José S Ramalho

Universidade NOVA de Lisboa | NOVA · Chronic Diseases Research Centre (CEDOC)

There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their pol...

CD2AP was identified as a genetic risk factor for late-onset Alzheimer’s disease (LOAD). However, how CD2AP contributes to LOAD synaptic dysfunction underlying AD memory deficits is unclear. We have shown that CD2AP loss-of-function increases β-amyloid (Aβ) endocytic production, but whether it contributes to synapse dysfunction is unknown. Because...

The intricate interplay between maternal immune response to SARS-CoV-2 and the transfer of protective factors to the fetus remains unclear. By analyzing mother:neonate dyads from second and third trimester SARS-CoV-2 infections, our study shows that neutralizing antibodies (NAbs) are infrequently detected in cord blood. We uncovered that this is du...

Familial Renal Glucosuria (FRG) is a co-dominantly inherited trait characterized by orthoglycaemic glucosuria. From 2003 to 2015 we have reported several cohorts validating SLC5A2 (16p11.2), encoding SGLT2 (Na+/glucose cotransporter family member 2), as the gene responsible for FRG. The aim of this work was to validate the variants identified in ou...

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and th...

Skin pigmentation is imparted by melanin and is crucial for photoprotection against ultraviolet radiation. Melanin is synthesized and packaged into melanosomes within melanocytes, and then transferred to keratinocytes. While the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer remain uncle...

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred – either in a membrane‐bound melanosome or as a melanosome core, i.e., melanocore. Here,...

Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentape...

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described a...

In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMCs homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cel...

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described a...

Exosomes are extracellular vesicles of endosomal origin released by virtually all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs and proteins between different cells, tissues or organs. However, the mechanisms that regulate the selective loading of cytosolic proteins into these...

Skin pigmentation relies on melanin and is crucial for photoprotection against ultraviolet radiation-induced toxicity. Melanin is synthesized and stored in melanosomes, within melanocytes and then transferred to keratinocytes. While the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer rema...

Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11...

Background: The molecular basis of familial non-medullary thyroid cancer (FNMTC) is still poorly understood, representing a limitation for molecular diagnosis and clinical management. In this study, we aimed to identify new susceptibility genes for FNMTC through whole-exome sequencing (WES) analysis of leukocyte DNAs of patients from a highly info...

Strategies targeting nucleolin have enabled a significant improvement in intracellular bioavailability of their encapsulated payloads. In this respect, assessment of the impact of target cell heterogeneity and nucleolin homology across species (structurally and functionally) is of major importance. This work also aimed at mathematically modelling t...

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred either in a membrane-bound melanosome or as a melanosome core, i.e. melanocore. Here we i...

Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11...

OBJECTIVE A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins in lysosomes. This event culminates with the loss of cell homeostasis, inflammation and cell death. In this study we propose to identify the chemical etiologica...

Introduction Age-related macular degeneration (AMD) is a degenerative retinal disease that affects central vision. Most of their phenotypical features are believed to be associated with the dysfunction of retinal pigment epithelium (RPE). The accumulation of damaged proteins in aged RPE is associated with disruption of proteolytic pathways and exoc...

Gap junctions (GJ) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct communication between adjacent cells. Cx43 ubiquitination has been suggested to induce the internalization of GJs, as well as the recruitment of the autophagy receptor p62 to mediate binding to LC3B and degradation by macroautophagy. In this report...

Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inacti...

Breast cancer is the first cause of cancer-related mortality among women worldwide, according to the most recent estimates. This mortality is mainly caused by the tumors’ ability to form metastases. Cancer cell migration and invasion are essential for metastasis and rely on the interplay between actin cytoskeleton remodeling and cell adhesion. Ther...

Rab and Arl guanine nucleotide-binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G prot...

The main aim of this work was to study the usefulness of human β-defensins 2 (BD-2) and 3 (BD-3), which are part of the innate immune system, in the treatment of infected ischemic skin flaps. We investigated the effect of transducing rat ischemic skin flaps with lentiviral vectors encoding human BD-2, BD-3, or both BD-2 and BD-3, to increase flap s...

