ABSTRACT In the context of increasing incidence of stroke (but also an increasing rate of survival), non-invasive brain stimulation techniques (NIBS) are more frequently used for patients with post-stroke aphasia (PWA) and post-stroke depression (PSD). NIBS techniques, modulating brain plasticity, might offer valid, alternative therapeutic strategies. The aim is to reach a better outcome because treatment of aphasia can also improve post-stroke depression and vice versa. Based on two literature reviews on NIBS effects on PSD and post-stroke aphasia the conclusion is that, although the field is relatively new, and many more investigations with larger samples of patients are required, transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) clinical application is well tolerated, safe, and feasible. Starting from these encouraging data, we used a combination of TMS and electroencephalography (EEG) to explore the excitability modulation before and after active (20 sessions) and sham (20 sessions) tDCS in a double-blind crossover experiment. Four chronic non fluent PWA underwent 8 weeks of verbal exercises coupled with tDCS over the perilesional areas close to the left inferior frontal gyrus. To evaluate changes induced by tDCS, TMS-EEG responses over Brodmann area 6 (BA6) were computed using five different parameters. In addition, these data were compared with those recorded from a matched control group. The results indicated a slight improvement after tDCS stimulation (as compared to sham) for patients with Broca’s aphasia, but not for those with global aphasia. Also, TMS-evoked EEG responses recorded from the ipsilesional hemisphere were abnormal in individuals with chronic post-stroke aphasia (slower and simple responses with higher amplitudes) when compared to responses from the contralesional hemisphere and from the control group. Critically, the Global Mean Field Power (GMFP), Local Mean Field Power (LMFP) and Natural Frequency values were modulated by anodal tDCS. Despite these interesting results, further data are needed in order the obtain more direct, stronger evidence linking behavioral tDCS effects and neurophysiological data.

Stroke is the main cause of adult motor disability. Nevertheless, recent meta-analyses show that the theoretical models conceived to explain the post-stroke brain reorganization are inaccurate and therefore misleading in laying the theoretical foundation for rehabilitation protocols. Mixed results are reported especially in works investigating the excitability properties of the stroke injured brain. Shedding light on the reasons that brought to such mixed results is the central topic of this doctoral thesis. In particular, this confounding evidence is here discussed and tackled in the light of recent works employing brain-state dependent stimulation protocols. These works have been of paramount importance, as they showed that the effects of non-invasive stimulation (TMS and/or rTMS) on the hand knob of the motor cortex depend on the instantaneous sensorimotor state. This local state is largely determined by the phase of the mu-alpha oscillations, with the negative peak representing a high excitability condition. Brain-state dependent results show that controlling for the local state at the moment of stimulation is crucial in order to reduce variability in studies investigating cortical excitability: an approach that has never been employed in stroke literature, so far. In this doctoral thesis, new evidence is provided on affected and unaffected hemispheres’ excitability properties depending on the local state at the moment of stimulation. This previously uncontrolled state-dependent variability is here proposed as one of the factors at the basis of the mixed results in stroke literature. Furthermore, the current models aimed at explaining post-stroke brain reorganization do not take into account factors that recent works suggest might contribute to stroke recovery. In fact, it is here suggested that: interhemispheric inhibition should not be interpreted as competition, structural reserve should be assessed also at the level of the corpus callosum, diaschisis processes should be taken into account and structural and functional connectivity patterns should be included in patients’ assessment. Finally, the excitability properties of the stroke brain have been often inferred comparing stroke patients’ with young healthy controls’. In this regard, it is here proposed that only healthy peers should be included in the control groups, as brain structural changes due to healthy aging have an impact on corticospinal