics lineages ST93, ST410 and ST457. Among companion animals undergoing antibiotic treatment due to urinary tract infection (UTI) or skin and soft tissue infection (SSTI), and their cohabiting humans, sharing of faecal ESBL/AmpC producing E. coli strains was observed in four households (PT= 2/43; UK= 2/22), with strains belonging to ST2015, ST617 and ST963. Additionally, one Portuguese household shared a multidrug-resistant (MDR) ACT-24-producing Enterobacter hormaechei subsp. hoffmannii strain. Notably, three other animals shared the clinical strains with their cohabiting humans: a ST556 Klebsiella pneumoniae, the high-risk clonal lineage ST131 E. coli strain and a ST2179 E. coli classified as Avian Pathogenic (APEC). CPE strains were only detected in animals under antibiotic treatment. These strains were positive for blaOXA-181 and blaNDM-5 genes, present in plasmids virtually identical to those found in isolates from humans, food, and the environment in other countries. MDR E. coli strains harbouring the plasmid-mediated colistin resistant gene, mcr-1, were recovered from faecal samples of companion animals and humans in Portugal, with no significant difference between healthy and infected groups (p-value=0.257). In two households with dogs diagnosed with SSTIs, sharing of mcr-1-positive ST744 E. coli strains with the owner were observed. The detection of MDR bacteria sharing between companion animals and their cohabiting humans, especially when it matched the animal's UTI/SSTI clinical strain, highlights the importance of considering household-level interventions in response to the spread of antimicrobial resistance in the community, reinforcing the need for active monitoring and stringent hygiene practices under a One Health approach
- Integrated control of avian gastrointestinal parasites : optimizing their coprological diagnosis and biocontrol using predatory fungiPublication . Lozano, João; Lozano, João Miguel Pestana; Carvalho, Luís Manuel Madeira de; Oliveira, Maria Manuela Castilho Monteiro de; Paz-Silva, AdolfoIn the last 30 years, several improvements were achieved in the control of animals’ gastrointestinal (GI) parasitism, including the development of more sensitive and precise coprological diagnosis techniques, like Mini-FLOTAC (MF), as well as the use of predatory fungi as a sustainable solution for parasite control. This project aimed to optimize the MF method for the diagnosis of avian GI parasitism, and to isolate predatory fungi and assess their potentialities in avian parasite control. First, the diagnosis of GI parasitism was performed in a poultry farm and three exotic bird collections, in Portugal mainland, using the MF technique. Eimeria spp. infections were identified in laying hens and peacocks, Capillaria spp., Trichostrongylus tenuis and Strongyloides pavonis in peacocks, as well as Libyostrongylus douglassii in ostriches and emus. Seven filamentous fungi were isolated from Galliformes feces, furtherly identified as Mucor circinelloides (n=6) and M. lusitanicus (n=1). All fungi were capable of destroying Eimeria spp. oocysts in vitro, with M. circinelloides isolate FR1 achieving the highest coccidicidal efficacy, 22%, after 14 days of exposure. All fungi were checked for potential in vitro susceptibilities to seven antiparasitic drugs (albendazole, fenbendazole, levamisole, ivermectin, lasalocid, amprolium and toltrazuril), and all were not susceptible to the tested antiparasitic drugs, independently of their concentrations. The characterization of the virulence profile of M. circinelloides FR1 revealed only the presence of genes coding for iron receptors (FOB1 and FOB2), iron permease (FTR1), ADP-ribosylation factors (ARF2, ARF6) and GTPase (CDC42), having the isolate only tested positive for lecithinase production. Oral administrations of spores of this fungal isolate were performed in laying hens and peacocks, which revealed that this fungus did not affect birds’ feces appearance and consistency (70 100% of normal feces), neither the relative abundances and alfa-diversities of their native gut bacteria and fungi. Finally, it was verified that the administration of this fungus spores to peacocks reduced significantly their Eimeria spp. fecal shedding up to 92%, after two months of trial. Results from this project point out the usefulness of MF in the coprological diagnosis of GI parasitism in birds, and provide the first report regarding the isolation of predatory fungi from bird feces. Finally, the safety and efficacy of using M. circinelloides FR1 in the integrated control of domestic and exotic birds GI parasitism was demonstrated
- Use of minimally invasive techniques in the diagnosis and characterization of lymphoid hematopoietic tumors in dogsPublication . Valente, Pâmela Cristina Lopes Gurgel; Duarte, António José de Freitas; Correia, Jorge Manuel de Jesus; Pomba, Maria Constança Matias FerreiraABSTRACT - Lymphoma is the most common hematopoietic tumor in dogs, presenting a variety of clinical and morphological forms originating from lymphoid cells. In contrast, multiple myeloma is considered rare. The introduction of minimally invasive techniques in human medicine has had a significant impact, allowing for the accurate diagnosis, screening, and monitoring of neoplasms in humans. The potential for applying these techniques to domestic animals, such as dogs with hematopoietic neoplasias, still lacks the necessary additional studies to assess their efficacy and applicability in veterinary medicine. The main objective of this work was the implementation and use of minimally invasive