The regulation of organelle transport by the cytoskeleton is fundamental for eukaryotic survival. Cytoskeleton motors are typically modular proteins with conserved motor and diverse cargo-binding domains. Motor:cargo interactions are often indirect and mediated by adaptor proteins, for example, Rab GTPases. Rab27a, via effector melanophilin (Mlph),...

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but...

Primers for qPCR gene expression analysis. (DOCX)

Ccbe1 knockdown does not seem to affect normal Brachyury expression pattern between days 0 and 6. qPCR analysis at days 0, 2, 4 and 6 of the general mesoderm marker brachyury (BraT). Analysis was performed in two individual Ccbe1 KD ESC clones (Clone 1 and Clone 2) and relative expression is compared to day 0 of each cell type. Mean ± SEM of three...

Fig S1. All CF1 primary cells express cytokeratins confirming their epithelial origin.

Fig S3. Treatment with 2‐FF does not induce cell death of CF1_T cell line.

Fig S5. TB2 primary breast IDC cells treated with 2‐FF compound loses functional E‐selectin ligands.

Fig S2. CF1_T cells continue to express E‐selectin ligands and α1,3/4‐FUTs after the immortalization process.

Fig S4. CF1_T cell line treated with 2‐FF compound loses the expression of E‐selectin ligands and sLeX/A glycans.

Breast cancer tissue overexpresses fucosylated glycans, such as sialyl-Lewis X/A (sLeX/A), and α-1,3/4-fucosyltransferases in relation to increased disease progression and metastasis. These glycans in tumor circulating cells mediate binding to vascular E-selectin, initiating tumor extravasation. However, their role(s) in breast carcinogenesis is st...

Melanin transfer from melanocytes to keratinocytes and subsequent accumulation in the supra-nuclear region is a critical process in skin pigmentation and protection against ultraviolet radiation. We have previously proposed that the main mode of transfer between melanocytes and keratinocytes is through exo/endocytosis of the melanosome core, termed...

Background: The great majority of thyroid carcinomas derive from the thyroid follicular cells and are designated as “Non-Medullary Thyroid Carcinomas” (NMTC). NMTC may present as a familial form, being designated as FNMTC (Familial Non-Medullary Thyroid Carcinoma). In FNMTC families, patients frequently have thyroid cancer together with benign lesi...

Microtubules and F-actin, and associated motor proteins, are considered to play complementary roles in long- and short-range organelle transport. However, there is growing appreciation that myosin/F-actin networks can drive long-range transport. In melanocytes myosin-Va and kinesin-1 have both been proposed as long-range centrifugal melanosome tran...

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We per...

Author Synucleinopathies are neurodegenerative diseases characterized by the abnormal accumulation of a neuronal protein called alpha-Synuclein (aSyn). The normal function of this protein in the cell remains unclear, but plays a role in synaptic function and plasticity, cell differentiation and vesicular trafficking. During disease, aSyn is believ...

Summary of the effect of traffic players on oligomerization and aggregation of aSyn. (DOCX)

Silencing of specific phosphotransferase genes does not affect aSyn oligomerization but alters the distribution of oligomers. Upon silencing of ALS2CR7 or PSPH, aSyn aggregates are seen within cells. Silencing of STK32B and PPP2R5E leads to reduced fluorescence in the nucleus. In addition, a ring of fluorescent signal surrounding the nucleus is obs...

Silencing of selected hits alters aSyn aggregation and cellular homeostasis. A. Quantification of the number of aSyn inclusions per cell. 3 different shRNAs per gene were used. The number of inclusions was divided in the following categories: no inclusions (gray), less than 10 inclusions (light green) and more than 10 inclusions (dark green). B. Cy...

Overexpression of Rab8b at different steps of aSyn aggregation. H4 cells with no aSyn or stable for aSyn-BiFC (green) were transfected with Rab8b-WT,–Q67L and–T22N constructs. To promote the formation of aSyn inclusions, cells were triple-transfected with aSynT, Synphilin-1 and the same constructs referred above. 48 h post-transfection, media with...