excitability. The aforementioned functional and structural issues are addressed in the following chapters by means of different techniques (i.e. TMS-EEG, MRI, MEG). In particular, in Chapter 1, a new framework of post-stroke brain reorganization is proposed, in which previously over-looked factors are suggested to be essential in the understanding of the potential plastic changes following stroke. Specifically, a new account where interhemispheric inhibition is interpreted in terms of integration and not competition, is supported. Moreover, the proposed framework includes recent pieces of evidence suggesting that structural reserve should be evaluated in the individual patient not only at the level of the cortex, but also in the different sections of the callosum. Finally, it is proposed that structural damage is not static, but rather dynamic as it continues also after the stroke episode through dischiasis processes. In Chapter 2, new knowledge is provided on the different excitability properties of the two hemispheres of stroke patients. In this chapter, TMS-EEG data of stimulation on both the affected and unaffected motor cortex in severe chronic strokes are analysed with a brain-state dependent approach. For the first time, it is shown that the excitability properties of the affected and unaffected hemispheres differ as the local state at the moment of stimulation influences the two hemispheres’ response differently. In particular, the strong and simplified TMS-evoked response in the affected hemisphere, previously reported in severe patients, is shown to depend on a disruption of the differentiation between the high and low excitability states of the motor cortex, determined by the instantaneous phase of alpha oscillations. This low differentiation between excitability states in the affected hemisphere should be systematically investigated, as it could be a potential feature of patients who experience poor recovery. Furthermore, in Chapter 3, connectivity at the individual alpha peak is investigated in a big cohort of healthy participants, in a resting state MEG dataset. This work was implemented because alpha connectivity networks have been shown to predict stroke recovery. For this reason, there is a necessity to reliably assess connectivity at alpha before and after rehabilitation, as this could be informative on the efficacy of rehabilitation. Specifically, it is shown that using complementary phase-coherence metrics is more effective to estimate connectivity patterns at source level. This compound approach is proposed as a tool to better control the modulatory effects of rehabilitation stimulation protocols, in order to identify which are the changes in activity patterns that are potentially responsible for recovery. Finally, in Chapter 4 brain structural changes associated with healthy aging are investigated in a big cohort of participants aged between 18 and 90 years old, both in terms of cortical thinning and cortical myelin concentration loss. In particular, given recent evidence on the relationship between cortical myelin content and corticospinal excitability, it is shown that age-dependent myelin loss occurs mostly at the level of the premotor, motor and sensory cortices. These structural changes need to be taken into account when stroke patients are compared with controls. In fact, since stroke patients are often in their elderly, these age-related structural changes need to be controlled by including only age-matched healthy participants in control groups, as this is not often a fulfilled criterion in stroke studies. To conclude, this doctoral thesis proposes that the current models’ inaccuracy depends on 1) patients’ individual structural and functional factors that have not been taken into account in previous models of brain reorganization post-stroke (Chapter 1), 2) brain-state dependent variability in stimulation effects that have not been controlled for in stroke literature (Chapter 2), 3) a lack of a systematic method to assess the effects of stimulation rehabilitation protocols (Chapter 3) and 4) structural brain changes due to healthy aging, that affect also the stroke brain, and that are not taken into account when patients are compared with young controls in corticospinal excitability studies (Chapter 4). To the author’s knowledge, this is the first work aimed at explaining mixed results in stroke literature from different perspectives and using different neuroimaging techniques for functional and structural anomalies, exploiting recent brain-state dependent approaches for the analysis of stroke patients’ data.