diagnostic techniques, such as cytology, cell block, immunocytochemistry and PCR for antigen receptor rearrangements (PARR), in the diagnosis of dogs with suspected lymphoid hematopoietic tumors. Initially, the research focused on evaluating and comparing various minimally invasive techniques used in cytological aspirates to diagnose multicentric lymphomas in 38 dogs. It was shown that these approaches make it possible to partially diagnose and characterise multicentric lymphoma in dogs, suggesting the feasibility of obtaining the necessary diagnostic material in a single consultation, which leads to significant gains in terms of cost and time savings without compromising diagnostic accuracy. This work then reinforces the importance of using cell blocks in the diagnosis of lymphoid cell neoplasms by demonstrating their potential in aiding the diagnosis of canine multiple myeloma (MM). Faced with the hypothesis of lymphoid neoplasms with a plasma cell phenotype in dogs, cell blocks from aspiration punctures of the spleen and bone marrow were taken for immunohistochemical analysis. The results of the immunophenotyping allowed the diagnosis of MM and excluded other lymphoid neoplasms. Finally, the applicability of the cytoblock and clonality assays in the diagnosis of a complex case of gastrointestinal lymphoma was evaluated. In this clinical case, the initial diagnosis observed by cytology and cytoblock techniques of liver aspirates was suggestive of lymphoma with hepatic dissemination and indicated rare co-expression of CD3 and CD20 positive lymphocytes, which was confirmed in histopathology with immunohistochemistry of endoscopic biopsies. The final diagnosis of T-cell lymphoma associated with type 2 enteropathy with hepatic infiltration was established by PARR following the detection of the monoclonal T-cell rearrangement. These results, therefore, demonstrate the utility and accuracy of minimally invasive methods in diagnosing and characterizing diferents types of lymphoid hematopoietic tumors in dogs
- Antibiotic-lipid based nanosystem as a tool to specifically target Staphylococcus aureus biofilmsPublication . Ferreira, M.C. Magda Sofia Catroga Ferreira; Aguiar, Sandra Isabel Rodrigues de; Gaspar, Maria Manuela de Jesus Guilherme; Bettencourt, Ana Francisca de Campos SimãoAbstract - Hospital acquired infections (HAIs) is one of the leading causes of death worldwide, with Staphylococcus aureus being among the most prevalent microorganisms implicated in these infections. The ability of S. aureus to form biofilms and evade the immune system, along with the emergence of multidrug-resistant strains (MDR), exacerbates the complexity of eradicating infections. Conventional therapies characterized by prolonged antibiotic regiments have a poor rate of success, mainly due to the reduced penetration of antibiotics through the biofilms and low accumulation levels at infected sites. The ineffectiveness of current treatments has stimulated extensive research into the development of innovative therapeutic approaches. To address this need, the aim of this thesis was the development of a nanosystem by incorporating an antibiotic into liposomes, taking advantage of the unique benefits offered by these nanotechnological platforms. For this purpose, we first explored the antibacterial activity of three common antibiotics in clinical use – levofloxacin (LEV), vancomycin (VCM) and rifabutin (RFB) – against a reference strain of S. aureus (ATCC®25923™) in both planktonic and biofilm states. Subsequently, the antibiotics were incorporated into liposomes with different lipid compositions, and their incorporations parameters were assessed. Free RFB displayed the most potent antibacterial effect with MIC and MBIC50 below 0.006 µg/mL, along with the highest antibiotic loading capacity when nanoformulated, preserving its antibacterial activity. Based on these results, RFB was selected for further in vitro studies and the influence of the different lipid compositions on bacterial biofilm interactions was assessed, using a biofilm transwell model and confocal scanning laser microscopy analysis. It was observed that the positively charged RFB liposomes (LIP3) exhibited the highest interaction with biofilms. Nevertheless, RFB incorporated in negatively charged liposomes with fusogenic properties (LIP1) displayed lower MBIC50 values. Preliminary safety assessment of RFB formulations towards osteoblast and fibroblast cell lines indicated that a reduction in cell viability was only observed for the LIP3. Taking this into account, LIP1 was selected to move forward. Following these findings, the potential of free RFB was validated in a collection of S. aureus clinical isolates to provide a more accurate reflection of the challenges faced in real-world settings, in both planktonic (n=114) and biofilm (n=40) states. Additionally, the antibacterial activity of RFB incorporated in our developed liposome (LIP1) was validated against a set of clinical isolates (n=40) in both states. In conclusion, all the work developed contributed to the pursuit of effective therapeutic strategies for planktonic and biofilm-associated S. aureus infections, by exploring the potential of antibiotic repurposing and incorporating them into liposomes