List of screened genes in the RNAi assay using aSyn-BiFC stable cells as readout. A. Human trafficking collection B. Human kinases / phosphatases collection. (DOCX)

Silencing of RAB27A alters aggregation of aSyn. A. Quantification of relative fluorescence intensity of aSyn-BiFC stable H4 cells submitted to silencing of RA27A. Three different shRNAs were tested. B. mRNA levels of cells submitted to silencing of the RAB27A normalized to control cells (cells transduced with scrambled shRNA). C. Immunoblotting ana...

Silencing of specific trafficking and kinase genes modifies aSyn oligomerization. A. Quantification of relative fluorescence intensity of aSyn-BiFC stable H4 cells submitted to silencing of RAB8B, RAB11A, RAB13, RAB39B, CAMK1 DYRK2, CC2D1A, CLK4 and SYTL5. Three different shRNAs were used per gene. B. mRNA levels of cells submitted to silencing of...

Overexpression of Rab11a at different steps of aSyn aggregation. H4 cells with no aSyn or stable for aSyn-BiFC (green) were transfected with constructs expressing Rab11a-WT, Q70L and -S25N. To promote the formation of aSyn inclusions, cells were triple-transfected with aSynT, Synphilin-1 and the same constructs referred above. 48 h post-transfectio...

Overexpression of Rab13 at different steps of aSyn aggregation. H4 cells with no aSyn or stable for aSyn-BiFC (green) were transfected with constructs expressing Rab13-WT, –67L and–T22N. To promote the formation of aSyn inclusions, cells were triple-transfected with aSynT, Synphilin-1 and the same constructs referred above. 48 h post-transfection,...

Overexpression of SLP5 at different steps of aSyn aggregation. H4 cells with no aSyn or stable for aSyn-BiFC (green) were transfected with (A) SLP5 or (B) empty vector. To promote the formation of aSyn inclusions, cells were triple-transfected with aSynT, Synphilin-1 and the same constructs referred above. 48 h post-transfection, media with no seru...

Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of influenza A genome, imposes a problem to assembly, as it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, event associated with influenza...

Striking evidence associates cancer stem cells (CSCs) to the high recurrence rates and poor survival of patients with muscle-invasive bladder cancer (BC). However, the prognostic implication of those cells in risk stratification is not firmly established, mainly due to the functional and phenotypic heterogeneity of CSCs populations, as well as, to...

Autophagy plays an important role in the defense against intracellular pathogens. However, some microorganisms can manipulate this host cell pathway to their advantage. In this study, we addressed the role of host cell autophagy during Plasmodium berghei liver infection. We show that vesicles containing the autophagic marker LC3 surround parasites...

Protein Kinase Domain Containing, Cytoplasmic (PKDCC) is a protein kinase which has been implicated in longitudinal bone growth through regulation of chondrocytes formation. Nevertheless, the mechanism by which this occurs remains unknown. Here, we identified two new members of the PKDCC family, Pkdcc1 and Pkdcc2 from Xenopus laevis. Interestingly...

The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic vaccination, but with limited succe...

Chaperone-Mediated Autophagy is a selective form of autophagy. Recently, the degradation of a newly identified CMA substrate, the HIF1A transcription factor, was found to be regulated by the ubiquitin ligase STUB1. In this study we show, for the first time, that K63 ubiquitination is necessary for CMA degradation of HIF1A in vitro and in vivo. Addi...

Research Day UA

In a variety of cells, secretory processes require the activation of both Rab27a and L-type channels of the Ca V 1.3 subtype. In the retinal pigment epithelium (RPE), Rab27a and Ca V 1.3 channels regulate growth-factor secretion towards its basolateral side. Analysis of murine retina sections revealed a co-localization of both Rab27a and Ca V 1.3 a...

Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an impo...

In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively [1-8]. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In...

Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is...

The Arf-like protein Arl13b has been implicated in ciliogenesis and Sonic hedgehog signaling. Furthermore, we have previously shown that it regulates endocytic recycling traffic and interacts with actin. Herein, we report that the non-muscle myosin heavy chain IIA, also known as Myh9, is an Arl13b effector. Moreover, we found that both proteins loc...