The use of biomarkers in Alzheimer’s disease (AD) has been fundamental for early diagnosis. Currently, biomarkers in use for clinical purposes assess the presence or quantify molecular markers of the disease, i.e., ß-amyloid or Tau protein, or quantify the medial-temporal atrophy caused by the disease. Neuroimaging techniques such as structural, functional and diffusion magnetic resonance imaging and positron-emission tomography have been essential in showing how Alzheimer’s disease pathology spreads within resting-state networks, ultimately impairing their functioning. However, neuroimaging techniques provide indirect measures that do not capture the physiological status of the affected cerebral tissues. Neurophysiological techniques, such as transcranial magnetic stimulation (TMS) and electroencephalography (EEG), are established techniques that can be used in combination to capture both the status of a target cortex and its connections through the brain through TMS-evoked potentials (TEPs). Therefore, TEPs have gained momentum as a possible novel AD biomarker. In the last decade, a specific five-phase framework for the development of novel AD biomarkers has been developed, with the goal of standardizing the steps needed to bring a measure from research to clinical practice. Phase 1 for TEPs, concerning the rationale of using them as a biomarker in AD, could be considered completed, while most of the research is now focusing on phase 2. In this phase, the ability of a measure to distinguish between healthy elderly individuals and AD patients is assessed, together with the reliability and replicability of the measure. In this thesis, we address several aims of phase 2 by testing whether early TEP responses could be used to differentiate between healthy elderly, prodromal, late-onset, and early-onset AD. Then, we test the sensitivity of TEPs to different preprocessing pipelines to assess their robustness. Third, we review the current literature on TEP reliability and describe which tests are missing for this measure to enter clinical practice. Finally, we propose a tool to promote replicability in noninvasive brain stimulation paradigms, such as TMS–EEG. We conclude that despite a solid rationale for the employment of TEPs in clinical practice, several methodological issues need to be addressed before TEPs can gain clinical utility.

Le interazioni sociali richiedono che un agente sia in grado di selezionare ed elaborare informazioni ambientali rilevanti, che sia situato in un contesto complesso, e che interagisca con altri agenti, rispettando le opportunità e i vincoli di contesto. Riconoscere noi stessi e gli altri come agenti intenzionali è un passaggio cruciale per il processo generale di comprensione sociale e, in particolare, per la nostra capacità di percepire le intenzioni e gli scopi altrui. Tali competenze sociali sostengono il nostro sviluppo fisico, cognitivo e affettivo promuovendo interazioni adattive. Di conseguenza, una disfunzione di tali competenze può compromettere gravemente l’autonomia e la qualità di vita. Si ritiene che un sistema distribuito medi la percezione di agentività e degli stati mentali altrui, ma la struttura interna dei processi che costituiscono la nostra capacità di comprendere i nostri simili e di interagire adeguatamente è tuttora per buona parte sconosciuta. Il progetto ha come obiettivo indagare le fasi iniziali di tali processi e, in particolare, l’elaborazione precoce di cues sociali (social affordances) per la detezione di agentività e opportunità d’interazione in contesti sociali. È strutturato in tre studi principali: il primo mira a esplorare i correlati elettrofisiologici (ERPs e dati di source localization) dell’elaborazione di informazioni visive per la detezione di agentività in interazione; il secondo mira a indagare possibili marcatori (ERPs) del profilo delle competenze di comprensione sociale associate alla sindrome di Williams; il terzo ha testato, tramite TMS, il ruolo causale di rTPJ nel mediare l’elaborazione pre-riflessiva di agentività e intenzionalità nel comportamento osservato. / Social interactions require an agent to be able to select and process relevant environmental information, to be situated in a complex context and to interact with other agents, according to the opportunities and boundaries of that context. Sensing ourselves and detecting others as intentional agents is a crucial step for the overall social understanding process and, in particular, for our ability to perceive others’ intentions and goals. Those social skills foster our physical, cognitive and affective development by promoting adaptive interactions. Consequently, a dysfunction of such skills can seriously affect the autonomy and quality of life. A distributed system is thought to subserve the perception of agency and others’ mental states, but the internal structure of processes that constitute our ability to understand our similars and interact adequately is still largely unknown. This project aimed at investigating early stages of those processes and, in particular, the initial elaboration of social cues (social affordances) for the detection of agentivity and opportunities for interaction in social situations. It is structured in three main empirical studies: the first one aimed at looking electrophysiological correlates (ERPs and source localization data) of visual information processing for the detection of agency in interactions; the second one aimed at looking for possible markers (ERPs) of the uneven profile of basic WS social understanding; the third one tested the causal role of rTPJ in mediating pre-reflective processing of agency and intentionality from observed behaviour by means of TMS.