- Characterization of insect bite hypersensitivity in a population of Lusitano horses : contribution for future implementation of skin prick tests (SPT) in IBH diagnosisPublication . Pessoa, Vera Purificação Carvalho; Tilley, Paula Alexandra Botelho Garcia de Andrade Pimenta; Ferreira, Manuel BrancoInsect Bite Hypersensitivity (IBH) is a recurrent seasonal pruritic dermatitis affecting many horses worldwide. It is mainly a type I hypersensitivity, but type IV may also occur, to allergens present in the saliva of Culicoides spp. It is a multifactorial disease affecting different breeds, with a variable prevalence. Treatment options are very limited, but being an allergic disease IgE-mediated, there is the possibility of developing specific immunotherapy. This study aimed to characterize IBH in Lusitano horses living in Portugal mainland, through a case control study involving 30 IBH-affected animals and 30 healthy controls. Questionnaires were performed for all the horses involved in the study in order to characterize the studied population. In vivo allergen testing was evaluated by performing skin tests, both skin prick tests (SPT) and intradermal tests (IDT), using 14 specific allergens, including 13 different recombinant (r-) proteins from Culicoides nubeculosus (Cul n) or Culicoides obsoletus (Cul o) salivary glands (termed Cul n 1 to Cul n 11, Cul o 1P and Cul o 2P) as well as Cul n whole body extract (WBE). Moreover, a cluster of 12 of these horses, 6 IBH-affected and 6 from the control group, were also tested with allergens produced in insect cells (Cul n 3Bac, Cul n 4Bac) and barley (Cul n 3 Bar, Cul n 4Bar) as well as Cul o 3 and Cul o whole body extract (WBE). Furthermore, in vitro diagnostic tests have been performed, namely, serum IgEs that were measured by ELISA for the allergens Cul n 3, 4, 9, 10 and Cul o 2, 3, as well as Cul o1P and Cul o2P and in vitro sulfidoleukotriene (sLT) release assay which were carried out with Cul n WBE and Cul o WBE. In our study SPTs presented a higher discriminatory diagnostic potential than IDTs, and should be preferred, being a potential breakthrough in the establishment of more suitable allergen avoidance measures in IBH diagnosis. Regarding the in vitro tests, even though serology measurement of specific IgEs showed that IBH-affected horses had higher serum IgE levels for the significant allergens, sLT release assay performed best. It was also possible to study and identify Culicoides spp found in the stud farms, which may be related to IBH in the studied horses. The most frequently found were Obsoletus group species, C. nubeculosus and C. imicola. This study contributed to improve IBH diagnosis, representing a step forward for the future implementation of locally relevant diagnostic allergen panels and eventually for the establishment of patient tailored, component resolved specific immunotherapy
- Development of novel antibody-drug conjugated molecules for treatment of B-cell maliganciesPublication . André, Ana Filipa Santos; Silva, Frederico Nuno Castanheira Aires da; Tavares, Luís Manuel Morgado; Gonçalves, João Manuel BrazCancer is one of the leading causes of death worldwide, and is expected to continue increasing, with a projected 28.4 million cases by 2040. Dogs are also significantly affected by cancer, including NHL, which is one of the most common hematological malignancies in both species. Dogs are considered excellent models to accelerate the translation of treatments for human patients due to their similarities with humans. While immunotherapies, particularly monoclonal antibodies (mAbs), have brought specificity to cancer therapies, they are still mostly used in combination with conventional chemotherapy, which remains the standard of care for both species. However, conventional treatments still do not fully cure and have numerous adverse effects, highlighting the need for further improvement. To address this need, using the promising advantages of rabbit-derived sdAbs, we aimed to develop a platform for a novel sdAb drug delivery system for NHL treatment using the dog as an animal model. For this purpose, we explored three different drug delivery methods: ADCs, immunotoxins and immunoliposomes. For the development of ADC, we explored the potential of rabbit derived single-domain antibodies (sdAb) to selectively conjugate a payload towards cysteine at position 80. First, a rabbit-derived sdAb library against canine B-cell lymphoma receptors was subjected to in vitro and in vivo phage display. Then, VL sdAb that specifically targeted canine lymphoma cells in vitro and presented a good tumor uptake was selected for SN-38 site selective payload conjugation via its Cys80 and generated a stable and homogeneous C5- DAB-SN-38. This study validated a platform to develop novel ADCs that combine rabbit sdAbs benefits with the advantages of canine lymphoma model. Furthermore, this previously characterized and validated VL sdAb was also used to develop a new immunotoxin for the treatment of canine B-cell lymphoma. For that purpose, VL sdAb was conjugated with the PE38 toxin truncated form and tested in vitro in a canine B-cell lymphoma cells and in in vivo in a xenograft mouse model of canine lymphoma. This study validated immunotoxins as a potential treatment for canine lymphoma. Lastly, to validate a new liposome for canine lymphoma, we aimed to develop a liposome-based nanocarrier for panobinostat, using folate-targeted and non-targeted formulations. Both formulations were evaluated in canine lymphoma cells, validating liposomes as an effective treatment for canine lymphoma. In the future, our goal is to conjugate our VL sdAb to the liposome to obtain an immunoliposome for canine B-cell lymphoma. In conclusion, all the work developed contributed to the understanding of the importance of using the dog as a model and how these animals can contribute for clinical translation in the immune-oncology field