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photo-protection against ultra-violet damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism...

Retinal degeneration disorders affect millions of people worldwide and constitute a set of incurable diseases that gradually progress to blindness. In many of these diseases several retinal cells layers are affected. In particular, the physiological function of the retinal pigment epithelium (RPE) is impaired. The RPE is a compact layer of pigmente...

Bypassing all the research advances in the last decades, cancer remains as a major public health problem affecting more than 1.5 million (M) new individuals each year just in the USA, and killing more than 0.5 M. Recent research emphasized the major role of cancer stem cells (CSCs) in the metastatic disease, the main cause of cancer patients mortal...

The transcription factor HIF1 is mostly regulated by the oxygen-dependent proteasomal degradation of the labile subunit HIF1A. Recent data showed degradation of HIF1A in the lysosome through chaperone-mediated autophagy (CMA). However the molecular mechanism involved has not been elucidated. This study shows that the KFERQ-like motif, that has been...

We have previously described the development of novel sterically stabilized F3-targeted pH-sensitive liposomes, which exhibited the ability to target both cancer and endothelial cells. Herein, the therapeutic potential of those liposomes was assessed upon encapsulation of a siRNA against a well-validated molecular target, PLK1. Treatment of prostat...

The main goal of this work was to assess in vitro the potential of Polo-like kinase gene (PLK-1) as a molecular target within the tumor microenvironment, namely in both cancer cells of tumors of different histological origin and endothelial cells from angiogenic blood vessels, upon silencing with anti-PLK-1 siRNA. In addition, the effect of Plk-1 d...

Aim: The design of novel F3-targeted liposomes with adequate features for systemic administration, to enable efficient intracellular delivery of siRNA toward both cancer and endothelial cells from angiogenic blood vessels. Materials & methods: Cellular association studies were performed by flow cytometry. Gene silencing was evaluated with eGFP-o...

Intracellular recycling pathways play critical roles in internalizing membrane and fluid phase cargo and in balancing the inflow and outflow of membrane and cell surface molecules. To identify proteins involved in the regulation of endocytic recycling, we used an shRNA trafficking library and screened for changes in the surface expression of CD1a a...

We attempted to find new proteins involved in the regulation of the endocytic recycling pathway used by CD1a, a MHC Class I-like lipid antigen-presenting molecule that follows an endocytic recycling pathway similar to that used by MHC Class I and other cargo internalized independently of clathrin. For that, a shRNA library comprising the main famil...

Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endo...

Rab14 localization in Plasmodium-infected macrophages. Macrophages were transfected with a plasmid encoding GFP-Rab14 and infected with P. berghei-RFP. After 15 minutes of incubation, cells were washed, chased for different time points and analyzed by confocal laser scanning microscopy. Representative images are shown for 5 (A), 15 (B) and 30 (C) m...

RT-qPCR primers. Primer sequences used to characterize Rab GTPases by RT-qPCR. (DOC)

Rab14 silencing increases phagocytosis of P. berghei and Rab14 overexpression decreases phagocytosis of the parasite. (A) Macrophages were transfected with siRNA for Rab14 or siRNA control for 48 h, and infected with GFP-P. berghei. Macrophages were transfected with GFP-Rab14 DNA (B) or GFP (C) for 8 h and then infected with RFP-P. berghei. (A) Col...

siRNA sequences. siRNA sequences of the siGenome Smartpool for Rab14 and Rab9a. (DOC)

The obligate intracellular liver stage of the Plasmodium parasite represents a bottleneck in the parasite life cycle and remains a promising target for therapeutic intervention. During this stage, parasites undergo dramatic morphological changes and achieve one of the fastest replication rates among eukaryotic species. Nevertheless, relatively litt...

The present work aimed at designing a lipid-based nanocarrier for siRNA delivery toward two cell sub-populations within breast tumors, the cancer and the endothelial cells from angiogenic tumor blood vessels. To achieve such goal, the F3 peptide, which is specifically internalized by nucleolin overexpressed on both those sub-populations, was used a...